En esta memoria se presenta la Tesis Doctoral que... DESARROLLO DE UNA PLATAFORMA DE Cucumis melo

En esta memoria se presenta la Tesis Doctoral que lleva por título:
DESARROLLO DE UNA PLATAFORMA DE
TILLING EN MELÓN (Cucumis melo L.)
Escrita por Mireia González To
Director: Jordi Garcia Mas
Tutora: Mª Carme Espunya
Esta Tesis ha sido realizada en el Programa de Genòmica i Biotecnologia del IRTA
que pertenece al Centre de Recerca en Agrigenòmica (CRAG) y dentro del programa
de doctorado del Departamento de Bioquímica de la UAB.
A Enero de 2014
T
T
A la meva mare que mha ensenyat a estimar i a mantenir lalegria
"Dijo Tennyson que si pudiéramos comprender una sola flor sabríamos quiénes somos y qué es el mundo"
Jorge L. Borges
Ara tot just intento pensar en el principi d’aquesta aventura i me n’adono que jo era una persona
totalment diferent. Els agraïments moltes vegades es queden curts, les paraules tants cops no poden
copsar tot el que ha significat un gest o la companyia d’una persona. Per això vull començar dient
que, segurament, aquestes paraules mai diguin tot el que volen dir. Aquest només és un intent de
reconèixer i sobretot, agrair-vos a tots els que m'heu acompanyat en aquesta aventura de 8 anys.
En primer lloc vull agrair a les persones que han fet possible aquest periple: a la Carme Espunya per
ser la professora que em va descobrir el meu amor científic cap a les plantes. Al Jordi Garcia-Mas per
confiar en una estudiant de pràctiques que arribava a l'IRTA de Cabrils amb moltes ganes d’aprendre
i al Pere Arús per recolzar-lo en aquesta decisió. Vull agrair als que han col.laborat en aquest
projecte perquè sense ells no s'hagués pogut dur a terme: a Fitó i al COMAV. En especial a la Belén
Picó per aconseguir publicar un article d'aquest treball amb la seva dedicació i persistència.
L'època a l’IRTA ha estat un dels períodes més feliços de la meva vida. Tots els que hi éreu hi vau
contribuir; cadascú a la seva manera i durada. Els becaris (Edu (i les guerres privades d'aigua al
laboratori quan ja no hi quedava ningú), Ibo, Iria, Juan (¡Ánimo, ya no queda nada!), Julio, Alí, Claudio,
Roger, Eudald...). A tots ells els hi agraeixo la companyia, el fet de poder compartir la mica de
desesperació que tantes vegades pateix un becari i poder riure dels nostres errors. Si un no
s'equivoca, no aprèn.
Des d’aquí, d'aquest petit espai meu i propi, vull fer un reconeixement a tots els tècnics, sense
vosaltres els laboratoris no avançarien. És d'aquestes coses que tothom sap, però que crec que a
vegades cal dir en veu alta i clara.
Ells són: Fuensi (gràcies per ensenyar-me tant i per tenir tanta paciència amb el nostre estimat LICOR), Àngel (gràcies per la teva disposició i la teva alegria incondicional) Joana, Ana, Patricia, Alicia,
Dani, Manoli... En especial a la Vane, sempre al meu costat, inseparables companyes de "poyata".
Gràcies per fer-me riure (i a vegades fer-me plorar), per fer-me ballar i treballar, per ensenyar-me les
tècniques i perquè fora de la feina m’has cuidat igual que cuides als teus. Gràcies per fer-me sentir
tan estimada.
Als companys de caseta (Montse, Francesc, Olga, Ainoha, Jonatan...) perquè allà vam ser
autodidactes, vam compartir cafès i coneixements. Potser la ciència no cal que sigui tan seria, a
vegades. A la Puri, pel “cachondeo” que m’animava les tardes com a gairebé ningú, encara que li
trepitgés els "fregaos".
A tots els investigadors (Werner, Jordi, Amparo, Momo, Montse M., Montse S., Wim, Txose, Gisela,
Torben, Toni M.) i especial al Mourad per ensenyar-me que la ciència pot ser molt creativa, plena de
colors inesperats (¡Esa eterna discusión con el salmón de los geranios!) i, sobretot, per acompanyarme en tantes tardes i nits de petita solitud a Arenys en les que em cuinaves cous-cous i peix al forn
com si fos la seva pròpia germana. Al Santi Vilanova, perquè sempre m'ha cuidat i perquè en els
moments que queia ell sempre m'oferia la mà per ajudar-me a aixecar. A la Marta Pujol, no només
per tot el que l’admiro com a professional i el que he après amb ella, sinó també com a persona.
Marta, sense tu, segur, que no hagués acabat aquest projecte. Gràcies Marta.
Un dels aventatges que té la investigació és que et permet viatjar i fer estades a laboratoris d’altres
països. A Evry vaig poder treballar al costat de l'Abdelhafid Bendahmane al qui vull agrair
l’ensenyament de la tècnica del TILLING. També a la Christelle Troadec, amb qui vam compartir hores
d'aprenentatge, llargues xerrades en franco-català-anglès i molts caramels. Merci beaucoup
Christelle. La estancia en Evry fue como un "mini Erasmus" gracias a dos latinos: Nilo y Silvia. A ellos
quiero agradecer que me acogieron en su piso como una más, me cocinaron a la italiana y me
prepararon Pisco-Sour para olvidarnos entre todos de los diseños de cebadores y de las plantas de
tomate. Nilo y Silvia siempre os recuerdo, gracias.
A pesar de haber acabado las prácticas de doctorado en Marzo de 2010 y de no haber presentado
la tesis, conseguí una beca en Italia, más concretamente en Roma para trabajar con Giovanni Giuliano
en el ENEA. Considero ese período como una ampliación del doctorado y de mi aprendizaje. Per
questo non vorrei lasciare di ringraziare a Marco, Silvia, Killy, Sara, Patrizia, Elio (DT!) e Giulia
l'incredibile esperienza di vivere e lavorare a Roma. Lavorare divertendomi così tanto: ballare e bere
la birra in laboratorio con Silvia al termine di lunghe giornate di lavoro tra i pomodori e PCR. Grazie a
Giuliano per la fiducia in me e nelle mie capacità. E a tutti per rendere questo periodo uno dei giorni
più felici della mia vita. Voglio tanto bene all'Italia e gli italiani!.
Finalment i no menys important, vull agrair als amics que m’acompanyeu des de sempre i que m'heu
vist créixer i madurar en aquest projecte. Que heu aguantat les llargues i constants (i segurament
pesades ;-) converses sobre aquest tema i sempre heu confiat en mi. Crec que si confies en algú
aquella persona també confia més amb ella mateixa per això vull dir-vos que és un regal preciós el
que m'heu fet: Club Bikini (Marta, Anna P., Anna A.), PSFs!! (Martina, Jess, Raquel B. -gràcies també
pel increïble disseny de la portada, sabia que faries alguna cosa que la sentiria pròpia, gràcies
artista!- Raquel Ll., Nayibe, Isa, Nurieta, Alba, Tere...), Claudia sempre aprop estiguis on estiguis,
Humanistes! (Marta, Eva, Laia) que m'heu acompanyat i juntes hem descobert tantes coses
fantàstiques de la vida com per exemple que el coneixement és poder. Sou totes fantàstiques, dones
increïbles. Us admiro a totes i de totes he après lliçons imprescindibles per créixer. Sou l'orgull del
gènere femení! ;-).
Al Jordi Carreras perquè sense ell probablement mai hagués iniciat aquesta aventura. Gràcies per
ajudar-me tant a creure en mi. I a tants amics que sempre em feu riure, pensar, imaginar, crear,
superar-me: Jorge, Aleix, Belén, La Peña!!!, Carme, Manolo, Chelo, Marisa, Alexis...Papa, gràcies per
ensenyar-me la disciplina, la constància i el sacrifici que han estat realment indispensables per
projectes com aquest.
Per acabar, vull agrair a la meva família tot l'esforç que han fet des de sempre i sobretot perquè sou
els meus fans incondicionals i em cuideu com ningú. Mama no tinc paraules, ets fantàstica, la meva
heroïna sense dubte. Marc, simplement t'adoro. Àvis (Eulàlia i Quimet) sou el meu exemple a seguir,
no he conegut persones més bones que vosaltres. Patrick, tempiternament, gràcies per TOT, per la
VIDA i per ensenyar-me que si un vol pot aconseguir els seus somnis. Nada por obligación, todo por
ilusión. Gràcies, Gracias, Grazie, Thanks, Merci,
Mireia González To
Barcelona, 07/01/2014
T
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,0
.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,2
.,.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,2
E@E@E@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@I
E@E@F@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@I
E@E@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@J
E@E@H
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L
E@E@I
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ED
E@E@I@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ED
C!-!0%+71(#,0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ED
C/!+0&,/*!#(:+%+71(#!@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EE
C0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EF
C0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EF
C(#/,*!1/(#%0$%@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EG
C
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EG
E@E@I@F@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EH
E@E@I@GA
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EH
.,/,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.3
E@F@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EJ
E@F@E@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EJ
C
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EK
@E%"($,0!)!0&,/*!01!21,*7/(#!0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EK
@F
+0%/#(,+%04$%)%#(,+%0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EL
C
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EL
@E%0!*(+!#(:+@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EL
@F%0-2/(+(5!#(:+@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EM
@G3($!#(:+@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EM
.,0,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,/-
E@G@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FD
E@G@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FD
E@G@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FE
E@G@G@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FF
C!0%0!+6),'!0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FF
C'%+1%0.28*(#,0*,$(&(#!$,/%0$%"!0%0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FG
C'%+1%0(+1%/#!)!+1%0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FH
E@G@G@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FH
C!$(!#(,+%0%)%#1/,*!'+71(#!0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FH
C!$(!#(,+%0(,+(5!+1%0@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FH
E@G@G@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FJ
C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FJ
CAB$%1/!+0&%/%+#(!C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FJ
C
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FJ
C21!'7+%0(0$(/('($!@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FK
.,1,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,/6
E@H@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@FM
E@H@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@GD
E@H@F@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@GF
E@H@F@F@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@GI
E@H@F@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@GJ
E@H@F@H
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@HE
E@H@F@I
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@HG
.,2,,,,,,,,,,,,,,,13
E@I@E
B(1,%+,%0!12/!0!C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@HJ
E@I@F$B
HC4!"$@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@HL
E@I@G
E@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@II
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,3.
/,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,30
+,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,30
/,.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,30
F@E@E
BDC@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@JG
F@E@F
EF@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@JI
F@E@F@E
E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@JI
F@E@F@F
F@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@JJ
F@E@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@JJ
F@E@G@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@JJ
F@E@G@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@JK
F@E@G@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@JL
/,/,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,36
F@F@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KD
F@F@E@E
BC@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KD
F@F@E@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KE
F@F@E@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KF
F@F@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KF
F@F@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KF
F@F@G@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KG
F@F@H
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KG
F@F@I
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KH
F@F@I@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KH
F@F@I@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KH
F@F@I@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KH
F@F@J
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KI
F@F@J@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KI
F@F@J@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KJ
F@F@J@G
?
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KK
F@F@K
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@KM
F@F@L
@@@@@@@@@@@@@@@@@@@@@KM
/,0,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,5-
F@G@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@LD
F@G@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@LD
+,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,5.
/,1,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,5.
/,2,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,5.
F@I@EB
C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@LE
F@I@F
0BC@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@LF
F@I@G
0
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@LG
/,3
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,50
F@J@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@LH
F@J@F
B
C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@LH
0,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,54
0,.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,54
G@E@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@LK
G@E@E@E
JFAEEG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@LK
G@E@E@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@MD
G@E@E@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@MF
G@E@E@H
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@MG
G@E@E@H@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@MG
G@E@E@H@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@MH
G@E@E@H@G
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@MH
G@E@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@MK
0,/
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,66
G@F@E
B
C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EDD
G@F@E@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EDE
G@F@E@F
>
@@@@@@@@@@@@@@@@@@@@@@@@EDI
G@F@E@G
GLHEEEI@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EDL
G@F@F$B
C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EEG
G@F@F@E
>
$
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EEH
G@F@F@F
G$@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EEK
G@F@G!"$B
HC@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EFE
G@F@G@E
>
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EFG
G@F@H#B
EC@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EFG
G@F@H@E
>
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EFH
G@F@I
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EFI
0,0 %,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,./5
G@G@E
#,
$@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EFL
G@G@F
#,
#@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EGJ
0,1,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.1-
G@H@E
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EHE
G@H@F
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EHF
G@H@G@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EHG
G@H@H
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EHI
1,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.22
1,.,,,,,,,,,,,,,,,,,.22
1,/,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.3.
H@F@E
B
C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EJE
H@F@F%
HB
HC@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EJF
H@F@G!"$@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EKD
H@F@H#B
C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EKE
H@F@I
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EKF
1,0 %,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.41
H@G@E%
HB
C@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EKH
H@G@F#B
EC@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@EKL
1,1,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.50
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.56
#$#%"&!',,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.6/
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,/..
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,/16
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,/24
T
TTTTT
m
'%30mGYG[m09$'!-m!-,m*-1m.0',!'.*#1m.0-"3!2-0#1m"#m+#*A,mm,'4#*m+3,"'*YYmYYYYYYYYYYYYYYYYYYYYYYYYYYmMm
'%30mGYH[m09$'!-m"#m*-1m.>1#1m!-,m+91m#6.-02!'A,m"#m+#*A,mm,'4#*m+3,"'*mmYYYYYYYYYYYYYYYYYYYYYYYmNm
'%30mGYI[m$#!2-m"#*mmm,'4#*m+-*#!3*0mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmHIm
'%30mGYJ[m0-2-!-*-m#+.*#"-m.0m#*m+<2-"-m"#mmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmIHm
'%30mGYK[m#2#!!'A,m"#m3,m+32!'A,m#,m3,m.- *!'A,m"#mm#,m+#*A,m#,m3,m%#*m"#m
!0'*+'"m4#02'!*m"#mjmJIFFYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmINm
'%30mGYL[m(#+.*-m"#m!304m"#m"'1-!'!'A,m"#m$0%+#,2-1m+.*'$'!"-1m7m0#,230*'8"-1YmYmJFm
'%30mGYM[m32m"#m1>,2#1'1m"#m!0-2#,-1m#,m.*,21YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmJMm
'%30mGYN[m#.0#1#,2!'A,m"#*m1'12#+m"#m0#1'12#,!'m#,m.*,21m 1"-m#,m*m',2#0!!'A,m%#,j
j%#,m^*-0ZmGOMG_YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmJOm
'%30mGYO[m3,!'-,+'#,2-m"#*m','!'-m"#m*m20"3!!'A,mmmmmmmmmmmmmmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmKHm
'%30mGYGF[m3,!'-,#1m.-123*"1m.0m#*mI#-)J>YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmKKm
'%30mGYGG[m1/3#+m"#*m!'!*-m"#m,%mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmKLm
'%30mGYGH[m1/3#+m"#*m;mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmKMm
'%30mHYG[m0-!#"'+'#,2-m.0m- 2#,#0m#*mm"#m*m.- *!'A,m"#mYmYYYYYYYYYYYYYYYYYYYYYYYYYYmLLm
'%30mHYH[m'1#@-mm"#m*1m.*!1m.--*YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmLMm
'%30mHYI[m1/3#+m"#*m.0-2-!-*-m"#mmmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmMIm
'%30mHYJ[m-,2(#m%#*mjJIFFmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmMLm
'%30mHYK[m'13*'8!'A,m"#m*1m+3#1201m#,m#*m%#*m"#mYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmMMm
'%30mHYL[m*,2'**m.0m*m"#!-,4-*3!'A,YYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmMNm
'%30mIYG[m-1'!'A,m"#m*-1m1m,*'8"-1m#,m*m*>,#mLHjGGIYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmOFm
'%30mIYH[m-0!#,2(#1m"#m%#0+',!'A,YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmOGm
'%30mIYI[mm1/3#+m"#*m$0%+#,2-m1#!3#,!'"-m"#*m%#,m>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmOKm
'%30mIYJ[m1/3#+m"#m*m0#%'A,m"#mm"#m+#*A,mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFHm
'%30mIYK[m#!3#,!'m.0-2#'!m"#*m%#,m"#m*mm#,m+#*A,mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFIm
'%30mIYL[m0 -*m$'*-%#,<2'!-m"#*m%#,m"#m*mm"#m+#*A,mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFJm
'%30mIYM[m1/3#+m"#*m$0%+#,2-m"#m*mm,*'8"-m+#"',2#mYmYYYYYYYYYYYYYYYYYYYYYYYYYYmGFKm
'%30mIYN[m0-+2-%0+1m"#m*1m"-1m$+'*'1m!-,m+32!'-,#1m#,m*m8-,m#6A,'!m"#m*m
mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFLm
'%30mIYO[m#.0#1#,2!'A,m"#m*m.02#m1#!3#,!'"m"#m*mmYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFMm
'%30mIYGF[m#.0#1#,2!'A,m"#m*-1mHFm+',-9!'"-1YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFNm
'%30mIYGG[m+%#,m"#m20#1m$032-1m"#m.*,21mHmmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFOm
'%30mIYGH[m*9,23*1mHm"#m*m$+'*'m+32,2#mINJYYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGGm
'%30mIYGI[m0-+2-%0+1m"#m*mmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGHm
'%30mIYGJ[m#.0#1#,2!'A,m"#*m%#,m>m#,m+#*A,m^(-0_YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGIm
'%30mIYGK[m0 -*m$'*-%#,<2'!-m"#*m%#,m>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGJm
'%30mIYGL[m*',#+'#,2-m#,20#m*1m.0-2#>,1m"#m>m7m#*mI#-)J>m"#m+#*AmmYYYYYYYYYYYYYYYYYYYYYYYYmGGLm
'%30mIYGM[m-"#*(#m"#m*m.0-2#>,m>m"#*m+32,2#mHYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGNm
'%30mIYGN[m-"#*(#m"#m*m.0-2#>,m>m#,mIm!-,m.-1' *#m*'%,"-m^+-*<!3*mKdm"#*m
+_YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGNm
'%30mIYGO[m-"#*(#m"#m*m.0-2#>,m>m#,mIm+#"',2#m#*m/.*/.O"#*m.0-%0+m7m"#*m
+32,2#mKYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGOm
'%30mIYHF[m/.*/.O"#*m.0-%0+m7m"#*m+32,2#mKmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGOm
'%30mIYHG[m-"#*(#m"#m*m.0-2#>,m>m#,mIm+#"',2#m#*m32.32m"#*m.0-%0+m7m
"#*m+32,2#mNmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHFm
'%30mIYHH[m-"#*(#m"#m*m.0-2#>,m>m#,mIm+#"',2#m#*m/.*/.O"#*m.0-%0+m7m"#*m
+32,2#mOmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHFm
'%30mIYHI[m-"#*(#m"#m*m.0-2#>,m#,mIm"#*m%#,m>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHGm
'%30mIYHJ[m1/3#+m"#*m%#,mI#-)J>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHHm
'%30mIYHK[m0 -*m$'*-%#,<2'!-m"#*m%#,mI#-)J>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHIm
'%30mIYHL[m1/3#+m"#*m%#,m;mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHIm
'%30mIYHM[m0 -*m$'*-%#,<2'!-m"#*m%#,m;mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHJm
'%30mIYHN[m#*m"#m!-m.0m#*m.0'+#0m+.*'!A,m"#m>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHOm
'%30mIYHO[m0-+2-%0+m"#m*1m40'#""#1mJMJm7mLHjGGImYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGIFm
'%30mIYIF[m#*m"#m!-m.0m#*m1#%3,"-m+.*'!A,m"#m>YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGIIm
'%30mIYIG[m0-+2-%0+1m"#mLHjGGIm7mmKFKLFHYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGILm
'%30mIYIH[m#*m"#m!-m.0m#*m+.*'!A,m"#*m%#,m;mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGIMm
'%30mIYII[m0-+2-%0+m"#*mmGm"#*m.0'+#0m#6A,m"#*m%#,m;mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGINm
'%30mIYIJ[m0-!#"'+'#,2-m.0m*m1#*#!!'A,m"#m+#*-,#1m!-,m$#,-2'.-1mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGJJm
'%30YJYG[m*9,23*m!-,m$#,-2'.-m* ',-mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGLHm
'%30mJYH[m*',#+'#,2-m+#"',2#m&/-.&m"#m*m.0-2#',m#JmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGLJm
'%30mJYI[m/.*/.O"#*m.0-%0+mm"#*m%#,m#JmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGLMm
'%30mJYJ[m*',#+'#,2-m+#"',2#m*312*m"#m>m7mI#-)J>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGMGm
'%30mJYK[m0"3!!'A,mm.0-2#>,m"#*m+m"#*m%#,m#JYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGMMm
'%30mJYL[m1203!230m"#m*-1m"-+','-1m'"#,2'$'!"-1m#,m;m"#m,#)*-#-mYYYYYYYYYYYYYYYYYYYYYYYYYYYmGMNm
'%30mJYM[m*',#+'#,2-m+B*2'.*#m"#m*-1m%#,#1m;m#,m,#)*-#-Zm2-+2#m7m+#*A,m7m*-1m
%#,#1m>Fm?m7mm"#m2-+2#YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGNGm
'%30mJYN[m!!#1'-,#1m#,m*1m/3#m1#m&,m"#2#!2"-m#*mmGmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGNHm
'%30mJYO[m,$-0+!'A,m"'1.-,' *#m#,m*m 1#m"#m"2-1m" mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGNJm
'%30mJYGF[m#,-2'.-1m"#m$032-m',2#0#1,2#1mm,'4#*m!-+#0!'*mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGNKm
'%30mJYGG[m#,-2'.-1m'"#,2'$'!"-1m#,m.*,2m#,m-201m.- *!'-,#1m"#mYmYYYYYYYYYYYYYYYYYYmGNMm
'%30mJYGH[m#,-2'.-1m'"#,2'$'!"-1m#,m$032-m#,m-201m.- *!'-,#1m"#mmmYYYYYYYYYYYYYYYYYYYYYmGNNm
m
*mGYG[m#.0#1#,2!'A,m"#m*1m$-0+1m232-+<0'!1mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGMm
*mGYH[m#13+#,m"#m*-1m+329%#,-1m+91m& '23*#1mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmHHm
*mGYI[m- *!'-,#1m"#mm"#100-**"1m#,m,'+*#1m7m.*,21mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmIJm
*mHYG[m-1'1m"#mm32'*'8"1m.0m+320m*m.- *!'A,mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmLJm
*mIYG[m-!*'8!'A,m"#m1YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmNNm
*mIYH[m-1'1m"#mm31"1m.0m+320m*1m1#+'**1YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmOHm
*mIYI[m *m!-+.02'4YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmOJm
*mIYJ[m2-1m32'*'8"-1m.0m- 2#,#0m*1m1#!3#,!'1m"#m*-1m%#,#1m!,"'"2-1mYYYYYYYYYYYYYYYYYYYYYmGFFm
*mIYK[m32,2#1m#,!-,20"-1m#,m#*m$0%+#,2-m"#mGGNNm. m"#*m%#,mmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFMm
*mIYL[m#0+',!'A,m"#m*m1#+'**1mHm#,m*1m$+'*'1mINJm7mGGGKYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGFOm
*mIYM[m#13*2"-1m"#m*m1#%0#%!'A,m"#m*m+32!'A,m#,m*1m$+'*'1m+32,2#1m"#m*mYYmGGHm
*mIYN[m32,2#1m"#*m.0'+#0m"#*m%#,m>YYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGKm
*mIYO[m32,2#1m#,m#*m1#%3,"-m+.*'!A,m"#*m%#,m>YYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGGKm
*mIYGF[m0!2#0>12'!1m"#*m+32,2#m#,!-,20"-m#,m#*m%#,mI#-)J>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHIm
*mIYGG[m$'!'#,!'m"#m+32!'A,m"#m.- *!'-,#1m"#mm+#*A,YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHKm
*mIYGH[m11m"#m+32!'A,m"#mm"#*mm7m#*mYmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGHMm
*mIYGI[m1m"#2#!2"-1m#,m#*m.0'+#0m+.*'!A,m"#*m%#,m>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGIGm
*mIYGJ[m1m"#2#!2"-1m#,m#*m1#%3,"-m+.*'!A,m"#mO>mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGIJm
*mIYGK[m.*-2'.-1m#,!-,20"-1m+#"',2#m#*m,9*'1'1m.-0m!-mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGIKm
*mIYGL[m-*'+-0$'1+-1m"#2#!2"-1m#,m#*m.0'+#0m#6A,m"#*m%#,m;mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGINm
*mIYGM[m *m!-,m*-1m&.*-2'.-1m"#2#!2"-1m.0m#*m%#,m;mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGIOm
*mIYGN[m-0!#,2(#1m"#m%#0+',!'A,m#,m*1mJFFm$+'*'1mHm- 1#04"1YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGJGm
*mIYGO[m#,-2'.-1m#,!-,20"-1m#,m#12"'-m"#m.*9,23*mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGJHm
*mIYHF[m#,-2'.-1m#,!-,20"-1m#,m#12"'-m"#m.*,2m"3*2mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGJIm
*mIYHG[m#,-2'.-1m#123"'"-1m#,m$032-1mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGJKm
*mJYG[m-0!#,2(#m"#m 1#1m131!#.2' *#1m*m+329%#,-mmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGKMm
*mJYH[m *m0#13+#,m"#m*-1m+32,2#1m#,!-,20"-1m#,m!"m.- *!'A,mYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGKOm
*mJYI[m#13*2"-1m"#*m#123"'-m"#m1#%0#%!'A,m"#*m%#,m#JmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGLOm
*mJYJ[m *m!-+.02'4m"#m*-1m.-*'+-0$'1+-1m!-YmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGMKm
*mJYK[m#,-2'.-1m"#m*1m!!#1'-,#1m/3#m.0#1#,2,m#*mGmYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYmGNHm
T
TTT
m
[m!'"-m#1-6'00' -,3!*#'!-m
[m+.*'$'#"m0%+#,2m#,%&2m-*7+-0.&'1+m
[m!2#0'*m02'$'!'*m&0-+-1-+#m
[m1'!m-!*m*'%,+#,2m#0!&m--*m
[m-"-,1m.2'+'8#"m2-m'1!-4#0m#*#2#0'-31m#1'-,1m
[m#,20-m"#m-,1#04!'A,m7m#(-0m"#m*m%0-"'4#01'""m*#,!',m
[m#,20#m"#m#!#0!m#,m%0'%#,A+'!Zm0!#*-,m
>[m!2-0m"#m','!'!'A,m"#m*m20"3!!'A,mJm
[m2'*m#2,-m3*$-,2-m
[m6.0#11#"m#/3#,!#m%m
[m2&7*#,#m#!#.2-0m
[m--"m,"m%0'!3*230#m0%,'82'-,m
[m>,#1m- *#m.*-'"#1m
[m','12#0'-m"#m%0'!3*230Zm#1!m7m*'+#,2!'A,Ym
[m#*-,m#!0-2'!m.-2m'031m
F[m#+'**1m+32%#,#'8"1m.0m%#0+',0m7m.0-"3!'0m*m%#,#0!'A,mGm
G[m0'+#0m%#,#0!'A,m"#m.*,21m!0#!'"1mm.02'0m"#m*1m1#+'**1mFm
H[m#%3,"m%#,#0!'A,m"#m.*,21m!0#!'"1mm.02'0m"#m*1m1#+'**1m"#m*1m.*,21mG
[m2'-,*m#,2#0m$-0m'-2#!&,-*-%7m,$-0+2'-,m
1[m>,#1m!1'm1-%<,'!1m^#0m1-%#,'!m',#1_mm
. [m.0#1m"#m 1#1m
[m#!!'A,m#,m!"#,m"#m*m.-*'+#01m^-*7+#01#m&',m#!2'-,_m
[m'2-#,-m#12301m
[mmGLGIMKm&-,%5,m&0+'m
[m'#*m"#m.-mGGGm
[m3,2'22'4#m0'2m-!31m
[m,"-+m+.*'$'#"m-*7+-0.&'!mm
[m#120'!2'-,m0%+#,2m#,%2&m-*7+-0.&'1+m
[m-02',%m,2-*#0,2m0-+m-*#0,2m
[m',%*#m3!*#-2'"#m-*7+-0.&'1+m
[m'+.*#m#/3#,!#m#.#2m
[m0%#2',%m,"3!#"m-!*m#1'-,1mm#,-+#1m
[m,'2<m"#m#!&#0!&#m#,m<,-+'/3#m<%#2*#Zm407m^0,!'_m
G
m
Hm
T
m
*m20 (-m"#1!0'2-m#,m#12m2#1'1m"-!2-0*m1#m#,+0!m"#,20-m"#*m.0-7#!2-mm
^HFGIjHFHIMZm HFFJjHFFM_m 3,-m "#m !37-1m - (#2'4-1m #0m *m !-,1203!!'A,m "#m 3,m
.*2$-0+m"#mm#,m+#*A,Ym
m "'1.-,' '*'""m "#m 0#!301-1m %#,<2'!-1m 7m %#,A+'!-1m .0m #*m +#*A,m 1#m &m
',!0#+#,2"-mm*-m*0%-m"#m*-1mB*2'+-1m@-1m#m',!*31-m*m1#!3#,!'m"#*m%#,-+m#129m
"'1.-,' *#Ym ',m #+ 0%-Zm *1m &#00+'#,21m %#,A+'!1m .0m "#2#0+',0m *m
$3,!'-,*'""m "#m *-1m %#,#1m #,m #12m #1.#!'#m 1-,m 2-"4>m *'+'2"1Ym *m m
^0%#2',%m ,"3!#"m -!*m #1'-,1m m #,-+#1_m #1m 3,m +<2-"-m "#m %#,<2'!m ',4#01m
+#"',2#m #*m !3*m 1#m .3#"#,m !0#0m #m '"#,2'$'!0m ,3#41m +32!'-,#1m #,m %#,#1m
!,"'"2-1m 7m - 1#040m 13m #$#!2-m $#,-2>.'!-Ym "#+91Zm 2+ '<,m 1#m .3#"#,m "#2#!20m
$#,-2'.-1m "#m ',2#0<1m .0m .-12#0'-0+#,2#m '"#,2'$'!0m *m +32!'A,m 0#1.-,1 *#m "#m *-1m
+'1+-1Ym
,m #12#m 20 (-m 1#m &m "#100-**"-m 3,m .- *!'A,m "#m m #,m //'#-O '&)m m
.02'0m "#m *m *>,#m "- *#m &.*-'"#m "#*m 2'.-m b'#*m "#m .-cm LHjGGIYm .*'!,"-m
','!'*+#,2#m#*m+329%#,-m/3>+'!-mm^2'*m#2,-m3*$-,2-_m1- 0#mGMYFFFm1#+'**1m
1#m &m "#100-**"-m 3,m !-*#!!'A,m "#m +32,2#1m !-+.3#12m .-0m IYHLNm $+'*'1m HYm m
.02'0m "#m JFFm $+>*'1m Hm 1#m - 1#040-,m "'4#01-1m $#,-2'.-1m +32,2#1m #,m *1m
"'$#0#,2#1m #2.1m "#m !0#!'+'#,2-m "#m *m .*,2Ym *%3,-1m "#m #12-1m +32,2#1m 1#m &,m
"#1!0'2-m !-,m "#2**#m %#,#0,"-m 3,m !29*-%-m "#m *-1m $#,-2'.-1m - 1#04"-1m #,m !"m
$+'*'Zm*%3,-1m"#m*-1m!3*#1m.3#"#,m2#,#0m',2#0<1m!-+#0!'*Ymm
0m +#"'0m *m 21m "#m +32!'A,m "#m *m .- *!'A,m 1#m &,m ,*'8"-m !320-m %#,#1Zm *m
$'2-#,-m"#12301mZm*-1m$!2-0#1m"#m','!'!'A,m"#m*m20"3!!'A,m>m3OI#-)J>O
7m#*m0#!#.2-0m"#m#2'*#,-m;Ym-1m+32,2#1m"#2#!2"-1m&,m.#0+'2'"-m!*!3*0m*m21m
"#m +32!'A,m "#m *m .- *!'A,Zm 3,m +32!'A,m !"m GZKm Ym ,m #*m !1-m "#m m 1#m &m
.-"'"-m- 2#,#0m3,m+32,2#m!-,m$#,-2'.-m* ',-Zm"#+-120,"-m1>m*m#$#!2'4'""m"#*m
+<2-"-mm#,m+#*A,Ym"#+91Zm.0*#*+#,2#m1#m,*'80-,m*-1m+'1+-1m%#,#1m
#,m-20m.- *!'A,m+32%#,'8"m',"#.#,"'#,2#Ym
m+ '<,m 1#m 0#*'8Am 3,m #123"'-m "#m !-m !-,m GGIm !!#1'-,#1m "#m +#*A,7m 1#m
'"#,2'$'!0-,m GOm &.*-2'.-1m "'12',2-1m #,m #*m %#,m>Zm 3,-m "#m #**-1Zm !-,m 3,m m /3#m
.0-4-!m3,m!+ '-m"#m+',-9!'"-m7m/3#m1A*-m1#m#,!3#,20m#,m*m!!#1'A,mmKFKLFHm7m
"-1m &.*-2'.-1m #,m #*m %#,m ;Zm 3,-m "#m *-1m !3*#1m 2+ '<,m !-,2'#,#m 3,m m /3#m
.0-4-!m3,m!+ '-m"#m+',-9!'"-Ymm
I
*m m $!'*'209m *-1m #123"'-1m "#m %#,<2'!m ',4#01m #,m +#*A,m 7m 0#m *m .3#02m m
$3230-1m #123"'-1m "#m %#,A+'!m $3,!'-,*m #,m 3,m %#,-+m 0#!'#,2#+#,2#m 1#!3#,!'"-Ym
12#m20 (-m&m0#.0#1#,2"-m2+ '<,m#*m.0'+#0m.1-m.0m.-"#0m"#100-**0m,3#41m
.- *!'-,#1m"#mm#,m+#*A,m!-,m+7-0#1m211m"#m+32!'A,m7m#,m-20-1m$-,"-1m
%#,<2'!-1Ym
T
Jm
&O'&9(O3O&O!(9'#O&O'&9(O
(.,)/#9(O
T
T
BMTT
T
BMBTTT
T
BMBMBTT
m #1.#!'#m //'#-O '&)m Em .#02#,#!#m m *m $+'*'m "#m *1m 3!30 '29!#1Zm m *m 20' 3m
#*-2&0'##Zm 13 20' 3m 3!3+#0',#Zm %<,#0-m //'#-Ym m .02#m "#m #12#m %<,#0-m /3#m
',!*37#m 2+ '<,m #*m .#.',-m ^//'#-O -.#0/-JO ^
#$$0#7Zm GOOF_Zm #,!-,20+-1m -20-1m "-1m
%<,#0-1m /3#m %03.,m #1.#!'#1m "#m '+.-02,!'m #!-,A+'!m m ,'4#*m +3,"'*m !-+-m
#.,/&&/-O &(./-m ^1,">_m 7m //,#.O **)m ^!* 8m 7m !* !>,_m ^
#$$0#7Zm GOOF_Ym *m
%<,#0-m //'#-O 1#m "'4'"#m #,m "-1m 13 %<,#0-1m /3#m 1#.0,m 91'!+#,2#m *1m #1.#!'#1m
$0'!,1m "#m *1m 1'92'!1Ym *m .0'+#0m 13 %<,#0-Zm "#,-+',"-m &)Zm ',!*37#Zm #,20#m
-20-1Zm *m +#*A,Ym *m 1#%3,"-m 13 %<,#0-Zm //'#-Zm ',!*37#Zm #,20#m -20-1Zm *m .#.',-m
^
#$$0#7Zm GOOF_Ym 6'12#,m "'12',21m !*1'$'!!'-,#1m "#*m +#*A,Zm 1',m #+ 0%-Zm 3,m "#m *1m
+91m !-+.*#21m #1m *m .0-.3#12m .-0m '202m 7m !-* -0"-0#1m ^HFFF_m /3#m "'4'"#m *m
#1.#!'#m#,mGLm%03.-1ZmKm"#m*-1m!3*#1m1#m%03.,m#,m*m13 #1.#!'#O!,-.#-m7mGGm#,m*m
13 #1.#!'#m'&)Ym
*m+#*A,m#1m3,m#1.#!'#m!-,m3,m%0,m40' '*'""m%#,<2'!m7m#62#,1+#,2#m!3*2'4"m
#,m2-"-m#*m+3,"-Ym,m0#!'#,2#m#123"'-m"#m1#!3#,!'1m"#mm,3!*#0m7m.*1+>"'!1m
#,m GFFm !!#1'-,#1m "#m //'#-m "#m "'12',21m 8-,1m %#-%09$'!1m ^$0'!Zm 3120*'m 7m
1'_m &m 13%#0'"-m /3#m #*m +#*A,m 2'#,#m 3,m -0'%#,m 1'92'!-m 7m /3#m .0- *#+#,2#m 13m
,2#!#1-0m1'*4#120#m1#m-0'%',Am#,m*m,"'m^# 12',ZmHFGF_Ymm
T
BMBMCTT
m .*,2m "#*m +#*A,m #1m ,3*m 7m &#0 9!#m 7m .3#"#m 1#0m "#m .-02#m 0120#0-m -m 20#."-0m
"#.#,"'#,"-m "#m *1m !-,"'!'-,#1m "#m !3*2'4-m m *1m !3*#1m 1#m 1-+#2Ym 3m 1'12#+m
0"'!3*0m #1m 3,",2#Zm +37m 0+'$'!"-m 7m "#m 09.'"-m "#100-**-Ym -1m 2**-1m
.0',!'.*#1m#129,m0#!3 '#02-1m"#m$-0+!'-,#1m.'*-11m7m.0#1#,2,m,3"-1m#,m*-1m/3#m1#m
"#100-**,m &-(1Zm 80!'**-1m 7m $*-0#1Ym -1m ,3#4-1m 2**-1m 0-2,m "#m *1m 6'*1m "#m *1m
&-(1Ym m &-(m 2'#,#m $-0+m "#m *'+ -m -4"-Zm & '23*+#,2#m .#,2%-,*m 7m !-,m *-1m
+90%#,#1m "#,2"-1Ym 1m &-(1m 2+ '<,m 1-,m 4#**-11m .-0m #*m #,4<1Ym 1m $*-0#1m 1-,m "#m
!-*-0m+0'**-m7m.3#"#,m1#0m+1!3*',1Zm$#+#,',1m-m&#0+$0-"'21Ym1m.0'+#01m#,m
K
(.,)/#9(
El melón y la genómica de melón
"#100-**01#m1-,m*1m$*-0#1m+1!3*',1m#,m*-1m#,20#,3"-1m.0'+0'-1m+'#,201m/3#m*1m
$#+#,',1m7m&#0+$0-"'21m.0#!#,m+91m20"#m#,m*1m0+'$'!!'-,#1m.-12#0'-0#1Ymm
.-*','8!'A,m #1m #,2-+A$'*_m 7m +7-0'20'+#,2#m 0#*'8"m .-0m #(1m ^,"#*Zm GONH\m
-,$-02#m7m!-*YZmHFFJ_Ym
m $-0+m "#*m $032-m #1m +37m 40' *#m ^#1$<0'!Zm #*>.2'!m 3m -4"_Ym #m &,m .-"'"-m
'"#,2'$'!0m %#,#1m /3#m .0#!#,m #120m 0#*!'-,"-1m !-,m *m $-0+m "#m $032-m .-0m 13m
'+.*'!!'A,m#,m*m"#2#0+',!'A,m"#m*m$-0+m"#m*m$*-0m-m"#*m-40'-Zm#,20#m#**-1Zm#*m%#,m
m^+-,-'!-_m7m#*m%#,m*m^.#,29+#0-_m^#0',m7m!-*ZmHFFH_Ym
0m *m !-02#8m "#*m $032-m #6'12#m 2+ '<,m 3,m %0,m 40' '*'""m 2,2-m "#m !-*-0#1m
^4#0"#Zm +0'**-Zm ,0,("-m -m *,!-_m !-+-m "#m 2#62301Ym ,!-,20+-1m .'#*#1m *'11Zm
0#2'!3*"1m-m#120'"1Ymm.3*.m.3#"#m1#0m *,!Zm+0'**Zm,0,("m-m4#0"-1Ym*m
$032-m "#*m +#*A,m .3#"#m +"300m "#m +,#0m !*'+2<0'!m -m ,-m !*'+2<0'!m 1#%B,m *m
40'#""m +"30!'A,m !*'+2<0'!m 1#m !0!2#0'8m .-0m 3,m 3+#,2-m "#m *m 21m "#m
0#1.'0!'A,m7m#1m','!'"m.-0m3,m.'!-m#,m*m!-,!#,20!'A,m"#m#2'*#,-Ym',m#+ 0%-Zm*m
+"30!'A,m,-m!*'+2<0'!m1#m!0!2#0'8m.-0m3,m"'1+',3!'A,m!-,2',3m#,m*m21m"#m
0#1.'0!'A,m 7m #,m *m .0-"3!!'A,m "#m #2'*#,-m ^--0#m 7m !-*Zm HFFH_Ym m '"#,2'$'!!'A,m "#m
*%3,-1m /(.#..#0O ,#.O )/-O ^1_m 0#1.-,1 *#1m "#m #12#m !09!2#0m (3,2-m !-,m *m
*-!*'8!'A,m "#m %#,#1m !,"'"2-1m "#m $'0+#8m "#*m $032-m #,m *>,#1m "#m ',20-%0#1'A,m
^1_m - 2#,'"1m "#m *-1m .0#,2*#1m m GLGIMKm 7m GGGZm #,20#m -20-1m #123"'-1Zm &,m
.#0+'2'"-m !-+#,80m m !-+.0#,"#0m !A+-m $3,!'-,m #12#m +#!,'1+-m ^-0#,-m 7m !-*YZm
HFFN_Ym121m!0!2#0>12'!1m&,m&#!&-m/3#m#*m+#*A,m1#m!-,1'"#0"-m!-+-m3,m"#m*1m
#1.#!'#1m +-"#*-m .0m .-"#0m !-+.0#,"#0m #*m .0-!#1-m "#m +"30!'A,m "#*m $032-m 1>m
!-+-m13m0#%3*!'A,mm,'4#*m%#,<2'!-m7m+-*#!3*0Ym
T
BMBMDTTTTT
*m +#*A,m 1#m!3*2'4m #,m 8-,1m 20-.'!*#1m 7m 13 20-.'!*#1m 1#!1Ym,m 30-.Zm 1#m !3*2'4m
#12!'-,*+#,2#m#,m4#0,-m7m.0'+4#0Ym#%B,m*-1mB*2'+-1m"2-1m"#m*mZm#,mHFGGm
&',m #0m #*m .0'+#0m .0-"3!2-0m +3,"'*m !-,m GIYFNKYKFFm 2-,#*"1Zm 1#%3'"-m .-0m 09,m
!-,m GYNFGYNIFm 7m .-0m 30/3>m !-,m GYLJMYOOFZm +'#,201m /3#m 1.@m /3#"m #,m 1<.2'+-m
*3%0m!-,m3,m.0-"3!!'A,m"#mNLOYJMKm2-,#*"1m^'%30mGYG_Ym
Lm
&O'&9(O3O&O!(9'#O&O'&9(O
(.,)/#9(O
m
m
(&30 TBMBOT09$'!-m!-,m*-1m.0',!'.*#1m.0-"3!2-0#1m"#m+#*A,mm,'4#*m+3,"'*Ym1.@m1#m#,!3#,20m#,m#*m
1<.2'+-m*3%0m^,2mGFFFW[m.0#!'-m+#"'-m"#m2-"1m*1m+-,#"1m',2#0,!'-,*#1m2-+,"-m#*m"A*0m+#0'!,-m
!-+-m0#$#0#,!'\m[m-,#*"m+#20'!_Ym3#,2#mZm&22.[ii$-122Y$-Y-0%i1'2#iIJHi"#$3*2Y1.6m^HFGI_Ym
m
m
'm ,*'8+-1m *1m #6.-02!'-,#1Zm 1.@m #1m #*m .0'+#0m #6.-02"-0m +3,"'*m !-,m
IMLYGFIm 2-,#*"1Zm 1#%3'"-m "#m 32#+*m 7m -,"301m ^'%30m GYH_Ym 12#m "2-m
!-00- -0m #*m &#!&-m "#m /3#m #,m 1.@m #*m +#*A,m #1m 3,m .0-"3!2-m ,-m 1A*-m +37m
"#+,""-m .-0m .02#m "#*m !-,13+'"-0Zm 1'm ,-m /3#m "#+91Zm 2'#,#m +3!&m "#+,"m m
,'4#*m ',2#0,!'-,*Zm 7m .-0m *-m 2,2-Zm 3,m '+.-02,2#m ..#*m #,m *m #!-,-+>m "#*m .>1Ym
#m "#!'0m /3#m #,m 1.@m #*m +#*A,m #1m *m 1#%3,"m #1.#!'#m &-02>!-*m +91m !3*2'4"m
"#1.3<1m "#*m 2-+2#Ym ,m #*m HFGGm 1#m !3*2'40-,m IHYFFFm m 7m 1#m .0-"3(#0-,m NLOYJMKm
2-,#*"1m"#m+#*A,Ymm+7-0m.02#m"#m*m13.#0$'!'#m!3*2'4"m"#m+#*A,m1#m#,!3#,20m
#,m*1m0#%'-,#1m"#m30!'Zm12'**m*m,!&m7m,"*3!>m^&22.[ii$-122Y$-Y-0%_Ym
M
(.,)/#9(
El melón y la genómica de melón
m
m
(&30 TBMCOT09$'!-m"#m*-1m.>1#1m!-,m+91m#6.-02!'A,m"#m+#*A,mm,'4#*m+3,"'*Ym1.@m1#m#,!3#,20m#,m
.0'+#0m*3%0Ymm3#,2#mZm&22.[ii$-122Y$-Y-0%i1'2#iIJHi"#$3*2Y1.6m^HFGI_Ym
m
BMBMET
TTTT
m'+.-02,!'m#!-,A+'!m"#*m+#*A,m#,m,3#120-m.>1m(312'$'!m/3#m#6'12,m#+.0#11m
.0-"3!2-01m "#m 1#+'**1m ',2#0#1"1m #,m +#(-00m #12m #1.#!'#m 7m ".20*m m *1m
,#!#1'""#1m"#m*-1m.0-"3!2-0#1m7m*-1m!-,13+'"-0#1Ym*%3,-1m"#m*-1m- (#2'4-1m"#m#12m
+#(-0m %#,<2'!m 1-,m - 2#,#0m .0-"3!!'-,#1m !-,m 3,m *2-m 0#,"'+'#,2-Zm ',!-0.-00m
0#1'12#,!'1m m .*%1m -m #,$#0+#""#1m 7m *m +#(-0m "#*m $032-m 2,2-m #,m !0!2#0#1m
+-0$-*A%'!-1m ^.-0m #(#+.*-m *m $-0+m "#*m $032-_m !-+-m -0%,-*<.2'!-1m ^.-0m #(#+.*-m #*m
0-+m -m *m "3*830_Ym 1m 0#1'12#,!'1m 31!"1m 1-,m .0',!'.*+#,2#m $0#,2#m m 4'031m
!-+-m.-0m#(#+.*-Zm#*m4'031m"#*m+-1'!-m"#*m.#.',-Zm//',O)-#O#,/-m^_m-m
#*m 4'031m "#m *1m +,!&1m ,#!0A2'!1m #*m +#*A,Zm &)(O ,).#O *).O #,/-m ^_m 7m
$0#,2#m m &-,%-1m !-+-m #*m ->"'-m ^))-*",O 2(."##FO)&)0#()'3-O #"),,/'_m -m m
310'3+m ^/-,#/'O )23-*),/'O $Ym 1.Ym '&)(#-_Ym 1'+'1+-Zm 1#m .0-!30m -.2'+'80m *m
.0-"3!!'A,m !-,m 40'#""#1m /3#m -$0#8!,m 3,m *2-m 0#,"'+'#,2-m #,m !-,"'!'-,#1m
"4#011m "#m 1#/3>m 7m #*#4"m 1*','""m -m /3#m .#0+'2,m 3,m "#!3"m !*'""m #,m
.-12!-1#!&Y
,2#1m "#*m "#1!3 0'+'#,2-m "#m *-1m +0!"-0#1m +-*#!3*0#1m #12#m 2'.-m "#m +#(-0m
"#.#,">m "'0#!2+#,2#m "#m *m #4*3!'A,m +#"',2#m *m - 1#04!'A,m "'0#!2m "#*m
$#,-2'.-Ym ',m #+ 0%-Zm *m #6.0#1'A,m $#,-2>.'!m #1m .0-"3!2-m "#m *m ',2#0!!'A,m "#*m
Nm
&O'&9(O3O&O!(9'#O&O'&9(O
(.,)/#9(O
%#,-2'.-m !-,m #*m + '#,2#m 7m ,-m 2-"-1m *-1m $!2-0#1m + '#,2*#1m .3#"#,m 1#0m
!-,20-*"-1Zm&#!&-m/3#m"'$'!3*2m#*m.0-!#1-m"#m1#*#!!'A,m^**0"ZmGOOO_Ym
0!'1m m *1m ,3#41m 2#!,-*-%>1m #,m
'-*-%>m +-*#!3*0m 1#m &,m "#100-**"-m
&#00+'#,21m !-+-m *-1m +0!"-0#1m +-*#!3*0#1m /3#m .#0+'2#,m *m 1#*#!!'A,m 1'12'"Ym
,m*>,#1m%#,#0*#1Zm*-1m+0!"-0#1m%#,<2'!-1m.#0+'2#,m0120#0m!-,m0.'"#8m#*mm
"#m*m.*,2m/3#m1#m#129m1#*#!!'-,,"-m.#0+'2'<,"-,-1m1 #0m1'm#*m*#*-m"#m3,m%#,m1#m
#,!3#,20m-m,-m#,m*m.*,2Ymm
-1m +0!"-0#1m %#,<2'!-1m 1-,m .-*'+-0$'1+-1m &#0#" *#1m "#m $-0+m +#,"#*',m
1#,!'**m^20!&,m7m#"ZmGOOO_Ym3m40' '*'""m.3#"#m1#0m"#2#!2"m7m32'*'8"m#,m#*m
#123"'-m "#m "'4#01-1m !0!2#0#1m %#,<2'!-1Ym ,m *m +#(-0m !*91'!m 1#m 32'*'8 ,m *-1m
+0!"-0#1m +-0$-*A%'!-1m /3#m ,-0+*+#,2#m 1#m .0-"3!>,m m .02'0m "#m 3,m +32!'A,m
"#m3,m%#,m","-m!-+-m0#13*2"-m#*m$#,-2'.-m"'$#0#,!'"-Ym-12#0'-0+#,2#m1#m310-,m
*-1m +0!"-0#1m 1"-1m #,m '1-#,8'+1m -m 40',2#1m +-*#!3*0#1m "#m 3,m +'1+m
.0-2#>,m ^2#
',1m 7m --"7Zm GONO\m !'**',m 7m !,"*'-1Zm GONI_Ym "-m /3#m !"m
'1-#,8'+m2'#,#m3,m"'12',2m!-+.-1'!'A,m#,m+',-9!'"-1m2+ '<,m0#13*2m"'12',2m13m
!0%m #*<!20'!Ym 12m !0!2#0>12'!m 1#m 31 m .0m 1#.00*1m +#"',2#m #*#!20-$-0#1'1m
4'13*'8,"-m #*m .20A,m "'$#0#,!'*m "#m !"m ',"'4'"3-Ym -12#0'-0+#,2#Zm 1#m
"#100-**0-,m *-1m +0!"-0#1m +-*#!3*0#1m 1"-1m #,m *-1m .-*'+-0$'1+-1m "#m Ym
-1m-.,#.#)(O,!'(.O(!".O)&3'),*"#-'O^1_m1-,m+0!"-0#1m 1"-1m#,m*1m
"'$#0#,!'1m"#m*-,%'23"m"#m$0%+#,2-1m"#mm- 2#,'"-1mm.02'0m"#m*m"'%#12'A,m"#*m
%#,-+Ym-1m$0%+#,2-1m1#m"'%'#0#,m!-,m"'12',21m#,8'+1m"#m0#120'!!'A,m/3#m!-02,m
#*mm.-0m*3%0#1m#1.#!>$'!-1m"#m*m1#!3#,!'m^"',1m"#m0#120'!!'A,_Ymm.0#1#,!'m
"#m.-*'+-0$'1+-1m.3#"#m$#!20mm*1m"',1m"#m0#120'!!'A,m'+.'"'#,"-m/3#m*m#,8'+m
"'%'#0m !-00#!2+#,2#m 7m .-0m *-m 2,2-m /3#m !"m %#,-2'.-m +3#120#m 3,m .20A,m
"'$#0#,!'*m^3"0!!-j0,1ZmGOOL\m*'4#0ZmHFFG_Ymm
1'+'1+-Zm *-1m +0!"-0#1m 1"-1m #,m *m 2<!,'!m "#m *m #!!'A,m #,m "#,m "#m *m
-*'+#01m ^_m &,m 1#04'"-Zm #,20#m -201m 32'*'""#1Zm .0m 1#%3'0m !-,12037#,"-m 7m
1230,"-m *-1m +.1m %#,<2'!-1Ym 7m +3!&-1m 2'.-1m "#m +0!"-0#1m "#0'4"-1m "#m *m
2<!,'!m "#m *m Zm !-+-m *-1m '*&# #O ,!'(.O (!".O )&3'),*"#-'O ^1_Zm *-1m
()'O '*&# #O )&3'),*"#O m ^1_Zm *-1m +'!0-12<*'2#1m -m #'*&O +/(O
*.-m^1_m-m*-1m1m^#(!&O/&).#O)&3'),*"#-'JEO-1m1m1#m 1,m#,m*m
"'%#12'A,m !-,m #,8'+1m "#m 0#120'!!'A,m "#*m m %#,A+'!-m 7m .-12#0'-0+#,2#m 13m
+.*'$'!!'A,m +#"',2#m m ^-1m 7m !-*YZm GOOK_Ym -1m 1m 1#m %#,#0,m .-0m *m
+.*'$'!!'A,m*#2-0'm"#m1#%+#,2-1m"#mm%#,A+'!-mm.02'0m"#m3,m1-*-m!# "-0m
"#m 1#!3#,!'m !-02m 7m 0 '200'm ^'**'+1Zm GOOF_Ym -1m 1m 1-,m $0%+#,2-1m "#m
1#!3#,!'1m "-,"#m &7m 3,m 0#.#2'!'A,m #,m 29,"#+m "#m HjLm ,3!*#A2'"-1Zm 3,/3#m #12m
O
(.,)/#9(
El melón y la genómica de melón
*-,%'23"m.3#"#m40'0m#,m$3,!'A,m"#m*m40'#""Ym0!'1mm#12m40'!'A,m1#m.3#"#,m
"#100-**0m +0!"-0#1m
1"-1m #,m *m "#2#!!'A,m "#*m ,B+#0-m "#m 0#.#2'!'-,#1m
.0#1#,2#1m#,m*-1m%#,-2'.-1m,*'8"-1m^+"m7m!-*YZmGONH\m-,8*-m7m!-*YZmHFFK_Ym
m.#10m"#m#6'12'0m3,m%0,m40'#""m"#m+0!"-0#1m+-*#!3*0#1Zm#,m*-1mB*2'+-1m@-1m
1#m &m &#!&-m 3,m 31-m +91m #6&312'4-m "#m *-1m +0!"-0#1m 1Ym 12#m 2'.-m "#m
.-*'+-0$'1+-m #1m +37m 3,",2#m #,m #*m m ^0--)#1Zm GOOO_m 7m #6'12#,m 40'1m
2#!,-*-%>1m 09.'"1m 7m #$'!'#,2#1m /3#m .#0+'2#,m 13m "#1!3 0'+'#,2-m 7m 13m %#,-2'."-m
^+'2&m 7m !-*YZm HFGH_Ym *%3,-1m #(#+.*-1m "#m 2<!,'!1m .0m 13m "#1!3 0'+'#,2-m 1-,m *m
&' 0'"!'A,m+#"',2#m+'!0-+20'!#1m^-0#4'28m7m!-*YZmHFFI_Zm#*m,9*'1'1m#(O-#&#)mm.02'0m
"#m 1#!3#,!'1m "#m 1m ^2*,--O +/(O !_m ^#&'1m 7m !-*YZm HFGH_m -m *m
0#1#!3#,!'!'A,m "#m +.*'!-,#1m ^&-'m 7m !-*YZm HFFM_Ym !23*+#,2#Zm #,m #1.#!'#1m !-,m #*m
%#,-+m 1#!3#,!'"-m 1#m .3#"#,m - 2#,#0m !'#,2-1m "#m +'*#1m "#m 1m m .02'0m "#m
0#1#!3#,!'!'A,m "#m 40'#""#1Ym 0m 13m %#,-2'."-m 1#m &,m "#100-**"-m 2<!,'!1m
!-+-m#*m31-m"#m1-,"1m/,Zm#,20#m+3!&1m-201Ym#!'#,2#+#,2#m1#m&m1230"-m#*m
+.m %#,<2'!-m "#m +#*A,m 201m #*m "#100-**-m "#m +0!"-0#1m +-*#!3*0#1m 2'.-m 1m
^0!'j1m7m!-*YZmHFGH_Ym
m
BMBMFTTTTTT
BMBMFMBTTTTT
,m#*mHFFKm1#m0#*'8Am*m.0'+#0m0#3,'A,m',2#0,!'-,*m"#m%#,A+'!m"#m3!30 '29!#1m
#,m0!#*-,Zm"-,"#m1#m#12 *#!'Am#*m(.,(.#)(&O//,#.O()'#-O(#.#.#0m^3_Zm
/3#m 2-+Am "#!'1'-,#1m "#m !'#02m 0#*#4,!'m .0m *m ',4#12'%!'A,m #,m #12m #1.#!'#Ym ,m
"#m #**1m $3#m - 2#,#0m 3,m #6&312'4m !-*#!!'A,m "#m 1#!3#,!'1m "#m 1Ym 20m "#!'1'A,m
0#*#4,2#m$3#m3,'$'!0m*-1m+.1m%#,<2'!-1m/3#m&12m#,2-,!#1m1#m& >,m"#100-**"-m
"#m3,m+-"-m',"#.#,"'#,2#m#,m"'12',2-1m* -02-0'-1Ym#m#12#m+-"-m1#m- 2#,"0>m3,m
+.m %#,<2'!-m !-,1#,1-m +3!&-m +91m 1230"-m ^'8m 7m !-*YZm HFGG_Ym ',*+#,2#Zm 1#m
"#!'"'Am#* -00m3,mB,'!m.9%',m5# m"-,"#m2-"-1m*-1m"2-1m%#,<2'!-1m7m%#,A+'!-1m
$3#0,m"#m!!#1-m.B *'!-m^&22.[ii555Y'!3%'Y-0%_Ym
m
_mmmm
,m +.m %#,<2'!-m -m +.m "#m *'%+'#,2-m #1m 3,m +.m "#m +0!"-0#1m .-*'+A0$'!-1m
"-,"#m1#m"#$',#,m*1m"'12,!'1m#,20#m*-1m+0!"-0#1m#,m$3,!'A,m"#m*m$0#!3#,!'m"#m
0#!-+ ',!'A,m /3#m &m & '"-m #,20#m #**-1Ym #.0-"3!#,m *m #1203!230m "#m *-1m
!0-+-1-+1m+-120,"-m*1m.-1'!'-,#1m"#m*-1m%#,#1m^#,m!1-m"#m/3#m#*m+0!"-0m#12<m
GF
&O'&9(O3O&O!(9'#O&O'&9(O
(.,)/#9(O
,!*"-m#,m3,m%#,_m-m"#m+0!"-0#1m,A,'+-1m 1"-1m#,m$0%+#,2-1m"#m1#!3#,!'1m
,-m!-"'$'!,2#1Ymm
,m +.m %#,<2'!-m 1#m - 2'#,#m %#,#0*+#,2#m m .02'0m "#m .- *!'-,#1m 1#%0#%,2#1m
- 2#,'"1m "#m !038+'#,2-1m #,20#m "-1m *>,#1m .0#,2*#1Ym 3,2-m +91m *#("-1m
$'*-%#,<2'!+#,2#m1#,m*-1m.0#,2*#1Zm+91m',$-0+2'4-m.-"09m1#0m#*m!038+'#,2-Zm7m
/3#m #*m %0"-m "#m .-*'+-0$'1+-m 1#09m +91m #*#4"-Ym 6'12#,m "'12',2-1m +.1m %#,<2'!-1m
"#m +#*A,m ^3"0!!-j0,1Zm GOOL\m <0',m 7m !-*YZm HFFH_Ym #0-m !"m +.m %#,<2'!-m &m
1'"-m #* -0"-m !-,m "'12',21m *>,#1m .0#,2*#1m -m "'12',2-1m 2'.-1m "#m .- *!'-,#1Ym -1m
2'.-1m "#m +0!"-0#1m /3#m 1#m &,m .-1'!'-,"-m #,m !"m +.m %#,<2'!-m 2+ '<,m 1-,m
"'12',2-1Ym*'4#0m7m!-*Ym^HFFG_m#* -00-,m3,m+.m%#,<2'!-mm.02'0m"#m3,m.- *!'A,m
Hm- 2#,'"m"#*m!038+'#,2-m#,20#m"-1m*>,#1m.0#,2*#1Zm3,m#*m2'.-m()),/-mb'#*m"#m
1.-cmGGGm7m*m!!#1'A,m!-0#,m-,%5&,m&0+'ZmmGLGIMKYm12#m+.m1#m%#,#0Am
','!'*+#,2#m !-,m JGGm +0!"-0#1m 1Ym -12#0'-0+#,2#m -,8*-m 7m !-*Ym ^HFFK_m
%#,#00-,m 3,m .- *!'A,m "#m *>,#1m "- *#m &.*-'"#1m ^1_Zm m .02'0m "#*m +'1+-m
!038+'#,2-m ,2#0'-0Ym m 4#,2(m "#m *1m 1m #1m /3#m !-,12'237#,m 3,m .- *!'A,m
',+-02*m 7m /3#m 1-,m 2-2*+#,2#m &-+-!'%A2'!1m 7m .-0m *-m 2,2-Zm #,m *1m %#,#0!'-,#1m
.-12#0'-0#1m - 2#,'"1m .-0m 32-$#!3,"!'A,Zm 1#m - 2'#,#,m .*,21m '"<,2'!1m m *1m
.0#,2*#1m ^-,8*-m 7m !-*YZm HFFK_Ym m .02'0m "#m #12m .- *!'A,m 1#m %#,#0Am 3,m ,3#4-m
+.m %#,<2'!-m 1"-m #,m #*m ,2#0'-0Zm 7m $',*+#,2#m 1#m $31'-,Am *m ',$-0+!'A,m "#m
+ -1m +.1Ym *m 0#13*2,2#m !-,2#,>m .0-6'+"+#,2#m JMOm +0!"-0#1m !-,m 3,m
"#,1'""m "#m HYKm !i+0!"-0m ^-,8*-m 7m !-*YZm HFFK_Zm *m !3*m 1#m @"'#0-,m ,3#4-1m
+0!"-0#1m2'.-m1m7m1m#,m.-12#0'-0#1m20 (-1m^#*#3m7m!-*YmHFFOZm#0,9,"#8j
'*4m 7m !-*YZm HFFN_Ym 30,2#m #*m HFGGm 7m "#,20-m "#*m 3m 1#m .3 *'!Am 3,m ,3#4-m +.m
!-,1#,1-m 1"-m #,m *m $31'A,m "#m *m ',$-0+!'A,m /3#m & >,m .-02"-m -!&-m +.1m
%#,<2'!-1m .3 *'!"-1m ,2#0'-0+#,2#Ym 12#m ,3#4-m +.m !-,1#,1-m !-,2'#,#m GKOHm
+0!"-0#1m ^LJFm 1Zm IIFm 1Zm HKHm 1Zm HIOm 1Zm NOm 1m 7m GGm !0!2#0#1m
+-0$-*A%'!-1Zm#,20#m-20-1_m!-,m3,m"#,1'""m+#"'m"#mFZMHm!+i+0!"-0m^'8m7m!-*YZm
HFGG_Ym,m#12#m+.m!-,1#,1-m2+ '<,m1#m&,m.-1'!'-,"-m2-"-1m*-1m1m"#1!0'2-1m
#,m+#*A,m&12m*m$#!&YYm
m
_mmm
20m &#00+'#,2m $0#!3#,2#+#,2#m 31"m #,m '-2#!,-*-%>m #1m *m 20,1$-0+!'A,m
%#,<2'!m.0m*m- 2#,!'A,m"#m.*,21m20,1%<,'!1Ym,m#*m!1-m"#*m+#*A,Zm3,/3#m1#m
&m !-,1#%3'"-m *m 20,1$-0+!'A,m !-,m !,).,#/'O ./' #(-FO #1m 3,m +<2-"-m
',#$'!'#,2#Ym m 20,1$-0+!'A,m #,m +#*A,m 1#m 32'*'8Am .-0m .0'+#0m 4#8m !-,m <6'2-m .-0m
,%m 7m 03+#2m #,m GOOFm .#0-m *m 21m "#m #$'!'#,!'m $3#m +37m (Ym ,-m "#m *-1m
GG
(.,)/#9(
El melón y la genómica de melón
',!-,4#,'#,2#1m #,m *m 20,1$-0+!'A,m "#m *1m .*,21m "#m +#*A,m $3#m #*m "#1!3 0'+'#,2-m
"#m /3#m !'#02-1m #6.*,2#1m .0-"3!>,m ,'4#*#1m +91m *2-1m "#m #2'*#,-m "30,2#m *m
',-!3*!'A,m!-,m!,).,#/'O^830m7m!-*YZmHFFF_Ym12m.0-"3!!'A,m"#m#2'*#,-m',&' >m
*m#6.0#1'A,m"#*m%#,m0#,m7m!-,1#!3#,2#+#,2#m*m20,1$-0+!'A,m^-,)m7m!-*YmHFFN_Ym
!23*+#,2#m2-"4>m1#m#129,m'"#,2'$'!"-m*-1m$!2-0#1m.-0m*-1m!3*#1m*m#$'!'#,!'m"#m
20,1$-0+!'A,m#,m+#*A,m#1m (m^&-4#*-,m7m!-*YZmHFGG_Ymm
_mm
0m .-"#0m 0#*'80m #6.#0'+#,2-1m !-+-m #*m !*-,(#m .-1'!'-,*m "#m %#,#1m 7m .0- 0m *m
$3,!'-,*'""m "#m !'#02-1m %#,#1Zm &m 1'"-m ',"'1.#,1 *#m "'1.-,#0m "#m !-*#!!'-,#1m "#m
1m ^0-+-1-+1m 02'$'!'*#1m !2#0',-1_Ym -1m 1m 1-,m 2+ '<,m "#m %0,m
32'*'""m.0m.-"#0m"#100-**0m+.1m$>1'!-1Zm-m&!#0m#123"'-1m"#m+'!0-1',2#,'m!-,m
-201m #1.#!'#1Zm #,20#m -201m .*'!!'-,#1m ^3"'+,m 7m !-*YZm HFFF\m #*#3m 7m !-*YZm HFFM\m
-,89*#8m 7m !-*YZm HFGF_Ym 1m .0'+#01m %#,-2#!1m "#m 1m #,m +#*A,m $3#0-,m
!-,1203'"1m m .02'0m "#m *m *>,#m jGm ^3-m 7m !-*YZm HFFG_Ym -12#0'-0+#,2#m 1#m &,m
%#,#0"-m -201m %#,-2#!1m /3#m 1#m &,m 31"-m .0m "#2#!20m %#,#1m "#m ',2#0<1m /3#m
!-,$'#0#,m0#1'12#,!'1mm.2A%#,-1m^'#2-m7m!-*YZmHFFL_m-m%#,#1m/3#m"#2#0+',,m#*m1#6-m
#,m *1m $*-0#1m "#m *1m .*,21m "#m +#*A,m ^-3*#+m 7m !-*YZm HFFN_Ym "#+91Zm *-1m 1m
.#0+'2#,m *-!*'80m +0!"-0#1m +-*#!3*0#1m "#m 3,m +.m %#,<2'!-m 7m "#m #12#m +-"-m
0#*'80m+.1m$>1'!-1m"#m*m#1.#!'#Zm.0-.-0!'-,,"-m,!*(#1m.0m*m*-!*'8!'A,m"#m
%#,#1m !,"'"2-1m 7m .0m #*m #,1+ *(#m "#m %#,-+1m ^-,89*#8m 7m !-*YZm HFGF _Ym
#"',2#m!*-,(#m.-1'!'-,*m1#m&,m'1*"-m*-1m%#,#1m"#m0#1'12#,!'m.FO(-0m7m)'N<m
$0#,2#mm*%3,1mm#,$#0+#""#1m/3#m13$0#m#*m!3*2'4-m"#*m+#*A,m^20,1+'1'A,m"#m4'031m
.-0m 9$'"-1Zm m ^&)(O ,).#O *).O #,/-_m 7m /-,#/'O )23-*),/'O EO -*Em +#*-,'1m
08m FZGZm 0#1.#!2'4+#,2#_m ^3/3#2m 7m !-*YZm HFFJ\m '#2-m 7m !-*YZm HFFL\m -- #30m 7m !-*YZm
HFFJ_Ym
_mm
*m!-,-!'+'#,2-m"#m1#!3#,!'1m"#m1m#1m!*4#m.0m*m1#*#!!'A,m"#m%#,#1m!,"'"2-1m
.0m !0!2#0#1m "#m ',2#0<1m %0-,A+'!-Ym -,8*#8j #1m 7m !-* -0"-0#1m #,m HFFMm
^,#6-m_m.3 *'!0-,mIFYFFFm,3#41m1#!3#,!'1m"#m1m- 2#,'"1mm.02'0m"#m-!&-m
%#,-2#!1m"#m!m#* -0"1mm.02'0m"#m+m#620>"-m"#m+3#1201m"#m$032-m#,m
"'12',2-1m #12"'-1m "#m "#100-**-m 7m "#m 2#('"-1m ',$#!2"-1m !-,m .2A%#,-1Ym -,m *m
- 2#,!'A,m "#m 1#!3#,!'1m "#m 1m 1#m .3"'#0-,m "#2#!20m ,3#4-1m .-*'+-0$'1+-1m 7m m
.02'0m"#m#**-1m"#100-**0m,3#4-1m+0!"-0#1m+-*#!3*0#1m3+#,2,"-m*m1230!'A,m
"#*m+.m%#,<2'!-m^#*#3m7m!-*YZmHFFO_m^,#6-m _Ym,mHFGGm-20-m#123"'-mm.02'0m"#mGKm
%#,-2#!1m "#m !m "#m $032-1Zm $*-0#1Zm &-(1Zm 0>!#1Zm !-2'*#"-,#1Zm 7m !**-1m "#m !320-m
GH
&O'&9(O3O&O!(9'#O&O'&9(O
(.,)/#9(O
%#,-2'.-1m"#m+#*A,Zm.#0+'2'Am- 2#,#0mMGYKMMm1mm.02'0m"#m%#,-2#!1m#,0'/3#!'"1mm
7mHHYGMOm1mm.02'0m"#m%#,-2#!1m"#m!m#129,"0Ymm',2#%0!'A,m"#m#12-1m1m
(3,2-m !-,m *-1m 1m "'1.-,' *#1m ,2#0'-0+#,2#m .#0+'2'Am - 2#,#0m 3,m 2-2*m "#m HJYJJJm
3,'%#,#1m ^*#.#2m 7m !-YZm HFGG_Ym #!'#,2#+#,2#m 1#m &,m %#,#0"-m 2+ '<,m "2-1m "#m
j1#/m m .02'0m "#m "'12',2-1m %#,-2'.-1m 7m 2#('"-1m "#m +#*A,Zm 32'*'8,"-m
.'0-1#!3#,!'!'A,m JKJm ^*,!m 7m !-*YYm HFGG\m -0 !&-m 7m !-*YYm HFGI\m -02,-7m 7m !-*YZm
HFGG_m-m1#!3#,!'!'A,m+#"',2#m'm^*,!m7m!-*YZmHFGH_Ym
_mmmmm
m#* -0!'A,m"#m+'!0-m+20'!#1m"#mm^'#,),,3-_m&m1'"-m-20m&#00+'#,2m/3#m
1#m&m"#100-**"-m#,m+#*A,m7m/3#m.-02m',$-0+!'A,mm,'4#*m20,1!0'.2A+'!-m-m"#m
#6.0#1'A,m"#m%#,#1m#,m2#('"-1m7m!-,"'!'-,#1m#1.#!>$'!1Ymm+'!0-m+20'8m"#m+#*A,m1#m
"'1#@Am!-,mGMYKGFm3,'%#,#1m"#0'4"-1m"#mIIYJGNm1m^1!0#**j0#31m7m!-*YZmHFFO_Ym
12-1m1m$3#0-,m'1*"-1mm.02'0m"#m"'12',2-1m#12"'-1m"#m"#100-**-m"#m*m.*,2m-m
#,m 1'23!'-,#1m + '#,2*#1m #1.#!>$'!1m ^-,89*#8j #1m 7m !-*YZm HFFM_Ym 0m 4*'"0m *m
+'!0-m+20'8m1#m0#*'80-,m20#1m#6.#0'+#,2-1m!-,m"'12',21m+3#1201m"#m+#*A,Ym,m*m
.0'+#0Zm 1#m !-+.0Am *m #6.0#1'A,m #,m $032-m ',+"30-m ^GKm ">1m "#1.3<1m "#m *m
.-*','8!'A,_m!-,m*m#6.0#1'A,m"#m3,m+3#120m"#m$032-m+"30-m^JLm">1m"#1.3<1m"#m
*m .-*','8!'A,_m #,m *m 40'#""m GGGm /3#m .#0+'2'Am !-+.00m *-1m !+ '-1m
20,1!0'.!'-,*#1m "30,2#m #*m "#100-**-m "#m $032-Ym ,m 3,m 1#%3,"m &' 0'"!'A,m 1#m
!-+.0Am 2#('"-m "#m 0>8m "#1.3<1m "#m *m ',-!3*!'A,m !-,m #*m &-,%-m )()-*),-/-O
(()(&&/-m!-,m2#('"-m"#m0>8m,-m',$#!2"-m#,m*m40'#""m0#1'12#,2#m//'#-m'&)O--*O
%0#12'1m 2NGEm ',*+#,2#m #,m *m 2#0!#0m &' 0'"!'A,Zm 1#m !-+.0Am 3,m +3#120m "#m
2#('"-m"#m!-2'*#"-,#1m',$#!2"-1m.-0m#*m4'031m"#*m+-1'!-m"#*m.#.',-m^_m!-,m3,m
+3#120m"#m2#('"-m,-m',$#!2"-m"#m*m40'#""m#,"0*Ym^1!0#**j0#31m7m!-*YZmHFFO_Ym
#!'#,2#+#,2#m *m +'!0-m +20'8m "#m m 1#m &m 32'*'8"-m .0m "#1!3 0'0m !+ '-1m
20,1!0'.!'-,*#1m1-!'"-1mm*m0#1'12#,!'m*m4'031m"#*m+-1'!-m"#m*m1,">m^_m
#,m*m!!#1'A,mjGKKGm^-,8*#8j #1m7m!-*YZmHFGH_m-m*m0#1'12#,!'mm)()-*),-/-O
(()(&&/-m^-'%m#2m*mHFGI_Ym
_mm
,m *-1m B*2'+-1m @-1m 1#m &,m "#100-**"-m 3,m !-,(3,2-m "#m 2<!,'!1m .*'! *#1m m *m
%#,A+'!m$3,!'-,*Ym,m"#m*1m+91m'+.-02,2#1m#1m#*m"#100-**-m"#m.*2$-0+1m.0m
*m!0!2#0'8!'A,m"#m+32,2#1m+#"',2#mYm1m.- *!'-,#1m"#mm1-,m
.- *!'-,#1m"#m+32,2#1m#* -0"1mm.02'0m"#m*m+32%#,'8!'A,m"#m*1m1#+'**1m-m
#*m .-*#,Zm !-,m 3,m %#,2#m +329%#,-Ym m .0'+#0m .03# m "#m +32%#,#'8!'A,m m %0,m
#1!*m #,m +#*A,m 1#m &'8-m #,m *m *>,#m -7m '80#j#*Zm 3,m +#*A,m 2'.-m *'Zm "#m *m /3#m 1#m
GI
(.,)/#9(
El melón y la genómica de melón
- 234'#0-,m IFFFm $+'*'1m HYm -1m #123"'-1m $#,-2>.'!-1m "#2#!20-,m +32!'-,#1m #,m
"'12',2-1m#12"'-1m"#m"#100-**-m7m2#('"-1Zm',!*3'"-1m!-2'*#"-,#1Zm&-(1Zm$*-0#1m7m$032-1m
^"+-0m 7m !-*YZm HFFM_Ym ',m #+ 0%-Zm *m .0'+#0m .- *!'A,m "#m m %#,#0"m #,m
#12m#1.#!'#m#1m*m/3#m1#m.0#1#,2m#,m#12#m20 (-Zm- 2#,'"mm.02'0m"#m3,m*>,#m"- *#m
&.*-'"#m LHjGGIm "#m +#*A,m 2'.-m b'#*m "#m .-cm !-,m 3,m 2-2*m "#m IHLNm $+'*'1m Hm
^-,8*#8j-m 7m !-*YZm HFGG_Ym ,m HFGFm 1#m .3 *'!Am 3,m ,3#4m .- *!'A,m #,m *m !3*m 1#m
32'*'8Am 3,m 40'#""m "#m +#*A,m 2'.-m ",(.#-m ^&0-,-_m .0m "#100-**0m 3,m
.*2$-0+m "#m m "#m JFHIm $+'*'1m HYm ,m #12m .- *!'A,m 1#m 31!0-,m
+32,2#1m #,m GGm %#,#1Zm '+.*'!"-1m #,m #*m .0-!#1-m "#m +"30!'A,m "#m $032-m 7m #,m *m
"#2#0+',!'A,m"#*m1#6-m"#m*1m$*-0#1m^&+,'m7m!-*YZmHFGF_m
T
BMBMFMCTTTTT
*m +#*A,m #1m 3,m #1.#!'#m "'.*-'"#m !-,m 3,m ,B+#0-m 91'!-m "#m !0-+-1-+1m ,SGHm
^H,SH6SHJ_m^&'2)#0ZmGOML_Ym*m2+@-m"#*m%#,-+m#1m"#m.0-6'+"+#,2#mJKFm m
^03+3%,2&,ZmGOOG_Zm2+@-m1'+'*0m*m%#,-+m"#*m00-8m^JGOm _m7m!1'm!320-m
4#!#1m#*m%#,-+m"#m,#)*-#-O."&#(m^GHKm _Ym,m#*m@-mHFFOm1#m','!'Am#*m.0-7#!2-m
m!-,m#*m- (#2'4-m"#m- 2#,#0m3,m -00"-0m"#m*m1#!3#,!'m"#*m%#,-+m
"#*m +#*A,Ym !23*+#,2#Zm #*m %#,-+m "#*m +#*A,m 7m &m 1'"-m .3 *'!"-Zm & '<,"-1#m
.-"'"-m #,1+ *0m .0-6'+"+#,2#m 3,m NIZIPm "#*m %#,-+Zm /3#m !-,2'#,#m HMYJHMm
%#,#1m^0!'j1m7m!-*YZmHFGH_Ymm
m
BMBMFMDTTTST
,m *m B*2'+m "<!"Zm 1#m &m .-"'"-m - 2#,#0m *m 1#!3#,!'m "#*m %#,-+m "#m "'12',2-1m
-0%,'1+-1m ^&22.[iim 555Y,! 'Y,*+Y,'&Y%-4i#,-+#1_Ym ',m #+ 0%-Zm 2-"4>m &-7m 1#m
#129,m "#100-**,"-m &#00+'#,21m .0m .-"#0m #6.*-20m #12m ',$-0+!'A,Ym -1m
%#,-+1m#129,m1#!3#,!'"-1m.#0-m2-"4>m,-m1#m1 #m!-,m#6!2'23"m!A+-m'"#,2'$'!0m
*m $3,!'-,*'""m "#m 2-"-1m *-1m %#,#1m /3#m &,m 1'"-m ,-2"-1Ym -0m *-m 2,2-Zm #,m *m
+7-0>m "#m *-1m %#,-+1m 1#!3#,!'"-1m #6'12#m 3,m *2-m .-0!#,2(#m "#m %#,#1m ,-2"-1m
'-',$-0+92'!+#,2#m 1',m $3,!'A,m "#+-120"Zm "#,-+',"-1m b.322'4#cm -m
b3,),-5,cm ^A.#8j0m 7m !-*YZm HFFL_Ym 0m .-"#0m ,-20m 3,m %#,-+m 1#m !-+ ',,m
91'!+#,2#m20#1m+<2-"-1[mG_m<2-"-1mO#(#.#)m"#m.0#"'!!'A,m"#m%#,#1m/3#m.#0+'2#,m
.0#"#!'0m *m .0#1#,!'m "#m 3,m %#,Ym H_m #"',2#m &-+-*-%>m !-,m 1m "#m *m +'1+m
#1.#!'#YmI_m#"',2#m&-+-*-%>m!-,m.0-2#>,1m"#m*1m!3*#1m7m1#m&m"#2#0+',"-m13m
$3,!'A,m7m/3#m#129,m.0#1#,2#1m#,m*1m 1#1m"#m"2-1Ymm0m**#%0mm"#1!'$00m*m$3,!'A,m
"#m !"m %#,m ,-2"-m #,m #*m %#,-+m &!#m $*2m "#100-**0m -20-1m +<2-"-1m !-+-m
.3#"#,m1#0m*1m2<!,'!1m"#m%#,<2'!m',4#01Ymmm
GJ
&O'&9(O3O&O!(9'#O&O'&9(O
(.,)/#9(O
"#+91Zm!-,m*m1#!3#,!'!'A,m"#m%#,-+1m1#m&m.-"'"-m- 1#040m/3#m3,m%0,m.02#m
"#*m %#,-+m !-,2'#,#m 1#!3#,!'m ,-m !-"'$'!,2#Ym -0m #(#+.*-Zm #,m #*m %#,-+m &3+,-m
1A*-m #*m GZKPm #1m !-"'$'!,2#m .0m .0-6'+"+#,2#m HKYFFFm %#,#1m ^-*$1 #0%Zm HFGG_Ym
12-m**#4Am&!#m3,-1m@-1mm"#$','0m#*mbm 130cm-m#*mb(3,)mcYm#mm"#,-+',Am
1>m m 2-"1m /3#**1m 1#!3#,!'1m "#*m %#,-+m /3#m ,-m !-"'$'!,m .0m 3,m .0-2#>,m -m
1#!3#,!'1m 0#%3*"-01Zm +7-0'20'+#,2#m 20,1.-1-,#1m -m #*#+#,2-1m 0#.#2'2'4-1Ym
,'!'*+#,2#Zm 1#m .#,1 m /3#m #12m .02#m "#*m %#,-+m ,-m 2#,>m ,',%3,m $3,!'A,Zm 1',m
#+ 0%-Zm1#m&m"#+-120"-m/3#m.02#m"#m#121m1#!3#,!'1m1#m20,1!0' #,m7m*%3,1m"#m
#**1m 0#%3*,m *m #6.0#1'A,m "#m *-1m %#,#1m ^&#m m ^7!*-.#"'m $m m
*#+#,21_m 0-(#!2m -,1-02'3+Zm HFFJ_m -m /3#m 1-,m '+.-02,2#1m .0m #12 '*'80m !'#021m
#1203!2301m!-+-m1-,m*-1m2#*A+#0-1m7m*-1m!#,20A+#0-1m^#$$#0,'!)ZmHFFO_Ymm
GK
(.,)/#9(
Mutaciones en el ADN
m
T
BMCTTT
T
BMCMBTTTT
#$','+-1m +32!'A,m !-+-m 3,m !+ '-m #,m *m ',$-0+!'A,m %#,<2'!m ^_Ym 1m
+32!'-,#1m 2'#,#,m '+.-02,!'m .-0/3<m .-02,m 40',2#1m *<*'!1m 7m 40',2#1m
$3,!'-,*#1m.0-4-!,"-m$#,-2'.-1m"'12',2-1m"#*m$#,-2'.-m1'*4#120#Ymm+32!'-,#1m1-,m
&#0#" *#1Zm 3,/3#m #,m -0%,'1+-1m +3*2'!#*3*0#1m &7m /3#m "'12',%3'0m #,20#m
+32!'-,#1m.0-"3!'"1m#,m*-1m%+#2-1m7m*1m.0-"3!'"1m#,m*1m!<*3*1m1-+92'!1Ym1m
+32!'-,#1m .0-"3!'"1m #,m *-1m %+#2-1m 1#m 20,1$#0'09,m m *m 1'%3'#,2#m %#,#0!'A,m
+'#,201m/3#m*1m.0-"3!'"1m#,m*1m!<*3*1m1-+92'!1m1A*-m$#!20,m*m',"'4'"3-m/3#m
*1m."#!#m7m,-m2#,"09,m3,m#$#!2-m&#0#"'20'-Ym
m$0#!3#,!'m"#m+32!'A,m#,m*-1m%#,#1m"#.#,"#m"#m40'-1m$!2-0#1Zm3,/3#m*-1m+91m
'+.-02,2#1m 1#,m *m *-,%'23"m "#*m %#,m 7m #*m &#!&-m "#m !-,2#,#0m 8-,1m !-,m +91m
.0- '*'""m"#m1#0m+32"1m^.-0m2#,#0m3,m*2m.0- '*'""m"#m+32!'A,m"# '"-mm
13m !-+.-1'!'A,m /3>+'!_Zm !-+-m 1-,m *1m 0#%'-,#1m .m ^03#, 3+m 7m !-*YZm GONGZm
-,2#*-,#Zm GOON_Ym m $0#!3#,!'m !-,m *m /3#m *m m .-*'+#01m ',1#02m 3,m 1#m
#/3'4-!"m #,m ++>$#0-1m #1m "#m G6GFjOm ^3+0m 7m !-*YZm HFFM_m +'#,201m /3#m *m
$0#!3#,!'m #,m *-1m 4'031m "#m m #1m "#m G6GFjLm^0-+1m 7m *#,Zm HFGF_Ym ,m .*,21m ,-m
.0#!#m& #0m3,m#123"'-m.0#!'1-m"#m#12-1m4*-0#1Ymm
1m +32!'-,#1m ,230*#1m .-02,m 40',2#1m *<*'!1m /3#m .3#"#,m .-020m ,3#41m
.0-.'#""#1m m 3,m #1.#!'#Ym ,m #(#+.*-m +.*'+#,2#m #123"'"-m #,m .*,21m 1-,m *1m
40',2#1m*<*'!1m"#*m%#,m>Ym,m+#*A,Zm1>m!-+-m#,m.'+'#,2-m^3$$#*m7m!-*YZmHFFH_Zm
%3'1,2#m^-m7m!-*YZmHFFJ_m7m-201m#1.#!'#1m1#m&,m"#2#!2"-m+32!'-,#1m,230*#1m#,m
#*m %#,m >m 0#1.-,1 *#1m "#m *m 0#1'12#,!'m m "'12',2-1m 4'031Zm !-+-m 4#0#+-1m +91m
"#*,2#Ym ,m 2-"-1m *-1m !1-1m #*m 4'031m ,-m .3#"#m ',2#0!!'-,0m !-,m #*m $!2-0m "#m
','!'!'A,m"#m*m20"3!!'A,mJm7m.-0m*-m2,2-m,-m.3#"#m0#.*'!01#m#m',$#!20m*m.*,2m
^'#2-m7m!-*YZmHFFL_Ymm
T
BMCMBMBTTTTT
1m
1#1m ,'20-%#,"1m 1-,m 131!#.2' *#1m m 13$0'0m *#1'-,#1m #1.-,29,#1m #,m 13m
!-,$-0+!'A,m /3>+'!Ym 121m *2#0!'-,#1m .3#"#,m .0-"3!'0m 40'-1m #$#!2-1m #,m *m
!"#,m +-*"#m $#!2,"-m m *m !-+.*#+#,20'#""m "#m *m +'1+m -m .0-4-!,"-m -20-1m
2'.-1m"#m*2#0!'-,#1m!-+-m*1m',1#0!'-,#1m-m*1m"#*#!'-,#1Ymm
m
GL
/.#)(-O(O&OOO
(.,)/#9(O
_mmmm
MBT$!(#.1T T+ 1T%.0, 1T2 32.,:0(" 1T
"m 3,m "#m *1m 1#1m "#*m m .3#"#m #6'12'0m #,m *m ,230*#8m #,m "-1m $-0+1m
232-+<0'!1Ym 1m "'$#0#,!'1m /3>+'!1m #,20#m *1m "-1m $-0+1m 232-+<0'!1m !-,1'12#,m
#,m #*m "#1.*8+'#,2-m "#m 92-+-1m "#m &'"0A%#,-m /3#m +'%0,m &!'m .-1'!'-,#1m +91m
',#12 *#1Ym -0+*+#,2#Zm #,m #*m m 1#m #,!3#,20m *m $-0+m .)Zm +'#,201m /3#m *1m
$-0+1m#'#()m-m()&m1-,m&12mGFYFFFm4#!#1m+91m',#12 *#1m^-,2#*-,#ZmGOON_Ym6'12#,m
"-1m2'.-1m"#m0#!!'-,#1m232-+<0'!1Zm*m#2-k#,A*'!m^"-,"#m3,m%03.-m!#2-m^S_m1#m
!-,4'#02#m#,m3,m%03.-m#,-*m^j__m-m+',j+',m^"-,"#m#*m%03.-m+',m^S_m1#m
!-,4'#02#m #,m 3,m %03.-m +',-m ^jH__Ym 1m $-0+1m 232-+<0'!1m ',#12 *#1m 2'#,#,m
#1.#!>$'!+#,2#m *2#0"1m *1m .0-.'#""#1m "#m !-+.*#+#,20'#""m #,m *m !"#,m "#m
m$#!2,"-m#,m13m0#.*'!!'A,Ym,m*m *mGYGm.3#"#m- 1#0401#m!-+-m!+ 'm*m
!-+.*#+#,20'#""m"#m*1m 1#1Y
T
!+ T BMBOT #.0#1#,2!'A,m "#m *1m $-0+1m 232-+<0'!1m "#m !"m 1#m /3#m $-0+m #*m m 7m "#m *m
!-+.*#+#,20'#""m/3#m"/3'#0#,Ym
m
m
-0+m
32-+<0'!m
"-.2m
.0-.'#""#1m
"#m
-+.*#+#,2m
!-,m
gm
m
m
gm
m
m
gm
m
m
gm
m
m
m
'm#,m*m!"#,m+-*"#m/3#m1#m#129m0#.*'!,"-m&7m3,m$-0+m232-+<0'!m"#m3,m 1#m
1#m.3#"#m.0-"3!'0m3,m#00-0m"#m!-+.*#+#,20'#""m.0-4-!,"-m*m',!-0.-0!'A,m"#m
3,m 1#m /3#m ,-m #1m *m !-00#1.-,"'#,2#m m *m 1#!3#,!'m -0'%',*m 7m #,m !-,1#!3#,!'m
.0-"3!'0m3,m+32!'A,Ym12-1m!+ '-1m.3#"#,m1#0m20,1'!'-,#1m 1"-1m#,m#*m!+ '-m
"#m3,m.30',mm-20m.30',m^Z_m-m"#m3,m.'0'+'"',mm-20m.'0'+'"',m^Zm_Ym1m
!-,1#!3#,!'1m"#m#12-1m!+ '-1m"#.#,"#,m"#m*m.-1'!'A,m7m"#*m2'.-m"#m!+ '-m/3#m1#m
.0-"38!m 1>m !-+-m "#m 13m *-!*'8!'A,m ^#,m 0#%'A,m #6A,'!m -m ',20A,'!_Ym 'm *m 1#m
!+ '"m1#m#,!3#,20m#,m0#%'A,m#6A,'!m7m,-m.0-4-!m3,m!+ '-m"#m+',-9!'"-m#,m
*m 20"3!!'A,m "#*m !-"A,Zm <12#m 1#09m 3,m !+ '-m 1'*#,!'-1-Ym -0+*+#,2#Zm #12#m 2'.-m "#m
!+ '-m 1#m .0-"3!#m !3,"-m +32m *m 2#0!#0m 1#m "#*m !-"A,m .3#12-m /3#m #*m !A"'%-m
GM
(.,)/#9(
Mutaciones en el ADN
%#,<2'!-m#1m"#%#,#0"-Ym'm#*m!+ '-m"#m 1#m.0-4-!m3,m!+ '-m"#m+',-9!'"-m1#m
"#,-+',m !+ '-m 1312'232'4-m -m ,-m 1',A,'+-EO ',*+#,2#Zm 1'm #*m !+ '-m "#m 1#m
.0-"3!#m3,m!-"A,m12-.m1#m"#,-+',m!+ '-m1',m1#,2'"-m""-m/3#m.-"0>m"#2#,#01#m
*m20"3!!'A,m**>m"-,"#m1#m"#m*m+32!'A,m7m#,m!-,1#!3#,!'Zm,-m.-"#0m1',2#2'80m*m
.0-2#>,m #,m 13m 2-2*'""Ym -1m !+ '-1m 1312'232'4-1m 7m 1',m 1#,2'"-O1-,m *-1m +91m %04#1m
7m/3#m.3#"#,m$#!20m1#0'+#,2#mm*m#1203!230m7m.-0m*-m2,2-mm*m$3,!'-,*'""m"#m
*m.0-2#>,Ym
MCT-1$0"(.-$1T7T#$+$"(.-$1T
,m "#m *1m +32!'-,#1m #1.-,29,#1m +91m & '23*#1m #,m 1#!3#,!'1m 0#.#2'"1m *0%1m
1-,m*1m"#*#!'-,#1m"#m$0%+#,2-1Ym20#'1',%#0m7m5#,m^GONK_m$-0+3*0-,m3,m+-"#*-m
/3#m #6.*'!m *-1m !+ '-1m "#m $1#m "30,2#m *m 1>,2#1'1m "#m Zm *-1m !3*#1m !+ ',m *m
.32m "#m *m *#!230m "#m *m .0-2#>,Ym 'm "30,2#m *m 0#.*'!!'A,m #,m *m !"#,m +-*"#m
2#,#+-1m3,m1#!3#,!'m!-,m3,m0#.#2'!'A,m"#m,3!*#A2'"-1m.3#"#,m-!300'0m"-1m!1-1Ym
*m .0'+#0-m #1m /3#m "30,2#m *m 0#.*'!!'A,m 1#m !0##m 3,m *8-m #,m *m !"#,m 0#.*'!"Ym m
0#.*'!!'A,m 1#%3'09m 13m !301-m 7m $',*+#,2#Zm .3#12-m /3#m #*m *8-m &m .0-4-!"-m 3,m
!-00'+'#,2-m"#m*m!"#,m0#.*'!"Zm1#m@"'09m3,m,3!*#A2'"-m+91m"#m*-1m/3#m&7m#,m
*m!"#,m+-*"#Ym*m1#%3,"-m!1-m#1m1'm#*m*8-m-!300#m#,m*m!"#,m+-*"#Ym*m!0#01#m
3,m !-00'+'#,2-m #,m *m !"#,m +-*"#Zm *m !"#,m /3#m 1#m #129m 0#.*'!,"-m 2#,"09m 3,m
,3!*#A2'"-m+#,-1m.3#12-m/3#m"-,"#m1#m&m.0-"3!'"-m#*m*8-m,-m1#m&m@"'"-m*m 1#m
!-+.*#+#,20'Ym ,m #*m .0'+#0m !1-m 1#m - 2#,"09m 3,m !"#,m !-,m 3,m ,3!*#A2'"-m "#m
+91Zm .-0m *-m 2,2-m 1#m & 09m .0-"3!'"-m 3,m ',1#0!'A,Ym ,m #*m 1#%3,"-m !1-m *m !"#,m
2#,"09m3,m,3!*#A2'"-m"#m+#,-1m7m.-0m*-m2,2-m1#m& 09m.0-"3!'"-m3,m"#*#!'A,Ym
m
_mmmmmm
T
MBT$1 ,(- "(>-T
-1m ,3!*#A2'"-1m .3#"#,m .#0"#0m #*m %03.-m +',-m .-0m "#1+',!'A,m -6'"2'4m
.0-4-!,"-m 3,m !-+.*#+#,20'#""m #00A,#m #,m *m 0#.*'!!'A,m "#*m Ym "-m /3#m
*m "#1+',!'A,m "#m !'2-1',m .0-"3!#m 30!'*-Zm *m !<*3*m 2'#,#m 1'12#+1m "#m 0#.0!'A,m
.0m0#.00m#12#m#00-0Ym1m%*3!-1'*11m"#m30!'*-m"#mm0#!-,-!#09,m*-1m30!'*-1m
#,m *m !"#,Zm *-1m #1!',"'09,m 7m #*m &3#!-m 1#09m 0#**#,"-m .-0m *m m .-*'+#01m m 7m
$',*+#,2#m*'%"-m.-0m3,m*'%1Ym'm#*m1'12#+m"#m0#.0!'A,m,-m"#2#!2m7m0#.0m#12#m
#00-0Zm #,m *m !"#,m +-*"#m .0#!#09m 3,m 30!'*-m /3#m .-12#0'-0+#,2#m #+.0#(09m !-,m
3,m"#,',m#,m4#8m"#m*m%3,',m/3#m*#m!-00#1.-,"#0>m^* #021m7m!-*YZmHFFL_Ym
T
GN
/.#)(-O(O&OOO
(.,)/#9(O
T
MCT$1/30(-(8 "(>-T
m"#1.30','8!'A,Zm'+.*'!m*m',2#003.!'A,m"#*m#,*!#m%*3!-1>"'!-m#,20#m*m 1#m7m*m
"#1-6'0' -1m7m*m.<0"'"m.-12#0'-0m"#m3,m0#1'"3-m"#m%3,',m-m"#,',m"#*mYm'm
#12m *#1'A,m .#01'12#Zm !-,"3!#m m 3,m "@-m %#,<2'!-m '+.-02,2#m .-0/3#m *-1m 1'2'-1m 1',m
.30',m /3#m 1#m -0'%',,m ,-m .3#"#,m #,*80m !-,m 13m 1#m !-+.*#+#,20'm "30,2#m *m
0#.*'!!'A,Ym ',m #+ 0%-Zm 1'12#+1m "#m 0#.0!'A,m #$'!!#1m #*'+',,m *-1m 1'2'-1m 1',m
.30',1Ym(-m!'#021m!'0!3,12,!'1m1#m.3#"#m',1#020m3,m 1#m#,m3,m1'2'-m1',m.30',\m
!-,m$0#!3#,!'m#12-m"m!-+-m0#13*2"-m3,m+32!'A,m^* #021m7m!-*YZmHFFL_Ym
MDT6(# "(>-T
*m +#2 -*'1+-m #0- '-m %#,#0Zm !-+-m 13 .0-"3!2-Zm !'#021m #1.#!'#1m !2'41m "#m
-6>%#,-Zm!-+-m0"'!*#1m13.#0A6'"-m^Hj_Zm.#0A6'"-m"#m&'"0A%#,-m^HH_m7m0"'!*#1m
&'"0-6'*-m^j_Ym-"1m#**1m!31,m*#1'-,#1m-6'"2'41m#,m#*mm/3#m",m*3%0mm
+32!'-,#1Zm*1m+91m$0#!3#,2#1m1-,mmmm.-0m-6'"!'A,m"#m*m%3,',m^* #021m7m!-*YZm
HFFL_YT
m
GO
(.,)/#9(
Mutagénesis inducida
m
m
BMDTTT
m
BMDMBTTTT
#m "#,-+',m +32%<,#1'1m m *m .0-"3!!'A,m ',2#,!'-,"m "#m 40' '*'""m %#,<2'!m m
204<1m "#m +<2-"-1m $>1'!-1m -m /3>+'!-1Ym m 32'*'8!'A,m "#m #12-1m +<2-"-1m .0-4-!m
!+ '-1m *m 80m #,m #*m Zm 7m #,m !-,1#!3#,!'Zm 2+ '<,m #,m *-1m %#,#1m 7m #,m #*m
-0%,'1+-Ym,m.*,21Zm*m+32%<,#1'1m%#,#0*+#,2#m1#m.*'!m#,m*1m1#+'**1m3,/3#m
#,m *%3,1m #1.#!'#1m !-+-m #*m +>8m 1#m &m .*'!"-m #,m #*m .-*#,Ym 12#m +<2-"-m '+.*'!m
/3#m &!#m $*2m #1.#00Zm *m +#,-1Zm m *m 1#%3,"m %#,#0!'A,m .0m - 1#040m #$#!2-1m
$#,-2>.'!-1m^-"7+m7m$8ZmHFFI_Ymm
m +32%<,#1'1m .3#"#m 1#0m "'0'%'"m -m ',"3!'"Ym *m .0'+#0m 2<0+',-m &!#m 0#$#0#,!'m m
/3#**1m +32!'-,#1m .0#!'11m /3#m 1#m .0-4-!,m #,m 3,m %#,m "#1#"-Ym *m 1#%3,"-Zm #,m
!+ '-Zm&!#m0#$#0#,!'mm*m+32%<,#1'1m',"3!'"m.-0m+329%#,-1m/3>+'!-1m-m$>1'!-1m
/3#m.0-4-!,m!+ '-1m*m80m7m"#m3,m+,#0m%#,#0*'8"m#,m2-"-m#*m%#,-+Ym-1m
.0'+#0-1m ',4#12'%"-0#1m /3#m .0-"3(#0-,m 02'$'!'*+#,2#m +32!'-,#1m #,m #*m m
$3#0-,m 3**#0m 7m 2"*#0m #,m #*m GOHMm 7m GOHNm 0#1.#!2'4+#,2#m !-,m *m "#+-120!'A,m "#*m
#$#!2-m+32%<,'!-m"#m*-1m07-1mm#,m+>8Zm!# "m7m,)-)*"#&O'&()!-.,m^* #021m7m
!-*YZmHFFL_Ym
m
BMDMCTTTTT
TT
m +#(-0m %#,<2'!m 1#m 1m #,m - 1#040m 7m 1#*#!!'-,0m *-1m +#(-0#1m ',"'4'"3-1m #,m
$3,!'A,m "#m *-1m !0!2#0#1m "#m ',2#0<1m .0m .-12#0'-0+#,2#m $'(0m #1#m !09!2#0m #,m 3,m
.- *!'A,Ym#m#12#m+-"-m1#m"#100-**,m.- *!'-,#1m!-,m!0!2#0#1mA.2'+-1m.0m3,m
- (#2'4-m!-+#0!'*Ymm+#(-0m%#,<2'!m31m!-+-m&#00+'#,2m*m40' '*'""m%#,<2'!m
#6'12#,2#m#,20#m.- *!'-,#1m7m#,20#m',"'4'"3-1Ym12m$3#,2#m"#m40' '*'""m1#m%#,#0m
#1.-,29,#+#,2#m!A+-m0#13*2"-m"#m#00-0#1m.0-4-!"-1m#,m*m"'4'1'A,m!#*3*0m7m#,m
#*m 1'12#+m "#m 0#.0!'A,m "#*m m ^*m 0"'!'A,m 1-*0m 2'#,#m 3,m '+.-02,2#m ..#*m #,m
#12-1m #00-0#1_Ym 1m +32!'-,#1m ,230*#1m ,-m 1-,m 3,",2#1m 7m +#,-1m 3,m !3,"-m
**#%,mm$#!20mm*m$3,!'-,*'""m"#m*1m.0-2#>,1m^#,m&3+,-1m*m21m"#m+32!'A,m
#1m "#*m -0"#,m "#m GFjLm +32!'-,#1i*-!31i%#,#0!'A,Zm 3,/3#m .3#"#m 40'0m "#,20-m "#*m
0,%-m"#mGFjJm&12mGFjMm"#.#,"'#,"-m"#*m2+@-m"#*m%#,m^-*%',ZmHFFO__Ym1m.-0m#**-m
/3#m m .0',!'.'-1m "#*m 1'%*-m m 1#m #+.#80-,m m .*'!0m 2<!,'!1m "#m +32%<,#1'1m .0m
.0-"3!'0m+91m40' '*'""m7m"#m3,m+-"-m+91m09.'"-Ymm.0',!'.'-1m"#m*-1m@-1mKFm1#m
HF
/.!6(-#-O#(/#O
(.,)/#9(O
#+.#80-,mm310m!-+#0!'*+#,2#m!3*2'4-1m+#(-0"-1mm204<1m"#m*m+32%<,#1'1Ymm
m ^))O (O !,#/&./,O ,!(#4.#)(_m #+.#8Am m "-!3+#,20m 2-"-1m *-1m !3*2'4-1m
- 2#,'"-1m .-0m +32%<,#1'1m 7m #,m GOOJm *m *'12m !-,2#,>m +91m "#m GNFFm !3*2'40#1m
^555Y$-Y-0%_Ym!23*+#,2#m*m+7-0>m"#m!3*2'4-1m#6.*-2"-1m#,m#*m+3,"-m1-,m7i-m
.0-4'#,#,m"#m!038+'#,2-1m"#m*-1m.0'+#0-1m!3*2'40#1m+32"-1Ym-0m#(#+.*-Zm#,mGOOJm
*m m "#2**Am /3#m #*m MFPm "#m 20'%-m "30-m ^,#.#/'O -.#0/'_m !3*2'4"-m #,m 2*'m
.0-4#,>m"#m!3*2'4-1m+32"-1Ymm
m+32%<,#1'1m/3>+'!m7m*m0"'!'A,m'-,'8,2#m&,m1'"-m32'*'8"1m#$'!'#,2#+#,2#m
.0m%#,#00m40' '*'""m%#,<2'!m/3#m.-12#0'-0+#,2#m&m1'"-m32'*'8"m#,m*m+#(-0m
%#,<2'!Ym 121m +#2-"-*-%>1m 2'#,#,m *m 4#,2(m "#m 1#0m +91m #!-,A+'!1m 7m *m +'1+-m
2'#+.-Zm +91m !#.2"1m 1-!'*+#,2#m /3#m *m 2#!,-*-%>m 20,1%<,'!Ym !23*+#,2#Zm m
204<1m "#m #12-1m +<2-"-1m #6'12#,m HYJHNm 40'#""#1m "#m !3*2'4-1m +32%#,'8"-1m #,m #*m
+3,"-ZmKFGm"#m#**-1m#,m00-8m^&22.[ii555j+4"Y'#Y-0%i_Ym
T
BMDMDTTTTT
,m+329%#,-m#1m3,m.0-"3!2-m,230*m-m#* -0"-m.-0m#*m&-+ 0#m/3#m.3#"#m*2#00m*m
#1203!230m -m *m 1#!3#,!'m "#*m Ym -"#+-1m !*1'$'!0m #12-1m %#,2#1m +329%#,-1m #,m
$3,!'A,m "#m !A+-m .0-"3!#,m *1m *#1'-,#1m 7m 1#m +#"',2#m 3,m +#!,'1+-m $>1'!-m -m
/3>+'!-Ymm+32%<,#1'1m+#"',2#m+32,2#1m/3>+'!-1m7m0"'!'-,#1m'-,'8,2#1m1#m&m
31"-m #,m .*,21m #,m %#,<2'!m "'0#!2m ^&'0*#7m 7m !-*YZm GOOH_Ym 12#m 2'.-m "#m +<2-"-1m
2'#,#,m !'#021m 4#,2(1m #,m !-+.0!'A,m !-,m *m +32%<,#1'1m ',1#0!'-,*Zm 7m /3#m
%#,#0,m "'4#011m 1#0'#1m *<*'!1Zm ',"3!#,m +32!'-,#1m !-,m *2m $0#!3#,!'m 7m 1-,m
.*'! *#1m m "'4#011m #1.#!'#1m ^."&77m 7m !-*Zm HFGG_Ym -0m *-m %#,#0*Zm *m
+32%<,#1'1m/3>+'!m.0-"3!#m+32!'-,#1mm"'12',2-1m,'4#*#1Zm7m1#,m+32!'-,#1m1',m
1#,2'"-m -m +32!'-,#1m 1312'232'41Ym ,m !+ '-Zm *m +32%<,#1'1m +#"',2#m 0"'!'A,m
'-,'8,2#m!-123+ 0mm.0-"3!'0m"#*#!'-,#1m!0-+-1A+'!1Ym-0m*-m2,2-Zm*m1#*#!!'A,m
"#*m +329%#,-m "# #m
101#m #,m *m #$'!'#,!'m 7m *m #1.#!'$'!'""m .0m ',"3!'0m
+32!'-,#1m 7m #,m *m *2m .0- '*'""m "#m #,!-,200m #*m 2'.-m "#m +32!'A,m "#1#"m
^ *m GYH_Ym + '<,m #1m '+.-02,2#m !-,-!#0m #*m 2'.-m "#m +32!'A,m /3#m .0-"3!#m #*m
+329%#,-m.0m.-"#0m1#*#!!'-,0m#*m+<2-"-m"#m"#2#!!'A,m+91m#$'!'#,2#m^--0,,##$m7m
!-*ZmGONH_Ymm
m
m
m
m
HG
(.,)/#9(
Mutagénesis inducida
m
!+ TBMCOm#13+#,m"#m*-1m+329%#,-1m+91m& '23*#1m!-,m13m+#!,'1+-m"#m!!'A,m7m#*m2'.-m"#m+32!'A,m
/3#m.0-"3!#,Ym
m
31 R,329&$-.T
$" -(1,.T
(/.T#$T,32 "(>-T/0.#3"(# T
00-0#1m"#m0#.*'!!'A,m7m"#m
1.-,29,#m
*-1m+#!,'1+-1m"#m
-"-m2'.-m"#m+32!'-,#1m
0#.0!'A,m"#*mm
Hj+',-.30',m
1#m,9*-%m
jmmjm7mjmmjm20,1'!'-,#1m
Kj 0-+30-30!'*-m
1#m,9*-%m
jmmjm7mjmmjm20,1'!'-,#1m
2'*m#2,-m
%#,2#m*/3'*,2#Zm%#,#0m
3*$-,2-m^_m
Lj+#2'*%3,',m
^H_m
jmmjm20,1'!'-,#1m
7-0+#,2#m!+ '-1mjmmjm
"'!'A,mm
>+#0-1m"#m.'0'+'"',m
20,1'!'-,#1Zm"#*#!'-,#1Zm!+ '-1m"#m
.32m"#m*#!230m^+#,-1m$0#!3#,2#_mm
7-1mm
"'!'A,m'-,'8,2#m
0-+30-m"#m#2'"'-m
%#,2#m',2#0!*,2#m
#*#!'-,#1m!0-+-1A+'!1m
+ '-1m"#m.32m"#m*#!230Zm
"3.*'!!'-,#1m7m"#*#!'-,#1m
m
m
m
BMDMDMBTTT
#.#,"'#,"-m "#*m +-"-m "#m !!'A,m -m +#!,'1+-1m "#m *-1m %#,2#1m /3>+'!-1m
+32%<,'!-1Zm.-"#+-1m"'12',%3'0m20#1m%03.-1m"'12',2-1[m
_mmm
1m
1#1m ,9*-%1m 1-,m #1203!2301m /3>+'!1m +37m 1#+#(,2#1m m *1m
1#1m
,'20-%#,"1m 7m 1#,m .30',1m -m .'0'+'"',1m 7m /3#m .-0m *-m 2,2-m m .3#"#,m 1312'23'0m m
<121m#m',!-0.-001#m#,m#*mm"30,2#m*m0#.*'!!'A,m7m.-12#0'-0+#,2#m.0-4-!0m3,m
+*m #+.0#(+'#,2-m "#m 1#1Zm .#0.#23,"-m 3,m +32!'A,m #,m *m 1#!3#,!'Ym -1m
#(#+.*-1m"#m#12-1m%#,2#1m1-,[m
jKj 0-+30-30!'*-[m !-+.3#12-m 02'$'!'*m 31"-m #,m ',4#12'%!'A,Ym 3m
1#+#(,8mm*m 1#m,'20-%#,"m2'+',m&!#m/3#m#+.0#(#m!-,m"#,',Ym
jHj+',-.30',[m +-*<!3*m 1#+#(,2#m m *m "#,',m 7m .-0m *-m 2,2-m .0-4-!m
#+.0#(+'#,2-m!-,m2'+',m-mm4#!#1Zm+#,-1m$0#!3#,2#+#,2#Zm!-,m!'2-1',m
^* #021m7m!-*YZmHFFL_Ym
m
m
HH
/.!6(-#-O#(/#O
(.,)/#9(O
_mmmmmm
6'12#,m 40'-1m %#,2#1m /3#m .0-4-!,m *2#0!'-,#1m #,m *m #1203!230m 7i-m #,m #*m
#+.0#(+'#,2-m"#m*1m 1#1m,'20-%#,"1Ym'20#+-1m*-1m#(#+.*-1m"#m/3#**-1m+91m
31"-1[mm
j!'"-m ,'20-1-[m .0-4-!m "#1+',!'A,m "#m !'2-1',m m 30!'*-Zm "#m
+#2'*!'2-1',mm2'+',m7m"#m%3,',mm-20m 1#m,'20-%#,"m*-m/3#m.0-4-!m
20,1'!'-,#1mYm
j%#,2#1m */3'*,2#1[m *-1m %#,2#1m */3'*,2#1m $3#0-,m *m .0'+#0m !*1#m "#m
+329%#,-1m #,m 1#0m "#1!3 '#02-1m !3,"-m 3#0 !&m 7m - 1-,m ^GOJL_m
"#1!3 0'#0-,m *-1m #$#!2-1m +32%<,'!-1m "#*m b%1m +-128cm 7m 131m
!-+.-,#,2#1m "30,2#m *m 1#%3,"m 3#00m 3,"'*Ym 6'12#,m 40'-1m %#,2#1m
*/3'*,2#1m !-+-m #*m +#2'*+#2,-m 13*$2-m ^_Zm 2'*m #2,-m 3*$-,2-m
^_Zm 7m *m ,'20-1-%3,'"',m /3#m $#!2,m *m Ym 12-1m %#,2#1m 1-,m
#$'!'#,2#1m +329%#,-1m 7m "#m $9!'*m 32'*'8!'A,Ym 1m .-0m #**-m /3#m 1-,m
& '23*+#,2#m 31"-1m #,m .*,21Zm #,m #1.#!'*m #*m Ym *m m */3'*m *1m
%3,',1Zm &!'#,"-m /3#m m #*m #+.0#(+'#,2-m 1#m !-,m *m 2'+',m 7m ,-m !-,m *m
!'2-1',m 7m .-0m *-m 2,2-m .0-4-!m 20,1'!'-,#1m "#*m 2'.-m ijim ^#%Zm GONJ_m
^'%30mGYI_Ym
m
m
m
m
(&30 TBMD[m$#!2-m"#*mmm,'4#*m+-*#!3*0Ym1m%3,',1m7m*1m2'+',1m1#m+#2'*,m7m#1#m!+ '-m/3>+'!-m
&!#m /3#m 1#m !-+.-02#,m !-+-m 3,m "#,',m 7m 3,m !'2-1',m 0#1.#!2'4+#,2#m 7m #,m !-,1#!3#,!'m
!-+.*#+#,2,m
!-,m
1#1m
"'12',21m
m
*1m
/3#m
"# #0>,m
!-+.*#+#,201#m
^$3#,2#[m
&22.[ii555Y3!+Y#1i',$-i%#,#2'!i%03.-"i32!'-,i+32!'-,Y&2+_YT
m
HI
(.,)/#9(
Mutagénesis inducida
m
_mmm
-1m %#,2#1m ',2#0!*,2#1m 1-,m +-*<!3*1m /3#m 2'#,#,m ,*-%>m !-,m *-1m ,3!*#A2'"-1m 7m
.-0m *-m 2,2-Zm 1-,m !.!#1m "#m "#1*'801#m #,20#m <12-1m #m ',2#0!*01#m #,m *m "- *#m &<*'!#m
&!'#,"-m/3#m*m.-*'+#01m1#m#/3'4-/3#m7m@"m-20m 1#m.0-"3!'#,"-m"#*#!'-,#1m
-m!-00'+'#,2-1m"#m*#!230Ym,m#(#+.*-m"#m#12-1m%#,2#1m1-,m*m.0-$*4',m7m*m,0,(m
"#m!0'"',Zm1>m!-+-m#*m 0-+30-m"#m#2'"'-m^* #021m7m!-*YZmHFFL_Ym
T
BMDMDMCTTT
T
_mmm
1m 0"'!'-,#1m #*#!20-+%,<2'!1m #129,m $-0+"1m .-0m *m !-+ ',!'A,m "#m !+.-1m
+%,<2'!-1m7m#*<!20'!-1m7m1#m.0-.%,mm204<1m"#*m#1.!'-m20,1.-02,"-m#,#0%>Ymm
0"'!'A,m#*#!20-+%,<2'!Zm!37m*-,%'23"m"#m-,"m#129m#,20#m*-1mJFFm7m*-1mGKm,+m1#m
"#,-+',mm^3*204'-*#2_Ymm*-,%'23"m"#m-,"m"#*mm!#0!,-m#129m#,20#m*-1mHFFj
JFFm,+Ym12m#1m*m+'1+m*-,%'23"m"#m-,"m/3#m 1-0 #m#*mZm!-,m3,mA.2'+-m#,m
HKJm ,+m /3#m #1m *m 1-0!'A,m +96'+Ym 12m !0!2#0>12'!m &!#m /3#m #*m m .3#"m
.0-4-!0m *#1'-,#1m #,m #*m Ym 1m +32!'-,#1m .0-"3!'"1m +91m !-+B,+#,2#m 1-,m
">+#0-1m "#m '+',1m 7m #121m "3.*'!!'-,#1m .0-4-!,m /3#m #,m *m 0#.*'!!'A,m *m m
.-*'+#01m',1#02#m3,m,3!*#A2'"-m',!-00#!2-m#,m#1m.-1'!'A,Ymm
_mmm
1m 0"'!'-,#1m '-,'8,2#1m 1-,m /3#**1m 0"'!'-,#1m !.!#1m "#m '-,'80m *m +2#0'Zm
#6207#,"-m *-1m #*#!20-,#1m "#m 131m #12"-1m #*#!20A,'!-1m /3#m *#1m +,2#,>,m *'%"-1m *m
92-+-Ym ,20#m *1m .-2#,2#1m #1.#!'#1m /3>+'!1m $-0+"1m .-0m *m 0"'!'A,m '-,'8,2#m 1#m
#,!3#,20,m*-1m0"'!*#1m*' 0#1Zm1'#,"-m#*m+91m'+.-02,2#m#*m0"'!*m&'"0-6'*-m^j_Zm
/3#m 0#!!'-,m !-,m +!0-+-*<!3*1Zm !-+-m #*m Zm 7m *1m ',!2'4m .0-"3!'#,"-m
0-2301m /3#m .3#"#,m "0m *3%0m m !+ '-1m #1203!230*#1m '+.-02,2#1Zm !-+-m 1-,m *1mm
+32!'-,#1m!0-+-1A+'!1m^&'0*#7ZmGOOH_Ym
m0"'!'A,m'-,'8,2#m&m1'"-m+.*'+#,2#m32'*'8"m.0m',"3!'0m+32!'-,#1m#,m*m
+#(-0m %#,<2'!m ^03%%#+,,m 7m !-*YZm GOOL\m #!!&','m 7m !-*YZm GOON\m &')8-,-m 7m !-*YZm
GOONm 7m HFFG_Ym ,m .*,21m 1#m &m - 1#04"-m /3#m #*m 31-m "#m 0"'!'-,#1m '-,'8,2#1m
.0-"3!#m +7-0+#,2#m "#*#!'-,#1m 7m +-"'$'!!'-,#1m !0-+-1A+'!1Ym 1m 0"'!'-,#1m
'-,'8,2#1m .3#"#,m .0-4#,'0m "#m 1312,!'1m /3#m #+'2#,m #121m 0"'!'-,#1m "#m $-0+m
#1.-,29,#m-m '#,m"#m%#,#0"-0#1m02'$'!'*#1m!-+-m.-0m#(#+.*-m*-1m%#,#0"-0#1m"#m
07-1mm-m*-1m!#*#0"-0#1m"#m.02>!3*1Ym
HJ
/.!6(-#-O#(/#O
(.,)/#9(O
j"'!'-,#1m++m7m07-1j[m1m0"'!'-,#1m!-,m07-1m%++j^v_m7m07-1j
m .0-"3!#,m "#*#!'-,#1m 7m -201m +32!'-,#1m !0-+A+'!1Zm 3,/3#m 1A*-m *%3,1m
"#m#**1m&,m1'"-m!0!2#0'8"1mm,'4#*m+-*#!3*0m^..#,&#'+#0m7m!-*YZmGOOG\m
&'0*#7m7m!-*YZmGOOH\m+ 0m7m!-*YZmGOOJ_Ym-1m07-1m%++j^v_m7m*-1m07-1jm
&,m1'"-m31"-1m.0m.0-"3!'0m+32,2#1m#,m,#)*-#-m^<"#'m7m-,!8ZmGOOH_m
7m#,m00-8m^,8--0m7m&##+ZmHFFI_Ym3m7m!-* -0"-0#1m^HFFK_m310-,m"-1m
"-1'1m "'$#0#,2#1m "#m 07-1m %++j^v_m ^HKFm 7m KFFm 7_m .0m .0-"3!'0m 3,m
!-*#!!'A,m "#m +32,2#1m #,m LJm ^40'#""m #(#m "#*m 00-8_m #,m *-1m !3*#1m
+'"'#0-,m #*m 2+@-m "#m *1m "#*#!'-,#1m ^#,m '*- 1#1_m !-+.0- ,"-m *1m
*2#0!'-,#1m!0-+-1A+'!1m^3m7m!-*YZmHFFK_Ym
m
j#320-,#1m 09.'"-1[m *m +<2-"-m "#m %#,<2'!m ',4#01m 1"-m #,m #*m -.O (/.,)(m
^',"3!2-0m"#m"#*#!'-,#1_m&m1'"-m"#100-**"-m.0m'"#,2'$'!0m7m'1*0m%#,#1m"#m
',2#0<1m #,m .*,21m ^'m 7m !-*YZm HFFG\m 'm 7m &,%Zm HFFH\m 3m 7m !-*YZm HFFK_Ym #m
!3#0"-m!-,m'm7m!-*Ym^HFFG_Zm#12#m+<2-"-m.0-"3!#m"#*#!'-,#1m!-,m#$'!'#,!'m7m
2+ '<,m 1#m .3#"#,m .0-"3!'0m *>,#1m +32,2#1m !-,m 3,m !- #0230m "#m +32!'A,m
+91m #$'!'#,2#m /3#m !-,m *m +32%<,#1'1m ',1#0!'-,*m /3#m -$0#!#m *m2#!,-*-%>m "#*m
jYm 12#m +<2-"-m &m 1'"-m 31"-m #,m 00-8Zm .0-"3!'#,"-m NZFFFm *>,#1m
+32,2#1m#,m*m40'#""m#(#mLJm^3m7m!-*YZmHFFK_Ymm
m
j-,#1m!#*#0"-1[m1,"-m'-,#1m"#m!0 A,m'-,'8"-1Zm1#m&,m!-,1#%3'"-m'1*0m
,3#4-1m+32,2#1m#,m,#)*-#-m^-.FO ,&;FO/0#;FO-/0;FO..;AZm7m..;B_m^,)m7m!-*YZm
GOOM\m 1#m 7m !-*YZm HFFF\m )+-2-m 7m !-*YZm HFFI\m &')8-,-m 7m !-*YZm HFFI_Ym
,*'8,"-m *m 1#!3#,!'m ,3!*#-2>"'!m "#m *-1m +32,2#1m 1#m "#1!3 0'Am /3#m *1m
+32!'-,#1m .0-"3!'"1m #0,m ',4#01'-,#1Zm 20,1*-!!'-,#1Zm 7m .#/3#@1m
"#*#!'-,#1m ^&')8-,-m 7m !-*YZm GOONZm HFFIZm )+-2-m 7m !-*YZm HFFI_Ym 123"',"-m
!-,m"#2**#m*m$0#!3#,!'m"#m#12-1m!+ '-1Zm1#m- 234-m/3#m*m$0#!3#,!'m"#m*1m
"#*#!'-,#1m #0m "#m LYGm Rm GFjKZm $0#!3#,!'m !-+.0 *#m m *m ',"3!'"m .-0m *-1m
,#320-,#1m 09.'"-1m ^'m 7m !-*YZm HFFG_Ym 12-1m 0#13*2"-1m ',"'!0-,m /3#m *m
+32%<,#1'1m+#"',2#m'-,#1m!#*#0"-1m.-">m1#0m31"m2,2-m.0m*m%#,<2'!m
"'0#!2m!-+-m.0m*m%#,<2'!m',4#01m"#m.*,21Ymm
m
T
T
T
T
T
HK
(.,)/#9(
Mutagénesis inducida
BMDMDMDTTTTT
m
_mm
-1m #*#+#,2-1m 20,1.-,' *#1m 2'#,#,m *m !.!'""m "#m "#1.*801#m .-0m #*m %#,-+m #m
',2#%001#m#,m3,m0#%'A,Ym'm*m0#%'A,m"-,"#m1#m',2#%0,m#1m3,m%#,Zm<12#m.3#"#m.#0"#0m
13m $3,!'-,*'""m "# '"-m m /3#m 1#m *2#0m 13m 1#!3#,!'Ym -1m 20,1.-1-,#1m 1-,m
+-*<!3*1m "#m m !-,m !.!'""m +A4'*Ym '#,#,m *m !.!'""m "#m 1*20m "#m 3,m
.-1'!'A,m m -20m "#*m %#,-+Ym -1m 20,1.-1-,#1m 1#m &,m !*1'$'!"-m #,m $3,!'A,m "#m 13m
+#!,'1+-m "#m 20,1.-1'!'A,Zm "#.#,"'#,"-m "#m 1'm #12#m .0-!#1-m 1#m "m !-,m 3,m .1-m
',2#0+#"'-m"#m20,1!0'.!'A,m^0#20-20,1.-1-,#1_m-m1'm1'+.*#+#,2#m1#m"#1.0#,"#,m7m1#m
!-.*,m#,m0#%'-,#1m"'4#011m"#*m%#,-+mI20,1.-1-,#1m"#mJEmm32'*'8!'A,m"#m
20,1.-1-,#1m !-+-m +<2-"-m "#m +32%<,#1'1m ',1#0!'-,*m &m 1'"-m 31"m !-+-m
2#!,-*-%>m"#m%#,<2'!m',4#01m^'0-!&')m7m!-*YZmHFFJ\m!027m7m!-*YZmHFFK_Ymm
m
_mjm^mm_m
!,).,#/'O./' #(-O#1m3,m !2#0'm/3#m.3#"#m',$#!20mm+91m"#mIIFm%<,#0-1m"#m
"'!-2'*#"A,#1m ^8/3'#0"-m 7m !-*YZm HFFG_Ym m !2#0'm .3#"#m ',$#!20m m 204<1m "#m 3,m
&#0'"m -m 3,m ',!'1'A,m 20,1$'0'#,"-m #,m #**m #*m .*91+'"-m #m ^',"3!2-0m "#m 23+-0#1_m 7m
.0-4-!9,"-*#m m *m .*,2m 3,m 23+-0m !**-1-m "#,-+',"-m b23+-0m "#m *m !-0-,cYm *m
.*91+'"-m#m+'"#m#,20#mGKFjHFFm m"#m*1m!3*#1mHFjHIm1#m',2#%0,m*#2-0'+#,2#m#,m
40'-1m *3%0#1m "#*m %#,-+m &3<1.#"Ym m #12#m m 1#m *#m "#,-+',m jm ^m
20,1$#0'"-_m^8/3'#0"-m7m!-*YZmHFFG_Em12m.0-.'#""m"#m',2#%0!'A,m#,m#*m%#,-+m1#m
&m31"-m.0m#*m'1*+'#,2-m"#m%#,#1m#,m.*,21Ym*mjm',20-"3!#m+32!'-,#1m*m
80m **#%,"-m m ',!2'40m *m $3,!'A,m "#m 3,m %#,Ym #"',2#m #12m 2#!,-*-%>m #1m .-1' *#m
'1*0m#*m%#,m/3#m#1m0#1.-,1 *#m"#m*m$*2m"#m$3,!'A,m!-,m1-,"1m#1.#!>$'!1m.0m#*m
jm7m*m+'1+-m2'#+.-Zm0#*!'-,0*-m!-,m3,m$#,-2'.-m+32,2#m^8/3'#0"-m7m!-*YZm
HFFG_Em12m2#!,-*-%>m"#m+32%<,#1'1m',1#0!'-,*m1#m&m32'*'8"-m2,2-m#,m,#)*-#-O
."&#(Zm
!-+-m
#,m
00-8m
^&22.[ii1'%,*Y#"3i!%'j ',i2",#6.0#11m
7m
&22.[ii1'%,*Y1*)Y#"3i!%'j ',i'!#_m 7m #,m +3!&1m -201m #1.#!'#1m ^,m 7m !-*YZm HFFI_Zm
#,20#m #**1m #,m 1,">Zm .0m - 2#,#0m 0#1'12#,!'1m m 4'031m !-+-m #*m 4'031m "#*m +-1'!-m
+0'**-m"#*m!* !>,m^_m^3m7m!-*YZmHFGG_Ymm
_mm
-1m 4'031m 1-,m +-*<!3*1m ',$#!2'41m /3#m .3#"#,m !*1'$'!01#m #,m $3,!'A,m "#m *m
,230*#8m "#m 13m +2#0'*m %#,<2'!-m ^+-,-m -m '!"#,m "#m m -m m 1#%B,m *m
!*1'$'!!'A,m"#m*2'+-0#m^*2'+-0#m7m+-0#ZmGOMG__Ym-1m4'031m',$#!2,mm*1m.*,21m
m 204<1m "#m .*%1m ^',1#!2-1_m 3m &-,%-1Ym m 32'*'8!'A,m "#m 4'031m #,m .*,21m !-+-m
HL
/.!6(-#-O#(/#O
(.,)/#9(O
4#!2-0#1m .3#"#m -$0#!#0m %0,"#1m 4#,2(1Ym #m +3*2'.*'!,m 09.'"+#,2#m 7m .3#"#,m
.0-"3!'0m3,m%0,m,B+#0-m"#m!-.'1m"#*m20,1%<,m^',1#02"-m.0#4'+#,2#m#,m#*m4'031_m
*!,8,"-m 4*-0#1m +96'+-1m #,m 3,m -m "-1m 1#+,1m "#1.3<1m "#m *m ',$#!!'A,m
^8/3'#0"-m 7m !-*YZm HFFG_Ym 3,/3#m 1#m &,m "#1!3 '#02-m 40'-1m 4'031m #,m .*,21m /3#m
.3#"#,m 3101#m !-+-m 4#!2-0#1m ^.-0m #(#+.*-m #*m 4'031m "#*m (1.#"-m "#*m 2 !-m ^_m
^%3"#*-j-+#0-m 7m !-*YZm HFFN__m 13m 32'*'8!'A,m ,-m #1m +37m $0#!3#,2#Ym *m +7-0m
.0- *#+m /3#m .0#1#,2,m *-1m 4'031m #,m .*,21m #1m /3#m 1'm #*m %#,m ',1#02"-m 13.#0m *m
'*- 1#Zm13m',#12 '*'""m#1m+37m*2m7m*m.*,2m*-m#*'+',m^8/3'#0"-m7m!-*YZmHFFG_Zm
^0'%&2m7m-721ZmGOON_Ym
0!'1m *m +#!,'1+-m ',$#!2'4-m "#m *-1m 4'031m !-+ ',"-m !-,m *-1m +#!,'1+-1m "#m
"#$#,1m "#m *1m .*,21m 1#m &m .-"'"-m "#100-**0m *m 2<!,'!m "#,-+',"m
'*#,!'+'#,2-m<,'!-m,"3!'"-m.-0m'031m^_m^3m7m!-*YZmHFFI_Ym3,"-m#*m4'031m
',$#!2m m *m .*,2m <12m "#1#,!"#,m 3,m +#!,'1+-m "#m "#$#,1m 1"m #,m *m
',2#0!!'A,m !-,m #*m +m "#*m 4'031m 7m #,m !-,1#!3#,!'m 1#m *-/3#m 13m 20,1!0'.!'A,Ym
',m #+ 0%-Zm 1'm 1#m 31m #*m 4'031m !-+-m 4#!2-0m ',1#029,"-*#m 3,m %#,m "#m *m .*,2m
&-1.#""-0m#*m.0-!#1-m'09m"'0'%'"-m!-,20m*-1m+m"#*m+'1+-m%#,m1'*#,!',"-m1>m
13m#6.0#1'A,m^3m7m!-*YZmHFFK_Ymm
_mmm
Una de las recientes tecnologías descritas de mutagénesis dirigida en plantas es la
denominada tecnología TALENs (Transcription Activator-Like Effector Nucleases).
Existen patógenos bacterianos del género Xanthomonas que infectan a una amplia
gama de especies y que durante la infección, proporcionan a las células de la planta
una serie de proteínas conocidas como Transcription Activator-Like Effectors (Boch y
Bonas, 2010). Estas proteinas se unen a promotores de genes diana de la planta y de
este modo modifican el transcriptoma de la planta huésped. Para unirse al ADN de
la planta, TALE contiene un dominio de unión compuesto por varias repeticiones de
monómeros de 34 aminoácidos cada uno de los cuales sólo dos residuos, que están
localizados en las posiciones 12 y 13, son hipervariables. Cada motivo hipervariable
TALE reconoce un par de bases en una secuencia de ADN.
TALENs se ha creado mediante la fusión de un dominio de unión (TALE) con una
nucleasa FokI. De este modo el complejo puede reconocer una secuencia concreta y
producir un corte de doble cadena en un sitio específico para inducir mutagénesis
dirigida, como por ejemplo la inserción de un fragmento exógeno de doble cadena
induciendo a la represión o activación del gen dependiendo de la función del
HM
(.,)/#9(
Mutagénesis inducida
dominio donde se ha localizado la inserción de TALENs. Esta tecnología de
mutagenesis dirigida ya se ha usado con éxito en varias especies como por ejemplo
en Arabidopsis thaliana (Cermak y col., 2011).
m
HN
O
(.,)/#9(O
m
m
BMETT
m
BMEMBTT
*m m ^,!.#(!O (/O )&O -#)(-O O ()'-_m #1m 3,m 2<!,'!m "#m %#,<2'!m
',4#01m/3#m!-+ ',m*m%#,#0!'A,m"#m+32!'-,#1m.3,2'$-0+#1m^-m1_m',"3!'"1mm
204<1m "#m 3,m %#,2#m +329%#,-m !-,m 3,m #$'!'#,2#m 2<!,'!m "#m 0120#-m +-*#!3*0Zm /3#m
.#0+'2#m '"#,2'$'!0m *-1m .-*'+-0$'1+-1m m ,'4#*m "#m 1#!3#,!'m ,3!*#-2>"'!m #,m "'4#011m
#1.#!'#1m 4#%#2*#1m ^!**3+m 7m !-*YZm HFFFm Zm _Ym 0!'1m m #12m 2<!,'!m #1m .-1' *#m
*-!*'80Zm#,m3,m.- *!'A,m!-,12'23'"m.-0m+'*#1m"#m',"'4'"3-1Zm3,m1#0'#m*<*'!m"#m
+32!'-,#1m.3,2'$-0+#1m',"3!'"1m#,m%#,#1m"#m',2#0<1m^'**m7m!-*YZmHFFI_Ym
m 2<!,'!m "#*m m $3#m 32'*'8"m .-0m .0'+#0m 4#8m .-0m !**3+m #,m *m .*,2m
+-"#*-m ,#)*-#-O ."&#(m ^!**3+m 7m !-*YZm HFFF_m 7m "#1"#m #,2-,!#1m &m 1'"-m 3,m
2<!,'!m32'*'8"m!-,m<6'2-m#,m40'-1m-0%,'1+-1m7m1#,m4#%#2*#1m^20'%-m"30-m^*"#m
7m !-*YZm HFFJ_\m !# "m ^*+=m 7m !-*YZm HFFN_\m 00-8m ^'**m 7m !-*YZm HFFM_\m +>8m ^'**m 7m !-*YZm
HFFJ_\m1-(m^--.#0m7m!-*YZmHFFN_\m%3'1,2#m^*+'1m7m!-*YZmHFFN_\m+#*A,m^&+,'j
0"1m 7m !-*Zm HFGF\m -,8*#8j-m 7m !-*YZm HFGG_\m )./-O $*)(#/-m ^#007m 7m !-*YZm HFFI\m
'*!&0'12m7m3%&,ZmHFFK__m-m,'+*#1m^.#8j8# 0m^'#,&-*"1m7m!-*YZmHFFI_\m,)-)*"#&O
'&()!-.,O ^',)*#0m 7m !-*YZm HFFK_\m (),"#.#-O &!(-m ^'*!&0'12m 7m !-*YZm HFFL__Zm
',"#.#,"'#,2#+#,2#m"#m*1m"'+#,1'-,#1m"#m*-1m%#,-+1Ymm
12m 2<!,'!m 2'#,#m *%3,1m !0!2#0>12'!1m /3#m *m &!#,m .#!3*'0m 7m 4#,2(-1Ym
#12!0#+-1m 20#1m .-0m 13m 0#*#4,!'Ym m .0'+#0Zm #1m /3#m ,-m 1#m 0#/3'#0#m #*m 31-m "#m
20,1$-0+!'A,m%#,<2'!m7m.-0m*-m2,2-m*-1m+32,2#1m- 2#,'"-1m,-m1-,m20,1%<,'!-1Ym
12m !0!2#0>12'!m #1m 3,m 4#,2(m .3#12-m /3#m *-1m 20,1%<,'!-1m ,-m #129,m !#.2"-1m
1-!'*+#,2#m #,m 30-.m 7m #12-m .#0(3"'!m *m !-+#0!'*'8!'A,m "#*m .0-"3!2-m "#m -0>%#,m
20,1%<,'!-Ym m 1#%3,"m #1m /3#m #1m 3,m 2<!,'!m /3#m .3#"#m 1#0m 32-+2'8"m 7m
-.2'+'8"m .#0+'2'#,"-m ,*'80m 3,m %0,m ,B+#0-m "#m +3#1201m #,m 3,m 2'#+.-m
+>,'+-Ym ',*+#,2#Zm %0!'1m m #12#m +<2-"-m 1#m .3#"#,m - 2#,#0m ,-m 1A*-m +32!'-,#1m
1',m1#,2'"-Zm1',-m2+ '<,m+32!'-,#1m"#*m2'.-m1312'232'4-m*1m!3*#1m.3#"#,m$#!20mm
*m$3,!'A,m"#*m%#,m1',m**#%0mm',!2'40*-Zm#1m"#!'0Zm!0#,"-m1#0'#1m*<*'!1Ym
#1"#m 3,m .3,2-m "#m 4'12m 2#A0'!-Zm *m 2<!,'!m m !-,1'12#m #,m ',"'4'"3*'80m *1m
40',2#1m *<*'!1m /3#m !-,2'#,#,m 3,m +32!'A,m 1'%,'$'!2'4m ^.-0m #(#+.*-Zm /3#m
.0-"38!,m !+ '-1m "#m +',-9!'"-_m +#"',2#m 3,m 0120#-m +-*#!3*0Ym "#+91Zm 1#m
HO
(.,)/#9(O
O
.3#"#,m1#*#!!'-,0m,3#4-1m%#,-2'.-1m/3#m.0-4-!,m$#,-2'.-1m/3#m.3#"#,m1#0mB2'*#1m
m,'4#*m!-+#0!'*Ym*mm#1m3,m2<!,'!m.*'! *#mm2-"-1m*-1m-0%,'1+-1m7m/3#m
13m#$'!'#,!'m,-m"#.#,"#m"#*m2+@-m"#*m%#,-+Zm,'m"#*m1'12#+m0#.0-"3!2'4-Zm,'m"#*m
!'!*-m '-*A%'!-m ^'*!&0'12m 7m 3%&,Zm HFFK_Ym 20m 4#,2(m #1m /3#m *1m +32!'-,#1m 1#m
.0-"3!#,m m 204<1m "#m +32%<,#1'1m /3>+'!m 7m .-0m *-m 2,2-Zm 1-,m +32!'-,#1m
'00#4#01' *#1m 7m "'120' 3'"1m *#2-0'+#,2#m m *-m *0%-m "#m 2-"-m #*m %#,-+Ym 1m
.- *!'-,#1m'"#*#1m1-,m/3#**1m#,m*1m/3#m1#m.3#"#,m'"#,2'$'!0m+32,2#1m.0m!"m
%#,m "',m ,*'8"-Ym "#+91Zm +#"',2#m #12m 2<!,'!m 1#m .3#"#,m ,*'80m 2+ '<,m
"'$#0#,!'1m %#,-2>.'!1m ,230*#1m #,20#m 40'#""#1m "#m 3,m +'1+m #1.#!'#m *-m /3#m 1#m
"#,-+',Zm +91m #1.#!>$'!+#,2#Zm !-m ^"#m ).3*m _m ^-+'m 7m !-*YZm
HFFJ_Ym
T
BMEMCTTTTT
m 2<!,'!m "#*m m 1#m "'4'"#m #,m 20#1m $1#1[m G_m 0-"3!'0m *m .- *!'A,m
+32%#,'8"ZmH_m#100-**0m*m.*2$-0+[m#620!!'A,m7m.0#.0!'A,m"#*mm.0m
#*m0120#-Zm7m
I_m,*'80m7m"#2#!20m*-1m+32,2#1m.0m*-1m%#,#1m"#m',2#0<1m#,m*m.- *!'A,Ym
,m .0'+#0m *3%0m 1#m "#100-**m *m .- *!'A,m +32,"-m *1m 1#+'**1m Fm 7m !0#!'#,"-m *m
%#,#0!'A,m Gm ^'%30m GYJ_Ym -12#0'-0+#,2#Zm m 204<1m "#m *m 32-$#!3,"!'A,m "#m *-1m
',"'4'"3-1m Gm 1#m - 2'#,#m *m %#,#0!'A,m HYm 1m 1#+'**1m "#m *m %#,#0!'A,m Hm 1#m
0#!-%#,m7m1#m*+!#,,m"#m+-"-m/3#m3,m4#8m'"#,2'$'!"1m*1m$+'*'1m+32,2#1m1#m
.3#",m4-*4#0mm.*,20m',"'4'"3-1mHm.0m!-,$'0+0m#*m$#,-2'.-m7m*m1#%0#%!'A,m
"#m *m +32!'A,Ym ,m 4#8m - 2#,'"m *m %#,#0!'A,m Hm 2+ '<,m 1#m .0-!#"#m m *m
#620!!'A,m "#*m +2#0'*m %#,<2'!-Ym 12#m +2#0'*m %#,<2'!-m #1m #*m 0#13*2"-m "#m *m
#620!!'A,m"#mm"#m*m+#8!*m"#m*1m+3#1201m"#m&-(m"#m"'#8m',"'4'"3-1m"#m!"m
$+'*'m HYm #1.3<1m "#m #620#0m #*m m "#m 2-"1m *1m $+'*'1m /3#m !-+.-,#,m *m
.- *!'A,Zm1#m-0%,'8m#,m.*!1m+#8!*,"-m#*mm"#m!"m!320-mm-!&-m$+'*'1m
.0m.-"#0m%'*'80m#*m.0-!#1-m"#m"#2#!!'A,m"#m+32,2#1m^+#8!*m"#m1m-m.--*1_Ym
0m #*m "#100-**-m "#m *%3,1m .- *!'-,#1m #12#m .0'+#0m .1-m #,m #*m /3#m 1#m - 2'#,#m #*m
m"#m*m.- *!'A,m&m40'"-m*'%#0+#,2#Ym*%3,-1m32-0#1m&,m#620>"-m#*mm
"#m 2,m 1-*-m 3,m .*,2m "#m *m %#,#0!'A,m Hm 7m ,-m "#m *m +#8!*m "#m "'#8m ',"'4'"3-1Zm
0#"3!'#,"-m 1>m !-,1'"#0 *#+#,2#m #*m 2'#+.-m "#m *m .0#.0!'A,m "#m *m .- *!'A,Zm
3,/3#m #,m #12#m !1-m 1#m 13#*#m 32-$#!3,"0m #*m ',"'4'"3-m Hm .0m .#0.#230m m *m
+32!'A,m#,m*m1'%3'#,2#m%#,#0!'A,Ym
IF
O
(.,)/#9(O
m !-,2',3!'A,m 1#m ','!'m #*m .0-!#1-m "#m "#2#!!'A,m "#m +32,2#1m #,m %#,#1m "#m ',2#0<1Ym
0m #**-Zm 1#m "'1#@,m !# "-0#1m "#m +-"-m /3#m +.*'$'/3#,m *1m 8-,1m "#*m %#,m /3#m 1#m
/3'#0#,m,*'80Ym#"',2#m#*m"'1#@-m"#m!# "-0#1m#1.#!>$'!-1m1#m.3#"#m.0-!#"#0mm*m
+.*'$'!!'A,m "#*m %#,m ^-m "#m 3,m 0#%'A,m "#*m %#,_m +#"',2#m Ym m m 1#m 0#*'8m
1- 0#m*m+#8!*m"#m1m+#,!'-,"m,2#0'-0+#,2#Zm.-0m*-m2,2-Zm+.*'$'!09m2-"-1m
*-1m*#*-1m/3#m1#m#,!3#,20#,m#,m#1#m+#8!*m^2,2-m#*m*#*-m1'*4#120#m!-+-m#*m+32"-_Ym
,m4#8m"#1,230*'8"-m#*m.0-"3!2-m"#m*mm1#m.0-!#"#mm*m0#,230*'8!'A,Ym,m
#*m !1-m "#m *m 0#,230*'8!'A,m #,20#m 3,m !"#,m +32"m !-,m 3,m 1'*4#120#m *m
!-+.*#+#,2!'A,m ,-m 1#09m 2-2*m 7m .-0m *-m 2,2-m 1#m .0-"3!'09m 3,m &#2#0-"B.*#6m #,m #*m
,3!*#A2'"-m +32"-Ym -12#0'-0+#,2#Zm 3,m #,"-,3!*#1m /3#m "'%'#0#m #6!*31'4+#,2#m
&#2#0-"B.*#6m ^&O _m 1#09m *m 0#1.-,1 *#m "#m !-020m *1m !"#,1m "-,"#m &7m 3,m
+32!'A,Ym #1.3<1m 1#m 4'13*'8,m *-1m $0%+#,2-1m "'%#0'"-1m #,m 3,m jm JIFFYm m
"#2#!!'A,m "#m *1m +32!'-,#1m -m 1m .3#"#m &!#01#m +#"',2#m -20-1m +<2-"-1m /3#m 1#m
!-+#,209,m .-12#0'-0+#,2#Zm !-+-m .-0m #(#+.*-Zm #*m m ^#!"O -)&/.#)(O &.#(!_Zm
3,/3#m#*m+<2-"-m+91m& '23*m#1m#*m31-m"#*mlmJIFFZm32'*'8"-m#,m#12#m20 (-Ym
0!'1mm#12m2#!,-*-%>m1#m.3#"#,m4'13*'80m"-1m!,*#1m"'12',2-1m#,m*-1m!3*#1m1#m
"#2#!2,m*-1m$0%+#,2-1m+.*'$'!"-1m"#1"#m!"m!# "-0mKam-mIam"#m*m!"#,m&12m
#*m.3,2-m"#m"'%#12'A,Ym#m#12#m+-"-Zm1#m.3#"#m!-,$'0+0m*m+32!'A,m.3#12-m/3#m*m
13+m "#*m 2+@-m "#m *-1m $0%+#,2-1m "'%#0'"-1m #,m !"m !,*m "# #m !-',!'"'0m !-,m *m
2-2*'""m "#*m 2+@-m "#*m $0%+#,2-m +.*'$'!"-m 1',m "'%#0'0m ^*m #6.*'!!'A,m "#m *m
4'13*'8!'A,m "#m *-1m $0%+#,2-1m "'%#0'"-1m #,m #*m jm #129m "#2**"m #,m *m '%30m
GYK_Ym
IG
(.,)/#9(O
O
T
(&30 T BME[m 0-2-!-*-m #+.*#"-m .0m #*m +<2-"-m "#m Ym ,m 4#8m +32"1m *1m 1#+'**1m ^F_m !-,m #*m
+329%#,-mZm1#m!0#!#m*m%#,#0!'A,mGYm#"',2#m*m32-$#!3,"!'A,m"#m#12m%#,#0!'A,m1#m- 2'#,#m*m
%#,#0!'A,m HYm #m !"m $+'*'m Hm 1#m !0#!#,m GFm ',"'4'"3-1m 7m 1#m *#1m 2-+m 3,m +3#120m "#m &-(Ym
-12#0'-0+#,2#Zm1#m#620#m#*mm"#m#1m+#8!*m7m"#m#12#m+-"-m1#m- 2'#,#m#*mm"#m!"m$+'*'mHYm#m
*m %#,#0!'A,m Hm 2+ '<,m 1#m *+!#,,m *1m 1#+'**1m .0m #6.#0'+#,2-1m $3230-1Ym -1m 1m "#m 2-"1m *1m
$+'*'1m1#m-0%,'8,m#,m.*!1m+#8!*9,"-*1m"#m!320-m#,m!320-Zm#,m,3#120-m!1-Y0m*m"#2#!!'A,m"#m*-1m
+32,2#1Zm.0'+#0-m1#m+.*'$'!m#*m%#,m"#m',2#0<1Ym#1.3<1m1#m"#1,230*'8,m*1m!"#,1m7m"#m#12#m+-"-m1#m
.0-4-!,m*-1m&#2#0-"B.*#6m/3#m"'%#0'09m*m#,"-,3!*#1m&EO',*+#,2#Zm*m"#2#!!'A,m"#m*-1m$0%+#,2-1m
"#m"'%#12'A,m1#m0#*'8m31,"-m#*mjmJIFFYm
m
BMEMCMBTTTTTT
1m.*,21m1-,m-0%,'1+-1m'"#*#1m.0m+32%#,'80m7m/3#m2'#,#,m*m!.!'""m"#m
+,2#,#0m*m-0%,'1+-m#,m3,m#12"-m*2#,2#m!-,m*m1#+'**Ym*m+329%#,-m'"#*m.0m
310m #,m #*m +<2-"-m "#m m #1m /3<*m /3#m ',"3!#m +32!'-,#1m *#2-0'+#,2#m
IH
T
O
(.,)/#9(O
0!,"-m 0#%'-,#1m !-"'$'!,2#1m 7m #,m 3,m *2m $0#!3#,!'m 7m 1#,m .3,23*#1Zm
.#/3#@1m "#*#!'-,#1m -m ',1#0!'-,#1Ym *m +329%#,-m /3>+'!-m m ^2'*m #2,-m
3*$-,2-_m %#,#0m 1m ^20,1'!'-,#1m [j[m ^0##,#m 7m !-*YZm HFFIZm *"#m 7m !-*YZm
HFFK__m 7m 1#m .3#"#m .*'!0Zm -.2'+'8,"-m *m "-1'1m #,m !"m #1.#!'#Zm .0m .0-"3!'0m
+32!'-,#1m 1',m .0-"3!'0m *#2*'""m "#m *1m 1#+'**1m -m "#m *1m .*,21m ^0##,#m 7m !-*YZm
HFFIZm --0,,##$m 7m !-*YZm GONH_Ym *m .0-!#"'+'#,2-m & '23*m 31"-m &12m &-0m .0m
.0-"3!'0m .*2$-0+1m "#m m &m 1'"-m ',20-"3!'0m *1m 1#+'**1m #,m 3,m 1-*3!'A,m
!-,m#*m+329%#,-m"'*3'"-m"30,2#m.0-6'+"+#,2#mGFjHJm&-01m7m#,m-1!30'""Zm1>m
1#m !-,1'%3#m +320m *1m !<*3*1m "#*m #+ 0'A,m /3#m +91m 20"#m .0-"3!'09,m *-1m 2#('"-1m
0#.0-"3!2'4-1Ym ,m *m +7-0>m "#m *-1m !1-1m *-1m ',"'4'"3-1m "#m *m %#,#0!'A,m Gm
2#,"09,m*m+32!'A,m#,m&#2#0-!'%-1'1m7m.0- *#+#,2#m1A*-m#,m3,m.02#m"#m*m.*,2Ym
0m 1#%300m *m .0- '*'""m "#m #,!-,200m *m +32!'A,m #,m &-+-!'%-1'1m &!#m $*2m
32-$#!3,"0m *m %#,#0!'A,m Gm 7m - 2#,#0m *m %#,#0!'A,m HYm #m #12#m +-"-m *m
.0- '*'""m"#m#,!-,200m3,m',"'4'"3-m&-+-!'%-2-m1'%3#m*m.0-.-0!'A,m"#m&#0#,!'m
+#,"#*',m G[H[Gm ^#,')-$$m 7m -+'Zm HFFI_Ym + '<,m #,m *%3,1m #1.#!'#1m !-+-m #,m
+>8m1#m&m.*'!"-m#*m+329%#,-m"'0#!2+#,2#m#,m#*m.-*#,m.0m*3#%-m',20-"3!'0*-m#,m
*m $*-0m $#+#,',m 1'*4#120#m "#m *m +'1+m 40'#""Ym 1m 1#+'**1m "#m *1m +8-0!1m
0#13*2,2#1m 1#m %#0+',,m .0m - 2#,#0m *m %#,#0!'A,m GYm -12#0'-0+#,2#m +#"',2#m
32-$#!3,"!'A,m1#m- 2'#,#m*mHm^'**m7m!-*YZmHFFI_Ym
#1"#m#*m@-mHFFFm1#m&,m"#100-**"-m.- *!'-,#1m"#mm#,m"'12',21m#1.#!'#1Ym
,-m "#m *-1m $!2-0#1m "#!'1'4-1m &m 1'"-m -.2'+'80m #*m .0-2-!-*-m "#m +32%#,'8!'A,m "#m
*1m1#+'**1m!-,m#*m- (#2'4-m"#m- 2#,#0m3,m"#,1'""m"#m+32!'A,m*2m7m*m4#8Zm/3#m
,-m$#!2#mm*m4' '*'""m"#m*1m.*,21m+32%#,#'8"1Ym0m"#2#0+',0m*m"-1'1m"#*m
+329%#,-m ^+7-0+#,2#m #*m _m #,m !"m #1.#!'#Zm &m 1'"-m ,#!#10'-m &!#0m
#6.#0'+#,2-1m .0#4'-1m .0m .-"#0m "#2#0+',0m *m "-1'1m A.2'+Ym 1m .-0m #**-m /3#m
#,!-,20+-1m "'12',2-1m 0,%-1m "#m "-1'1m #,m $3,!'A,m "#m *m #1.#!'#m ^ *m GYI_Ym 0m
,)-)*"#&O '&()!-.,m 1#m "#2#0+',Am /3#m *m !-,!#,20!'A,m A.2'+m #0m "#m KFm +m
^',)*#0m7m!-*YZmHFFK_Zm+'#,201m/3#m#,m ,#)*-#-O."&#(m#129m"#,20-m"#*m0,%-m"#m
HFjJFm +m ^0##,#m 7m !-*YZm HFFI_Ym #!'#,2#+#,2#m 1#m &,m .3 *'!"-m .- *!'-,#1m "#m
m#,m+#*A,m/3#m&,m.#0+'2'"-m(3120m*m"-1'1m"#m+329%#,-m.0m#12m#1.#!'#m
^&+,'j0"1m 7m !-*YZm HFGF\m -,8*#8j-m 7m !-*YZm HFGG_Ym 0m #*m +#*A,m 1#m &m
!-+.0- "-m /3#m *m "-1'1m "#m &12m #*m IPm "#m +329%#,-m .0-.-0!'-,m 3,m .- *!'A,m
!-,m3,m$0#!3#,!'m"#m+32!'A,m"#mGiKMIm m^&+,'j0"1m7m!-*YZmHFGF_Ymm
T
T
II
(.,)/#9(O
O
!+ TBMDOm- *!'-,#1m"#mm"#100-**"1m#,m,'+*#1m7m.*,21m.-0m-0"#,m!0-,-*A%'!-Ym#m.3#"#,m
- 1#040m *1m "'12',21m "-1'1m "#m +329%#,-m ^#,m *%3,-1m !1-1m #6.0#1"1m #,m +Zm #,m -201m #,m Pm "#m 4i4m
"#.#,"'#,"-m"#m*m.3 *'!!'A,_m32'*'8"1m#,m%#,-+1m"#m"'$#0#,2#m2+@-m7m!-,m"'$#0#,2#1m.*-'">1Ym,m
1-+ 0#"-m %0'1m *1m .- *!'-,#1m "#m +#*A,O "#100-**"1m &12m #*m +-+#,2-Zm 3,m "#m #**1m *m #* -0"m #,m
#12#m 20 (-m ^-,8*#8j-m 7m !-*YZm HFGG_Ym g[m j#2'*jj,'20-1-30#Zm gg8j[m !'"-m "#m 1-"'-m 7m +#2'*j
,'20-130#Zmggg[mj,'20-1-jj+#2'*30#Ym
m
1/$"($$
,)-)*"#&O
'&()!-.,O
(#)O,,#)O
,#)*-#-O
."&#(O
),/'O
0/&!,O
329&$-.T
.1(1T#$T
, =.T#$+T
,329&$-.T
&$-., T
+.(#; T
1 T#$T
,32 "(>-T
$%$0$-"( T
#,2*#7m7m
m
KFm+m
GNFm m
Hm
GiHFOm) m
!#,,,Zm
HFFFm
gm
IYFm+m
GZMFFm m
Hm
GiHIKm) m
m
HFjJFm+m
GHKm m
Hm
GiIFFm) m
m
HFjIFm+m
KZIFFm m
Hm
Gi m
'#,&-*"1m7m
!-*YZmHFFIm
0##,#m7m!-*YZm
HFFIm
*"5#**m7m!-*YZm
HFFJm
'**m7m!-*YZm
O'3-O
m
GmPm
HZKFFm m
Hm
GiKFFm) m
,34O-.#0O
m
FYNjGmPm
JIFm m
Hm
FYKi m
3m7m!-*YZmHFFKm
,34O-.#0O
m
GYLmPm
JIFm m
Hm
Gi m
3m7m!-*YZmHFFKm
m
FYMKjGPm
GHZFFFm m
Jm
GiJFm) m
m
FYMKjGYHPm
GMZFFFm m
Lm
GiHJm m
HFFJZm
,#.#/'O
./,!#/'O
-/-*EO
*"#m7m!-*YZm
HFFKm
/,/'O
,#.#/'O
-.#0/'O
,)-)*"#&O
'&()!-.,O
,)-)*"#&O
m
KFm+m
GNFm m
Hm
GiGKLm) m
m
GHKm+m
GNFm m
Hm
GiOFYKm) m
EO&!(-O
m
HKm+m
GFFm m
Hm
GiHOIm) m
O'3-O
m
FYFLHKmPm
HZKFFm m
Hm
O'3-O
m
FYFLHKmPm
HZKFFm m
Hm
'&()!-.,O
IJ
*"#m7m!-*YZm
HFFKm
',)*#0m7m!-*YZm
HFFKm
',)*#0m7m!-*YZm
HFFKm
'*!&0'12m7m!-*YZm
HFFLm
FZOIi) mm
#'*m7m-,"#Zm
MIm
HFFMm
HYGFi) m
#'*m7m-,"#Zm
HHm
HFFMm
O
#-/'O
-.#0/'O
,34O-.#0O
,34O-.#0O
(.,)/#9(O
0'/3#1m7m!-*YZm
m
Jm+m
JZIFFm m
Hm
GiLLOm) m
m
GYKmPm
JIFm m
Hm
GiHOJm) m
'**m7m!-*YZmHFFMm
JIFm m
Hm
GiHLKm) m
'**m7m!-*YZmHFFMm
8j
ggm
HFFMm
Gm+m8j
GKm+m
m
&3#(O'2O
gggm
HYKm+m
GZGGKm m
Hm
GiGJFm) m
--.#0ZmHFFNm
&3#(O'2O
m
KFm+m
GZGGKm m
Hm
GiHKFm) m
--.#0ZmHFFNm
&3#(O'2O
m
JFm+m
GZGGKm m
Hm
GiKKFm) m
--.#0ZmHFFNm
m
FZHmPm
GHKm m
Hm
GiJGKm m
m
FZOmPm
GIZFFFm m
Lm
GiIFm m
m
FZIm7mFZJmPm
HNIZNm m
Hm
GiLFm m
m
GPm
OKFm m
Hm
GiIHHm) m
m
FZMPm
OKFm m
Hm
GiKMJm) m
,#)*-#-O
."&#(O
0(O-.#0m
,--#O
,*m
)&(/'O
&3)*,-#/'m
)&(/'O
&3)*,-#/'m
$
O
,"#-O
"3*)!OO
&#(."/-O
((/-O$
$
$
m
BSDKm
EFAT!m
Cm
BRFHDT*!m
m
FZJm7mGZHmPm
jm
Jm
GiOLMm m
T
T
FZMmPT
ANFSBNFKT
IGNOm T
EFAT!T
HT
CT
GiJMKm T
BRTBNFT!T
31&m7m071,Zm
HFGFm
&5"#m7m!-*YZm
HFGFm
2#.&#,1-,m7m
!-*YZmHFGFm
',-'m7m!-*YZm
HFGFm
',-'m7m!-*YZm
HFGFm
', -(S
0# 1NTCABAm
,-**m7m!-*YZm
HFGGmm
#22m7m!-*YZm
HFGGmT
.-8 +$8S.T7T
".+MNTCABBT
m
BMEMCMCTTTTTTT
20-m $!2-0m /3#m .3#"#m *'+'20m *m '"#,2'$'!!'A,m "#m +32,2#1m #1m #*m "'1#@-m "#m *1m
+#8!*1m "#m 1m ^.--*1_Ym m "'$#0#,!'m #,20#m &!#0m +#8!*1m "#m !320-m $+'*'1m -m
+#8!*1m"#m-!&-m#1m#*m!-12#m7m#*m2'#+.-m,#!#10'-1m.0m*m"#2#!!'A,Ym-1m$!2-0#1m/3#m
!-,"'!'-,,m *m #$'!'#,!'m #,m *m "#2#!!'A,m "#m +32,2#1m #,m 3,m +#8!*m 1-,m *m !*'""m
"#*mm%#,A+'!-Zm*m.0#!'1'A,m"#m13m!3,2'$'!!'A,m7m#*m+<2-"-m31"-m.0m0#*'80m
*m "#2#!!'A,m "#m 1Ym ,m *1m "'4#011m .*2$-0+1m .3 *'!"1m 1#m &,m "'1#@"-m
& '23*+#,2#m+#8!*1m"#m"-1m&12m-!&-m$+'*'1Zm3,/3#m*mA.2'+m#1m*m+#8!*m"#m
-!&-m 7m /3#m 0#"3!#m &12m *m +'2"m #*m !-12#m 7m #*m 2'#+.-m "#m 0120#-m "#m *m .- *!'A,m
IK
(.,)/#9(O
O
0#1.#!2-m *m 31-m "#m +#8!*1m "#m !320-m $+'*'1Ym ,m 4#8m "#2#!2"m 3,m +32!'A,m #,m
3,m +#8!*m 1#m "# #m 0#.#2'0m #*m ,9*'1'1m #,m !"m m ',"'4'"3*m /3#m !-+.-,#m *m
+#8!*Ym12#m.0-!#1-m1#m"#,-+',m"#!-,4-*3!'A,m7m#1m,#!#10'-m.0m',"'4'"3*'80m
*m$+'*'m0#1.-,1 *#m"#m*m+32!'A,m^-* #02m7m!-*YZmHFFG_Ym,m*2#0,2'4m.0m*m
"#2#!!'A,m"#m+32,2#1m#1m0#*'80m+#8!*1m#,m"-1m"'+#,1'-,#1m"#m+,#0m/3#m!"m
+3#120m #129m .0#1#,2#m #,m "-1m B,'!-1m .--*1Ym 12m #1202#%'m 7m 1#m &m 31"-m m %0,m
#1!*m#,m#*m#4O-,0#O^&22.[ii%#,-+#Y.30"3#Y#"3i+'8#2'**',%i_Ym3,/3#m
"3.*'!0m *1m +#8!*1m 0#"3!#m *m 4#*-!'""m "#m *m "#2#!!'A,m "#m *-1m +32,2#1m 2+ '<,m
0#"3!#m*m',!#02'"3+ 0#m^"#1!02m$*1-1m.-1'2'4-1m-m02#$!2-1_Ym"#+91Zm#*m.1-m"#m
*m"#2#!!'A,m',"'4'"3*m"#m*m$+'*'m-m"#!-,4-*3!'A,m,-m#1m,#!#10'-Ym
T
BMEMCMDTTTT
-,m 40'1m *1m 2#!,-*-%>1m /3#m #6'12#,m .0m *m "#2#!!'A,m "#m 1m 7m /3#m &,m 1'"-m
31"1m.0m"#2#!20m+32!'-,#1m#,m.*2$-0+1m"#mYm#1"#m*m#*#!20-$-0#1'1m
!-,m 2',!'A,m "#m 0-+30-m "#m #2'"'-m ^-+'m 7m #,')-$$Zm HFFL_Zm #*m m ^#!"O
*, ),'(O &#+/#O ",)'.)!,*"3JO ^!**3+m 7m !-*YZm HFFF _Zm *m #1.#!20-1!-.>m "#m
+11Zm *m #*#!20-$-0#1'1m !.'*0m #,m %0"'#,2#m #,m !-,"'!'-,#1m "#1,230*'8,2#1m -m *m
"'%#12'A,m#,8'+92'!mm,'4#*m,3!*#-2>"'!-m^-+'m7m#,')-$$ZmHFFL_Zm&12m*1mB*2'+1m
2#!,-*-%>1m !-+-m *m #!"O -)&/.#)(O &.#(!m ^_m ^-,%m 7m !-*YZm HFFO_Zm *m
1#!3#,!'!'A,m +1'4m 2.O (,.#)(O +/(#(!m ^_m ^'%-*m 7m !-*YZm HFFO_m -m *m
32'*'8!'A,m"#mjm^.,#2N--#-.O-,O-),*.#)(M)(#4.#)(N#'O O&#!".JO
^&5"#mm7m!-*YZmHFGF_Ym3*/3'#0m"#m#121m2<!,'!1m1#m.3#"#m310m#,m#*mm
.0m *m "#2#!!'A,m "#m +32,2#1Zm 1',m #+ 0%-Zm #*m +<2-"-m 1#*#!!'-,"-m "# #m .#0+'2'0m
3,m,9*'1'1m+1'4-m7m09.'"-Zm1>m!-+-m"#m+96'+m1#,1' '*'""Ymm
-"-1m*-1m+<2-"-1m!'2"-1m,2#0'-0+#,2#m&,m1'"-m31"-1m.0m*m"#2#!!'A,m"#m1m
^!**3+m7m!-*YZmHFFFZm-* #02m7m!-*YZmHFFGZm'#,&-*"1m7m!-*YZmHFFI_m.#0-m#*m+<2-"-m
+91m !-+B,m .0m #*m m &m 1'"-m *m "#2#!!'A,m "#m *1m +32!'-,#1m m .02'0m "#m *m
"'%#12'A,m !-,m ,3!*#11m #1.#!>$'!1m "#m &#2#0-"B.*#6m ^'%30m GYK_Ym *m .0-!#"'+'#,2-m
!-+B,m #1m +.*'$'!0m 3,m 0#%'A,m "#*m %#,m "#m ',2#0<1m +#"',2#m m !-,m !# "-0#1m
#1.#!>$'!-1m7m+0!"-1m!-,m3,m$*3-0A$-0-m"'12',2-m.0m!"m#620#+-mKam^7#mMFF_m7m
Iam^7#mNFF_Ym*m.0-"3!2-m"#m*mm#1m"#1,230*'8"-m7m0#,230*'8"-m"#m+-"-m
/3#m *-1m *#*-1m +32"-1m 1#m +#8!*,m !-,m *-1m *#*-1m 1'*4#120#1m 7m $-0+,m *-1m
&#2#0-"B.*#6Ym *m ',!3 01#m !-,(3,2+#,2#m !-,m *m #,"-,3!*#1m #1.#!>$'!m "#m
&#2#0-"B.*#6m1#m"'%'#0#,m1A*-m/3#**-1m$0%+#,2-1m/3#m!-,2'#,#,m3,m+32!'A,Ymm
IL
O
(.,)/#9(O
m 4'13*'8!'A,m 1#m 0#*'8m +#"',2#m %#*#1m 4#02'!*#1m "#m !0'*+'"m #,m .*2$-0+1m
!-+-m#*mjmm,*78#0m2*m7m!-+-m.3#"#m4#01#m#,m*m'%30mGYKYm,m.0'+#0m
*3%0m #*m $0%+#,2-m "#*m %#,m "#m ',2#0<1m 1#*#!!'-,"-m #1m +.*'$'!"-m +#"',2#m 3,m
!# "-0m "'0#!2-m +0!"-m $*3-0#1!#,2#+#,2#m !-,m 7#m MFFZm 7m !-,m 3,m !# "-0m
0#4#01-m +0!"-m $*3-0#1!#,2#+#,2#m !-,m 7#m NFFYm #1.3<1m "#m *m $-0+!'A,m "#*m
&#2#0-"B.*#6m *1m +3#1201m 1-,m ',!3 "1m !-,m #*m .0Zm .-12#0'-0+#,2#Zm 1#0m
"#1,230*'8"1m ^"#2**"-m #,m *m '%30m GYJ_m 7m !0%"1m #,m 3,m %#*m 4#02'!*m "#m
!0'*+'"m .0m 4'13*'80*1m +#"',2#m #*m jm JIFFYm 0m !"m %#*m 1#m .0-"3!#,m
"-1m'+9%#,#1[m3,m.0-"3!'"m.-0m*m1#@*m/3#m#+'2#m#*m$*3-0A$-0-m',!-0.-0"-m#,m#*m
!# "-0m "'0#!2-m ^7#m MFFZm 0-(-_m 7m *m -20m *m 1#@*m .0-"3!'"m .-0m #*m $*3-0A$-0-m
',!-0.-0"-m #,m #*m !# "-0m 0#4#01-m ^7#m NFFZm 4#0"#_Ym -0m *-m 2,2-Zm #,m #*m !,*m "#*m
7#m MFFm .-"0#+-1m 4#0m #*m $0%+#,2-m /3#m 0!m *m *-,%'23"m "#1"#m #*m #620#+-m Kam
&12m #*m .3,2-m "#m *m "'%#12'A,m ^/3#m #1m #*m +'1+-m *3%0m "-,"#m 1#m &m $-0+"-m #*m
&#2#0-"B.*#6m 7m .-0m *-m 2,2-m 2+ '<,m "-,"#m #129m *m +32!'A,_Ym '#,201m /3#m #,m #*m
!,*m7#mNFFm.-"0#+-1m4#0m#*m$0%+#,2-m/3#m 0!m*m*-,%'23"m"#1"#m#*m#620#+-m
Iam&12m#*m.3,2-m"#m*m"'%#12'A,Ymm13+m"#*m2+@-m"#*m$0%+#,2-m"'%#0'"-m#,m#*m
!,*m7#mMFFm7m#*m2+@-m"#*m$0%+#,2-m"'%#0'"-m#,m#*m!,*m7#mNFFm2'#,#m/3#m
!-',!'"'0m!-,m#*m2+@-m2-2*m"#*m$0%+#,2-m+.*'$'!"-m2*m!-+-m.3#"#m- 1#0401#m
#,m#*m%#*m"#m*m'%30mGYKm^LFFQJFFSGFFFm. _Ym
m
IM
(.,)/#9(O
O
m
(&30 TBMFOm#2#!!'A,m"#m3,m+32!'A,m#,m3,m.- *!'A,m"#mm#,m+#*A,m#,m3,m%#*m"#m!0'*+'"m
4#02'!*m"#mjmJIFFYmm*m'8/3'#0"m1#m- 1#04m#*m%#*m4'13*'8"-m#,m#*m!,*mMFFZmm*m"#0#!&m#,m#*m
!,*m NFFYm ,m !"m !,*m 1#m 4'13*'8m #*m $0%+#,2-m "'%#0'"-m "#1"#m 3,-m "#m *-1m #620#+-1Zm 7m /3#m #*m
!# "-0m "'0#!2-m ^KaZm 0-(-_m **#4m #*m +0!(#m MFFm +'#,201m /3#m #*m !# "-0m 0#4#01-m ^IaZm 4#0"#_m **#4m #*m
+0!(#m.0m#*mNFFYmm13+m"#*m2+@-m"#m*-1m$0%+#,2-1m"#2#!2"-1m#,m!"m!,*m^MFF[mLFFm. m
QmNFF[mJFFm. _m"# #m!-',!'"'0m!-,m#*m2+@-m2-2*m"#*m$0%+#,2-m1',m"'%#0'0m4'13*'8"-m#,m#*m#620#+-m
13.#0'-0m"#*m%#*m^GFFFm. _Ym
m
0m#*m.0-!#1-m"#m"'%#12'A,m"#*m&#2#0-"B.*#6m1#m&,m"#1!0'2-m*%3,1m#,"-,3!*#11m
^3&0+,,m7m!-*YmHFFKZm-3'*m7m!-*YZmGOOKZm1&*m7m!-*YZmGOOKZm'**m7m!-*YZmHFFJ _Ymm
#,"-,3!*#1m +91m !-+3,+#,2#m 32'*'8"m .0m #*m m #1m &O#620>"m "#m .'-m
^&,3_m^*#7)-51)'m7m!-*YZmGOON_Ym1m3,m#,"-,3!*#1m/3#m!2Bm"'%'0'#,"-m#*mm
"#m !"#,m 1#,!'**Ym #m &m "#+-120"-m 2+ '<,m /3#m -201m #,"-,3!*#11m !-+-m *1m
#620>"1m "#m (3">m 1-,m #$'!'#,2#1m .0m "#2#!20m +32!'-,#1m +#"',2#m #*m +<2-"-m
m ^'**m 7m !-*YZm HFFJ _Ym m #*#!!'A,m "#m *m #,8'+m 2+ '<,m .3#"#m 1#0m 3,m $!2-0m
IN
O
(.,)/#9(O
*'+'2,2#m .0m *m 1#,1' '*'""m "#*m +<2-"-Zm 7m 1#m "#+-120Am /3#m *m 32'*'8!'A,m "#m
#620!2-m "#m &m (3,2+#,2#m !-,m #*m +<2-"-m "#m 4'13*'8!'A,m "#m jm JIFFm #0m #*m
+91m 1#,1' *#m .0m *m "#2#!!'A,m "#m 1m #,m +#8!*1m "#m Nm $+'*'1m ^"'*3!'A,m GiGLm "#*m
*#*-m+32"-m#,m3,m#1.#!'#m"'.*-'"#_m^0##,#m7m!-*YZmHFFI_Ymm#,8'+m&m0#!-,-!#m
*-1m &#2#0-"B.*#6m !-,m *m .0#$#0#,!'m im Um im Vm im Um iZm 7m 0#!-,-!#m !-,m +#,-0m
#1.#!'$'!'""mimVmimVmimTmimVmim7m2-"4>m!-,m+#,-1mim^*#7)-51)'m7m
!-*YZm GOON\m ,%m 7m !-*YZm HFFF_Ym *m +'1+-m 2'#+.-Zm #12m #,8'+m 2'#,#m 3,m 3#,m
#1.#!'$'!'""m #,m #*m 0#!-,-!'+'#,2-m "#m &#2#0-"3.*#6m #,m 3,m "'*3!'A,m *<*'!m "#m G[GLYm
1m .-0m #**-Zm /3#m 1#m 32'*'8m .0m *m "#2#!!'A,m "#m 1m #,m +#8!*1m "#m Nm $+'*'1m #,m
#1.#!'#1m "'.*-'"#1Ym ,m !+ '-Zm *m #,8'+m ()m ^!*-,"m m .02'0m "#m 3,m %#,m /3#m
!-"'$'!m.-0m3,m#,"-,3!*#1m'"#,2'$'!"m#,m,#)*-#-_Zm0#!-,-!#m2-"-1m*-1m2'.-1m
"#m +32!'-,#1m !-,m *m +'1+m #1.#!'$'!'""m #,m "'*3!'-,#1m *<*'!1m "#m G[LJm ^0'/3#1m 7m
!-*YZm HFFM_Ym ,m !3,2-m *m 1.#!2-m #!-,A+'!-Zm *m - 2#,!'A,m !-+#0!'*m "#m + 1m
#,"-,3!*#11m 2'#,#,m 3,m !-12#m *2-Zm 1',m #+ 0%-Zm .0m *m - 2#,!'A,m "#m &m 1#m &,m
.3 *'!"-m.0-2-!-*-1m.0m- 2#,#0m3,m#620!2-m"#m.'-m/3#m$3,!'-,m#$'!'#,2#+#,2#m7m
0#"3!#m #*m !-12#m !-+.0"-m !-,m *m - 2#,!'A,m "#m *m #,8'+m .30'$'!"m ^,%m 7m !-*YZm
HFFF_Ym 30,2#m*-1mB*2'+-1m@-1m 1#m &,m &#!&-m #123"'-1m !-+.0,"-m*m#$'!'#,!'m"#m
+ 1m#,"-,3!*#11m#,m*m"#2#!!'A,m"#m.-*'+-0$'1+-1m+#"',2#m!-Ym-1m
0#13*2"-1m - 2#,'"-1m 1-,m '"<,2'!-1m "#+-120,"-m /3#m *m #$'!'#,!'m #1m .09!2'!+#,2#m
'%3*m ^-,-m 7m !-*YZm HFGG_Ym -0m *-m 2,2-Zm *m 32'*'8!'A,m "#m 3,m 3m -20m #,"-,3!*#1m
"#.#,"#09m "#*m 2'.-m "#m m #1.#0"-m 1#%B,m #*m +329%#,-m 32'*'8"-Zm #*m !-12#m "#m *m
+'1+m7m"#m*m0#%'A,mm#123"'0m^#3,%m7m!-*YZmHFFK\m-"%1-,m7m#**1ZmGOMM_Ymm
,m 4#,2(m '+.-02,2#m "#*m +<2-"-m #,8'+92'!-Zm !-,$0-,2"-m !-,m *1m -201m 2<!,'!1m
!'2"1m,2#0'-0+#,2#Zm#1m/3#mm*m4#8m/3#m1#m"#2#!2m*m+32!'A,Zm2+ '<,m1#m"#2#!2m
13m *-!*'8!'A,m "#,20-m "#*m %#,Ym -12#0'-0+#,2#Zm +#"',2#m *m 1#!3#,!'!'A,m "#*m
$0%+#,2-m #,m *m $+'*'m +32,2#m 1#m .3#"#m 4'13*'80m #,m #*m !0-+2-%0+m 1'm *m
+32!'A,m1#m#,!3#,20m#,m&-+-!'%-1'1m-m&#2#0-!'%-1'1Ym
m.02'0m"#*m@-mHFFOm1#m#+.#8Amm310m-20-m+<2-"-m"#m"#2#!!'A,m"#m*-1m$0%+#,2-1m
+32"-1[m*m2#!,-*-%>m"#m#!"O-)&/.#)(O&.#(!m-m,9*'1'1m"#m!3041m"#m"'1-!'!'A,m
"#*mm^-,%m7m!-*YZmHFFO\m"7m7m!-*YZmHFFO_Ym12m2<!,'!m1#m 1m#,m+-,'2-0'80m*m
!',<2'!m "#m "'1-!'!'A,m "#m *-1m $0%+#,2-1m +.*'$'!"-1m 7m 0#,230*'8"-1m
.0#4'+#,2#m.0m.0-4-!0m*m$-0+!'A,m"#m&#2#0-"B.*#6Ym#"',2#m#12m.*'!!'A,m
1#m.3#"#m"#2#0+',0m*m+m^'*,./,O'&.#(!_m"#m*-1m+.*'!-,#1m.0m!-+.0- 0m
1'm&7m.0#1#,!'m"#m+32!'A,m-m,-Ymm!-+.*#+#,20'#""m#,20#m"-1m!"#,1m"#m3,m
',"'4'"3-m1'*4#120#m1#09m 1-*32m7Zm.-0m*-m2,2-Zm*m#12 '*'""m1#09m*2Zm.-0m*-m/3#m13m
2#+.#0230m"#m"'1-!'!'A,m1#09m+7-0Ym',m#+ 0%-Zm1'm*1m!"#,1m0#,230*'8"1m
IO
(.,)/#9(O
O
.-1##,m3,m&#2#0-"B.*#6Zm13m#12 '*'""m1#09m+#,-0m7m13m2#+.#0230m"#m"'1-!'!'A,m
1#09m +#,-0m 2+ '<,m ^#(#+.*-m #,m *m '%30m GYL_Ym 12m 2<!,'!m 7m &m 1'"-m 32'*'8"m #,m
20'%-m ^-,%m 7m !-*YZm HFFO_Zm 2-+2#m ^"7m 7m !-*YZm HFFO_m 7m ,#)*-#-O ^31&m 7m 071,Zm
HFGF_m 7m .3#12m m .3,2-m 0#!'#,2#+#,2#m #,m ,3#120-m * -02-0'-m ^#0%0#!&#Zm
!-+3,'!!'A,m.#01-,*_Ym
m
m
(&30 T BMGOm (#+.*-m "#m !304m "#m "'1-!'!'A,m "#m $0%+#,2-1m +.*'$'!"-1m 7m 0#,230*'8"-1Ym #m .3#"#m
"#2#0+',0m*m.0#1#,!'m"#m3,m&#2#0-"B.*#6m.-0/3#m.0#!#m3,m.'!-mm3,m2#+.#0230m+#,-0m^MMZLE_mm
*m "#2#0+',"m .0m *m +#8!*m 1'*4#120#m ^MOZKE_Ym 1m 3,'""#1m "#m 1#@*m "#m $*3-0#1!#,!'m 1#m +'"#,m #,m
$3,!'A,m"#m*m4#*-!'""m"#m!+ '-m"#m*1m3,'""#1m"#m$*3-0#1!#,!'m0#*2'4m^_m!-,m#*m2'#+.-m^_m^j"m
^_mim"_m^$3#,2#[m31&m7m071,ZmHFGF_Ym
m
m .#10m "#m *m #$'!'#,!'m .0- "m "#m *-1m +<2-"-1m +#,!'-,"-1m ,2#0'-0+#,2#Zm *m
2#,"#,!'m !23*m #1m "#m 310m -20-1m "-1m +<2-"-1m ,-m +#,-1m #$'!'#,2#1[m jm 7m
Ym m 2<!,'!m "#m jm .0#!#m #120m ","-m +37m 3#,-1m 0#13*2"-1m #,m
#1.#!'#1m"#m.*,21m!-+-m0(O-.#0m^&5"#m7m!-*YZmHFGF_Ym,m!3,2-mm*1m,3#41m
2#!,-*-%>1m"#m1#!3#,!'!'A,Zm*1m+91m31"1m#,mm1-,mJKJm()'O+/(,O
O #m ^-!&#m ..*'#"m !'#,!#_m #m &&/'#(O I-*#6JO ()'O (&34,Ym ,m 2-+2#m 1#m
&,m32'*'8"-m1'+3*29,#+#,2#m+91m"#mGHYFFFm+3#1201m.0m,*'80m"'12',2-1m%#,#1m
#,m3,m1-*m.*!mJKJ '!-'2#0m*2#m^_m^'%-* 7m!-*YZmHFFO_YmmB*2'+m',,-4!'A,m
#,m#12#m1#,2'"-m&m1'"-m32'*'80m*1m2#!,-*-%>1m"#m1#!3#,!'!'A,m+1'4m!-+ ',"1m
!-,m *m #* -0!'A,m "#m +#8!*1m #,m "'12',21m "'+#,1'-,#1m ^1'm 7m !-*YZm HFGG_Ym 0m
!-,$'0+0m 3,m +32!'A,m +#"',2#m 1#!3#,!'!'A,m &!#m $*2m 3,m !- #0230m "#*m
%#,-+m"#mHFjIFm#,m,#)*-#-O^11-51)7m7m!-*YZmHFGF_Ym,m00-8m^,34O-.#0_m7m#,m
20'%-m ^,#.#/'O /,/'_m 1#m &,m "#2#!2"-m #$'!'#,2#+#,2#m +32!'-,#1m +#"',2#m *m
1#!3#,!'!'A,m "#m $0%+#,2-1m "#m m !-,m **3+',m #,m 3,m 2-2*m "#m MLNm ',"'4'"3-1m
!-+ ',9,"-*-1m#,m+#8!*1m"#m&12mOJm',"'4'"3-1m^1'm7m!-*YZmHFGG_Ymm
JF
O
(.,)/#9(O
*m +<2-"-m m !-+ ',"-m !-,m *m 1#!3#,!'!'A,m +1'4m 7m *m .*'!!'A,m "#m
+#8!*1m#,m"'12',21m"'+#,1'-,#1m.0#!#m.0#1#,20m40'1m4#,2(1Ym,m.0'+#0m*3%0m
*m.*'!!'A,m"#m40'1m+#8!*1m.#0+'2#m*m!-,$'0+!'A,m"#*m+32,2#m7m"'1+',37#m*m
.0- '*'""m"#m#00-0Ym"#+91Zm!-,m*m'"#,2'$'!!'A,m"#m*m$+'*'m+32,2#m1#m- 2'#,#m
2+ '<,m*m',$-0+!'A,m"#*m2'.-m"#m!+ '-m,3!*#-2>"'!-m7m"#m13m.-1'!'A,Zm1>m!-+-m"#m
13m #$#!2-m #,m *m 1#!3#,!'m +',-!>"'!Ym 20m 4#,2(m #1m /3#m 1#m "'1+',37#m *m
,#!#1'""m"#m31-m"#m!# "-0#1m+0!"-1m!-,m$*3-0A$-0-1m,'m2+.-!-m#,"-,3!*#11Ym
',*+#,2#Zm #1m 3,m +<2-"-m #1!* *#m m "'12',2-1m ,'4#*#1m .3"'<,"-1#m .*'!0m .0m
2,2-1m %#,#1m !-+-m 1#m ,#!#10'-m - 2#,'#,"-m 3,m %0,m .0#!'1'A,m #,m *m "#2#!!'A,m "#m
+32!'-,#1m7m#,m3,m.#0>-"-m"#m2'#+.-m+37m0#"3!'"-m^1'm7m!-*YZmHFGG_Ymm
m
BMEMCMETTTTT
m 21m "#m +32!'A,m - 2#,'"m &m 1'"-m 40' *#m #,m *1m "'12',21m .*2$-0+1m "#m
m "#100-**"1m &12m &-0m ^ *m GYI_Zm #,m ,#)*-#-O &m 1'"-m '"#,2'$'!"m
3,m+32!'A,m!"mIFFm m^0##,#m7m!-*YZmHFFI_Zm#,m+>8m3,m!"mKFFm) m^'**m7m!-*YZm
HFFJZ_m7m#,m00-8m3,m!"mHOJm m^'**m7m!-*YZmHFFM_Zm+'#,201m/3#m#,m!# "m*m21m
"#m +32!'A,m #1m "#m Gm !"m m ^*"5#**m 7m !-*YZm HFFJ_Ym !23*+#,2#m *m "#,1'""m "#m
+32!'A,m+91m*2m&m1'"-m"#1!0'2m#,m20'%-m2#20.*-'"#m^GiJFm) _m7m20'%-m&#6.*-'"#m
^GiHJm ) _m ^*"#m 7m !-*YZm HFFK_Ym ,m !3,2-m *m +#*A,m 1#m &m "#100-**"-m 3,m .- *!'A,m
!-,m3,m21m"#m+32!'A,m"#mGiKMIm m^&+,'j0"1m7m!-*YZmHFGF_Ym
*m +<2-"-m m .#0+'2#m *m '"#,2'$'!!'A,m "#m *1m $+'*'1m +32,2#1Ym ,m 4#8m
'"#,2'$'!"1m *1m $+'*'1m &!#m $*2m 1#!3#,!'0m #*m $0%+#,2-m "-,"#m 1#m #,!3#,20m *m
+32!'A,m .0m .-"#0m .0#"#!'0m 131m #$#!2-1Ym # #0#+-1m ,*'80m 1'm 1#m 202m "#m 3,m
+32!'A,m #,m 0#%'A,m ',20A,'!m -m #6A,'!Ym ,m !1-m "#m *-!*'801#m #,m 3,m #6A,Zm
#6+',0#+-1m 1'm #*m !+ '-m "#m ,3!*#A2'"-m .0-"3!#m 2+ '<,m 3,m !+ '-m "#m
+',-9!'"-m -m 1'Zm .-0m #*m !-,200'-Zm #1m 1'*#,!'-1Ym ,m !1-m "#m .0-4-!0m 3,m !+ '-m "#m
+',-9!'"-m2+ '<,m"# #0#+-1m!-+.0- 0m1'm#*m!+ '-m.0-4-!"-m.3#"#m$#!20mm
*m#1203!230m20'"'+#,1'-,*m"#m*m.0-2#>,Ym*m!1-m+91m#620#+-m#1m/3<*m#,m#*m/3#m#*m
!+ '-m "#m +',-9!'"-m .0-4-!m 3,m !-"A,m b12-.cm m *m .0-2#>,m .0-"3!'#,"-m 3,m
',2#003.!'A,m "#m *m 20"3!!'A,Ym -0m *-m 2,2-Zm *1m +32!'-,#1m '"#,2'$'!"1m #,m 0#%'A,m
!-"'$'!,2#m"#m*m1#!3#,!'m,3!*#-2>"'!m.3#"#,m!*1'$'!01#m#,[m
jm +32!'-,#1m 1',m 1#,2'"-GO #,m *1m !3*#1m #*m !+ '-m ,3!*#-2>"'!-m .0-4-!m 3,m
!-,4#01'A,m"#m3,m+',-9!'"-mm3,m!-"A,m12-.Zm',2#003+.'#,"-m1>m*m!-00#!2m
20"3!!'A,m"#m*m.0-2#>,Ym
JG
(.,)/#9(O
O
jm+32!'-,#1m1312'232'41m-m,-m1',A,'+1[m#,m*1m!3*#1m#*m!+ '-m,3!*#-2>"'!-m
1-!'"-mm*m+32!'A,m.0-4-!m3,m!+ '-m"#m+',-9!'"-Ym12#m!+ '-m/3#"m
2+ '<,m!-,"'!'-,"-m.-0m*1m!0!2#0>12'!1m/3>+'!1m"#*m,3#4-m+',-9!'"-Zm
/3#m.3#"#m-!1'-,0m!+ '-1m!-,$-0+!'-,*#1m7i-m$3,!'-,*#1m"#m*m.0-2#>,Ym
jm+32!'-,#1m1'*#,!'-11m-m1',A,'+1[m#,m*1m!3*#1m*m+32!'A,m,3!*#-2>"'!m,-m
.0-4-!m3,m!+ '-m#,m#*m!-"A,m7m.-0m*-m2,2-m,-m&7m3,m!+ '-m+',-!>"'!-m
7m*m.0-2#>,m+32,2#m1#09m'"<,2'!mm*m1'*4#120#Ym
m
0m3,m2#!,-*-%>m 1"m#,m*m%#,<2'!m',4#01m!-+-m#*mZm*1m+32!'-,#1m
1',m 1#,2'"-m 7m 1312'232'41m 1-,m *1m !-,1'"#0"1m +91m '+.-02,2#1m 7m /3#m .3#"#,m
$#!20m .-2#,!'*+#,2#m m *m $3,!'-,*'""m "#m *m .0-2#>,m 7m .-0m *-m 2,2-Zm "0m *3%0m m
3,m,3#4-m$#,-2'.-Ym30,2#m*m!-,1203!!'A,m"#m*m.- *!'A,m0#13*2m#1#,!'*m!-,-!#0m
*m.0- '*'""m"#m/3#m#12-m.3#"m13!#"#0m#,m*m0#%'A,m,*'8"m^!**3+m7m!-*YZm
HFFF _Ym 1m '+.-02,2#m ,*'80m 3,m 0#%'A,m "#*m %#,m #,m *m !3*m .-"+-1m #,!-,200m
.-2#,!'*+#,2#m +91m +32!'-,#1m 2'.-m 1',m 1#,2'"-m 7m 1312'232'41Ym 0m #1!-%#0m 3,m
0#%'A,m A.2'+m 1#m "'1.-,#m "#m &#00+'#,21m !-+-m #*m .0-%0+m m ^))(O
*.#'#4O .)O ..O &.,#)/-O -#)(-_Zm /3#m .#0+'2#m !*!3*0m *m .0- '*'""m "#m
- 2#,#0m +32!'-,#1m .3,23*#1m #,m 3,m "#2#0+',"m 0#%'A,m 7m /3#m $#!2#,m m *m
#1203!230m "#m *m .0-2#>,m ^&22.[ii555Y.0-5# Y-0%i!-""*#_m ^'**m 7m !-*YZm HFFI_Ym
m#1m3,m.0-%0+m/3#m+3#120m2-"1m*1m+32!'-,#1m/3#m.-"0>,m-!300'0m#,m
3,m 1#!3#,!'m "#2#0+',"m #,m $3,!'A,m "#*m 2'.-m "#m +329%#,-m 32'*'8"-m 7m *m +'1+-m
2'#+.-Zm,*'8m#,m!"m.-1'!'A,m*m.0- '*'""m"#m$#!20mm*m$3,!'-,*'""m"#m*m
.0-2#>,m#,m!1-m"#m!+ '-Ym
,m 4#8m '"#,2'$'!"m 3,m +32!'A,m 1',m 1#,2'"-m .3#"#m #12'+01#m !A+-m $#!209m m *m
$3,!'-,*'""m "#m *m .0-2#>,Ym 0m .-"#0m 0#*'80m 3,m .0#"'!!'A,m "#*m #$#!2-m "#m *m
+32!'A,m #6'12#,m .0-%0+1m ',$-0+92'!-1m #1.#!>$'!-1m 1"-1m #,m *m &-+-*-%>m !-,m
1#!3#,!'1m.0-2#'!1m"#m-201m#1.#!'#1Ym,m#(#+.*-m#1m#*mm^,)$.O&#!(O
&.O +/(-O (O 0&/.O -_m ^&22.[ii555Y.0-5# Y-0%i.01#1,.i\m 7*-0m 7m
0##,#Zm HFFI\m '**m 7m !-*YZm HFFI_m 7m #*m .0-%0+m m ^),.#(!O(.)&,(.O,)'O)&,(._m
/3#m #$#!2B,m *m .0#"'!!'A,m #,m 1#m m 3,m jm ^)-#.#)(N*# #O .,.#0O -#O
)&O &#!('(.O ,"O ))&_m 7m #,m 1#m m *1m !0!2#0>12'!1m $>1'!-j/3>+'!1m "#m *-1m
+',-9!'"-1m^&22.[ii *-!)1Y$&!0!Y-0%i1'$2iY&2+*\m%m7m#,')-$$ZmHFFG_Ym
*mm.-0m*-m2,2-Zm.0-.-0!'-,[m
JH
O
(.,)/#9(O
jm32!'-,#1m',"'4'"3*#1m/3#m.3#"#,m.0-"3!'0m1#0'#1m*<*'!1m/3#m.0-4-/3#,m
,3#4-1m $#,-2'.-1Ym 12m %0"!'A,m $#,-2>.'!m *'%"m m 3,m 1#0'#m *<*'!m .3#"#m
.0-6'+0,-1mm!-,-!#0m*m$3,!'-,*'""m"#*m%#,Ym
jm#0+'2#m!0#0m7m!0!2#0'80m3,m,3#4m40'#""m%#,<2'!m#,m#*m%#0+-.*1+m
"#m#1.#!'#1m!3*2'4"1m"#m',2#0<1Ym
m
,m #*m !1-m "#m *-1m +32,2#1m '"#,2'$'!"-1m +#"',2#m m #1m ,#!#10'-m 32'*'80m
0#20-!038+'#,2-1m .0m - 2#,#0m *m +32!'A,m '1*"m #,m 3,m $-,"-m %#,<2'!-m "#1#"-Ym
# #m 2#,#01#m #,m !3#,2m /3#m *-1m +32,2#1m "#2#!2"-1m +#"',2#m m **#4,m
',!-0.-0"1m ,-m 1A*-m *m +32!'A,m "#2#!2"m #,m #*m %#,m "#m ',2#0<1m 1',-m 2+ '<,m
+3!&1m -201m +32!'-,#1m "'120' 3'"1m *m 80m #,m #*m 0#12-m "#*m %#,-+m ^*"#m 7m !-*YZm
HFFK_Ym 0m .-"#0m '"#,2'$'!0m !-,m +7-0m #6!2'23"m *m $3,!'-,*'""m "#m 3,m %#,m ,-m
12m!-,m#,!-,200m#*m+32,2#m7m- 1#040m#*m$#,-2'.-m1',-m/3#m"#+91Zm#1m,#!#10'-m
- 2#,#0m #*m +32,2#m '1*"-m "#m -201m .-1' *#1m +32!'-,#1m "'!'-,*#1Ym A*-m "#m #12#m
+-"-m1#m.-"0>m!-,$'0+0m!-,m!#02#8m/3#m*m+32!'A,m'"#,2'$'!"m#1m*m0#1.-,1 *#m
"#m !'#02-m $#,-2'.-Ym -0m *-m 2,2-m #,m *m $1#m $',*m "#*m .0-2-!-*-m "#m m 1#m "# #,m
#$#!230m 40'-1m 0#20-!038+'#,2-1Zm #,20#m #*m +32,2#m 7m #*m 1'*4#120#m "#m *m +'1+m
40'#""Zm.0m.-"#0m'1*0m*m+32!'A,m"#m',2#0<1m^*"#m7m!-*YZmHFFK_Ym
T
BMEMCMFTTTTT
6'12#,m .*2$-0+1m "#m m #,m ,3+#0-11m #1.#!'#1m 4#%#2*#1m 7m ,'+*#1Ym m
%0,m 4#,2(m "#*m m #1m /3#m .3#"#m 32'*'801#m #,m #1.#!'#1m "'.*-'"#1m -m
.-*'.*-'"#1Zm !-,m "'$#0#,2#1m 2+@-1m "#m %#,-+1m 7m /3#m .-1#,m !3*/3'#0m 2'.-m "#m
0#.0-"3!!'A,m ^0)*#7m 7m ,%Zm HFFN_Ym *m B,'!-m 0#/3#0'+'#,2-m #1m .-"#0m +320m *1m
1#+'**1m-m#*m.-*#,m.0m.-12#0'-0+#,2#m!0#!#0m*1m%#,#0!'-,#1m1'%3'#,2#1m7m#620#0m
#*m m .0m ,*'80*-Ym ,m *m *m GYIm 1#m +3#120m 3,m 0#13+#,m "#m *%3,1m "#m *1m
.- *!'-,#1m "#m m "#100-**"1m &12m #*m +-+#,2-m "#+-120,"-m /3#m #12m
2<!,'!m#1m.*'! *#m#,m"'$#0#,2#1m#1.#!'#1Ym #m"#12!0m2+ '<,m*m40' '*'""m"#*m
2+@-m "#m *-1m %#,-+1m #,m *-1m !3*#1m 1#m &m .*'!"-m *m 2<!,'!Ym -1m 2+@-1m #,m
-0%,'1+-1m"'.*-'"#1m 0!,m"#1"#m*1mGFFm m#,mEO&!(-m^'*!&0'12m7m!-*YZmHFFL_m
&12m*1mKZIFFm m"#m),/'O0/&!,m^*"5#**m7m!-*YZmHFFJ_Ymm2<!,'!m2+ '<,m&m
1'"-m #$'!'#,2#m #,m -0%,'1+-1m .-*'.*-'"#1m !-+-m ,#.#/'O ./,!#/'O 13 1.EO /,/'Zm
2#20.*-'"#m^*"#m7m!-*YZmHFFK_m7O,#.#/'O-.#0/'ZO&#6.*-'"#m^*"#m7m!-*YZmHFFK_Zm#,m
#12#m!1-m*-1m2+@-1m"#m%#,-+1m 0!,m&12m*1mGMYFFFm m"#m,#.#/'O-.#0/'Em
m21m"#m+32!'A,m#1m3,-m"#m*-1m4*-0#1m+91m'+.-02,2#1m.0m"#2#0+',0m1'm3,m
.- *!'A,m"#mm#1m#$'!'#,2#Ym39,2-m+7-0m1#m*m.0- '*'""m"#m#,!-,200m
JI
(.,)/#9(O
O
+32,2#1m#,m3,m.*2$-0+m"#mm+91m#$#!2'4m1#09Ym1m211m"#m+32!'A,m#,m
.- *!'-,#1m "#m m "#m .*,21m 0!,m 3,m +.*'-m 0,%-Ym ,m "'.*-'"#1m "#1"#m
GiLFm) m#,m,--#O,*m^2#.&#,1-,m7m!-*YZmHFGF_m&12mFZKiGm m#,m,34O-.#0m^3m
7m !-*YZm HFFK_m +'#,201m /3#m #,m #1.#!'#1m .-*'.*-'"#1m #,!-,20+-1m 211m "#1"#m GiHJm m
#,m,#.#/'O-.#0/'m^*"#mm7m!-*YZmHFFK_m&12mGiOLMm m#,m,"#-O"3*)!m^,-**m7m
!-*YZmHFGG_Ym# #m2#,#01#m#,m!3#,2m/3#m#,m#*m.0-!#"'+'#,2-m"#m!9*!3*-m,-m1'#+.0#m1#m
&,m 31"-m *1m +'1+1m 40' *#1m 7m .-0m *-m 2,2-Zm 0#13*2m "'$>!'*m &!#0m !-+.0!'-,#1m
#,20#m #1.#!'#1m #,m 2<0+',-1m %#,#0*#1Ym 0m *%3,1m .- *!'-,#1m "#m m 1#m&,m
"#100-**"-m #123"'-1m $#,-2>.'!-1m "#m *-1m +32,2#1m #,m "'12',2-1m 2#('"-1m 7m "'$#0#,2#1m
$1#1m "#m !0#!'+'#,2-Ym 1m 1#1m "#m "2-1m "#m #121m .- *!'-,#1m .#0+'2#,m - 1#040m
$#,-2'.-1m +32,2#1m #,m #*m "#100-**-m "#m *m .*,2Zm "#*m $032-Zm "#m *m $*-0m -m "#m *-1m
!-2'*#"-,#1Zm#,20#m-20-1Ym*mm^(#.6OO","O(O6()'#+/O6!.&_m"'1.-,#m
"#m 3,m 1#m "#m "2-1m ^" _m /3#m .#0+'2#m 1-*'!'20m 1#+'**1m "#m +32,2#1m "#m
"'12',21m
#1.#!'#1m
!-+-m
2-+2#Zm
!-*8Zm
%3'1,2#m
-m
+#*A,m
^&22.[ii555Y4#01'**#1Y',0Y$0i30%4i2'**',%Y&2+_Ym + '<,m #6'12#,m -201m !-+-m *m "#*m
!#,20-m )."'-.O-,"O(-.#./.m /3#m .0-.-0!'-,m +32,2#1m "#m 3,m .- *!'A,m "#m
m "#m 20'%-Ym 0m ,#)*-#-m #6'12#m 3,m 1#04'!'-m 5# O ^&22.[ii2'**',%Y$&!0!Y-0%_m
"-,"#m 1#m .3#"#m #,!-,200m 2-"m *m ',$-0+!'A,m !#0!m "#m *-1m +32,2#1m 7m
!0!2#0'8"-1m7m3,m1#04'!'-m"#m'"#,2'$'!!'A,m.0m%#,#1m"#m',2#0<1Ym+ '<,m#6'12#,m
*%3,1m #+.0#11m .0'4"1m /3#m &,m 32'*'8"-m #*m m !-+-m +<2-"-m .0m
"#100-**0m .0-"3!2-1m !-+#0!'*'8 *#1Ym 12#m #1m #*m !1-m "#m ,#O #)-#(-m
^&22.[ii555Y0!"' '-Y!-+i_Zm 3,m #+.0#1m ,-02#+#0'!,m /3#m &m '1*"-m
+#"',2#mm3,m2-+2#m+32,2#m"#m$032-m"#m*0%m4'"Zm!.8m"#m!-,1#0401#m
#,mA.2'+1m!-,"'!'-,#1m"30,2#m+3!&-1m+91m">1m/3#m3,m2-+2#m,-0+*Zm7m3,m2'.-m
"#m20'%-m.2-m.0m!#*>!-1m^&22.[ii555Y0!"' '-Y!-+i!#*'!1$#5&#2_Ym',*+#,2#m
+#,!'-,0m *m
1#m "#m "2-1m "#m 1-(m ^&22.[ii555Y1-7 #,2'**',%Y-0%i',"#6Y(1._m
0#*'8"m.-0m#*m"#.02+#,2-m"#m%#,A+'!m"#m.*,21m"#m*m,'4#01'""m"#m**',-'1m
^)/.",(O &&#()#-O (#0,-#.3O .O ,)(&_m *m !3*m #1m 3,m .*2$-0+m "#m m
#$'!'#,2#m .-0m 131m *21m 211m "#m +32!'A,m 7m .-0m -$0#!#0m *m .-1' '*'""m "#m 31!0m
+32,2#1m#,m$3,!'A,m"#m3,m$#,-2'.-m"#2#0+',"-m-m"#m3,m%#,m"#m',2#0<1m^--.#0m7m
!-*YZm HFFN_Ym ,m *%3,1m .- *!'-,#1m 1#m &,m .-"'"-m '"#,2'$'!0m +32!'-,#1m /3#m &,m
.0-.-0!'-,"-m3,m,3#4-m$#,-2'.-Ym-0m#(#+.*-Zm#,m2-+2#m*m+32!'A,mGJNKm#,m#*m
%#,m >;m .0-4-!m 3,m !+ '-m #,m #*m -*&##(!m "#*m %#,m .0-.-0!'-,,"-m 3,m +m
',!-+.*#2-m "#m +-"-m /3#m *m .0-2#>,m ,-m .3#"#m 1',2#2'801#m !-00#!2+#,2#Ym 12m
JJ
O
(.,)/#9(O
+32!'A,m!-,$'#0#m0#1'12#,!'mm"-1m.-2'4'031[m*m4'031mm"#m*m.22m^)..)O#,/-OZm
_m7m*m4'031m"#*m+-2#"-m"#*m.'+'#,2-m^**,O')..&O0#,/-Zm#_m^'0-,ZmHFGF_Ymm
,m +#*A,m *m 2<!,'!m ,-m 1-*-m &m .-"'"-m .0-.-0!'-,0m +32,2#1m $#,-2>.'!-1m
^&+,'j0"1Zm HFGF\m -,8*#8j-m 7m !-*YZm HFGG_m 1',-m /3#m "#+91Zm 1#m &m .-"'"-m
32'*'80m.0m!-,$'0+0m*m$3,!'A,m"#m*%3,-1m%#,#1Ym,m#(#+.*-m#1m*m!-,$'0+!'A,m
"#m*m$3,!'A,m"#*m%#,mGm^02',m7m!-*YZmHFFO_Ym#m1#*#!!'-,0-,m20#1m',"'4'"3-1m
+32,2#1m .0m #12#m %#,m "#,20-m "#m *m .- *!'A,m "#m Ym 1m +32!'-,#1m
^#0IFL&#Zm*7HJH0%m7m&#GOI#3_m #,m&-+-!'%-1'1m.0-"3!>,m*m$#+','8!'A,m"#m
*1m $*-0#1m #,m 2-"m *m .*,2m 7m .-0m *-m 2,2-Zm *m !-,4#01'A,m "#m .*,21m +-,-'!1m m
%',-'!1m^02',m7m!-*YZmHFFO_Ym
30,2#m *-1m B*2'+-1m @-1m &,m .0#!'"-m #123"'-1m +#2-"-*A%'!-1m /3#m &,m
.#0+'2'"-m .#0$#!!'-,0m *m 2<!,'!m 7m -.2'+'80*Ym ,m "#m #121m ,3#41m +#2-"-*-%>1m
#1m*m**+"mm^/&#O#O2.,.#)(O ,)'O,,3O#--/O ),O_Zm/3#m
&m-.2'+'8"-m#*m+-"-m"#m.0#.0!'A,m"#m*1m+#8!*1m"#mYm,m*m2<!,'!m!*91'!m
1#m +#8!* ,m *-1m 1m #620>"-1m m .02'0m "#m *m %#,#0!'A,m HYm ,m !+ '-Zm #12m
,3#4m 2<!,'!m +#8!*m "'0#!2+#,2#m *-1m !-2'*#"-,#1m "#m .*,21m HYm "#+91Zm *m
.0#.0!'A,m"#m*1m.*!1m"#m+#8!*m#1m"'12',2Ym#m"#,-+',mb.--*m#,mHcm7m!-,1'12#m
#,m0#*'80m"-1m2'.-1m"#m.*!1m"#m+#8!*[m3,1m 1"1m#,m*m+#8!*1m"#m*-1m1m
"#m*1m$'*1m"#m*1m.*!1m"#m*m.- *!'A,m7m*1m-201m!-,m*1m+#8!*1m"#m*-1m1m"#m
*1m!-*3+,1m"#m*1m.*!1m"#m*m.- *!'A,Ymm4#,2(m#1m/3#m3,m4#8m"#2#!2"-m3,m
+32,2#m,-m&!#m$*2m&!#0m*m"#!-,4-*3!'A,m7m/3#m!-,m#12m2#!,-*-%>m1#m!-,$'0+m
"'0#!2+#,2#m*m$+'*'m+32"m#,m!3#12'A,m^0##*)1&+'m7m!-*YZmHFFO_Ym
20m0#!'#,2#m.3 *'!!'A,m-.2'+'8m2-"4>m+91m#*m.0-2-!-*-m!*91'!-[m'm^31&m
7m071,ZmHFGF_Ym,m#12#m+<2-"-m#*m.0-!#1-m.0m- 2#,#0m*m%#,#0!'A,mHm#1m"'12',2-Ym
#1.3<1m "#m 32-.-*','80m *m %#,#0!'A,m Gm 7m !0#!#0*Zm 1#m 0#!-%#,m *1m 1#+'**1m HYm
',m #+ 0%-Zm #,m #12#m !1-m 1#m +#8!*,m *1m 1#+'**1m 7m .-12#0'-0+#,2#m 1#m &!#,m
%#0+',0m "'0#!2+#,2#m #,m .*!1m N*O I071,Zm HFFJ\m *0)m 7m 071,Zm HFFM_Ym
0!'1m m #12#m 2'.-m "#m .*!1Zm 3,m 4#8m %#0+',"1m *1m .*,21m 7m .0#!'"1m *1m
.0'+#01m&-(1m1#m.3#"#m0#*'80m*m#620!!'A,m"'0#!2+#,2#m#,m.*!Ymm"#2#!!'A,m"#m
+32,2#1m 1#m &!#m +#"',2#m *m 2#!,-*-%>m "#m ,9*'1'1m "#m *m !304m "#m "'1-!'!'A,m
^_Ym 12-1m !+ '-1m .#0+'2#,m 0#"3!'0m #*m 2'#+.-m "#m #1.#0m .0m 0#*'80m *m
"#2#!!'A,m"#m+32,2#1m7Zmm*m4#8Zm.#0+'2#,m1#*#!!'-,0m*1m.*,21m+32,2#1m,2#1m"#m
1#0m 201*""1m *m ',4#0,"#0-m !-,m *-m /3#m 1#m 0#"3!#m !-,1'"#0 *#+#,2#m #*m !-12#m "#*m
#6.#0'+#,2-m^31&m7m071,ZmHFGF_Ymm
m
JK
(.,)/#9(
Genes candidatos para el TILLINGO
m
m
BMFT T T T T T T T T
TTT
m
m#*#!!'A,m"#*m%#,m"',m.0m#*m,9*'1'1m7m.-0m*-m2,2-Zm*m"#2#!!'A,m"#m+32,2#1m#,m3,m
.*2$-0+m"#mm"#.#,"#m"#*m- (#2'4-m"#m*m',4#12'%!'A,m/3#m1#m#12<m0#*'8,"-Ym
0m#12#m20 (-m*m#*#!!'A,m"#m*-1m%#,#1m&m1'"-Zm.-0m3,m*"-Zm!-+.0- 0m*m#$'!'#,!'m"#m
*m .*2$-0+m 32'*'8,"-m #*m %#,m '2-#,-m "#12301m ^_m 7Zm .-0m -20-m *"-Zm .-"#0m
"#2#!20m+32,2#1m#,m%#,#1m"#m',2#0<1m%0-,A+'!-m!-+-m#1m#*m!1-m"#m;m0#*!'-,"-m
!-,m*m+"30!'A,m"#*m$032-m7m"#m*-1m$!2-0#1m"#m','!'!'A,m"#m*m20"3!!'A,mJ[m>O7m
13m '1-$-0+m I#-)J>m ',4-*3!0"-1m #,m 0#1'12#,!'1m 0#!#1'41m m 4'031Ym ,m #*m 1'%3'#,2#m
.02"-m 1#m .0#2#,"#m ',20-"3!'0m #12-1m %#,#1m 1>m !-+-m "0m *m ',$-0+!'A,m +-*#!3*0m 7m
$3,!'-,*m /3#m 1#m 2'#,#m "#m #**-1m 7m .-"#0m #,2#,"#0m 13m #*#!!'A,m .0m #*m ,9*'1'1m +#"',2#m
m#,m#12m2#1'1m"-!2-0*Ym
T
BMFMBTTP(2.$-.T$1 230 1 QT
mm#1m3,m#,8'+m!*4#m.0m*m.0-"3!!'A,m"#m!0-2#,-'"#1m^'3*',-m7m!-*YZmGOOI\mm
'm7m!-*YZmGOOL\m1!1-,m7m!-*YZmHFFH_Ym-1m!0-2#,-'"#1m1-,m.'%+#,2-1m-0%9,'!-1m/3#m1#m
#,!3#,20,m #,m .*,21m 7m -20-1m -0%,'1+-1m $-2-1',2<2'!-1Ym *m !-*-0m "#m #12-1m .'%+#,2-1m
.3#"#m40'0m"#1"#m#*m+0'**-m&12m#*m0-(-m.1,"-m.-0m3,m%0,m40'#""m"#m,0,(1Ym
*m!-*-0m#1m.0-4-!"-m.-0m13m#1203!230m+-*#!3*0m!-+.3#12m.-0m40'-1m"- *#1m#,*!#1m
"#m!0 -,-m/3#m.0-"3!#,m#*m.0-!#1-m"#,-+',"-m!-,(3%!'A,m^'3*',-ZmGOOI_Ym30,2#m
#12#m.0-!#1-m3+#,2,m*-1m"- *#1m#,*!#1m7Zm.-0m*-m2,2-Zm2+ '<,m3+#,2m*m*-,%'23"m
"#m -,"m m *m /3#m .#0!' #m *m *38m *m +-*<!3*Zm ","-m !"m 4#8m 3,m !-*-0m +91m 0-('8-Ym -1m
!0-2#,-'"#1m 2'#,#,m 40'1m $3,!'-,#1m "#.#,"'#,"-m "#*m -0%9,3*-m "A,"#m #12<,m
!3+3*"-1m^',"#,m7m!-*YZmGOOG_Ym,m#*m!*-0-.*12-Zm*-1m!0-2#,-'"#1m 1-0 #,m*m*38m#,m
#*m#1.#!20-m83*m^#,20#mJFFm7mLFFm,+_m7m*m#,#0%>m/3#m0#!' #,m*m20,1$'#0#,mm*m!*-0-$'*m
^'m 7m !-*YZm GOOL_Ym 1'+'1+-Zm *-1m !0-2#,-'"#1m 1-,m .0-2#!2-0#1m "#m *m -6'"!'A,m #,m *-1m
.0-!#1-1m$-2-0#!2'4-1m#,m-0%,'1+-1m$-2-1',2<2'!-1m^'3*',-ZmGOOI\m02*#7m7m!-*YZmGOOO_Ym
,m *-1m !0-+-.*12-1Zm "-,"#m *m !*-0-$'*m ,-m #129m .0#1#,2#Zm 1#m !3+3*,m #,m 2#('"-1m !-+-m
*-1m .<2*-1m "#m *1m $*-0#1m -m *-1m $032-1m #,m #12"'-1m 4,8"-1m "#100-**,"-m 3,m $3,!'A,m
91'!+#,2#m #1203!230*m 7m "#m 20!!'A,m .0m *m .-*','8!'A,m ^0-2#5-*"Zm HFFL_Ym m
',4#12'%!'A,m !-,m !*-0-.*12-1m "#m .*,21m 13.#0'-0#1m &m 73""-m m !-,-!#0m !-,m +91m
"#2**#m *m *-!*'8!'A,m "#m *m Ym ,m #).#(O./'O1#m .3"-m *-!*'80m *m m #,m 3,m
JLm
(.,)/#9(
Genes candidatos para el TILLING
MKPm #,m *1m +#+ 0,1m "#m 2'*!-'"#1Ym ,m .-0!#,2(#m +3!&-m +#,-0m $3#m "#2#!2"-m 2,2-m
#,m*m+#+ 0,m#62#0,m^KP_m!-+-m#,m#*m#120-+m^GLP_m^',"#,m7m!-*YZmGOOG\m*j '*'m7m
023,%Zm GOOO_Ym m 0#%3*!'A,m "#m #12m #,8'+m &m 1'"-m ',4#12'%"m #,m "'12',21m .*,21m
!-+-m#*m,0!'1-m^*j '*'m7m023,%ZmGOOO_m7m#*m2-+2#m^#!)#0m7m!-*YZmGOOH\m'3*',-m7m
!-*YZm GOOI_Ym m !-,!#,20!'A,m "#m !0-2#,-'"#1m 1>m !-+-m *m #6.0#1'A,m "#m *m m 7m *m
'2-#,-m ',21m ^_m 3+#,2,m #,m !-,"'!'-,#1m "#m ',2#,1'""m "#m *38m #,m .*9,23*1Zm 1',m
#+ 0%-Zm #*m 3+#,2-m #,m .*,21m "3*21m ,-m .0#!#m 1'%,'$'!2'4-Ym ,m *m 0>8Zm #121m
!-,!#,20!'-,#1m "'1+',37#,m !-,1'"#0 *#+#,2#m +'#,201m /3#m #,m $*-0#1m 3+#,2,m #,m
.<2*-1m7m,2#01m7m"'1+',37#,m#,m1<.*-1m7m!0.#*-1Ym30,2#m#*m"#100-**-m"#m*m$*-0m*m
#6.0#1'A,m "#m + 1m #,8'+1m 3+#,2m "'#8m 4#!#1m ',+#"'2+#,2#m ,2#1m "#m *m ,2#1'1m
^'3*',-m7m!-*YZmGOOI_Ymm
m '-1>,2#1'1m"#m!0-2#,-1m^'%30mGYM_m1#m','!'m!-,m20#1m!-,"#,1!'-,#1m0#*'8"1m.-0m
*m #0,'*%#0,'*j'$-1$2-m 1',21m - 2#,'#,"-m #0,'*%#0,'*j'$-1$2-m ^_Ym
-12#0'-0+#,2#m*m#,8'+m'2-#,-m1',21m^_m!2*'8m*m0#!!'A,m"#mmm$'2-#,-Ym
3#%-Zm 3,m 1#0'#m "#m "#1230!'-,#1m !-,4'#02#,m #*m $'2-#,-m #,m *'!-.#,-Ym -1m .0-"3!2-1m
',2#0+#"'-1m 2'#,#,m 3,m 0,%-m "#m !-*-0#1m !0!2#0>12'!-m "#m *-1m !0-2#,-1Zm "#*m 0-(-m "#*m
*'!-.#,-m&12m#*m+0'**-m"#m*m8#6,2',m^'3*',-m7m!-*YZmGOOI_m^'%30mGYM_Ym
m
T
m
(&30 T BMH[m 32m "#m 1>,2#1'1m "#m !0-2#,-1m #,m .*,21Ym m .02'0m "#*m #0,'*%#0,'*j'$-1$2-m ^_m &12m *m
- 2#,!'A,m"#m*32#>,m7m8#6,2',Zm.1,"-m.-0m*m$-0+!'A,m"#m*'!-.#,-m^$'%30m20"3!'"m7m".2"mm.02'0m
JMm
(.,)/#9(
Genes candidatos para el TILLINGO
"#m*m-0'%',*m"#m!&#*mYm)#*2-,m^)#*2-,m7m!-*YZmHFFL__Ym
m
m !-,4#01'A,m "#m $'2-#,-m m *'!-.#,-m 4'#,#m +#"'"m #,m %0,m .0-.-0!'A,m .-0m *m Ym 'm
#12m #,8'+m ,-m $3,!'-,m !-00#!2+#,2#m -m ,-m #129m .0#1#,2#Zm *m 1>,2#1'1m "#m !0-2#,-1m 1#m
"#2'#,#m .-0m !-+.*#2-m ^'01!& #0%m Zm HFFG_Ym m 31#,!'m "#m #12-1m .0-"3!2-1m &!#m /3#m #*m
2#('"-m,-m2#,%m.'%+#,2!'A,m*%3,m","-m!-+-m0#13*2"-m3,m.*,2m* ',Ymm4>m"#m
!0-2#,-1m .0-1'%3#m #,m *m $-0+!'A,m "#m 6,2-$'*1Ym 121m 1-,m *1m .0#!301-01m "#m *m
&-0+-,m"#m!0#!'+'#,2-m7m"#100-**-m"#m*m.*,2Zm#*m9!'"-m !>1'!-Ym-0m!-,1'%3'#,2#Zm*m
',2#003.!'A,m "#m *m #6.0#1'A,m "#m *m m 2+ '<,m .3#"#m .0-.-0!'-,0m $#,-2'.-1m #,,-1m
^',m7m!-*YZmHFFM_Ymm
,m0#13+#,Zm3,m+32,2#m2-2*m-m.0!'*m#,m#12#m%#,m.3#"#m.0-4-!0m$#,-2'.-1m$9!'*+#,2#m
"#2#!2 *#1m 1',m ,#!#1'""m "#m &!#0m 3,m !0!2#0'8!'A,m -m #,17-1m #6&312'4-1m m ,'4#*m
+#2 A*'!-m-m '-/3>+'!-Ymm4'13*'8!'A,m"#m#12-1m$#,-2'.-1m0#13*2m3,m!-,20-*m.0#!'1-m
7m09.'"-m.0m#6.#0'+#,2-1m!-+-m.-0m#(#+.*-m"#m20,1$-0+!'A,m-m"#m1'*#,!'+'#,2-m"#m
%#,#1m ^&#,%Zm HFGF_Ym ,m *%3,-1m #123"'-1m .0#4'-1m !-,m +32,2#1m "#m *m m 1#m & >,m
- 2#,'"-m .*,21m "#m ,#)*-#-m * ',1m 7m #,,1m ^',m 7m !-*YZm HFFM_m +#"',2#m #*m
1'*#,!'+'#,2-m -m *m +32!'A,m "'0'%'"m .-0m jYm #!'#,2#+#,2#Zm #,m 2-+2#m 1#m &m
1'*#,!'"-m *m m +#"',2#m m - 1#04,"-m 2+ '<,m 3,m "'1+',3!'A,m "#m *m
.'%+#,2!'A,m #,m &-(1m +'#,201m /3#m #*m 1'*#,!'+'#,2-m "#m m ,-m &m $#!2"-m m *m
.'%+#,2!'A,m#,m#*m$032-m^3"0,m7m!-*YZmHFGG_Ym
-0m#12#m+-2'4-m*mm0#13*2m3,m 3#,m!,"'"2-m.0m*m!-+.0- !'A,m"#m*m#$'!'#,!'m
"#m+32!'A,m"#m3,m.- *!'A,m"#mYm0!'1mm*m1#!3#,!'!'A,m"#*m%#,-+m"#*m
+#*A,m ^0!'j1m 7m !-*YZm HFGH_m 1#m &m "#2#0+',"-m /3#m #12m #1.#!'#m 1A*-m .0#1#,2m 3,m
!-.'m"#*m%#,m"#m*mZm"2-m0#*#4,2#m.0m#*m.0#1#,2#m#123"'-m"#mY
T
BMFMCT!TPTTTTTTEQT7T"#!T
1m .*,21m #129,m #,m !-,2!2-m !-,12,2#m !-,m -20-1m -0%,'1+-1m #6.-,'<,"-1#m m
',$#!!'-,#1[m 2/3#1m "#m ',1#!2-1Zm &-,%-1Zm !2#0'1m 7m 4'031Zm #,20#m -20-1m .2A%#,-1Ym m
',2#0!!'A,m #,20#m 3,m .*,2m 7m 3,m +'!0--0%,'1+-m .3#"#m 1#0m "#m 40'-1m 2'.-1m ^##,Zm
GOOF_Zm "#1"#m 0#*!'-,#1m .01'20'1m ^0##, #0%m 7m -Zm HFFJ_m &12m 1'+ 'A2'!1m ^A.#8j
9#8m 7m !-*YZm HFGF\m #0,9,"#8j.0'!'-Zm HFGF_Ym -+-m 0#13*2"-m "#m #12m !-,12,2#m
',2#0!!'A,Zm *1m .*,21m &,m '"-m "#100-**"-m +#!,'1+-1m "#m "#$#,1m !-,12'232'4-1m #m
',"3!' *#1m ^##,Zm GOOF_Ym -+-m #(#+.*-m "#m *m "#$#,1m !-,12'232'4Zm .-"#+-1m !'20m *1m
!0!2#0>12'!1m $>1'!-j/3>+'!1m "#m *m .0#"m !#*3*0m ^#,,7Zm GOOK_Ym ',m #+ 0%-Zm #6'12#m
-20-m +#!,'1+-m "#m "#$#,1m +91m !-+.*#(-m #m ',"3!' *#m 1"-m #,m *m 1>,2#1'1m "#m
!-+.3#12-1m /3>+'!-1m ^#,,7Zm GOOK_Ym 0m *m "#1!0'.!'A,m "#m #12#m 2'.-m "#m "#$#,1m 1#m &m
JNm
(.,)/#9(
Genes candidatos para el TILLING
13%#0'"-m *m #6'12#,!'m "#m 3,m +#!,'1+-m .-0m .02#m "#m *m .*,2m 1"-m #,m 0#!#.2-0#1m
!.!#1m"#m0#!-,-!#0m*m',$#!!'A,m7m!2'40m#*m1'12#+m"#m"#$#,1Ymm
Ym *-0m ^*-0Zm GOMG_m +#"',2#m #123"'-1m #,20#m *m ',2#0!!'A,m "#*m *',-m !3*2'4"-m ^#(/'O
/-#..#--#'/'Jm7m#*m&-,%-m&'*-),O&#(#m"#100-**Am*m2#-0>m"#*m%#,jj%#,m^'%30mGYN_Ym
,m#12m2#-0>m1#m#4'"#,!' m*m#6'12#,!'m"#m%#,#1m#,m*m.*,2m/3#m!-,$'#0#,m0#1'12#,!'m
m 3,m .2A%#,-m #1.#!>$'!-Ym !23*+#,2#Zm 1#m 1 #m /3#m *1m 0#1'12#,!'1m #,m .*,21m .3#"#,m
#120m%#,#0"1m.-0m3,m1A*-m%#,m^0#1'12#,!'m+-,-%<,'!_m-m.-0m#*m#$#!2-m"#m40'-1m%#,#1m
^0#1'12#,!'m .-*'%<,'!_Ym -12#0'-0+#,2#m 1#m &m .0-.3#12-m /3#m *-1m ',"3!2-0#1m "#m *m
',$#!!'A,m 1-,m %#,#1m **+"-1m 0,m ^$!2-0#1m "#m 4'03*#,!'_m .0#1#,2#1m #,m #*m .2A%#,-Zm
"9,"-*#m m *m &'.A2#1'1m "#m *-0m 3,m $3,"+#,2-m %#,<2'!-m ^0)#0m 7m -*#+,Zm GOOM_Ym -1m
%#,#1m0,m"#*m.2A%#,-m1#0>,m!.!#1m"#m',"3!'0m3,m0#1.3#12m"#m"#$#,1m#,m/3#**-1m
&3<1.#"#1m/3#m!-,2#,%,m*-1m%#,#1m"#m0#1'12#,!'m#1.#!>$'!-1m^_m7m/3#m0#!-,-!#,m*m%#,m
0,O^0)#0m7m-*#+,ZmGOOM_Ym'm#12m',2#0!!'A,m,-m1#m.3#"#m"0m.-0m*2#0!'A,m"#m3,-m
"#m*-1m"-1m%#,#1m^m-m0,_m#,2-,!#1m*m.*,2m0#13*2m',$#!2"Ym3,/3#m#*m+-"#*-m$3#m
"#100-**"-m .0m *m ',2#0!!'A,m &-,%-j.*,2Zm 2+ '<,m &m 1'"-m 49*'"-m .0m #*m
#,2#,"'+'#,2-m "#m -201m ',2#0!!'-,#1m $0#,2#m m -20-1m .2A%#,-1m !-+-m !2#0'1Zm 4'031Zm
,#+92-"-1m-m',1#!2-1m^##,ZmGOOF_m
m
m
m
m
m
TTTTTTTTTTT+ -2 TTTTT
2>&$-.T
R T
T
0RT
00T
#1'12#,2#m
31!#.2' *#m
31!#.2' *#m
31!#.2' *#m
m
(&30 T BMIOT #.0#1#,2!'A,m "#*m 1'12#+m "#m 0#1'12#,!'m #,m .*,21m 1"-m #,m *m ',2#0!!'A,m %#,jj%#,m ^*-0Zm
GOMG_Ym ,m *m 2 *m 13.#0'-0m 1#m 0#.0#1#,2m *m ',2#0!!'A,m #,m 1#m %#,<2'!Ym 0m 1'+.*'$'!0Zm 13.-,#+-1m /3#m *m
JOm
(.,)/#9(
Genes candidatos para el TILLINGO
.*,2m!-,2'#,#m#*m%#,m"#m0#1'12#,!'mm+'#,201m/3#Zm.-0m#*m!-,200'-Zm#*m%#,-2'.-m,m1'%,'$'!m/3#m*m.*,2m,-m
2'#,#m*m0#1'12#,!'Ym#m'%3*m+-"-m#*m.2A%#,-m#1m.-02"-0m"#m*m4'03*#,!'m^_m+'#,201m/3#m#*m%#,-2'.-mm,-m
#1m!.8m"#m',2#0!!'-,0m!-,mYm
m
!23*+#,2#m1#m32'*'8m#*m2<0+',-m4'03*#,2-m.0m/3#**-1m.2A%#,-1m/3#m1-,m!.!#1m"#m
',$#!20m*m&3<1.#"m+'#,201m/3#m*-1m/3#m,-m2'#,#,m<6'2-m1#m"#,-+',,m4'03*#,2-1Ym-,m
#121m 1#1m 1#m "#1!0' 'Am 3,m +-"#*-m "-,"#m 1#m .-">,m "0m ',$#!!'-,#1m !-+.2' *#1m #m
',!-+.2' *#1m ^,m "#*m *,)m 7m !-*YZm GOLK_Ym ,m ',$#!!'A,m !-+.2' *#m !-,1'12#m #,m *m
',2#0!!'A,m #,20#m 3,m .2A%#,-m 4'03*#,2-m 7m 3,m &3<1.#"m 131!#.2' *#Ym ,m ',$#!!'A,m
',!-+.2' *#m1#m"m#,20#m3,m.2A%#,-m4'03*#,2-m7m3,m&3<1.#"m0#1'12#,2#Zm"#m+-"-m/3#m
1'm#*m&3<1.#"m!-,2'#,#m#*m%#,m"#m0#1'12#,!'m!.8m"#m0#!-,-!#0m#*m$!2-0m"#m4'03*#,!'m
"#*m.2A%#,-Zm"#1#,!"#,m3,m.0-!#1-m"#m1#@*'8!'A,m.0m*m!2'4!'A,m"#m*m0#1.3#12m
"#m"#$#,1Ymm#12m0#1.3#12m1#m*m"#,-+',m0#1.3#12m"#m&'.#01#,1' '*'""m^_m^#,2Zm
GOOL_Ym',m#+ 0%-Zm1'm*m.*,2m,-m!-,2'#,#m#*m!-+.*#(-mm!.8m"#m0#!-,-!#0m#*m$!2-0m
0,O#,2-,!#1m #*m .2A%#,-m ',$#!2m *m .*,2m ^02',m 7m !-*YZm HFFI_Zm 7m .-"#+-1m !-,1'"#00m
#1#m$!2-0m"#m4'03*#,!'m!-+-m3,m$!2-0m4'03*#,2-Ymm
-0m *-m 2,2-m *-1m %#,#1m m "#1#+.#@,m 3,m ..#*m $3,"+#,2*m #,m #*m 0#!-,-!'+'#,2-m "#*m
%#,m 0,m 7m #,m !-,1#!3#,!'m #,m *m 0#1'12#,!'m "#m *m .*,2m $0#,2#m m .2A%#,-1Ym m 09.'"m
#4-*3!'A,m"#m*1m.*,21m#,m#12#m1#,2'"-m&m**#4"-mm3,m%0,m"'4#01'$'!!'A,m"#m%#,#1mm
^&'1&-*+m 7m !-*YZm HFFL_Ym 12-1m 1#m &,m .-"'"-m !*1'$'!0m #,m $3,!'A,m "#m *-1m "-+','-1m
#1203!230*#1m/3#m!-,2'#,#,m^#,2ZmGOOL\m,m-'(#,ZmHFFM_Ym#m&,m"#1!0'2-m!',!-m%03.-1[m
*m!*1#mm/3#m!-+.0#,"#m*-1m%#,#1m"#m0#1'12#,!'m/3#m!-"'$'!,m.0m.0-2#>,1m!-,m*m
+#,-1m 3,m "-+','-m #,m &<*'!#m 13.#0m #,0-**"Zm 3,m 1'2'-m "#m 3,'A,m m ,3!*#A2'"-1m 7m 3,m
"-+','-m 0'!-m #,m 0#.#2'!'A,m "#m #3!',m ^#3_m ^jj_Ym m 1#%3,"m !*1#m m
!-,2'#,#,m*m+#,-1m3,m"-+','-m"#m0#!#.2-0m2'.-m-**m7m*-1m-20-1m"-1m"-+','-1m'%3*#1mm
*m !*1#m m ^jj_Ym m !*1#m m ^,*.),N&#%O *,).#(_m 2'#,#,m 3,m 0#!#.2-0m "#m
/3',1m 1#0',j20#-,',m 7m 3,m "-+','-m #620!#*3*0m !-+.3#12-m .-0m 3,m 0#%'A,m 0'!m #,m
0#.#2'!'-,#1m"#m*#3!',m^#0i&0j_Ymm!*1#mm1-,m/3#**-1m%#,#1m!-,m3,m"-+','-m
/3',1m7m3,m0#%'A,m#620!#*3*0m0'!m#,m0#.#2'!'-,#1m"#m*#3!',m^j',_Ym',*+#,2#Zm
#*m B*2'+-m %03.-m ',!*37#m 2-"-1m /3#**-1m %#,#1m "#1!0'2-1m #,m /3#m 13m $3,!'-,+'#,2-m #1m m
204<1m "#m "'12',2-1m +#!,'1+-1m +-*#!3*0#1m !-+-m .-0m #(#+.*-m '&)m 7m -N;m ^31!&%#1Zm
GOOM\m 0,"5%2Zm HFFH_Ym -1m %#,#1m m &,m 1'"-m '1*"-1m #,m 40'1m #1.#!'#1m "#m .*,21Zm
+3!&-1m "#m #**-1m #,m *m $+'*'m "#m *1m 1-*,9!#1Zm "-,"#m 1#m &,m '1*"-m IIm %#,#1m "#m
0#1'12#,!'m^,m-'(#,ZmHFFM\m-+#0m7m!-*YZmHFFM_Ym',m#+ 0%-Zm2+ '<,m1#m&,m!*-,"-m
+3!&-1m-20-1m#,m-201m#1.#!'#1m!-+-m#,m,#)*-#-O."&#(m^HGmj%#,#1_m^#7#01m7m!-*YZm
KFm
Genes candidatos para el TILLING
(.,)/#9(
GOOO_Zm,34O-.#0m^00-8ZmJmj%#,#1_m^-,%m7m!-*YZmGOOK_Zm//'#-O'&)m^Gmj%#,_m^
-- #30m
7m!-*YZmHFFJ_m7m./O-.#0m^*#!&3%ZmGmj%#,_m^&#,m7m!-*YZmHFFH_Ym
-1m 4'031m 1-,m !.!#1m "#m "#100-**0m +#!,'1+-1m .0m 1#%300m 13m 13.#04'4#,!'m 7m
2'#,#,m *m !.!'""m "#m +320m 7m !-,1#%3'0m #4"'01#m "#m *-1m 1'12#+1m ',+3,'20'-1Ym
!-123+ 0,mm!-,12'23'01#mm.02'0m"#m20#1m-m!320-m.0-2#>,1Ym12m*'+'2!'A,m*#1m- *'%Zm
#,m+3!&1m-!1'-,#1Zmm32'*'80m*-1m0#!301-1m"#*m.0-.'-m&3<1.#"m.0m.-"#0m1- 0#4'4'0m7m
&!#0m#$#!2'4m*m',$#!!'A,Ym01#0m^01#0ZmGONL_m.0-.31-m/3#m*m.<0"'"m"#m!-+.-,#,2#1m
"#*m&3<1.#"m,#!#10'-1m.0m#*m!'!*-m"#m*m4'"m"#m*-1m4'031m.-">m1#0m2+ '<,m3,m2'.-m"#m
0#1'12#,!'Ym m #12m 0#1'12#,!'m 1#m *#m **+m 0#1'12#,!'m 0#!#1'4Ym 12m &'.A2#1'1m &m 1'"-m
!-,$'0+"Zm 1- 0#2-"-Zm !-,m .0-2#>,1m 0#*!'-,"1m !-,m *m +/3',0'm "#m 20"3!!'A,m
!#*3*0m^- %*'m7m0,2ZmHFFL_Ymm
*%3,-1m 1'12#+1m "#m 0#1'12#,!'m 0#!#1'4m #,m .*,21m &,m 1'"-m "#1!3 '#02-1m
0#!'#,2#+#,2#Ym,m#(#+.*-m1-,m*-1m$!2-0#1m"#m','!'!'A,m"#m*m20"3!!'A,m7m#,m.02'!3*0m
*m$+'*'m"#m.0-2#>,1m>m7m>O^'%30mGYO_m^'#2-m7m!-*YZmHFFLZm- %*'m7m0,2Zm
HFFLZm03,'%#0ZmHFFN_Ym
*m .0-!#1-m "#m 20"3!!'A,m "#m +m m .0-2#>,m !-,12m 91'!+#,2#m "#m 20#1m $1#1[m
','!'!'A,Zm#*-,%!'A,m7m2#0+',!'A,m^* #021m7m!-*YZmHFFL_Ymm$1#m"#m','!'!'A,m!-,1'12#m
#,m *m ',2#0!!'A,m #,20#m *1m "-1m 13 3,'""#1m "#*m 0' -1-+m 7m #*m !-"A,m "#m ','!'-m "#*m
+Ym30,2#m*m#*-,%!'A,m1#m"m*m1>,2#1'1m"#*m.<.2'"-m#,m$3,!'A,m"#m*m*#!230m"#m
*-1m !-"-,#1m "#*m +#,1(#0-m "#,20-m "#*m 0' -1-+Ym ',*+#,2#Zm *m 2#0+',!'A,m '+.*'!m *m
',2#0!!'A,m "#*m 0' -1-+m !-,m #*m !-"A,m -.)*m 7m *m "'1-!'!'A,m !-+.*#2m "#*m +m 7m #*m
!-+.*#(-m0' -1-+*Ymm
KGm
(.,)/#9(
m
Genes candidatos para el TILLINGO
m
(&30 TBMJ[m1/3#+m"#*m$3,!'-,+'#,2-m"#*m','!'-m"#m*m20"3!!'A,m"A,"#m*-1m$!2-0#1m"#m','!'!'A,m(3#%,m3,m
..#*m'+.-02,2#m#,m*m#12 '*'8!'A,m"#*m+m^$3#,2#[m#**'1m7m!-*YZmHFFH_Ym
m
,m*m$1#m"#m','!'!'A,m"#m*m20"3!!'A,m!-,20' 37#,m3,m1#0'#m"#m$!2-0#1m#3!0'-21m"#m
','!'!'A,m"#m*m20"3!!'A,m^_Ym,'!'*+#,2#m*m13 3,'""m0' -1A+'!mJF1m1#m!#0!m*mKam
"#*m +Ym #1.3<1Zm 3,m +-4'+'#,2-m *2#0*m -m -((#(!m !-,"3!#m *m 0#!-,-!'+'#,2-m "#*m
.0'+#0m !-"A,m m ^#*m !-"A,m ','!'"-0m "#m *m 20"3!!'A,_Ym -12#0'-0+#,2#m 1#m "m *m
$-0+!'A,m"#m*m13 3,'""m0' -1A+'!mLFm7m*m$1#m"#m#*-,%!'A,Ym*m$!2-0m>O3,#m*m
.02#m Kam !-,m *m .02#m Iam "#*m +Ym *m !-+.*#(-m >m #129m !-+.3#12-m .-0m "-1m
13 3,'""#1[m >m 7m "-1m .0-2#>,1m +91[O >m 7m >Ym m .0-2#>,m #Jm 1#m 3,#m *m
#620#+-mKam"#*m+mm204<1m"#m3,m.0-2#>,m"#m3,'A,mm^)&3OO#(#(!O,).#(_m
^'%30mGYO_Ym-0m-20-m*"-Zm>m#1m3,m!-+.*#(-m"#m.0-2#>,1m/3#m2'#,#m*m!.!'""m"#m
',2#0!!'-,0m !-,m -201m 0#%'-,#1m "#*m 1'12#+m "#m ','!'!'A,m "#m *m 20"3!!'A,m !-+-m .-0m
#(#+.*-m#*m#620#+-m.-*'mm^m204<1m"#m*m.0-2#>,m_m-m!-,m#*m$!2-0m>O^&#*'!1m
/3#m"#1#,0#"m#1203!2301m1#!3,"0'1_m7m2+ '<,m!-,m#*m$!2-0m=O^!-+.*#(-m"#m40'1m
13 3,'""#1m/3#m',2#0!!'-,m!-,m*m13 3,'""m0' -1A+'!mJF_m^#**'1m7m!-*YZmHFFH_Ym
KHm
Genes candidatos para el TILLING
(.,)/#9(
1m$3,!'-,#1m"#m#12-1m"-1m$!2-0#1m^>O7m>_m1-,m 91'!+#,2#m',2#0!!'-,0m!-,m#*m
+m 7m "'0'%'0*-m "#m +-"-m /3#m #*m 0#12-m "#m .0-2#>,1m .3#",m !230m 1- 0#m <*Ym 1>m .-0m
#(#+.*-m>m3,'"-mmm1#m#,!0%m"#m',2#0!!'-,0m!-,m#*m#620#+-mIam.0m.#0+'2'0m
*m 0#!'0!3*0'8!'A,m 1- 0#m 1'm +'1+-m "#*m +m +'#,201m /3#m *m .0-2#>,m >m
',2#0!!'-,m!-,m#*m#620#+-mKam^- %*'m7m0,2ZmHFFL_Ym
91m"#m*m+'2"m"#m*-1m%#,#1m,230*#1m"#m0#1'12#,!'mm.-2'4'031m#,m.*,21m1#m 1,m#,m
3,m +#!,'1+-m "#m 0#1'12#,!'m 0#!#1'4m +'#,201m /3#m #,m -20-1m 2'.-1m "#m 4'031m 1A*-m 1#m
#,!3#,20m #12#m 2'.-m "#m 0#1'12#,!'m #,m 3,m HFPm ^0-44'"#,2'm 7m +.2-,Zm GOOH_Ym -1m
.-2'4'031m 1-,m 4'031m "#m !"#,m 1'+.*#m .-1'2'4m "#m +Ym 12,m !-+.3#12-1m .-0m 3,m
.0-2#>,m 4'0*m ^%_m #,m #*m #620#+-m KaZm 3,m !"#,m "#m .-*'"#,',1m #,m #*m #620#+-m Iam 7m
-201m.0-2#>,1m4'0*#1m/3#m"#1.3<1m"#m*m',$#!!'A,m.1,mm1#0m$3,!'-,*#1m+#"',2#m*1m
.0-2#11m4'0*#1Ymm.0-2#>,m%m#1m*m!*4#m.0m*m',2#0!!'A,m!-,m#*m$!2-0m>Zm-m13m
'1-$-0+mI#-)J>Zm2*m7m!-+-m1#m&m"#+-120"-m#,m#6.#0'+#,2-1m"#m"- *#m&> 0'"-m#,m
*#4"301m ^'22+,,Zm 7m !-*YZm GOOM_Ym "#+91m -20-1m #123"'-1m +91m .0#!'1-1m "#$',#,m /3#m
+32!'-,#1m #,m *m .0-2#>,m %m '+.'"#,m *m ',2#0!!'A,m !-,m #*m $!2-0m I#-)J>m 7Zm .-0m *-m
2,2-Zm *m ',!.!'""m "#m ',$#!20m .-0m .02#m "#*m 4'031m ^<-,0"m 7m !-*YZm HFFF_Ym #m &m
"#+-120"-m 2+ '<,Zm /3#m +32,2#1m #,m #*m %#,m I#-)J>m .0-"3!'"-1m +#"',2#m m #,m
,#)*-#-m.'#0"#,m*m131!#.2' '*'""mmm^))O."O0#,/-_m^#**'1m7m!-*YZmHFFH_Ym
#m&m.-"'"-m'1*0m*m%#,m>m!-+-m0#1.-,1 *#m"#m*%3,1m0#1'12#,!'1m,230*#1m#,m
"'12',21m #1.#!'#1Ym *%3,-1m #(#+.*-1m 1-,m #*m %#,m ');m #,m *#!&3%m /3#m "m 0#1'12#,!'m *m
4'031m m ^../O ')-#O 0#,/-_m ^'!'1#m 7m !-*Zm HFFI_Zm #*m %#,m -';m #,m %3'1,2#m /3#m "m
0#1'12#,!'m m m ^O -N),(O ')-#O 0#,/-_m ^-m 7m !-*m Zm HFFJ_Zm #*m %#,m *).N;m #,m
2-+2#m /3#m "m 0#1'12#,!'m m m ^)..)O 0#,/-O _m ^3$$#*m 7m !-*Zm HFFK_m 7m $',*+#,2#m
2+ '<,m#,m+#*A,m#*m%#,m(-0m/3#m"m0#1'12#,!'m*m,')0#,/-mm^&)(O(,).#O-*).O
0#,/-_m^'#2-m7m!-*YZmHFFL_Ym-"-1m#**-1m1-,m&-+A*-%-1m"#m>m7m!-,2'#,#,m+32!'-,#1m
#1.#!>$'!1m/3#m"#2#0+',,m*m0#1'12#,!'Ym-"1m*1m+32!'-,#1m"#1!0'21m#,m#12-1m%#,#1m
#129,m *-!*'8"1m #,m 8-,1m ,-m !-,1#04"1m "#*m %#,Ym 0!'1m m *m .0#"'!!'A,m "#m *m
#1203!230m Im 1#m &m .-"'"-m "#1!0' '0m *m *-!*'8!'A,m "#m *1m +32!'-,#1m !#0!m "#m *m
.0-2#>,m"#m3,'A,m%.m"#*m+m-m '#,m#,m8-,1m#62#0,1m"#m#12m^- %*'m7m0,2Zm
HFFL_Ym '#2-m 7m !-* -0"-0#1m #,m #*m HFFLm "#100-**0-,m 3,m 20 (-m #,m +#*A,m "-,"#m
"#+-1200-,m +#"',2#m !*-,(#m .-1'!'-,*m 7m +'!0-1',2#,'m !-,m ,#)*-#-m /3#m #*m *-!31m
"#*m%#,m(-0m/3#m" m0#1'12#,!'m*m4'031mm!-00#1.-,">m*m%#,m/3#m!-"'$'! m.-0m
#*m $!2-0m "#m ','!'!'A,m "#m *m 20"3!!'A,m ^>_Ym -+.*#+#,20-,m #*m 20 (-m !-,m
#6.#0'+#,2-1m"#m#6.0#1'A,m!-,$'0+,"-m/3#m#*m%#,m(-0m#0m>m7m"#+91m1#m'"#,2'$'!Am
*m +32!'A,m 0#1.-,1 *#m "#m *m 0#1'12#,!'m m m /3#m .0-4-!4m 3,m !+ '-m #,m *m
.-1'!'A,mHHNm^'1j#3_m"#m*m1#!3#,!'m.0-2#'!Ym20-m#123"'-m"#m!-Zm^'#2-m7m
KIm
(.,)/#9(
Genes candidatos para el TILLINGO
!-*YZmHFFM_m!-,$'0+Am/3#m*m+32!'A,m"#1!0'2m#1m*m/3#m!-,$#0>m0#1'12#,!'mmm#,m
40'1m "#m *1m 40'#""#1m ,*'8"1Ym -12#0'-0+#,2#Zm #12m ',$-0+!'A,m $3#m +.*'"m
',4#12'%,"-m #*m +#!,'1+-m .-0m #*m !3*m #*m $!2-0m >m 0#%3* m *m 0#1'12#,!'m m Ym
-0m 3,m*"-m1#m+.Am*m0#%'A,m#,m#*mIam/3#m"#2#0+', m*m^_4'03*#,!'m+#"',2#m
4'031m /3'+<0'!-1m ^4'03*#,2-1m 7m 4'03*#,2-1_m 7m .-0m -20m .02#Zm 1#m '"#,2'$'!Am #*m $0%+#,2-m
**+"-mIajm^*O(*((.O,(-&.#)(&O("(,_m#,m#*mIam!-+.0- ,"-m/3#m
#0m "#!'1'4-m .0m *m 4'03*#,!'m "#*m 4'031Ym *m 4'031m ,-m .-">m ',$#!20m .-0/3#m ,-m 1#m " m
3,m!-00#!2m',2#0!!'A,m#,20#m#12m.02#m"#*mm4'0*m7m#*m!-+.*#(-m"#m','!'!'A,m"#m*m
20"3!!'A,m7m.-0m*-m2,2-Zm*m.*,2m0#13*2 m0#1'12#,2#m^03,'%#0m7m!-*YZmHFFN_Ymm
,m ,#)*-#-O 1#m !-,$'+Am /3#m I#-)J>m 7m >m 2'#,#,m 3,m JLPm "#m 1#+#(,8m #,m *m
1#!3#,!'m+',-!>"'!m^-"0>%3#8m7m!-*YZmGOON_Ym3,/3#m1#m&m"#+-120"-m/3#m+ 1m
.0-2#>,1m1#m#,!3#,20,m#,m*m+'1+m!-,!#,20!'A,m^-"0>%3#8m7m!-*YZmGOON_m,-m1#m1 #m
#6!2+#,2#m !A+-m $3,!'-,m #*m +#!,'1+-m +#"',2#m #*m !3*m 1#m "#!'"#m !39*m "#m *1m "-1m
1#09m *m 0#1.-,1 *#m "#m ',2#0!!'-,0m !-,m *m .0-2#>,m %m "#*m m 4'0*Ym ,m 1'"-m
.0-.3#121m 20#1m .-1' *#1m $3,!'-,#1m .0m I#-)J>m ^'%30m GYGF_m %0!'1m *m #123"'-m #,m
+32,2#1m!-,m.<0"'"m"#m131!#.2' '*'""m#,m,#)*-#-O^#**'1m7m!-*YZmHFFH_Ymm
#1.#!2-m *m %#,O >m "# #m !-+#,201#m /3#m 7m 1#m &,m #,!-,20"-m +32,2#1m +#"',2#m
m#,m2-+2#m7m!# "m^*+<m7m!-*YZmHFFN_Ym,m2-+2#m1#m'"#,2'$'!Am3,m+32,2#m
/3#m.#0">m*m!.!'""m"#m-*&##(!m#,20#m#*m1#%3,"-m7m2#0!#0m#6A,m7m*m.0-2#>,m,-m.-">m
#6.0#101#m!-00#!2+#,2#Ym#m!0!2#0'8Am*m',+3,'""m"#m#12#m+32,2#m#6.-,'<,"-*-mm
"'12',2-1m4'031m"#+-1209,"-1#m/3#m#0m0#1'12#,2#mm"-1m"#m#**-1[m*m4'031mm"#m*m.22m
^)..)O #,/-O Zm _m 7m *m 4'031m "#*m +-2#"-m "#*m .'+'#,2-m ^**,O ')..&O 0#,/-Zm #_m
^'0-,m7m!-*YZmHFGF_Ymm
0!'1mm*m0#!'#,2#m.3 *'!!'A,m"#*m%#,-+m"#*m+#*A,m^0!'j1m7m!-*YZmHFGH_m1#m&m
.-"'"-m !-+.0- 0m /3#m #6'12#m 1A*-m 3,m !-.'m "#*m %#,m >m 1>m !-+-m "#m 13m '1-$-0+m
I#-)J>Ym
O
KJm
Genes candidatos para el TILLING
(.,)/#9(
m
m
(&30 TBMBA[m3,!'-,#1m.-123*"1m.0m#*mI#-)J>YmQT,'!'"-0m"#m*m20"3!!'A,mm.-0m13m$3,!'A,m"#m3,'A,mm
*m.0-2#>,m%m"#*m+m4'0*YmQm12 '*'8"-0m"#*m%#,-+Ymm3,'A,m#,20#mI#-)J>jm%m.3#"#m.0-2#%#0m
*m +m 4'0*m "#m *m !!'A,m "#m .0-2#>,1m b"#!."-01cm !-+-m *m j*')#m -m #6-,3!*#11m /3#m "#%0",m #*m
m !-+-m *m j*')#Ym Qm 09$'!-m ',20!#*3*0Ym I#-)J>Zm ',2#0!!'-,m !-,m I#-)J>m /3#m **#4m "&#0'"-1m
+'!0-2B 3*-1m!-,m!2'4'""m"#m"&#1'A,Ym#m#12#m+-"-mI#-)J>m3,#m*1m.0-2#>,1m4'0*#1m!-,m-201m.0-2#>,1m
!-,m!2'4'""m20,1.-02"-0m^$3#,2#[m#**'1m7m!-*YZmHFFH_Ym
T
BMFMDTTTTBTT
*m #2'*#,-m ^HJ_m #1m 3,m &-0+-,m /3#m #,20#m -201m +3!&1m $3,!'-,#1m #,m .*,21m !2Bm
!-+-m!2'4"-0m"#m*m+"30!'A,m"#m*-1m$032-1m!*'+2<0'!-1Ymm.0-"3!!'A,m"#m#2'*#,-m
!-+.-02m 3,m !+ '-m #,m *m !-,1'12#,!'m "#*m $032-m ^03++#**m 7m 0.12#0Zm HFFG_Zm #,m #*m
!-*-0m "#m *m .'#*m ^*-0#1m 7m !-*YZm HFFH_m 7m #,m *m .0-"3!!'A,m "#m 0-+1Zm #,20#m -20-1m #$#!2-1m
^#7+-30m7m!*11-,ZmGOOI_Ym 1>,2#1'1m"#m#2'*#,-m4'#,#m0#%3*"m.-0m3,m+#!,'1+-m
"#m 0#20-*'+#,2!'A,m !-,12'23'"-m 91'!+#,2#m .-0m #*m !'!*-m "#m ,%m ^'%30m GYGG_m
(3,2+#,2#m!-,m*m.0-"3!!'A,m"#m#2'*#,-mm.02'0m"#mm^j"#,-17*+#2&'-,',#_m7m"#m
*m"'$31'A,m"#*m#2'*#,-mm204<1m"#*m$032-m^#,0"m7m-3 *#ZmHFFK_Ym#0-m*m0#%3*!'A,m"#*m
#2'*#,-m .3#"#m 1#0m 2,2-m m ,'4#*m '-1',2<2'!-m !-+-m m ,'4#*m 0#!#.2'4-Ym #m !-,-!#m "#m *m
!!'A,m *'+'2,2#m 1- 0#m *m .0-"3!!'A,m "#m #2'*#,-m "#m *%3,1m #,8'+1m !-+-m *m m
-6'"1m 7m *m m 1',21m ^'%30m GYGG_Ym #0-m *m 0#%3*!'A,m m ,'4#*m "#m 0#!#.!'A,m #1m +91m
KKm
(.,)/#9(
Genes candidatos para el TILLINGO
"#1!-,-!'"Ym*m&#!&-m"#m/3#m#*m#2'*#,-m1A*-m!2B#m#,m2#('"-1m#1.#!>$'!-1m7m/3#m1#m3,m
+-*<!3*m%1#-1m,-1m',"'!m/3#m13m0#!#.!'A,m#1m3,m.0-!#1-m!-+.*#(-Ym
m
m
m
(&30 TBMBB[m1/3#+m"#*m!'!*-m"#m,%m7m*m4>m"#mjmHJm&12m*m$-0+!'A,m"#m#2'*#,-Ym0'-1m$!2-0#1m
#62#0,-1m0#%3*,m#,m.02#m*m1>,2#1'1m"#m#2'*#,-m7m1#mm,'4#*m.-1'2'4-m!-+-mm,'4#*m,#%2'4-m^$3#,2#[m- #0A,m7m
!-*YZmHFFK_m
m
m 20'.*#m 0#1.3#12m *m #2'*#,-m 1#m
1m #,m 20#1m !+ '-1m +-0$-*A%'!-1m "30,2#m *m
%#0+',!'A,m #,m !-,"'!'-,#1m "#m -1!30'""m 7m .0#1#,!'m "#m #2'*#,-[m &'.-!-2'*-1m 7m 0>!#1m
+91m !-021m 7m $',1m 7m 3,m !0#!'+'#,2-m !0!2#0>12'!-m #,m $-0+m "#m %,!&-m ^*##!)#0Zm GOOO\m
38+,m 7m !)#0Zm GOOF_Ym ,m 1'"-m '"#,2'$'!"-1m 40'-1m +32,2#1m #,m ,#)*-#-O /3#m ,-m
+3#120,m*m20'.*#m0#1.3#12m*m#2'*#,-m7m/3#m.-0m*-m2,2-Zm.0#1#,2,m',1#,1' '*'""mm#12m
&-0+-,Ym*m'1*+'#,2-m"#m*-1m%#,#1m0#1.-,1 *#1m"#m#12-1m$#,-2'.-1m&,m1'"-m31"-1m
.0m#,2#,"#0m#*m+#!,'1+-m"#m0#!#.!'A,m"#*m#2'*#,-m1>m!-+-m13m1#@*m"#m20,1"3!!'A,m
^&,%m 7m !-*YZm GOOI\m #&+,m 7m !-*Y\m GOOL_Ym ,-m "#m #12-1m %#,#1Zm /3#m !-"'$'!m .0m 3,m
0#!#.2-0m"#*m#2'*#,-Zm#1m#*m;Ymm
KLm
Genes candidatos para el TILLING
(.,)/#9(
*m ;m $-0+m .02#m "#m 3,m $+'*'m "#m %#,#1m /3#m #,m ,#)*-#-m #129m !-+.3#12m .-0m
!',!-m 0#!#.2-0#1[m ;FO ;FO >FO <Zm 7m <m ^3m 7m !-*YZGOOK_Ym -"-1m #**-1m 1-,m
0#!#.2-0#1m "#*m #2'*#,-m 7Zm m *m 4#8Zm 0#%3*"-0#1m ,#%2'4-1m "#m <12#Ym ,m ,#)*-#-m 1#m &m
"#+-120"-m /3#m +32,2#1m "#$'!'#,2#1m #,m #121m .0-2#>,1m 1-,m ',1#,1' *#1m *m #2'*#,-m
'*)',1-,m 7m !-*YZm GOOK_Ym 1203!230*+#,2#m 2-"-1m 1-,m +37m 1'+'*0#1m 1'#,"-m .0-2#>,1m
20,1+#+ 0,*#1m 3,/3#m #,m #*m !1-m "#m ;FO ;m 7m >m !-,2'#,#,m "#+91m 3,m
"-+','-m 0#!#.2-0m j2#0+',*m m !-,2',3!'A,m "#*m "-+','-m '12'"',m /3',1m ^&#,m 7m
**'#ZmHFGF_Ym
m .0-2#>,m ;m !-,2'#,#m 3,m 0#%'A,m "#m *2m $','""m 7m "#m 3,'A,m *m #2'*#,-m ^'%30m
GYGH_Ym,m,#)*-#-m#1m3,m">+#0-m"#mHm.0-2#>,1m',2#%0*#1m"#m+#+ 0,m!-,m!2'4'""m
&'12'"',m)',1m7m!.!'""m32-$-1$-0'*,2#Ymm3,'A,m"#*m#2'*#,-mm13m0#!#.2-0m',"3!#m
13m 32-$-1$-0'*!'A,m m ,'4#*m "#m 0#1'"3-1m "#m &'12'"',m 7m *3#%-m *m 20,1$#0#,!'m "#m #12-1m
$-1$2-1m &!'m 0#1'"3-1m "#m 1.022-Ym *m 0#!#.2-0m 1>m !2'4"-m ','!'m 3,m !1!"m "#m
1#@*'8!'-,#1m &!'m -201m .0-2#>,1m #$#!2-01m ^!1!"m "#*m 2'.-m m /3',1m !-,m
"#12',-m$',*mm,'4#*m"#*m_m^- #0A,m7m!-*YZmHFFK_m^'%30mGYGH_Ymm
m
m
m
(&30 TBMBC[m 1/3#+m "#*m ;m 7m "#*m +#!,'1+-m "#m 0#!#.!'A,m "#*m #2'*#,-m #,m ,#)*-#-Ym ^$3#,2#[m - #0A,m 7m
!-*YZmHFFK_Ym
m
6'12#,m +32!'-,#1m "#1!0'21m #,m ,#)*-#-m #,m #*m #620#+-m +',-m &'"0-$A '!-m /3#m
0#"3!#,m -m ,3*,m *m 3,'A,m *m #2'*#,-m ^!&**#0m 7m *##!)#0Zm GOOK_Ym 19,"-1#m #,m #121m
.03# 1m40'-1m32-0#1m&,m!-,!*3'"-m/3#m#*m%#,m;m2'#,#m3,m!-+#2'"-m'+.-02,2#m#,m
*m 0#!#.!'A,m "#*m #2'*#,-m ^#,m 7m !-*YZm GOOKZm &-,%m 7m !-*YZm HFFNZm 12%,m 7m !-*YZm HFFM_Ym
1'+'1+-Zm #,m ,#)*-#-m #129m "#1!0'2m *m +32!'A,m !-00#1.-,"'#,2#m *m +',-9!'"-m
71JK70m "#m *m 0#%'A,m j2#0+',*m /3#m .0-4-!m 3,m $#,-2'.-m !-,m 3,m ',2#,1m
KMm
(.,)/#9(
Genes candidatos para el TILLINGO
',1#,1' '*'""m *m #2'*#,-m ^*##!)#0Zm GOOO_Ym -0m -20-m *"-Zm -20-1m #123"'-1m ^'!)m 7m 3*2#0Zm
GOKL_m "#+-1200-,m /3#m #*m +32,2#m ,m ^0,O ,#*m _m "#m 2-+2#m 2#,>m $032-1m /3#m ,-m
+"30 ,Ym&-0m1#m1 #m/3#m#12#m+32,2#m,m#1m',1#,1' *#m*m#2'*#,-m^,&,m7m!-*YZm
GOOJ_m 7m /3#m #*m *-!31m /3#m .0-4-!m #*m $#,-2'.-m ,m 2'#,#m &-+-*-%>m !-,m #*m %#,m ;m "#m
,#)*-#-Em m +32!'A,m 0#1.-,1 *#m #,m 2-+2#m #1m 3,m !+ '-m "#m 1#m /3#m .0-4-!m 3,m
!+ '-m+',-!>"'!-m"#m.0-*',mm*#3!',Ym12-1m,2#!#"#,2#1mm',"'!,m/3#m;m#1m3,m
3#,m !,"'"2-m .0m *'+'20m *m 0#!#.!'A,m "#m #2'*#,-m 7m #,m !-,1#!3#,!'Zm 3,m %#,m "',m
',2#0#1,2#m.0m1#0m,*'8"-m"#,20-m"#m3,m.- *!'A,m"#mZm#1.#!'*+#,2#m#,m
+#*A,Zm 3,m #1.#!'#m +-"#*-m .0m #123"'0m #*m .0-!#1-m "#m +"30!'A,m .3#12-m /3#m 1#m
#,!3#,20,m 40'#""#1m !*'+2<0'!1m 7m ,-m !*'+2<0'!1Ym -0m #(#+.*-Zm *1m 40'#""#1m "#*m
2'.-m (.&/*(-#-m .0#1#,2,m 3,m .'!-m #,m *m .0-"3!!'A,m "#m #2'*#,-m /3#m !-',!'"#m !-,m *m
+"30#8m "#*m $032-m 7m !-,m 13m 1!'1'A,m ^&#**'#m 7m *4#'2Zm GOOI_Ym ',m #+ 0%-Zm *1m
40'#""#1m"#*m2'.-m()),/-m,-m.0#1#,2,m3,m.'!-m"#m#2'*#,-m7m,-m1#m.0-"3!#m 1!'1'A,m
"#*m$032-Zm","-m$032-1m"#m*0%m4'"m^#,"**ZmGONN_Ym,m3+#,2-m"#m*m!-,!#,20!'A,m
"#m#2'*#,-m#,m$032-m"#m+#*A,m",(.#-m.0-4-!m3,m"'1+',3!'A,m"#m*m"30#8m#,m*m.'#*m
^-1#m7m"$'#*"ZmGOON_Ym,m-20-m#123"'-m1#m&m"#+-120"-m/3#m3,m202+'#,2-m"#*m$032-m
"#m +#*A,m !-,m Gj+#2'*!'!*-.0-.#,-m ^Gj_Zm 3,m ',&' '"-0m "#m *m !!'A,m "#m #2'*#,-Zm
.0-4-!m 3,m 0#"3!!'A,m "#*m *,"+'#,2-m "#m *m .'#*m "#+-120,"-m *m '+.*'!!'A,m "#*m
#2'*#,-m #,m *-1m !+ '-1m "#m *m .0#"m !#*3*0m "30,2#m #*m .0-!#1-m "#m +"30!'A,m #,m
40'#""#1m !*'+2<0'!1m ^'1&'7+m 7m !-*YZm HFFM_Ym *m +0'**#-m 7m #*m !-,2#,'"-m "#m
!0-2#,-'"#1m#,m*m.'#*Zm*m$-0+!'A,m"#m*m8-,m"#m 1!'1'A,m.#"3,!3*0m7m*m0#1.'0!'A,m
!*'+2<0'!m #,m #*m +#*A,m (.&)/*#m ",(.#-O 1-,m .0-!#1-1m "#.#,"'#,2#1m "#m #2'*#,-m
^*-0#1Zm HFFG\m 3@#8j*#,'31m 7m !-*YZm HFFLZm HFFN_Ym ',m #+ 0%-Zm -20-1m .0-!#1-1m /3#m
-!300#,m"30,2#m*m+"30!'A,m!-+-m*m!3+3*!'A,m"#m8B!0#1Zm*m!'"#8Zm#*m.#1-m"#*m
$032-Zm #*m 2+@-Zm #*m ,B+#0-m "#m 1#+'**1m 7m #*m !-,2#,'"-m "#m !0-2#,-'"#1m #,m *m !0,#m "#*m
$032-m"#*m+#*A,m1#m&,m"#1!0'2-m!-+-m.0-!#1-1m',"#.#,"'#,2#1m"#m*m1>,2#1'1m"#m#2'*#,-m
^3@#8j*#,'31m7m!-*YZmHFFN_Ym
-1m%#,#1mm1',21m7mm-6'"1m2+ '<,m&,m1'"-m"#1!0'2-1m!-+-m0#%3*"-0#1m#,m
*m #6.0#1'A,m "#m #2'*#,-m #,m 40'1m #1.#!'#1m 7m 2#('"-1m ^0#+ ',1)'m 7m &#-*-%'1Zm GOOJ\m
'-4,,-,'Zm HFFM_Ym + -1m 3+#,2,m 13m !2'4'""m !-',!'"'#,"-m !-,m #*m +-+#,2-m "#m
+96'+m .0-"3!!'A,m "#m #2'*#,-m 7m +"30!'A,m "#*m $032-m ^*##!)#0m 7m #,"#Zm HFFF_Ym 3,m
1>Zm#*m.0-!#1-m#1m+3!&-m+91m!-+.*#(-m7m1#m1 #m/3#m#,m*m0#%3*!'A,m',2#04'#,#,m-20-1m
%#,#1m #,20#m #**-1Zm $!2-0#1m "#m 20,1!0'.!'A,Zm Zm m 7m *m $+'*'m "#m 0#!#.2-0#1m "#m
#2'*#,-m^830m7m!-*YZmHFFF_Ymm
',*+#,2#Zm""-m/3#m#,m,#)*-#-m*m$+'*'m"#m0#!#.2-0#1m^;FO;FO<FO>m7m
<_m&m1'"-m '#,m'"#,2'$'!"m7m"#1!0'2m^2#.,-4m7m!)#0ZmHFFF_m7m1#m&m#4'"#,!'"-m
KNm
Genes candidatos para el TILLING
(.,)/#9(
*m .02'!'.!'A,m "#m #12-1m 0#!#.2-0#1m #,m *m +"30!'A,m "#m 40'1m $0321m !*'+2<0'!1Zm 1#m
"#!'"'Am /3#m ;m #0m 3,m 3#,m !,"'"2-m .0m #*m Ym *m ',2#0<1m .-0m "#1!3 0'0m #*m
+#!,'1+-m+-*#!3*0m0#1.-,1 *#m"#m*m0#%3*!'A,m"#m*m+"30!'A,m#1m#4'"#,2#m2,2-m
m,'4#*m!-+#0!'*m!-+-m!'#,2>$'!-Ym-1m+32,2#1m#,m#*m%#,m;m.-"0>,m.-020m,3#4-1m
!-,-!'+'#,2-1m1- 0#m*m0#!#.!'A,m"#*m#2'*#,-m7m#,m!-,1#!3#,!'Zm1- 0#m*m+"30!'A,m"#*m
$032-Zm 3,/3#m !-+-m 7m 1#m &m +#,!'-,"-m ,2#0'-0+#,2#Zm 2-"-1m *-1m .0-!#1-1m "#m
+"30!'A,m 7m #*m !0!2#0m !*'+2<0'!-m ,-m 1-,m #6!*31'4+#,2#m "#.#,"'#,2#1m "#m #12m
&-0+-,m^'-4,,-,'mZmHFFG_Ymm
m
KOm
(.,)/#9(
Genes candidatos para el TILLINGO
T
LFm
$.#0)-
T
T
-1m- (#2'4-1m.0-.3#12-1m.0m#12m2#1'1m"-!2-0*m1-,m*-1m1'%3'#,2#1[m
m
jm#100-**0m*m.0'+#0m.*2$-0+m"#m+32,2#1mm.0m*m#1.#!'#m//'#-O'&)m
m.02'0m"#m*m40'#""m"#m+#*A,m2'.-m'#*m"#m1.-mLHjGGIYmT
T
jm -,#0m m .3,2-m #*m +<2-"-m m 7m "#2#0+',0m *-1m .09+#20-1m /3#m -.2'+'8#,m *m
"#2#!!'A,m "#m +32,2#1m 7m +','+'8#,m #*m ,B+#0-m "#m $*1-1m .-1'2'4-1Zm 32'*'8,"-m *m
"#2#!!'A,m"#m+32,2#1m+#"',2#m"'%#12'A,m"#m&#2#0-"3.*#6m!-,m#,"-,3!*#11YT
T
jm 2#,#0m *1m 1#!3#,!'1m "#m *-1m %#,#1m 1#*#!!'-,"-1m Zm >FO I#-)J>m 7m ;m m
.02'0m"#mLHjGGIYm#*'80m*m B1/3#"m"#m+32,2#1m.0m!"m%#,m#,m*m.- *!'A,m"#m
m!-,m#*m- (#2'4-m"#m#,!-,200m+32,2#1m#,m!"m3,-m"#m#**-1m7m"#2#0+',0Zm1'm#1m
.-1' *#Zm*-1m#$#!2-1m$#,-2>.'!-1m"#m#121m+32!'-,#1m1>m!-+-m!-+.0- 0m13m1#%0#%!'A,m
#,m%#,#0!'-,#1m.-12#0'-0#1Ym
m
jm 12 *#!#0m 3,m !-+.0!'A,m #,20#m "-1m .- *!'-,#1m "#m Zm *m - 2#,'"m #,m #12#m
20 (-m "#m 2#1'1m "-!2-0*m 7m *m "#*m m !-,m #*m - (#2'4-m "#m !-+.0- 0m *-1m .09+#20-1m
A.2'+-1m .0m *m - 2#,!'A,m "#m 3,m .- *!'A,m "#m m #$'!'#,2#m !-,m 3,m 21m "#m
+32!'A,m!#.2 *#Ym
m
jm #*'80m 3,m #123"'-m "#m !-m #,m *-1m %#,#1m >m 7m ;O #,20#m LHjGGIm 7m GGIm
!!#1'-,#1m"#m+#*A,Zm/3#m0#.0#1#,2,m*m40' '*'""m"#m*m#1.#!'#Zm.0-.-0!'-,"1m.-0m
#*mET
m
jm,'!'0m*m!-,1203!!'A,m"#m3,m!29*-%-m"#m+32,2#1m$#,-2>.'!-1m#,m+#*A,Zm!2*-%,"-m
*1m +32!'-,#1m #,!-,20"1m #,m JFFm $+'*'1m HZm !-,m *1m "#1!0'.!'-,#1m "#m *-1m $#,-2'.-1m
+32,2#1m#,!-,20"-1YT
T
LG
.,#&O3O'6.))-
Creación y desarrollo de una población TILLINGO
m
m
LH
m
Creación y desarrollo de una población TILLING
.,#&O3O'6.))-
m
CMTTTT
T
TOTTTT
T
CMBMTTTTTTT
m
0m *m #* -0!'A,m "#m *m .- *!'A,m "#m m #,m +#*A,m 1#m #1!-%'Am *m *>,#m
"- *#m &.*-'"#m "#m *m 40'#""m "#m +#*A,m 2'.-m b'#*m "#m .-cm LHjGGIYm 12m *>,#m
.0-4'#,#m "#m *m #+.0#1m '#&&-O #.9Fm *m !3*m .02'!'.Am #,m #*m .0-7#!2-m m
^HFFIjHFHIMm"#*m_m"A,"#m#129m#,+0!"-m#12#m20 (-Ym*m%#,2#m/3>+'!-mm
^2'*m #2,-m 3*$-,2-_m $3#m #*m +329%#,-m #1!-%'"-m .0m #* -00m #12m .- *!'A,m "#m
m ""-m /3#m .0-4-!m +32!'-,#1m .3,23*#1Zm */3'*,"-m *1m %3,',1m 7m
.0-4-!,"-m /3#m #,*!#,m !-,m *1m 2'+',1m 7m ,-m !-,m 13m 1#m !-+.*#+#,20'Zm !'2-1',m
^#%ZmGONJ_Ym
T
CMBMBTTTTTTTPAQT
m+32!'A,m"#m*1m1#+'**1m1#m&'8-m#,m20#1m#6.#0'+#,2-1m"'12',2-1Zm7m/3#m#*m,B+#0-m"#m
1#+'**1m#0m+37m#*#4"-m^GMYFFFm1#+'**1m.0-6'+"+#,2#_m7m201m*m+32%<,#1'1Zm*1m
1#+'**1m "# >,m 0#.02'01#m #,m "-1m !#,20-1m !-* -0"-0#1m "-,"#m .-12#0'-0+#,2#m 1#m
0#*'80>m#*m!0#!'+'#,2-m"#m*1m.*,21Ym,m*m.0'+#0m2,"m^@-mHYFFK_m1#m+320-,mNYFFFm
1#+'**1m.0-6'+"+#,2#mm3,m!-,!#,20!'A,m"#mGPmm7m1#m+,"0-,mm*m#+.0#1m
'#&&-O#.9O.0m13m!0#!'+'#,2-Ym,m*m1#%3,"m2,"m^@-mHYFFK_m1#m+320-,m-201mNYFFFm
1#+'**1m .0-6'+"+#,2#Zm .#0-m #12m 4#8m !-,m 20#1m !-,!#,20!'-,#1m "'12',21m "#m m
^FZKPZm GPm 7m GZKP_m 7m #,m #12m -!1'A,Zm 1#m +,"0-,m *m %03.-m !-* -0"-0m "#m *#,!'m
^jm ^,'4#01'""m -*'2<!,'!m "#m *#,!'__Ym ',*+#,2#Zm #,m #*m HYFFLm 1#m
+320-,m .0-6'+"+#,2#m GYFFFm 1#+'**1m #,m 3,m 2#0!#0m 2,"Zm 7m /3#m +3!&1m "#m *1m
.*,21m /3#m 1#m !0#!'#0-,m #,m *#,!'m "30,2#m #*m @-m HFFKm +30'#0-,m "# '"-m m 3,m
',!'"#,!'m#,m!+.-m"#*m!-*.1-m"#*m+#*A,Ym,m#12mB*2'+m2,"m1#m31Am3,mB,'!m"-1'1m
"#m GYKm Pm "#m Ym *m .0-2-!-*-m "#m +32!'A,m "#m *1m 1#+'**1m 1#m -0%,'8Am #,m 20#1m ">1m
!-,1#!32'4-1Ym *m .0'+#0m ">m 1#m &'"020-,m *1m 1#+'**1Ym 0m #**-m 1#m .31'#0-,m #,m
0#!'.'#,2#1m "#m .*912'!-m #,m 3,m .0-.-0!'A,m "#m HKm +*m "#m %3m !"m GFFm 1#+'**1Ym 1m
1#+'**1m 1#m "#(0-,m #,m %'2!'A,m 134#m "30,2#m GLm &-01Ym *m 1#%3,"-m ">m 1#m @"'Am #,m
LI
.,#&O3O'6.))-
Creación y desarrollo de una población TILLINGO
!+.,m#620!2-0m#*m%#,2#m+329%#,-mYm#m.*'!0-,m!-,!#,20!'-,#1m"#mFYKPZmGPm
7m GYKPm ^ *m HYG_m #,m *-2#1m "#m HFFm 1#+'**1m .-0m 0#!'.'#,2#m !-,m KFm +*m "#m %3Ym *m
202+'#,2-m1#m.*'!Am"30,2#mGNm&-01m#,m*m!+.,m#620!2-0m!-,m%'2!'A,m134#Ym
m
!+ TCMBOm2-1m!-00#1.-,"'#,2#1mm*1m"-1'1m"#mm32'*'8"1m.0m+320m*m.- *!'A,Zm*-1m$032-1m- 2#,'"-1m
#,m*m%#,#0!'A,mGZm#*m,B+#0-m7m.-0!#,2(#m"#m*1m$+'*'1m!-,m1#+'**1m4' *#1m7m,-m4' *#1m#,m*m%#,#0!'A,m
HYm',*+#,2#m#*m,B+#0-m"#m$+'*'1mHm',!*3'"1m#,m*1m.*!1m.--*m/3#m$-0+,m*m.- *!'A,m"#mYm
m
-1'1mm
FZKPm
m
GPm
m
GYKPm
m
GPm
m
m
+'*'1m!-,m
1#+'**1mHm,-m
4' *#1
+'*'1m!-,m
1#+'**1mHm!-,m
.0'#,!'m4' *#
+'*'1mHm#,m
.--*1m"#mm
HFJm
GNm^NYNHP_
GNLm^OGYGNP_
GMMm
HFOm
HKm^GGYOLP_
GNJm^NNYFJP_
GKGm
IOHm
KKm^GJYFIP_
IIMm^NKYOMP_
HLOm
HYIIKm
IOOm^GMYFN_
GYOILm^NHYOHP_
GYMMGm
IYGJFm
JOMm
HYLJIm
HYILNm
Em"#m
$032-1mGm
m
*m2#0!#0m">m1#m0#!-%'#0-,m*1m1#+'**1m!-,m.0#!3!'A,m#,m3,m+**m"#m3,m.-0-m',$#0'-0m
*m 2+@-m "#m *m 1#+'**m !#009,"-*m !-,m 3,m 0'"Ym #m .*'!0-,m 20#1m *4"-1Ym 0m #*m
.0'+#0-m 1#m ',20-"3(-m *m +**m !-,m *1m 1#+'**1m "#,20-m "#m 3,m 41-m "#m .0#!'.'2"-1m "#m Km
*'20-1m7m1#m"#(Am#,m%'2!'A,m"30,2#mJm&-01Ym0m#*m1#%3,"-m*4"-m1#m0#.'2'Am#*m+'1+-m
.0-!#"'+'#,2-m .#0-m #,m #12m -!1'A,m "30,2#m Gm &-0Ym ',*+#,2#Zm #*m B*2'+-m *4"-m 1#m
0#*'8Am"#(,"-m*m+**m#,m3,m.'*m (-m%3m"#*m%0'$-m"30,2#mGm&-0Ym
*m +2#0'*m "#m * -02-0'-m 31"-m .0m *-1m *4"-1m "#m *1m 1#+'**1m 1#m *#m @"'Am 3,m
1-*3!'A,m "#m Gm "#m m .0m ',!2'40m #*m Ym *m +'1+-m ">m 1#m .0#.00-,m *1m
1#+'**1m.0m*m%#0+',!'A,Ym#m0#.02'#0-,mKFm1#+'**1m#,m!"m ,"#(m"#m.*912'!-m!-,m
3,m 1#m "#m ..#*m "#m $'*20-m &B+#"-m 7m 1#m !3 0'#0-,m !-,m .*912'!-m 20,1.0#,2#m .0m
+,2#,#0m *m &3+#""Ym #m <12#m +-"-m 1#m "#(0-,m 2-"m *m ,-!&#m #,m !9+0m .0m *m
%#0+',!'A,m #,m !-,"'!'-,#1m A.2'+1m "#m *38m ^GHjHFm &-01m *38m !-,2',3_Zm 2#+.#0230m
^HNEjIFE_m7m%0"-m"#m&3+#""m"#!3"-1m^&'"02!'A,m.0#4'm"#m*1m1#+'**1m7m..#*m
LJ
Creación y desarrollo de una población TILLING
.,#&O3O'6.))-
&B+#"-m #,m *m 1#m "#*m 0#!'.'#,2#_Ym *m ">m 1'%3'#,2#Zm *1m 1#+'**1m %#0+',"1m $3#0-,m
201*""1mm131m0#1.#!2'4-1m"#12',-1m.0m1#0m.*,2"1Ym
T
CMBMCTTTTTBTTCT
,m 2-2*m "#m GMYFFFm 1#+'**1m 1#m 0#.02'#0-,m #,m *-1m "-1m "#12',-1m !-+-m 1#m &m "#1!0'2-m
,2#0'-0+#,2#Ym*m!0#!'+'#,2-m#,m 0#0m"#m0m#,m'#&&-O#.9m1#m&'8-m#,m',4#0,"#0-m
7m#,m!-,"'!'-,#1mA.2'+1m.0m#*m+#*A,m^DmHKjIFEm7mKKjLKPm"#m&3+#""_Ym
,m *#,!'m 1#m !0#!'Am *m .0'+#0m 2,"m "#*m HFFKm #,m !+.-Zm .#0-m 234'#0-,m 3,m %04#m
',$#!!'A,m.-0m!-*.1-m7m+30'#0-,m%0,m.02#m"#m*1m.*,21m1- 0#4'4'#,"-m3,m.#/3#@m
.02#m "#m *1m NYFFFm 1#+'**1m .*,2"1m ','!'*+#,2#Ym -0m #12#m +-2'4-m .-12#0'-0+#,2#Zm 1#m
.*,20-,m GYFFFm 1#+'**1m +91Zm 202"1m *m GZKPm "#m Zm #12m 4#8m #,m ',4#0,"#0-m .0m
!-,20-*0m+91m*1m!-,"'!'-,#1m+ '#,2*#1m7m1- 0#2-"-m#4'20m',$#!!'-,#1Ymm
T
CMBMCMBTTTTTBTTT
12#m 20 (-m $3#m 0#*'8"-m .-0m #*m .#01-,*m "#m 1-.-02#m "#m !"m !#,20-m "-,"#m $3#0-,m
"'120' 3'"1m *1m 1#+'**1m ^m 7m '#&&-O #.9_Ym m +#"'"m /3#m *1m .*,21m $3#0-,m
!0#!'#,"-m1#m' ,m.-","-m"#m+,#0m/3#m1A*-m/3#"0m3,m0+m.0',!'.*m7m*%3,1m
1#!3,"0'1m "A,"#m 3,m 4#8m .0#!'"1m *1m $*-0#1m 1#m .-*','80-,m +,3*+#,2#Ym #m
32-$#!3,"0-,m3,m2-2*m"#mIYGJFm.*,21mGm^ *mHYG_Ym
0m *m .-*','8!'A,Zm .0'+#0-m 1#m #*'%'Am 3,m $*-0m $#+#,',m 7m 1#m *#m #620(#0-,m *-1m .<2*-1m
!-,m!3'""-m"#(,"-m#*m%',#!#-m*' 0#m7m1- 0#2-"-m*m.02#m13.#0'-0m"#*m-40'-m^#12'%+_m
!!#1' *#Ym-,m*m73"m"#m3,m!*4-m$'*"-m1#m0#!-%'Am3,m.-!-m"#m.-*#,m"#m*m,2#0m"#m
3,m$*-0m+1!3*',m"#m*m+'1+m.*,2Zm!-,m!3'""-m#m',2#,2,"-m/3#m<12#m.#0+,#!'#0m
*-m+91m1#!-m.-1' *#Ym#m.*'!Am#12#m.-*#,m#,!'+m"#*m#12'%+m',2#,2,"-m2+ '<,m/3#m#*m
-40'-m #1234'#0m *-m +91m 1#!-m .-1' *#m 7m /3#m !-,m *m &3+#""m #*m .-*#,m ,-m $#!3,"m
#$'!'#,2#+#,2#Ym
-12#0'-0+#,2#Zm 1#m !3 0'Am *m $*-0m !-,m 3,m 1- 0#m "#m ..#*m .0m #4'20m .-*','8!'-,#1m
!038"1m7m1#m0-23*Am!-,m*m$#!&m.0m.-"#0m!-,20-*0m*-1m">1m"#m!3("-Ym+ '<,m1#m
!-020-,m*1m0+1m*2#0*#1m/3#m#+#0%>,m"#m*m+'1+m0+m#,m*m!3*m1#m#,!-,20 m*m
$*-0m.-*','8"Ym#m#12#m+-"-m*m.*,2m.-">m!-,!#,200m+91m0#!301-1m#,m#*m!3("-m"#*m
$032-Ym #1.3<1m "#m 3,m 1#+,m .0-6'+"+#,2#m 1#m 0#2'0 m #*m 1- 0#m "#m ..#*m .0m
!-,$'0+0m #*m !3("-Ym -0m *-m %#,#0*m 1#m - 234-m 3,m $032-m .-0m .*,2Ym -1m +#*-,#1m 1#m
"#(0-,m!0#!#0m"30,2#mKKm">1m^_m.0-6'+"+#,2#Ym
m
m
LK
.,#&O3O'6.))-
Creación y desarrollo de una población TILLINGO
m
CMBMCMCTTTTTCT
,m2-2*m"#mHYILNm$032-1mGm.0-"3(#0-,m1#+'**mHm4' *#m^ *mHYG_Ymm#12-1m$032-1m1#m
*#1m #620(#0-,m 7m *'+.'0-,m *1m 1#+'**1Ym -12#0'-0+#,2#m 1#m %#0+',0-,m "'#8m 1#+'**1m "#m
!"m$+'*'m7m"#m!"m.*9,23*m1#m#620(-m3,m"'1!-m"#m&-(Ym-1m"'#8m"'1!-1m"#m&-(m1#m
+#8!*0-,m.0m#620#0m#*mm/3#m.-12#0'-0+#,2#Zm1#m-0%,'8Am#,m.*!1Ym*m0#12-m"#m
1#+'**1m"#m!"m$+'*'mHm1#mm%30"Am.0m$32301m.*'!!'-,#1m^'%30mHYG_Ym
m
m
m
(&30 TCMB[m1/3#+m"#*m.0-!#"'+'#,2-m32'*'8"-m.0m- 2#,#0m#*mm"#m*m.- *!'A,m"#mYm,m.0'+#0m
*3%0m1#m+320-,m.0-6'+"+#,2#mGMYFFFm1#+'**1m*-m/3#m.#0+'2'Am*m32-$#!3,"!'A,m"#mIYGJFm.*,21mGm
.0m*m- 2#,!'A,m"#mHYILNm$+'*'1mHYm#m!"m$+'*'mHm1#m%#0+',0-,m"'#8m',"'4'"3-1m7m1#m#620(-m#*mm
"#m !"m 3,m "#m *1m "'#8m .*,21m .0m $-0+0m #*m .--*m "#m Ym 12#m m #1m *m 1#m "#m *m .*2$-0+m "#m
Ymm
m
CMBMDTTTTT
T
CMBMDMBTTTTTTTT
m#620!!'A,m"#*mm1#m&'8-m#,m#*mm!-,m#*m'2m!-+#0!'*m#17mOLm.*,2m','m
'2m ^HKF_m ^_m "#m +-"-m /3#m 1#m .3"'#0-,m &!#0m 3,m %0,m ,B+#0-m "#m #620!!'-,#1m
!-,m3,m*2-m0#,"'+'#,2-m7m#,m3,m.#0>-"-m"#m2'#+.-m0#"3!'"-Ym,m4#8m#620>"-m#*mZm
1#m +,"Am m ,3#120-m * -02-0'-m "#*m m #,m 0'*1m "-,"#m 1#m 4#0'$'!Am *m !*'""m 7m 1#m
!3,2'$'!Am +#"',2#m %#*#1m "#m %0-1Ym #1.3<1m 1#m 20,1$'0'#0-,m *-1m 1m "#m 23 -1m m
.*!1m"#mOLm^'%30mHYH_Zm- 2#,'#,"-m$',*+#,2#m3,m2-2*m"#mHKm.*!1m12-!)Ym
m
LL
Creación y desarrollo de una población TILLING
.,#&O3O'6.))-
m
m
(&30 TCMC[m1/3#+m"#*m"'1#@-m1#%3'"-m.0m#* -00m*1m.*!1m.--*Ym%[mm%#,A+'!-Ym,'!'*+#,2#m1#m
',20-"3(#0-,m 2-"-1m *-1m 1m "#m !"m $+'*'m Hm #,m .*!1m .)%m !-,m 3,m !-,!#,20!'A,m +#"'m "#m KFm ,%ix*Ym
"m .-!'**-m !-,2'#,#m #*m m "#m 3,m $+'*'m Hm !-+.3#12m .-0m GFm ',"'4'"3-1Ym -12#0'-0+#,2#m 1#m
!-,12037#0-,m*1m.*!1m"#m"'*3!'A,Zm"'*37#,"-mmKm,%ix*m#*mm"#m!"m$+'*'Ym',*+#,2#m1#m-0%,'80-,m
*1m.*!1m.--*m+#8!*,"-m*1m$+'*'1m"#m!320-m#,m!320-m#,m#*m-0"#,m/3#m+3#120m*m$'%30Ym
m
#m 0#*'80-,m !-.'1m "#m *1m .*!1m 12-!)m .#0-m #12m 4#8m !-,m #*m m "'*3'"-m GiGFYm #m
@"'#0-,mHFmx*m"#*mm12-!)m!-,mGNFmx*m"#m%3m.0m- 2#,#0m3,m4-*3+#,m2-2*m"#m*m
"'*3!'A,m"#m!"m$+'*'m"#mHFFmx*Ym0m*m!-,1203!!'A,m"#m*1m.*!1m.--*m1#m+#8!*0-,m
#/3'+-*0+#,2#m *-1m 1m "#m !"m !320-m $+'*'1Ym *m "'1#@-m 1#m +3#120m #,m *m '%30m
HYHY
T
CMBMDMCTTTTTT
*m.0-2-!-*-m31"-m#129m 1"-m#,m#*m.0-2-!-*-m.3 *'!"-m.-0m-7*#m,"m-7*#m^GYOOF_m
*'%#0+#,2#m+-"'$'!"-Ym*m+2#0'*m"#m.02'"m$3#m3,m.#/3#@-m"'1!-m"#m&-(m#,m#12"'-m
"#m.*9,23*Ym*m"'1!-m"#m&-(m1#m',20-"3(-m#,m3,m**(), m"#mGZOm+*m#*m!3*m1#m13+#0%'Am
#,m,'20A%#,-m*>/3'"-m*-m+91m09.'"+#,2#m.-1' *#Ym-12#0'-0+#,2#m1#m0#2'0Am#*m**(), m7m
LM
.,#&O3O'6.))-
Creación y desarrollo de una población TILLINGO
1#m 20'230Am #*m +2#0'*m !-,m *m 73"m "#m 3,m .#/3#@m 0-!m .0m "#4-*4#0m *m +3#120m
20'230"m *m ,'20A%#,-Ym -12#0'-0+#,2#Zm 1#m *#m @"'Am KFFm x*m "#m 2+.A,m "#m #620!!'A,m
-7*#m ^,#6-m Y$_m .0#!*#,2"-m m LKEm +#8!*9,"-m 134#+#,2#m .0m "#1.3<1m
',20-"3!'0*-m*m @-m+0>mmLKEm"30,2#mIFm+',32-1Ym
#m @"'#0-,m KFFm x*m "#m !*-0-$-0+-m '1-+>*'!-m #,m .0-.-0!'A,m HJ[G\m 1#m +#8!*Am 7m 1#m
!#,20'$3%Am m GGYFFFm 0.+m m Dm + '#,2#m "30,2#m GFm +',32-1Ym #m #1m !#,20'$3%!'A,m 1#m
0#!3.#0Am *m $1#m !3-1m 7m 1#m *#m @"'#0-,m IKFm x*m "#m Hj.0-.,-*m ^'1-.0-.,-*_m $0>-m
!#,20'$3%,"-*-m Km +',32-1m m GFYFFFm 0.+m .0m .-"#0m .0#!'.'20m *-1m 9!'"-1m ,3!*#'!-1Ym *m
m .0#!'.'2"-m 1#m *#m @"'#0-,m HFFm x*m "#m 2+.A,m "#m *4"-m "#m -7*#m ^,#6-m Y%_m
"#(9,"-*-m0#.-10m3,m+>,'+-m"#mHFm+',32-1mmjHFEZm"#1.3<1m1#m!#,20'$3%AmIm+',32-1mm
GFYFFFm 0.+m 7m m Dm + '#,2#Ym ',*+#,2#m 1#m #620(-m #*m 1- 0#,",2#m .0m "#(0m #*m .#**#2m
1#!9,"-1#m"30,2#mIFm+',32-1Ym,m4#8m1#!-m1#m0#131.#,"'Am#*m.#**#2m!-,mKFmx*m"#m%3m
Zm 7m 1#m "#(0-,m *1m +3#1201m m JEm 2-"m *m ,-!&#m .0m $!'*'20m 3,m 3#,m
0#131.#,1'A,m"#*mYm
CMBMDMDTTTTTT
0m *m !-+.0- !'A,m "#m *m !*'""m 7m !,2'""m "#*m m "#m *1m #620!!'-,#1Zm 1#m
1#.00-,m *-1m $0%+#,2-1m #,m %#*#1m "#m %0-1Ym -1m %#*#1m !-,2#,>,m 3,m GPm "#m %0-1m
^m "#m 0-,"'1_m #,m m FYHK6m ^,#6-m Y'_m 7m Gm x*iGFFm +*m "#m 0-+30-m "#m #2'"'-Ym m
#*#!20-$-0#1'1m"#m0#*'8Am!-,m2+.A,mZm7m1#m!0%0-,m*1m+3#1201m#,m%3m(3,2-m!-,m
#*m !-*-0,2#m "#m !0%m ^GiK_m m ^,#6-m Y&_Ym 3,2-m !-,m *1m +3#1201m 1#m !0%Am 2+ '<,m #*m
+0!"-0m "#m .#1-m +-*#!3*0m "#.#,"'#,"-m "#*m2+@-m "#*m $0%+#,2-m m "#2#!20m ^m
"#*m$%-mwm-m*m+'6_Ym',*+#,2#Zm1#m.*'!Am3,m4-*2(#m"#m#,20#mNKjGFFmYmm
LN
.,#&O3O'6.))-
Detección e identificación de los mutantes
CMCTTTTTTT
m
,m4#8m#*#%'"-m#*m%#,m"',m7m- 2#,'"m13m1#!3#,!'m#1.#!>$'!m"#m*m40'#""mLHjGGIZm
!-,m*m/3#m&#+-1m"#100-**"-m*m.- *!'A,m"#mZm1#m.0-!#"'Amm*m"#2#!!'A,m"#m
*-1m+32,2#1Ym*m.0-2-!-*-m#129m 1"-m#,m!320-m$1#1[mG_m+.*'$'!!'A,m"#*m$0%+#,2-m
"#*m %#,m +#"',2#m Zm H_m #1,230*'8!'A,m 7m 0#,230*'8!'A,m "#m *-1m +.*'!-,#1Zm I_m
'%#12'A,m"#m*-1m&#2#0-"B.*#6ZmJ_m'13*'8!'A,m"#m*-1m$0%+#,2-1m"#m"'%#12'A,m#,m%#*#1m
"#m!0'*+'"m4#02'!*#1m^'%30mHYI_Ym
m
m
m
(&30 T CMD[m 1/3#+m "#*m .0-2-!-*-m "#m Ym _m -.O Zm 1#%3,"m +.*'$'!!'A,m ^+91m #1.#!>$'!_m "#*m
$0%+#,2-m m ,*'80m !-,m !# "-0#1m +0!"-1m !-,m $*3-0#1!#,!'Ym _m #1,230*'8!'A,m "#m *-1m $0%+#,2-1m 7m
0#,230*'8!'A,m .0-4-!,"-m *-1m &#2#0-"B.*#6m #,20#m *-1m $0%+#,2-1m ,-m !-+.*#+#,20'-1m .-0m !-,2#,#0m 3,m
LO
.,#&O3O'6.))-
Detección e identificación de los mutantesO
+32!'A,Ym!_m'%#12'A,m!-,m*m#,"-,3!*#1m#1.#!>$'!m"#m&#2#0-"B.*#6m7m.-0m*-m2,2-m"#m*-1m$0%+#,2-1m/3#m
!-,2'#,#,m +32!'A,Ym -1m *#*-1m 1'*4#120#1m ,-m $-0+09,m &#2#0-"B.*#6m 7m .-0m *-m 2,2-Zm ,-m 1-,m "'%#0'"-1Ym "_m
#1,230*'8!'A,m 7m 4'13*'8!'A,m "#m *-1m $0%+#,2-1m #,m %#*#1m "#m !0'*+'"Ym ,m #*m jm JIFFm .-"#+-1m
4'13*'80m*-1m"-1m!,*#1m^mMFFm7mNFF_m.-0m1#.0"-m7m!-,$'0+0m*1m+32!'-,#1m+#"',2#m#*m!9*!3*-m"#*m
2+@-m "#*m $0%+#,2-m "'%#0'"-Ym -,m *m 13+m "#m *-1m $0%+#,2-1m "'%#0'"-1m 7m 4'13*'8"-1m #,m + -1m !,*#1m
"# #m- 2#,#01#m#*m$0%+#,2-m,-m"'%#0'"-m-0'%',*Ymm
m
CMCMBTTTTTTT
0m .*'!0m #*m +<2-"-m "#m m .0'+#0-m &!#m $*2m "#$','0m #*m %#,m "',m /3#m
/3#0#+-1m #123"'0m 7m "#*m !3*m /3#0#+-1m #,!-,200m +32,2#1Ym ,m 4#8m #*#%'"-m #*m %#,m
&!#m$*2m1#!3#,!'0*-m#,m*m#1.#!'#m-m40'#""m!-,m*m/3#m1#m&7m- 2#,'"-m*m.- *!'A,m
"#mm.0m3,m#$'!'#,2#m"'1#@-m"#m*-1m!# "-0#1Ym
0m*m+.*'$'!!'A,m"#m*-1m%#,#1mZmI#-)J>m7O;m1#m 31!0-,m1m#,m*m 1#m"#m
"2-1m "#m m ^!23*+#,2#m 1#m .3#"#m #,!-,200m #12m ',$-0+!'A,m #,m
555Y'!3%'Y-0%_m +'#,201m /3#m .0m #*m %#,m >m *-1m !# "-0#1m 7m #12 ,m "'1#@"-1m
,2#0'-0+#,2#m ^'#2-m 7m !-*YZm HFFLJYm 0m !"m 3,-m "#m #**-1m 1#m #,!-,20Am 3,m 3,'%#,m
^555Y'!3%'Y-0%_m7mm.02'0m"#m#1#m$0%+#,2-m1#m"'1#@0-,m*-1m!# "-0#1m.0m#+.#80mm
1#!3#,!'0m#*m%#,m#,2#0-m-m0#%'-,#1m!-,!0#21m"#*m+'1+-Ym0m#*m"'1#@-m"#m!# "-0#1m1#m
31Am #*m .0-%0+m .0'+#0Im ^&22.[ii$0-"-Y5'Y+'2Y#"3_m 7m #,m "#2#0+',"1m -!1'-,#1m 1#m
"'1#@0-,m*-1m!# "-0#1m+,3*+#,2#Ym
!23*+#,2#Zm 7m %0!'1m m *m "'1.-,' '*'""m "#m *m 1#!3#,!'m "#*m %#,-+m "#*m +#*A,Zm ,-m
1#0>m ,#!#10'-m 1#!3#,!'0m *-1m 1m .3#12-m /3#m 7m "'1.-,"0>+-1m "#m *m 1#!3#,!'m
#1.#!'$'!m"#m!"m%#,m#,m*m#1.#!'#m"#m#123"'-Ym
T
CMCMBMBTTTTTPQT
,m 4#8m "'1#@"-1m *-1m !# "-0#1Zm 1#m -.2'+'80Am *m +.*'$'!!'A,m +#"',2#m m #,m
%0"'#,2#m "#m 2#+.#0230m .0m "#2#0+',0m *m 2#+.#0230m "#m &' 0'"!'A,m A.2'+Ym 12m
m!-,1'12#m#,m.0#.00m*m+'1+m+#8!*m^"#mm7m!# "-0#1_m7m.0- 0*mm"'12',21m
2#+.#02301m"#m&' 0'"!'A,m.0m.-"#0m- 1#040m#,m!3*m#1m+91m#1.#!>$'!Ym
0m.0#.00m*mm#129,"0m1#m32'*'80-,m"#mGFjKFm,%m"#mZm2+.A,m mGFm^ m
G[m %*Hm GYKm +Zm *m KFm +Zm 0'1j*m GFm +m .m NYIZm %#*2',m FYFFGP_Zm FYJm +m "#m
!"m,3!*#A2'"-ZmFYJmxm"#m!"m!# "-0Zm7m#,m$3,!'A,m"#m!"m.0#(m"#m!# "-0#1Zmm"#m
GYKm+mmIYKm+m"#m%*Hm7mHm"#m/m.-*'+#01Ym*m4-*3+#,m$',*m#129,"0m32'*'8"-m
#0m"#mHKmx*Ymm0#!!'A,m1#m0#*'8 m#,m2#0+-!'!*"-0#1m#,#m+.mm712#+mOMFFZm
MF
Detección e identificación de los mutantes
.,#&O3O'6.))-
HMHFm &#0+*m 7!*#0m ^..*'#"m '-1712#+1_m 7m jHFFm #*2'#0m &#0+*m 7!*#0m ^
m
#1#0!&_m.0mm*1m0#!!'-,#1m"#mm#,m%0"'#,2#1m"#m2#+.#0230Ym
*m .0-%0+m #12,"0'8"-m !-,1'12>m "#m 3,m .0'+#0m !'!*-m "#m "#1,230*'8!'A,m "#m Hm
+',32-1m m OJEZm 7m IKm !'!*-1m "#m "#1,230*'8!'A,m "#m *m !"#,m ^HFm 1#%3,"-1m m OJE_Zm
&' 0'"!'A,m"#m*-1m!# "-0#1m^2#+.#0230m#1.#!>$'!m.0m!"m.0#(_Zm7m#*-,%!'A,m"#*m
$0%+#,2-m ^"#m IFm 1#%3,"-1m m Gm +',32-m #,m $3,!'A,m "#*m 2+@-m "#*m +.*'!A,m m MHm E_Ym
',*+#,2#m 1#m @"'Am 3,m !'!*-m "#m Km +',32-1m m MHEm .0m ! 0m *m #*-,%!'A,m 7m 1#m
+,234-m*m0#!!'A,mmGHEYm
m
CMCMBMCTTTT
-1m $0%+#,2-1m 0#13*2,2#1m $3#0-,m .0'+#0+#,2#m .30'$'!"-1m !-,m #*m #!"O /,O ,)/.O
/,# #.#)(O #.m ^-!&#_m .0m .-12#0'-0+#,2#m 1#0m 1#!3#,!'"-1Ym ,m !"m 0#!!'A,m "#m
1#!3#,!'!'A,m 1#m @"'#0-,m Im x*m ^GFjHKm ,%_m "#*m $0%+#,2-m "#m m .30'$'!"-m /3#m
"#1# m1#!3#,!'01#ZmHmx*m"#m2+.A,mm^HFFm+m0'1j*ZmOYF\mKm+m%*H_ZmHmx*m
"#m '%m 7#m ^#!3L,'#(.),O 0;E;O 3&O +/(#(!O #._m 7m $',*+#,2#m Im x*m "#m %3m
m .0m !-+.*#20m #*m 4-*3+#,m 2-2*m "#m GFm x*Ym 0m 1#!3#,!'0m #*m $0%+#,2-m 1#m &'8-m
3,mm"#m1#!3#,!'!'A,m/3#m!-,12 m"#mHKm!'!*-1Zm"#m20#1m.1-1\m3,m.0'+#0m.1-m.0m
*m "#1,230*'8!'A,m m OLEm "30,2#m GFm 1#%3,"-1Zm 3,m .1-m "#m &' 0'"!'A,m "#m *-1m
!# "-0#1m m KFEm "30,2#m Km 1#%3,"-1m 7m 3,m 2#0!#0m .1-m "#m #*-,%!'A,m m LFEm "30,2#m Jm
+',32-1Ym
-12#0'-0+#,2#m1#m.0-!#"'Amm*m.0#!'.'2!'A,m"#*mm#,m.*!YmmGFmx*m"#*m4-*3+#,m"#m
*m0#!!'A,m"#m1#!3#,!'!'A,m1#m*#m@">,mGFmx*m"#m%3m7m.-12#0'-0+#,2#ZmHmx*m"#mm
GHKm+ZmHmx*m"#m!mIm.mJZLZm7mKFmx*m"#m2m 1-*32-Ym#1.3<1m"#m+#8!*0*-m1#m
"#( m.0#!'.'20m#*mm"30,2#mGKm+',32-1m7m1#m!#,20'$3% mmIYFFFm0.+m"30,2#mIFm
+',32-1Zm 1#m ',4#02>m *m .*!m 7m 1#m #*'+', m #*m 1- 0#,",2#Ym 3#%-m 1#m 0#*'8 m 3,m
!#,20'$3%!'A,m!-02mmGYFFFm0.+m.0m"#1.3<1m@"'0*#mGKFmx*m"#m2m*mMFmPYm,m4#8m
+#8!*"m *m .*!m .-0m ',4#01'A,m 1#m !#,20'$3% m m IYFFFm 0.+m "30,2#m GFm +',32-1m
#*'+',,"-m .-0m 1-0!'A,m #*m 1- 0#,",2#Ym ',*+#,2#m 1#m .-">m &!#0m 3,m
!#,20'$3%!'A,m!-02m!-,m*m.*!m',4#02'"m#,!'+m"#m..#*m 1-0 #,2#m.0m1#%300m3,m
1#!"-m +91m ',2#,1-m "#*m .0#!'.'2"-Ym ,m 4#8m .0#!'.'2"1m *1m 1#!3#,!'1m 1#m
0#131.#,"'#0-,m !"m 3,m "#m #**1m !-,m GHm x*m "#m $-0++'"m ''m 7m 1#m !0%0-,m #,m #*m
1#!3#,!'"-0mIGIF*Ym
m
m
MG
.,#&O3O'6.))-
Detección e identificación de los mutantesO
CMCMBMDTTTTT
1m 1#!3#,!'1m $3#0-,m ,*'8"1m & '23*+#,2#m !-,m #*m .0-%0+m +/(",O >EAEO #m
!"m %#,m 1#m 32'*'8Am #*m -02A*-%-m "#m *m #1.#!'#m +91m !#0!,m /3#m &3 '#0m "'1.-,' *#m #,m *m
1#m"#m"2-1m!-+-m+-"#*-Ym#m#12#m+-"-m.3"'+-1m- 2#,#0m*m1#!3#,!'m#6!2m"#m!"m
%#,m -m "#*m $0%+#,2-m "#m ',2#0<1m #,m *m 40'#""m LHjGGIm 7m *-!*'80m *m .-1'!'A,m "#m
',20-,#1m7m#6-,#1Ym
T
CMCMCTTTTTTTTT
,m 4#8m - 2#,'"m *m 1#!3#,!'m #1.#!>$'!m "#m *-1m %#,#1m #,m *m 40'#""m LHjGGIZm
.0-!#"'+-1m *m "'1#@-m "#m !# "-0#1m /3#m +.*'$'!0>,m *m 0#%'A,m "#m ',2#0<1m .0m
.-12#0'-0+#,2#m 1#0m ,*'8"m +#"',2#m #*m +<2-"-m Ym 0m *m "#2#!!'A,m "#m *-1m
+32,2#1m1#m0#/3'#0#m3,m+.*'$'!!'A,m+37m#1.#!>$'!Zm#1m.-0m#**-m/3#m1#m"'1#@0-,m"-1m
.0#(1m "#m !# "-0#1m .0m !"m 0#%'A,Ym ,m +.*'$'! m 3,m $0%+#,2-m +7-0m "#m *m
0#%'A,m "#m ',2#0<1m +'#,201m /3#m *m -20m .0#(m "#m !# "-0#1m #12 m "'1#@"m m 3,m
"'12,!'m "#m #,20#m GFm 7m HFm . m +91m "#*,2#m "#m *m .0#(m ,2#0'-0Zm &!'#,"-m 1>m 3,m
+.*'$'!!'A,m "#m 3,m $0%+#,2-m .0#4'+#,2#m 7m +.*'$'!"-m -m "- *#m +.*'$'!!'A,m
^-.O_Ym
0m *m #*#!!'A,m "#*m $0%+#,2-m m +.*'$'!0m 1#m 32'*'8Am #*m .0-%0+m m ^))(-O
*.#'#4O .)O #-)0,O &.,#)/-O -#)(-m ^&22.[ii555Y.0-5# Y-0%i',.32i__Ym m
.#0+'2#m'"#,2'$'!0m*1m8-,1m"#*m%#,m/3#m3,m4#8m+32"1m2'#,#,m+91m.0- '*'""m"#m
40'0m #*m +',-9!'"-m #,m 8-,1m !-,1#04"1m "#m *m .0-2#>,m 7m .-0m *-m 2,2-Zm +91m
.0- '*'""m"#m$#!20mm*m$3,!'-,*'""m"#m*m+'1+Ym*m.0-%0+m.#0+'2#m',20-"3!'0m
40' *#1m!-+-m*m#1.#!'#m/3#m1#m+32Zm7m#*m+<2-"-m7i-m%#,2#m/3>+'!-m!-,m#*m/3#m1#m&m
0#*'8"-m*m+32!'A,m.0m.-"#0m0#*'80m#*m!9*!3*-Ym,m4#8m'"#,2'$'!"m#12m0#%'A,m1#m
"'1#@0-,m *-1m !# "-0#1m #1.#!>$'!-1m !-,m Zm .0'+#0Im -m +#"',2#m "'1#@-m +,3*m
^,#6-mY_Ym
m
CMCMDTTTTTT
0!'1m*m"'1#@-m"#m"-1m.0#(1m"#m!# "-0#1m7mm*m-.Om1#m- 2#,>m3,m.0-"3!2-m
+3!&-m +91m #1.#!>$'!-Ym 0m *m .0'+#0m m ^.02"-m HYHYGYG_m 1#m 32'*'80-,m *-1m
!# "-0#1m1',m+0!(#Ymm0#!!'A,m"#mm1#m0#*'8Amm.02'0m"#m*m+#8!*m#12,"0'8"m
+-120"m#,m*m *mHYHYm
m
MH
.,#&O3O'6.))-
Detección e identificación de los mutantes
!+ TCMC[m-+.-1'!'A,m"#m*m0#!!'A,m"#mm#,m#*m.0-2-!-*-m"#mm^g,#6-mY Yggm.0-!#"#,2#m"#m
!"m.-!'**-m"#m*1m.*!1m.--*_m
m
.-"$-20 "(>-T
(-("( +T
.-"$-20 "(>-T
%(- +T
R0$ ""(>-T
GFm+m
Gm+m
HYKmx*m
HKm+m
Km+m
GFm+m
GFm+m
HZKm+m
FZHm+m
FZJm+m
FZJm+m
HYKmx*m
Gmx*m
Gmx*m
Gmx*m
Km3ix*m
Km3m
Gmx*m
ggm
Km,%ix*m
^.0-6_m
GFm,%m
Hmx*m
-*3+#,m
2-2*m
m
m
HKmx*m
.,/.-$-2$T
+.A,m"#m
gm
%*Hm
"1m
# "-0mm
# "-0mm
/m
-*'+#01m
m
CMCMDMBT$
T
0m*m1#%3,"mm^-.O_m^'%30mHYIY_m1#m32'*'8Am#*m.0-2-!-*-m,2#0'-0m3,/3#m
#,m #12#m !1-m 1#m @">,m *-1m !# "-0#1m +0!"-1m .0m jZm "#m +-"-m /3#m *-1m
!# "-0#1m "'0#!2-1m #129,m +0!"-1m !-,m #*m $*3-0-!0-+-m MFFm ^0-(-_m 7m *-1m !# "-0#1m
0#4#01-1m !-,m #*m $*3-0-!0-+-m NFFm ^4#0"#_Ym *m m @"'"-m #0m #*m .0-"3!2-m "#m *m
.0'+#0m Ym *m .0-%0+m "#m m 32'*'8"-m #0m #*m +'1+-m /3#m #,m *m .0'+#0m
+.*'$'!!'A,m 3,/3#m !+ ',"-m *m 2#+.#0230m "#m &' 0'"!'A,m .0m !"m .0#(m "#m
!# "-0#1Ym ,m #121m +.*'$'!!'-,#1m "# >m 2#,#01#m .0#!3!'A,m 7m 1#m "# >,m #,4-*4#0m *-1m
23 -1m !-,m ..#*m "#m *3+','-m #,m 2-"-m +-+#,2-m 7m /3#m *-1m $*3-0-!0-+-1m m 1#m
"#%0",m*m#6.-,#01#mm*m*38Ym
',*+#,2#Zm*m4'13*'8!'A,m"#*m.0-"3!2-m"#mm1#m0#*'8Am#,m%#*#1m"#m%0-1m*mGPm7m
2',!'A,m !-,m 0-+30-m "#m #2'"'-Ym #m 31Am +0!"-0m "#m .#1-m +-*#!3*0m "#!3"-m m !"m
$0%+#,2-m ^*O #2m .0m $0%+#,2-1m "#m GFFjGFFFm . m 7m wm "'%#0'"-m !-,m #(Cm
.0m $0%+#,2-1m "#m +91m "#m GFFFm . _m .0m !-+.0- 0m /3#m #*m +.*'!A,m - 2#,'"-m #0m #*m
!-00#!2-Ym
CMCMETTTT
12#m.1-m#1m,#!#10'-m.0m*m"#2#!!'A,m"#m*m+32!'A,m^'%30mHYIY _Ym"m.-!'**-m"#m
*1m .*!1m .--*m !-,12m "#m m "#m Jm $+'*'1m Hm "'12',21m ^!"m 3,m "#m #**1m !-,m GFm
',"'4'"3-1Zm.-0m*-m2,2-m#,m!"m.-!'**-m&7mJFm',"'4'"3-1m0#.0#1#,2"-1_Ym*mm#129m
+32"-m *m 80m 7m #,m !"m .-!'**-m .3#"#m & #0m .0#1#,!'m "#m *#*-1m 1'*4#120#1m 7m *#*-1m
MI
.,#&O3O'6.))-
Detección e identificación de los mutantesO
+32"-1Ym m $-0+!'A,m "#*m &#2#0-"B.*#6m 1A*-m 1#m "09m #,20#m $0%+#,2-1m !-,m *%B,m
!+ '-m"#m 1#m-mZm#1m"#!'0Zm#,20#m*#*-1m"'12',2-1m^+32"-m7m,-m+32"-_Ym
0m*m$-0+!'A,m"#*m&#2#0-"B.*#6m1#m1-+#2'#0-,m*1m1m!-,m+0!(#m^-.O_mm
3,m!'!*-m"#m"#1,230*'8!'A,m7m*3#%-m3,m0#,230*'8!'A,m*#,2Ym0m#**-m1#m',20-"3(-m
*m -.O O #,m #*m 2#0+-!'!*"-0m HFFm !-,m 3,m .0-%0+m "#m OJEm "30,2#m GFm
+',32-1m7m*3#%-m3,m%0"'#,2#m"#mOJEmmNEm"'1+',37#,"-mjFYGEi1m7m$',*'8,"-mmJEYm
m
CMCMFTTTT
T
CMCMFMBTTTTTTTTT
0m #*#%'0m !-00#!2+#,2#m *m "'*3!'A,m "#m *m #,8'+m ()O 1#m 31Am *m ',$-0+!'A,m
.0-.-0!'-,"m .-0m #*m * -02-0'-m "#*m Zm /3#m $3#m #*m !#,20-m /3#m ,-1m .0-.-0!'-,Am *m
#,8'+m !-,!#,20"Ym ,m #*m ,#6-m Y!m .3#"#m 4#01#m #*m %09$'!-m #* -0"-m .-0m #12#m
* -02-0'-m"A,"#m1#m.3#"#m"#2#0+',0m*m"'*3!'A,m"#m#,8'+m,#!#10'm#,m$3,!'A,m"#m*m
!-,!#,20!'A,m"#mm- 2#,'"-m#,m#*m.0-"3!2-m"#m*mYm
m !-,!#,20!'A,m +#"'m "#m m - 2#,'"m #,m ,3#120-m * -02-0'-m #,m *1m !-,"'!'-,#1m
!'2"1m,2#0'-0+#,2#m#0m"#*m0,%-m"#mJFjKFm,%Zm.-0m#12#m+-2'4-m7m& '#,"-m0#*'8"-m
.03# 1m 2#12m .0m !-00- -00m *m ',$-0+!'A,m "#*m %09$'!-m 1#m 32'*'8Am *m "'*3!'A,m GiKFFFm
^,#6-mY!_m"#m*m#,8'+m()Em
m
CMCMFMCTTTTTTTTT
0m!"m0#!!'A,m"#m"'%#12'A,m^'%30mHYIY!_m^7m2#,'#,"-m#,m!3#,2m/3#m*m!,2'""m"#m
.0-"3!2-m"#mm1'#+.0#m-1!'* m#,20#mJFm7mKFm,%_m1#m@"'AmIx*m"#*m.0-"3!2-m"#mZm
Im x*m "#m 2+.A,m .0m *m "'%#12'A,m ^,#6-m Y#_Zm Im x*m "#m ()m ^GiKYFFF_m 7m HGm x*m "#m %3m
m.0m**#%0mm*-1mIFmx*m"#m4-*3+#,m2-2*Ym12m0#!!'A,m1#m',!3 mmJKEm"30,2#m
HFm+',32-1Ym-12#0'-0+#,2#Zm.0m"#2#,#0m*m0#!!'A,m"#m"'%#12'A,Zm1#m@"'#0-,mKmx*m"#m
mFYGKmm+,2#,'#,"-m*m0#!!'A,m#,m&'#*-m7m.0-2#%'"m"#m*m*38Ym
T
CMCMFMDTTTTTTT
0m*m.30'$'!!'A,m.0'+#0-m"# >m.0#.001#m*m.*!m"#m.30'$'!!'A,m**#,,"-m*m.*!m
"#m!0%m!-,m*"2m#,m.-*4-m^#.&"#6mjKFm3.#0$',#Zm+#01&+m'-1!'#,!#1_Zm7m
#6.,"'0m !-,m *m #1.923*m /3#m **#4m "(3,2m *m .*!m "#m !0%m #.&"#6m KFm
^FOLJKZm '**'.-0#_Ym ,m 4#8m '#,m **#,-1m *-1m .-!'**-1m 7m *m .*!m *'+.'m 1#m ',4'02'Am
1- 0#m 3,m .*!m $'*20-m '**'.-0#m ^3*2'!0##,j_m "#(,"-m /3#m #*m *"2m !7#0m #,m
!"m.-!'**-Ym@"'+-1mIHKmx*m"#m%3mmm!"m.-!'**-m"#m*m.*!m$'*20-m.0m/3#m#*m
MJ
Detección e identificación de los mutantes
.,#&O3O'6.))-
*"2O .-*'+#0'80m 7m $-0+0m *m 0#1',m /3#m .30'$'!0>m #*m .0-"3!2-m "#m *m "'%#12'A,Ym
,m4#8m@"'"m#*m%3m&'8-m$*2m#1.#00m3,m+>,'+-m"#m3,m&-0m.0m/3#m*m0#1',m
1-0 '#0m13$'!'#,2#+#,2#m#*m%3Ym
#1.3<1m "#m 3,m &-0m 1#m !#,20'$3%Am *m .*!m $'*20-m !-,m 3,m .*!m "# (-m ^'!0-.*2#m
0#',#0m "#m OLm .-!'**-1_m m HYFFFm 0.+m "30,2#m Hm +',32-1m .0m 1#!0m *m 0#1',Ym ,m 4#8m
#6.3*1"m #*m %3m *m .*!m #12 m *'12m .0m !0%0m *-1m IKm x*m "#m *m "'%#12'A,m "#m
&#2#0-"B.*#6Ym 0m '1*0m *m +3#120m 1#m !-*-!Am *m .*!m "#m 0#!-*#!!'A,m ^*!m _Zm
& '#,"-m!0%"-m.0#4'+#,2#mKmx*m"#m83*m!-,m$-0++'"m#,m!"m.-!'**-Zm"# (-m"#m*m
.*!m $'*20-m ^.0#12,"-m 2#,!'A,m m /3#m *m -0'#,2!'A,m "#m *1m "-1m .*!1m 1#m *m +'1+_Ym
-12#0'-0+#,2#m1#m@"'#0-,m*-1mIKmx*m"#m*m"'%#12'A,m#,m!"m.-!'**-m7m1#m!#,20'$3%Am-20m
4#8mHm+',32-1mmHYFFFm0.+Zm#*37#,"-m1>m*m+3#120Ym0m#*m1#!"-m7m!-,!#,20"-m"#m*m
+3#120m1#m"#.-1'2Am*m.*!m#,m#*m*OO^
-3,mmGFFO_mmLKEm"30,2#mJKm+',32-1Ym
#m #12#m +-"-m 1#m 0#"3(-m #*m 4-*3+#,m "#m %3m 7m 1#m - 234-m *m +3#120m !-,!#,20"m 7m m
.3,2-m.0m*m!0%m#,m#*m%#*m"#m!0'*+'"m4#02'!*Ym
m
CMCMGT T T T T T T T T
TTT
m
CMCMGMBTTTTTT
0m*m1#.0!'A,m"#m*1m+3#1201m&#+-1m32'*'8"-m#*m1#!3#,!'"-0mjmJIFFYm0m
*m .0#.0!'A,m "#*m %#*Zm .0'+#0-m 1#m +-,20-,m "#!3"+#,2#m *-1m !0'12*#1m "#*m jm
^!0'12*#1m202"-1m#1.#!>$'!+#,2#m.0m*m!-00#!2m!.2!'A,m7m#*m,9*'1'1m"#*m*91#0m"#m*m
+'1+m+9/3',_Ym,20#m*-1m"-1m!0'12*#1m1#m!-*-!0-,m*-1m#1.!'"-0#1m"#mHKm++m"#mj
Zm7m1#m#,!(0-,m*-1m!0'12*#1m!-,m*1m.'#81m.0m#12#m+-,2(#Ym,m4#8m+-,2"-1m1#m
.0#.0Am *m +#8!*m !-,m HKm +*m "#m !0'*+'"m ^/&O;BG;MO+/(#(!O,HO.#)(&O
#!()-.#-_Zm HFm x*m "#m m 7m GFFm x*m "#m m ^.#013*$2-m "#m +-,'-_Ym m +#8!*m 1#m
%'2Am*'%#0+#,2#m7m1#m 1-0 'Am#,m3,m(#0',%m"#mKFm+*m.0m',7#!20*m',+#"'2+#,2#m
#,m #*m +-,2(#m "#m *-1m !0'12*#1Ym ',*+#,2#m 1#m .31-m #*m .#',#m #,m *m .02#m 13.#0'-0m "#*m %#*m
.0m +0!0m #*m #1.!'-m "A,"#m 1#m !0%0>,m .-12#0'-0+#,2#m *1m +3#1201Ym ,m 4#8m
.-*'+#0'8"-m#*m%#*m^#,20#mIFm7mJKm+',32-1_m1#m+-,2Am"#,20-m"#*mjmJIFFYm-"1m*1m
.'#81m1#m#,!(0-,m2*m7m!-+-m+3#120m*m'%30mHYJYm#m31AmmGFm"'*3'"-mm.02'0m"#*m
12-!)m !-+#0!'*m ^m 3$$#0m !)Zm j_m !-+-m 2+.A,m .0m !-00#0m *1m
+3#1201Ym
1m !-,"'!'-,#1m "#m .0#!*#,2+'#,2-m "#*m %#*m 31"1m #,m #*m jm JIFFm $3#0-,m *1m
#12,"0'8"1m .0m #*m m /3#m !-00#1.-,"#,m m GKFFm Zm JFm +Zm JFm m 7m KFEm
MK
.,#&O3O'6.))-
Detección e identificación de los mutantesO
"30,2#mGKm+',32-1Ym,m4#8m&#!&-m#*m.0#!*#,2+'#,2-m"#*m%#*m1#m.0-!#"'Amm!0%0m*1m
+3#1201Ym
m
m
m
(&30 TCMEOT#.0#1#,2!'A,m"#*m+-,2(#m,#!#10'-m.0m!-00#0m#*m%#*m#,m#*mjmJIFFYm*m%#*m1#m',20-"3!#m#,m*m
+9/3',mjmJIFFm7m1#m!-,20-*m+#"',2#m3,m!-+.32"-0m /3#m!-,20-*m*1m!-,"'!'-,#1m "#m#*#!20-$-0#1'1m
^'+%#,m#620>"m"# 555Y*'!-0Y!-+_
m
CMCMGMCTTTTTTTTTTT
1m +3#1201m !-,!#,20"1m +#"',2#m #*m *O 0m 1#m "#1,230*'80-,m #,m 3,m
2#0+-!'!*"-0m m OJEm "30,2#m Im +',32-1m #m ',+#"'2+#,2#m 1#m .31'#0-,m #,m &'#*-Ym *m
+0!"-0m "#m MFFm . m ^jm '8#m 2,"0"m KFjMFFm .m 7#hm MFFm 7m NFF\_m .0m #*m j
m 2+ '<,m "# >m "#1,230*'801#Ym 1m +3#120m 1#m !0%0-,m #,m 3,m .*!m "#m !0%m
^B@&&O',(O,3FODB@FOO"O&O)'*(3_m .0m 1>m .-12#0'-0+#,2#m 1-0 #0m
*1m +3#1201m !-,m *-1m .#',#1m "#1#!& *#1m ^
B@O O "O &O )'*(3_Ym #1.3<1m "#m "-1m
+',32-1m^#,m*-1m/3#m*m+3#120m1#m 1-0 #m '#,m#,m#*m.#',#_m1#m!-*-!Am#*m.#',#m#,m*m.02#m
13.#0'-0m "#*m %#*m 7m 1#m "#(0-,m !-00#0m *1m +3#1201m "30,2#m Jm +',32-1m !-,m #*m .#',#m
#,!("-Ym ',*+#,2#Zm 1#m "#234-m #*m 4-*2(#m .0m /3'20m #*m .#',#m "#1#!& *#m "#(,"-m
!-00#0m*1m+3#1201m#*m2'#+.-m,#!#10'-m#,m$3,!'A,m"#*m2+@-m"#*m+.*'!A,m^.0mGFFFm
. ZmJjLm&-01_m^'%30mHYIY"_Ym
m
T
ML
Detección e identificación de los mutantes
.,#&O3O'6.))-
CMCMGMDTTTTTOTT
-1m +32,2#1m 1#m '"#,2'$'!0-,m $',*+#,2#m #,m *-1m %#*#1m "#m !0'*+'"Ym 0m "#2#!20m 3,m
+32,2#m&!#m$*2m&!#0m3,m,9*'1'1m"#2**"-m"#m*m'+%#,m#,m!"m!00'*m7m0#!-00#0*-m
+,3*+#,2#m.0m.-"#0m"#2#!20m*-1m.#/3#@-1m.3,2-1m/3#m.0#!#,m!-+-m0#13*2"-m"#m
*m"'%#12'A,m"#m3,m&#2#0-"B.*#6m!-,m*m#,8'+m()Ym
0m #*m ,9*'1'1m "#*m %#*m 1#m ,*'8Am *m '+%#,m ^!-,m *m 73"m "#*m .0-%0+m ").)")*_m
+.*'9,"-*m &12m 0!0m 2-"-1m *-1m !00'*#1m .0m #,!-,200m *-1m !-02#1m "#m *-1m
&#2#0-"B.*#6Ym m '+%#,m 1#m ,*'8Am !-,(3,2+#,2#m #,m *-1m "-1m !,*#1m ^MFFm 7m NFF_m 7m
.-12#0'-0+#,2#m 1#m 4#0'$'!Am #,m !"m !,*m #*m .3,2-m "#m '"#,2'$'!!'A,m "#*m &#2#0-"B.*#6m
!-+.0- 9,"-1#m /3#m *m 13+m "#*m 2+@-m "#*m !-02#m #,m *-1m "-1m !,*#1Zm !-',!'">m !-,m #*m
2+@-m2-2*m"#*m$0%+#,2-m,*'8"-m^'%30mHYK_Ym
m
m
(&30 T CMF[m '13*'8!'A,m "#m *1m +3#1201m #,m #*m %#*m "#m Ym m "'%#12'A,m "#m 3,m &#2#0-"B.*#6m 1#m .3#"#m
4'13*'80m +#"',2#m "-1m .3,2-1m #,m #*m +'1+-m !,*Ym ,-m ^0-(-_m .#02#,#!#m *m $0%+#,2-m "'%#0'"-m "#1"#m #*m
!# "-0m "'0#!2-m ^+0!"-m !-,m m MFF_m &12m #*m &#2#0-"B.*#6Zm 7m #*m -20-m $0%+#,2-m !-00#1.-,"#m "#1"#m #*m
MM
.,#&O3O'6.))-
Detección e identificación de los mutantesO
!# "-0m',4#01-m^+0!"-m!-,mmNFF_m&12m#*m&#2#0-"B.*#6Ym#m#12#m+-"-m*m13+m"#*m2+@-m"#m*-1m"-1m
$0%+#,2-1m"# #m"0m#*m2-2*m"#*m2+@-m"#*m+.*'!A,Zm/3#m!-00#1.-,"#m*m&-+-"B.*#6m^ ,"m+0'**m"#m*m
.02#m 13.#0'-0m "#*m %#*_Ym 0m 4'13*'80m !-,m +91m "#2**#m 7m 1#%300m *m #1.#!'$'!'""m "#*m !-02#m 1#m .3#"#m
4'13*'80m2+ '<,m#*m%#*m#,m!"m!,*m',"#.#,"'#,2#Zm#,m#12#m!1-m1A*-m4#0#+-1m#*m$0%+#,2-m+0!"-m.0m
#1#m!,*m^'+9%#,#1m#,m *,!-m7m,#%0-mm*m"#0#!&_Ym
m
,m4#8m'"#,2'$'!"-1m*-1m!00'*#1m/3#m!-,2#,>,m3,m"'%#12'A,m1#m.0-!#"'Am.0'+#0-mm*m
'"#,2'$'!!'A,m"#*m.-!'**-m"#m*m.*!m+#8!*m7m.-12#0'-0+#,2#mm'"#,2'$'!0m*m$+'*'mHm
!-,!0#2m /3#m !-,2#,>m *m +32!'A,m "#,20-m "#m *m +#8!*m ^0#!-0"#+-1m /3#m *1m +#8!*1m
#129,m$-0+"-1m.-0m!320-m$+'*'1_Ymm#12#m.0-!#1-m1#m*#m**+m"#!-,4-*3!'A,Ym
0m*m"#!-,4-*3!'A,m1#m#* -0Am3,m.*,2'**m^'%30mHYL_m"A,"#m1#@*,"-m*m.-1'!'A,m
"#*m .--*m +32,2#Zm 1#m .-">,m '"#,2'$'!0m !-,m 1#%30'""m *1m !320-m $+'*'1m Hm /3#m *-m
$-0+ ,Zm%0!'1mm*m#1203!230m7mm*-1m!-*-0#1m"#m*m+'1+Ym
,m4#8m'"#,2'$'!"1m*1m!320-m$+'*'1m1#m0#.#2>m#*m.0-2-!-*-m^"#1"#m*m.0'+#0mZm
-.O Zm $-0+!'A,m "#m &#2#0-"B.*#6Zm "'%#12'A,Zm .30'$'!!'A,Zm 7m %#*m "#m !0'*+'"_m
.0m.-"#0m'"#,2'$'!0m!39*m"#m*1m!320-m$+'*'1mHm#0m*m/3#m!-,2#,>m*m+32!'A,Ymm
m
m
(&30 T CMG[m *,2'**m "'1#@"m .0m -.2'+'80m *m "#!-,4-*3!'A,Ym 0'+#0-m 1#m +0!m *m .-1'!'A,m #,m *m .*!m
"-,"#m 1#m &m "#2#!2"-m #*m .--*m +32,2#Ym + '<,m 1#m !-*-0#09m !-,m #*m !-*-0m !-00#1.-,"'#,2#m m *m .*!m .--*m
"A,"#m&#+-1m#,!-,20"-m#*m+32,2#m^#,m#*m#(#+.*-m*m.-1'!'A,m"#*m+32,2#m"#2#!2"-m#1mJm"#m*m.*!m.--*mLm
^!-*-0m $3!1'__Ym -12#0'-0+#,2#m 1#m .0-!#"#m m '"#,2'$'!0m *m .*!m "'*3!'A,m "#m *m /3#m .0-4'#,#m #*m +32,2#[m
"#1.*89,"-,-1mm*m"#0#!&m^$*#!&mGm%0'1_m"#1"#m,3#120-m#(#+.*-m^JZm$3!1'_m2#,#+-1m*m$'*m"#2#0+',"m7m
,-1m"#2#,"0#+-1m#,m*m!-*3+,m"#*m+'1+-m!-*-0mYm#m#12#m+-"-m.-"0#+-1m'"#,2'$'!0m*m.*!m"'*3!'A,m"-,"#m
1#m#,!3#,20m#*m+32,2#m^#,m#*m#(#+.*-m*m.*!m"'*3!'A,m#1m*mHHm^HH__Ym,m4#8m'"#,2'$'!"m*m.*!m"'*3!'A,m
#1m,#!#10'-m'"#,2'$'!0m*m.-1'!'A,m"#m*1m!320-m$+'*'1m/3#m!-+.-,#,m#*m.--*m#,m*m.*!m"'*3!'A,Ym0m#**-Zm
32'*'8+-1m *m $*#!&m Hm 7m ,-1m "#1.*8+-1m m *m '8/3'#0"m "#1"#m *m *-!*'8!'A,m "#m ,3#120-m +32,2#Ym
,+#"'2+#,2#m1 #+-1m/3#m*1m$+'*'1m/3#m!-+.-,#,m#*m.--*m+32,2#m#129,m#,m*1m.-1'!'-,#1m^jjj_m
^%0!'1mm*m$*#!&mH_m"#m*m!-*3+,mJm"#m*m.*!m"#m"'*3!'A,mHHYm
T
T
T
MN
Detección e identificación de los mutantes
.,#&O3O'6.))-
CMCMHTTTTTTT
0m '"#,2'$'!0m #*m !+ '-m "#m 1#m .0-"3!'"-m .-0m #*m m &'8-m $*2m 1#!3#,!'0m *1m
$+'*'1mHm+32,2#1Ym0m#**-m1#m+.*'$'!Am+#"',2#m-.O-Om"#m*1m$+'*'1m
+32,2#1m"#m!"m%#,m,*'8"-Ymm+.*'$'!!'A,m1#m!-+.0- Am+#"',2#m%#*m"#m%0-1m
7m #*m $0%+#,2-m 1#m .30'$'!Am +#"',2#m #.&"#6Ym 0m *m 1#!3#,!'!'A,m 1#m 32'*'8Am #*m
.0-2-!-*-m"#1!0'2-m#,m#*m.02"-m,2#0'-0m^HYHYGYH_Ym
,m 4#8m - 2#,'"1m *1m 1#!3#,!'1m 1#m ,*'80-,m !-,m #*m .0-%0+m +/(",O >EAm 7m 1#m
31!Am #,m #*m !0-+2-%0+m *m +32!'A,m #,m $-0+m "#m "- *#m .'!-m ^7m /3#m !"m $+'*'m
!-,2'#,#m 1m "#m GFm ',"'4'"3-1m 7m #,m !-,1#!3#,!'m 1#m 202m "#m 3,m +#8!*m "#m *#*-1m
+32,2#1m 7m 1'*4#120#1_Ym ,m 4#8m "#2#!2"m *m +32!'A,m 1#m ,*'8Am !-,m #*m +'1+-m
.0-%0+m1'm*m+32!'A,m#12 m*-!*'8"m#,m8-,m',20A,'!m-m#6A,'!m7m#,m#12#mB*2'+-m
!1-m 1'm !-+.-02 m 3,m !+ '-m "#m +',-9!'"-Ym 0m #**-Zm 1#m 20"3(-m *m 1#!3#,!'m
- 2#,'"m"#*m+32,2#m7m1#m!-+.0Am!-,m*m1#!3#,!'m"#m*m.0-2#>,m-0'%',*Ym1>m1#m.3"-m
4#0'$'!0m 1'm & >m 3,m !+ '-m "#m +',-9!'"-m 7m !A+-m "#m "0912'!-m .-">m 1#0m #*m !+ '-m
+#"',2#m,9*'1'1m!-,m#*m.0-%0+mYm
m
CMCMIT T T T T T T T T T
TT
12m.02#m"#*m.0-2-!-*-m1A*-m.3"-m0#*'801#m!-,m*-1m+32,2#1m"#m*m'2-#,-m"#12301Ym
',m#+ 0%-Zm20 (-1m.-12#0'-0#1m0#*'8"-1m.-0m-20-1m!-* -0"-0#1m"#*m* -02-0'-m&,m
.#0+'2'"-m/3#m2#,%+-1m"2-1m"#m1#%0#%!'A,m"#m-20-1m+32,2#1m"#*m%#,m>m^<12-1m1#m
.0#1#,20,m.-12#0'-0+#,2#m#,m#*m.02"-mJYHYH_Ym
,m 4#8m '"#,2'$'!"m *m $+'*'m Hm +32,2#m 1#m %#0+',0-,m HKm 1#+'**1m "#m *m +'1+m
$+'*'m ^.0-.-0!'-,"1m .-0m jZm *#,!'Zm "-,"#m 1#m &**,m *+!#,"1_Ym
121m 1#m %#0+',0-,m .0m - 1#040m 1#%0#%!'A,m "#m *m +32!'A,m 7m *%B,m $#,-2'.-Ym ,m
#12"'-m"#m.*9,23*m1#m.0-!#"'Amm&!#0m3,m#620!!'A,m"#mm"#m*1m.*,21m+32,2#1m
.0m*mm1#%B,m#*m.0-2-!-*-m"#1!0'2-m#,m#*m.02"-mHYGYIYHm.0m.-"#0m1#!3#,!'0m7m
,*'80m ^1#%B,m .02"-m HYHYGYH_m *m 0#%'A,m "-,"#m 1#m #,!-,20 m *m +32!'A,Zm 7m "#m #12#m
+-"-m !-+.0- 0m /3#m & >m 1#%0#%!'A,m "#*m *#*-m +32,2#m #m '"#,2'$'!0m 3,m ',"'4'"3-m
!-,m#*m*#*-m+32,2#m#,m&-+-8'%-1'1Ymm
m
MO
.,#&O3O'6.))-
EcoTILLING
CMDTT
T
CMDMBTTTT
0m#*m!-m1#m.0#.0Am#*mm"#mGGIm40'#""#1m"#m+#*A,m/3#m1#m0#!' '#0-,m
"#*m ,!-m "#m %#0+-.*1+m "#*m m ^,#6-m Y(_Ym #m !"m 3,-m "#m #**-1m 1#m &'8-m *m
!3,2'$'!!'A,m +#"',2#m ,-"0-.m ^(),)*LO *.,)*").)'.,O N;E:::_Ym ,m 4#8m
"#2#0+',"m *m !-,!#,20!'A,m "#m m "#m !"m 40'#""Zm 1#m "'*37Am m KFm ,%ix*m 7m 1#m
@"'Am #*m 4-*3+#,m "#!3"-m "#m m "#m LHjGGIm ^m "#m 0#$#0#,!'_m .0m - 2#,#0m
3,m+#8!*m#/3'+-*0m#,m*m/3#m+ -1m1m#1234'#0,mmKFm,%ix*Ym
#m"'1#@Am3,m.*!m"-,"#m!"m.-!'**-m!-,2#,>m*m+#8!*m"#m*1m"'$#0#,2#1m40'#""#1m
"#m+#*A,m!-,m#*mm"#m0#$#0#,!'Ym0m"#2#0+',0m*1m"'$#0#,!'1m*<*'!1m^&.*-2'.-1_m
#,20#m*1m40'#""#1m1#m0#*'8Am#*m.0-2-!-*-m"#mm!-,m*-1m%#,#1m>m7m;Em,m
*-1m %#*#1m "#m !0'*+'"m 7m *1m 1#!3#,!'!'-,#1m .-12#0'-0#1m 1#m .3"'#0-,m '"#,2'$'!0m *-1m
"'12',2-1m&.*-2'.-1m.0m!"m%#,Ym
m
CMDMCTTTTT
0m *m "#2#0+',!'A,m "#m *-1m &.*-2'.-1m 1#m 1#*#!!'-,0-,m *1m 40'#""#1m /3#m
.0#1#,2 ,m &#2#0-"B.*#6m #,m *-1m %#*#1m 7m 1#m 1#!3#,!'0-,m !-,m #*m +'1+-m .0-2-!-*-m
',"'!"-m ,2#0'-0+#,2#Ym ,m 4#8m - 2#,'"1m *1m 1#!3#,!'1m 1#m ,*'80-,m !-,m #*m
.0-%0+m +/(",O >EAm 7m 1#m '"#,2'$'!0-,m *-1m &.*-2'.-1m "'4#0%#,2#1m m *m 40'#""m
0#$#0#,!'Ym ',*+#,2#Zm 1#m !-+.00-,m *-1m "2-1m $#,-2>.'!-1m .0-!#"#,2#1m "#*m m
.0m !"m 40'#""m !-,m *-1m &.*-2'.-1m .0m ',2#,20m "#2#0+',0m 1'm #6'12>m *%3,m
!-00#*!'A,m#,20#m*m$3,!'A,m"#*m%#,mm7m13m$#,-2'.-Ymm
T
NF
,).))&)-O!(,&-
.,#&O3O'6.))-OO
m
TOTTTT
-1m .0-2-!-*-1m "#1!0'2-1m m !-,2',3!'A,m &,m 1'"-m 32'*'8"-1m $0#!3#,2#+#,2#m "30,2#m #*m
.#0>-"-m "#m #12m 2#1'1m 3,/3#m ,-m #129,m "'0#!2+#,2#m 0#*!'-,"-1m !-,m #*m .0-2-!-*-m "#m
Y
TT
CMETTTTT
#m 310-,m ,3#1201m "#m .3*.m "#m $032-m "#m +#*A,m GGGm GKm ">1m 7m JLm ">1m .-12,2#1'1Ym *m
.0-2-!-*-m32'*'8"-m!-00#1.-,"#m*mjm^_Ym
#m2-+0-,mHm%m"#m!"m+3#120m"#m.3*.m7m1#m.3*4#0'80-,m!-,m,'20A%#,-m*>/3'"-m#,m3,m
+-02#0-m .0#4'+#,2#m #12#0'*'8"-Ym ,m 4#8m .3*4#0'8"-Zm #*m +2#0'*m 1#m 20,1$'0'Am m 3,m
23 -m*!-,m"#mKFm+*m7m1#m*#m@"'AmHFm+*m"#mjYm-12#0'-0+#,2#m1#m0#.02'Am
*m+#8!*m#,m.02#1m'%3*#1m#,m23 -1m*!-,m"#mKFm+*Zm7m1#m*#1m@"'Am-20m4#8mm!"m3,-m
FZHm+*m.-0m+*m"#m4-*3+#,m"#mjYm#m%'2Am7m1#m!#,20'$3%Am*m+#8!*mmIYFFFm
0.+m"30,2#mJKm+',32-1mmJEYm-12#0'-0+#,2#m1#m0#!3.#0Am*m$1#m!3-1m7m1#m0#.02'Am
#,m 23 -1m #.#,"-0$m "#m GZKm +*m @"'<,"-*#m m !"m 3,-m FZKm +*m "#m '1-.0-.,-*m
!#,20'$3%9,"-*-1m m NYFFFm 0.+m "30,2#m IFm +',32-1m m JEYm #m #12#m +-"-m 1#m - 234-m 3,m
.#*#2Zm /3#m 1#m "#(Am 1#!0m #,20#m GFjGKm +',32-1m m 2#+.#0230m + '#,2#m .0m
.-12#0'-0+#,2#m 0#131.#,"#0*-m !-,m HFm x*m "#m %3Ym m !-+.0- !'A,m "#m *m !,2'""m 7m
!*'""m "#*m m 1#m &'8-m +#"',2#m %#*m "#m %0-1Ym 0#4'+#,2#m 1#m &'!'#0-,m 40'-1m
*4"-1m"#mm^G_m*m+2#0'*m"#m#*#!20-$-0#1'1m.0m#4'20m*m!!'A,m"#m11Ym
T
CMFTTTTT
T
CMFMBTTP$$$QT
,m4#8m'"#,2'$'!"-m3,m.-*'+-0$'1+-m#,m3,m1#!3#,!'m.3#"#m,*'801#m1'm#,m<*m#6'12#m
*%3,m"',m"#m#,8'+m"#m0#120'!!'A,Ym#m#12#m+-"-Zm1'm1#m0#*'8m*m+.*'$'!!'A,m"#*m
$0%+#,2-m 7m .-12#0'-0+#,2#Zm *m "'%#12'A,Zm #*m .20A,m - 2#,'"-m 1#09m "'12',2-m #,20#m #*m
%#,-2'.-m /3#m 2'#,#m #*m .-*'+-0$'1+-m 7m #*m /3#m ,-Ym 12m "'$#0#,!'m "#m .20A,m 1#m .-"09m
4'13*'80m #,m 3,m %#*m "#m %0-1m ^-,'#!8,7m 7m 313 #*Zm GOOI_Ym m !-,!#,20!'A,m "#m
%0-1m"#m#12-1m%#*#1m"#.#,"#09m"#*m2+@-m#1.#0"-m"#m*-1m$0%+#,2-1m"#m"'%#12'A,Ym
m +#,-0m 2+@-m "#m $0%+#,2-Zm +91m "#,1'""m "# #m 2#,#0m #*m %#*Zm **#%,"-m m 3101#m
,-0+*+#,2#m%#*#1m"#m%0-1m*mHPYm,m4#8m+.*'$'!"1m*1m0#%'-,#1m"#*m$0%+#,2-m
NG
.,#&O3O'6.))-
,).))&)-O!(,&-O
,*'8"-Zm1#m.0-!#"#mm.0#.00m*1m"'%#12'-,#1m!-,m*1m#,8'+1m"#m0#120'!!'A,Ym121m1#m
0#*'80-,m & '23*+#,2#m #,m 3,m 4-*3+#,m 2-2*m "#m HFm x*m /3#m !-,2'#,#,m Hm x*m "#m 2+.A,m
GF6m^!-00#1.-,"'#,2#mm!"m#,8'+m"#m0#120'!!'A,_ZmHYKmm"#m#,8'+ZmGFmx*m"#*m.0-"3!2-m
+.*'$'!"-Ymm+#8!*m1#m',!3 mm*m2#+.#0230mA.2'+m"#m!"m#,8'+m^IMEjLKE_m
"30,2#m Hm &m ^2+ '<,m 40' *#m #,m $3,!'A,m "#m *m #,8'+_Ym *m .0-"3!2-m "#m "'%#12'A,m 1#m
4'13*'8m201m#*#!20-$-0#1'1m#,m%0-1YT
T
CMFMCTTT1TP$$
QT
*m .0-2-!-*-m 31"-m #,m #*m * -02-0'-m .0m *m +.*'$'!!'A,m "#m +0!"-0#1m 1m #12 m
.3#12-m m .3,2-m !-,m +0!(#m 0"'-!2'4-m ^28'0m 7m !-*YZm GOOL_Ym 0m *m 0#!!'A,m "#m
+.*'$'!!'A,m 1#m 31Am #*m 2+.A,m "#m m G6m ^0'1j*m MKm +m .m NYNZm ^J_Hm HFm Zm
5##,jHFmFYFGmP_Zm%*HmIYKm+ZmFYLmxm"#m!"m!# "-0m^"#.#,"'#,"-m"#*m_ZmGLLm
xm "#m ,3!*#A2'"-1m "Zm "Zm "Zm Hm xm "Zm FYGm x*m "#m tIIm j"m ^IYFFFm
'i++-*_ZmHmm+m.-*'+#01m7mLFm+%m"#mm+-*"#m#,m3,m4-*3+#,m2-2*m"#mGKmx*Ym1m
!-,"'!'-,#1m "#m +.*'$'!!'A,m $3#0-,m 3,m .0'+#0m !'!*-m "#m "#1,230*'8!'A,m m OJEm
"30,2#m Gm +',Zm .-12#0'-0+#,2#m IKm !'!*-1m "#m +.*'$'!!'A,m "#m IFm 1m m OJm EZm IFm 1m "#m
&' 0'"!'A,m m KOEm 7m IFm 1m "#m #*-,%!'A,m m MHEm 7m .-0m B*2'+-Zm 3,m #62#,1'A,m $',*m "#m Km
+',mmMHEYm
-12#0'-0+#,2#m m *m +3#120m 1#m *#m @"'Am GFm x*m "#m 2+.A,m "#m !0%m ^OKPm $-0++'"Zm
m GFm +Zm 83*m "#m 0-+-$#,-*m FYFGm PZm 6'*#,!',-*m FYFGP_Ym 1m +3#1201m $3#0-,m
"#1,230*'8"1m "30,2#m GFm +',32-1m m GFFEm .0m .-"#0m .0-!#"#0m m *m 1#.0!'A,m
+#"',2#m#*#!20-$-0#1'1m4#02'!*m#,m!-,"'!'-,#1m"#1,230*'8,2#1m#,m%#*#1m"#m!0'*+'"m
"30,2#mHjIm&-01Ym
#1.3<1m "#m @"'0*#m m *m +3#120m #*m 2+.A,m "#m !0%m 7m .*'!0*#m #*m .0-!#1-m "#m
"#1,230*'8!'A,Zm 1#m !0%0-,m Jm x*m "#m *-1m .0-"3!2-1m "#m +.*'$'!!'A,m #,m #*m %#*m "#m
!0'*+'"Ym*m!-,2#,'"-m"#m!0'*+'"m32'*'8"-m.0m*-1m1m#1m& '23*+#,2#m"#m3,m
LPm "#m !0'*+'"m ^GO[Gm !0'*+'"i '1!0'*+'"_Zm 2+.A,m m G6Zm 30#m MYKm Zm FYFKPm
m ^.#013*$2-m "#m +-,'-_m 7m FYGPm m ^ZZaZaj#20+#2&7*#,#"'+',_Ym ,m
4#8m .-*'+#0'8"-m #*m %#*Zm 1#m !*#,2Am &12m *!,80m *-1m KFEm .0m .-12#0'-0+#,2#m !0%0m
*1m+3#1201m7m.*'!0m3,m4-*2(#m"#mLFmm"30,2#mHjIm&-01Ym
#1.3<1m#*m%#*m$3#m20,1$#0'"-mm3,m..#*m&2+,mImZm1#!"-m*m4!>-m7m#6.3#12-m
#,m3,m$'*+m$-2-%09$'!-mm30'6mHm&12m#*m">m1'%3'#,2#Ym
m
T
NH
,).))&)-O!(,&-
.,#&O3O'6.))-OO
CMFMDTTT1TTTTTT
12m 2<!,'!m 1#m 1m #,m #*m .0-2-!-*-m "#*m '2m !-+#0!'*m m 0'1+m 1&-2m ""m
0'+#0m62#,1'-,m'2m^..*'#"m'-1712#+1_Ym-0+*+#,2#m$3#m31"m#,m*-1m!1-1m#,m/3#m
,-m 1#m "#2#!2 m ,',%3,m #,8'+m "#m 0#120'!!'A,m /3#m .3"'#1#m "'$#0#,!'0m + -1m *#*-1m
.0m "'1#@0m 3,m +0!"-0m 2'.-m Ym 0m ','!'0m #*m .0-2-!-*-m "# >m - 2#,#01#m #*m
.0-"3!2-m "#m m !-,m *-1m !# "-0#1m #1.#!>$'!-1m m 7m !-+.0- 0m *m !,2'""m 1>m !-+-m *m
!*'""m "#m *m +.*'$'!!'A,Ym 0m #12#m .0-2-!-*-m #0m ,#!#10'-m "'1#@0m 3,m !# "-0m
',2#0,-m "#m 3,1m HFm . m /3#m 2#0+',0m *m .-1'!'A,m Iam (312-m ,2#1m "#*m ,3!*#A2'"-m
.-*'+A0$'!-Ymm
#m 2-+0-,m KFm ,%m "#*m .0-"3!2-m +.*'$'!"-m 7m 1#m *#m @"'#0-,m Hm x*m "#m *m #,8'+m 6-j
jm^Zm*#4#*,"Zm&'-_m.0m',!3 0m*m+#8!*m"30,2#mGKm+',32-1mmIMmEYmm12m
#,8'+m !-,2'#,#m 3,m #6-,3!*#1m m 7m 3,m $-1$21m *!*',m "#m %+ m /3#m 1-,m
&'"0-*>2'!1m 7m 1#m 32'*'8,m .0m #*'+',0m *-1m 0#12-1m "#m "1m 7m !# "-0#1m /3#m /3#",m
"#1.3<1m "#m 0#*'80m *m Zm "#m +-"-m /3#m ,-m .3#",m ',2#0$#0'0m #,m *-1m .1-1m 1'%3'#,2#1m
"#*m.0-2-!-*-Ym-12#0'-0+#,2#m1#m',!2'4Am*m#,8'+mmNFmEm"30,2#mGKm+',32-1Ymm
#m#12m0#!!'A,m1#m32'*'80-,mHKm,%m!-+-m+-*"#m.0m*m#62#,1'A,Zm(3,2+#,2#m!-,mHYKm
x*m "#m ,1&-2m #"7m #!2'-,m '6m 7m Hm .+-*m "#*m !# "-0m ',2#0,-m .0#4'+#,2#m
"'1#@"-Zm#,m3,m4-*3+#,m2-2*m"#mKmx*Ymm0#!!'A,m"#m#62#,1'A,m!-,1'12'Am#,mHKm!'!*-1m"#m
3,m.0'+#0m"#1,230*'8!'A,mmOLEm"30,2#mGFm1Zm*m&' 0'"!'A,m"#*m!# "-0m',2#0,-mm
KFEm"30,2#mIFm1m7m*m#62#,1'A,mmLFEm"30,2#mIFm1Ym',*+#,2#m1#m+,234-m*m0#!!'A,m
mJmEYm-12#0'-0+#,2#m1#m202Am*m0#!!'A,m!-,mGmm"#m$-1$21m',2#12',*m -4',m^-!&#_m
mIMEm"30,2#mGm&Zm.0m"#%0"0m#*m!# "-0m',2#0,-m/3#m.3"'#0m/3#"0m#,m#6!#1-Ym#m
',!2'4Am*m#,8'+m**#4,"-m*m0#!!'A,mmMKEm"30,2#mGKm+',Ym3#%-m1#m@"'AmKFFm
!-+-m +0!"-0m "#m .#1-m +-*#!3*0Ym *m !# "-0m #62#,"'"-m 1#m 4'13*'8Am #,m 3,m
1#!3#,!'"-0mm0'1+mIGFFm^..*'#"m'-1712#+1_m#,m*1m!-,"'!'-,#1m0#!-+#,""1m#,m
#*m.0-2-!-*-m"#m1&-2Ym
m
CMGT
TTT
0m #*m !*-,(#m "#m m #,m .*91+'"-m 1#m 32'*'80-,m *1m !#.1m "#m EO )&#m m GFOm
!-+.#2#,2#1m .-0m !&-/3#m 2<0+'!-m /3#m 4#,>,m "(3,21m *m '2m "#*m 4#!2-0m "#m !*-,(#m
.jm b17m #!2-0m 712#+cm ^0-+#%_Ym 0m ','!'0m #*m !*-,(#m 1#m +.*'$'!Am #*m
$0%+#,2-m#,m!3#12'A,m+#"',2#mYm-1m!# "-0#1m32'*'8"-1m$3#0-,m#,m!"m!1-m*-1m
#1.#!>$'!-1m"#m*m0#%'A,mm+.*'$'!0Ym-12#0'-0+#,2#m1#m234-m/3#m.30'$'!0m#*m$0%+#,2-m
"#m m +#"',2#m #*m '2m !-+#0!'*m -!&#m ^#!"O/,OO,)/.O/,# #.#)(O#._Ym ,m
NI
.,#&O3O'6.))-
,).))&)-O!(,&-O
4#8m &#!&m *m !-+.0- !'A,m "#m *m !*'""m 7m !,2'""m "#*m .0-"3!2-m +#"',2#m %#*m "#m
%0-1Zm 1#m 0#*'8Am *m *'%!'A,m 2*m 7m !-+-m ',"'!m #*m $ 0'!,2#m "#*m '2m .jm 17m
#!2-0m712#+mYm-12#0'-0+#,2#m1#m0#*'8Am*m20,1$-0+!'A,m#,m*1m!<*3*1m!-+.#2#,2#1m
mGFOm/3#m2+ '<,m',!*3>m#*m'2m!-+#0!'*Ym
CMGMBT T T T T T T
T
,20#mGjImx*m"#m*m0#!!'A,m"#m*'%!'A,m$3#m@"'"mmKFmx*m"#m!<*3*1m!-+.#2#,2#1m/3#m
#1234'#0-,m.0#4'+#,2#m',!3 "1m#,m&'#*-m"30,2#mIFm+',Ym*m!&-/3#m2<0+'!-m1#m0#*'8Am
mJHEm"30,2#m.0-6'+"+#,2#mKFm1m7m09.'"+#,2#m1#m',!3 Am#,m&'#*-m"30,2#mHm+',Ym
3#%-m 1#m 20,1$'0'#0-,m *1m !<*3*1m m 3,m 23 -m #.#,"-0$m !-,m OKFm x*m "#m m ^GPm !2-j
20'.2-,ZmFYKmPm#620!2-m"#m*#4"30ZmFYKmPm*m.mMYF_m.0m',!3 0*1m"30,2#mGm&mm
IMEm7m!-,m3,m%'2!'A,m"#mHFFm0.+Ym
1m!<*3*1m20,1$-0+"1m$3#0-,m1#*#!!'-,"1m#,m.*!1mm%0m^ZmGYKmPm !2-j%0_m
!-,m,2' 'A2'!-m"#m0#1'12#,!'m+.'!'*',m^KFmx%i+_Zmj*m^Kj 0-+-jJj!&*-0-jIj',"-*'*jm
uj%*!2-1'"1_m ^HFm ,%i+_m #m m ^'1-.0-.'*2'-jm ujm j%*!2-1'"1_m ^JFm x%i+_Ym 1m
.*!1m 1#m "#(0-,m ',!3 0m 2-"m 3,m ,-!&#m m IMEm .0m .-"#0m 1#*#!!'-,0m *1m !<*3*1m
20,1$-0+"1Ym
m
CMGMCTTTTTPQT
0m *m .30'$'!!'A,m "#*m m .*1+>"'!-m 1#m 31Am #*m '2m '%#,hm ','.0#.m ^'%#,_Zm
1"-m#,m*m*'1'1m*!*',m"#m*1m!<*3*1m !2#0',1m^'0, -+m7m-*7ZmGOMO_m!-,m*%3,m
+-"'$'!!'A,Ymm#m32'*'80-,mGZKm+m"#m!3*2'4-m !2#0',-m/3#m1#m& >m!0#!'"-m"30,2#m
2-"m*m,-!&#m,2#0'-0mmIMEYm-12#0'-0+#,2#Zm1#m!#,20'$3%AmmNYFFFm0.+m"30,2#mIm+',m
"#m +-"-m /3#m #*m .0#!'.'2"-m $3#m 0#131.#,"'"-m !-,m *m 1-*3!'A,m m (3,2-m !-,m 1m ^GFFm
x%i+_Ym -12#0'-0+#,2#m 1#m @"'#0-,m JFFm x*m "#m *m 1-*3!'A,m m ^/3#m .0-"3!#m *m *'1'1m
!#*3*0_m 7m 1#m ',!3 Am #,m &'#*-m "30,2#m Km +',m !-,m 3,m 0#4#m %'2!'A,m .0m ,-m "@0m #*m
m .*1+>"'!-Ym *m @"'0m IFFm x*m "#m 1-*3!'A,m m 1#m ,#320*'8Am *m m *'1'1m ^.3#12-m /3#m
!-,2'#,#m3,m*2m!-,!#,20!'A,m"#m1*#1m7m#121m.#0+'2#,m*m0#,230*'8!'A,m"#*mm
.*1+>"'!-m7m13m#12 '*'8!'A,m#,m*m1-*3!'A,_Ym#1.3<1m3,m!#,20'$3%!'A,mmGIYFFFm0.+m
"30,2#m Mm +',m m Jm Em .#0+'2'Am *m .0#!'.'2!'A,m "#m *-1m 0#12-1m !#*3*0#1Ym *m m
.*1+>"'!-m !-,2#,'"-m #,m #*m 1- 0#,",2#m 1#m 20,1$'0'Am m 3,m 23 -m ,3#4-m #4'2,"-m #*m
.0#!'.'2"-Ym#m*#m@"'Am3,m4-*3+#,m"#m$#,-*j!*-0-$-0+-m7m1#m!#,20'$3%Am"30,2#mKm+',m
m GIYFFFm 0.+Ym #m 0#!3.#0Am *m $1#m !3-1m @"'<,"-*#m KFFm x*m "#m '1-.0-.,-*m 7m
NJ
,).))&)-O!(,&-
.,#&O3O'6.))-OO
+#8!*9,"-*-m '#,m.-0m',4#01'A,Ym#m!#,20'$3%AmGFm+',mmGIYFFFm0.+m7m1#m*'+.'Am#*m.#*#2m
- 2#,'"-Ym ',*+#,2#m 1#m *4Am #*m .#*#2m !-,m #2,-*m *m MFPm !#,20'$3%9,"-*-m 2+ '<,m
"30,2#mKm+',mmGIYFFFm0.+Ym3#%-m1#m"#(Am1#!0m#*m.#*#2m7m1#m0#131.#,"'Am#,mKFmx*m"#m
%3m"#1'-,'8"m#12<0'*m- 2#,'#,"-m3,m0#,"'+'#,2-m"#mGjJmx%m"#mm.-0mGZKm+m"#m
!3*2'4-Ym
m
T
NK
)(-.,/#9(OO/(O*&. ),'OO
Resultados
m
m
NL
m
)(-.,/#9(OO/(O*&. ),'OO
Resultados
m
DMTT
T
T
DMBT T T T T T T T
Tm
30,2#m *-1m B*2'+-1m @-1m *m 2<!,'!m "#*m m 1#m &m 31"-m +.*'+#,2#m .0m #*m
.-7-m "#m *m +#(-0m %#,<2'!m #,m .*,21m ^,%Zm HFFL_Zm 3,/3#m #,m +#*A,m 2-"4>m ,-m 1#m
& >m "#100-**"-m ,',%3,m .- *!'A,m "#m +32,2#1Ym +.0#11m .0'4"1m !-+-m '#&&-O
#.9m1#m& >,m+-120"-m',2#0#1"1m#,m73"0mm"#100-**0m&#00+'#,21m/3#m73"0,m
m -.2'+'80m *m 1#*#!!'A,m 7m +#(-0m "#m #12m #1.#!'#Ym *m "#100-**-m "#m 3,m .*2$-0+m "#m
+32,2#1m0#.0#1#,20>m3,m!-*#!!'A,m"#m',"'4'"3-1m!-,m,3#4-1m*#*-1m/3#m.-"0>,Zm#,m
*%3,-1m!1-1Zm.0-.-0!'-,0m,3#4-1m!0!2#0#1m"#m',2#0<1m.0m*m#1.#!'#Ymm/3#m/3>m1#m
"#1!0' #m #1m *m .0'+#0m .- *!'A,m "#m m #* -0"m #,m //'#-O '&)m ^3,/3#m
"30,2#m*-1m!320-m@-1m"#m#12#m20 (-m7m1#m&m- 2#,'"-m-20m^&+,'j0"1m7m!-*YZm
HFGF__Zm /3#m &m 0#/3#0'"-m 3,m *0%-m .0-!#1-m "#m .3#12m m .3,2-m 7m -.2'+'8!'A,m "#*m
.0-2-!-*-Ym 12#m 20 (-m 1#m #,!3#,20m #,+0!"-m "#,20-m "#*m .0-7#!2-m m
^HFGIjHFHIMZm HFFJjHFFM_m /3#m 2#,>m !-+-m - (#2'4-m "#100-**0m &#00+'#,21m
%#,A+'!1m.0m#*m+#*A,m7m/3#m!-,2Am!-,m*m!-* -0!'A,m"#m*m#+.0#1m'#&&-O#.9m7m#*m
m^,12'232-m"#m-,1#04!'A,m7m#(-0m"#m*m%0-"'4#01'""m*#,!',_m.0m*m
.3#12mm.3,2-m"#m*m.*2$-0+m"#mYm
m
DMBMBTTTTTTT
DMBMBMBTTTTTTTTGCSBBDT
0m"#100-**0m*m.- *!'A,m"#mm#,m+#*A,m1#m"#!'"'Am310m*m*>,#m.30m"- *#m
&.*-'"#m ^_m "#m +#*A,m 2'.-m b'#*m "#m 1.-cm 40'#""m ()),/-m LHjGGIYm m #+.0#1m
'#&&-O#.9O$3#m*m#,!0%"m"#m13 +','1200m*1m1#+'**1m"#m#12m*>,#m.0m%#,#00m*m
.- *!'A,m"#mYm
0m !-00- -00m /3#m *m *>,#m #0m 0#*+#,2#m &-+-!'%-2Zm 1#m ,*'8Am !-,m 3,m 2#0>m "#m
+'!0-12<*'2#1m ^1_m 2,2-m *m *>,#m -0'%',*m LHjGGIm !A+-m *1m*>,#1m .0#,2*#1m 31"1m
$0#!3#,2#+#,2#m#,m#*m* -02-0'-Zm*m*>,#mb'#*m"#m.-cmGGGm7m*m!!#1'A,m!-0#,mm
GLGIMKYm ,*'8,"-m Hm 1m .-0m %03.-m "#m *'%+#,2-m ^ *m IYGm 7m '%30m IYG_m *-1m
0#13*2"-1m +-1200-,m /3#m #*m %#,-2'.-m "#m *m *>,#m 31"m #0m '%3*m /3#m #*m %#,-2'.-m "#m
NM
)(-.,/#9(OO/(O*&. ),'OO
Resultados
GGGZm 7m "'12',2-m "#*m m 7m m 13m 4#8Zm .3"'+-1m !-,$'0+0m /3#m #0m &-+-!'%-2m .0m *-1m HJm
1m#,17"-1Ym
m
T
!+ TDMB[m#1!0'.!'A,m"#m*-1m1m32'*'8"-1m.0m*m!-+.0- !'A,m"#m*m&-+-!'%-1'1m"#m*m*>,#mLHjGGIYmm
m
-+ 0#m
+0!"-0m
m
m
NKm
NKm
m
Jm
Jm
m
IOm
IOm
m
LNm
LNm
m
IFm
IFm
m
GHKm
GHKm
m
GFNm
GFNm
m
Lm
Lm
m
KHm
KHm
m
Hm
Hm
m
GNm
GNm
m
JGm
JGm
m
m
NN
m
m
# "-0m"'0#!2-i0#4#01-m
#$#0#,!'m
iLHj
GGIm
m
#0,9,"#8j
'*4m7m!-*YZm
HFFNm
GFMm
GGFgm
-,8*-m7m!-*YZm
HFFKm
GHLm
GIFm
-m.3 *'!"-m
GJFm
GLFm
-,8*-m7m!-*YZm
HFFKm
GNFm
GOKm
-,8*-m7m!-*YZm
HFFKm
GMNm
GKFgm
#0,9,"#8j
'*4m7m!-*YZm
HFFNm
GIKm
GGFgm
#0,9,"#8mj
'*4m7m!-*YZm
HFFNm
GGIm
GHFgm
-,8*-m7m!-*YZm
HFFKm
GJFm
GKFm
-,8*-m7m!-*YZm
HFFKm
GHFm
GJFm
-,8*-m7m!-*YZm
HFFKm
GLKm
GMKm
-,8*-m7m!-*YZm
HFFKm
GJFm
HGFm
-,8*-m7m!-*YZm
HFFKm
GHFm
GJKm
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
+@-m*#*-1m
^. _m
m
m
m
)(-.,/#9(OO/(O*&. ),'OO
m
MKm
MKm
m
GMHm
GMHm
m
Hm
Hm
m
KKm
KKm
m
JMm
JMm
m
HHm
HHm
m
MGm
MGm
m
Nm
Nm
m
LIm
LIm
m
NOm
NOm
m
HOm
HOm
m
IIm
IIm
Resultados
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
-,8*-m7m!-*YZm
HFFKm
GKFm
GMKm
#0,9,"#8j
'*4m7m!-*YZm
HFFNm
GGGm
GIFgm
-,8*-m7m!-*YZm
HFFKm
GKHm
GJFgm
#0,9,"#8j
'*4m7m!-*YZm
HFFNm
GGGm
GHFgm
,',j-*#%m7m
!-*YZmHFFFm
GKJm
GJKgm
-,8*-m7m!-*YZm
HFFKm
GLLm
GMFm
-,8*-m7m!-*YZm
HFFKm
GLKm
HFKm
-,8*-m7m!-*YZm
HFFKm
GJFm
GKKm
#0,9,"#8j
'*4m7m!-*YZm
HFFNm
GFFm
GIFgm
-,8*-m7m!-*YZm
HFFKm
GINm
GMKm
-,8*-m7m!-*YZm
HFFKm
GIMm
GHFgm
-,8*-m7m!-*YZm
HFFKm
GGFm
GHKm
m
[email protected]'+"-m"#2#0+',"-m+#"',2#m4'13*'8!'A,m"#m*-1m%#*#1Ymm
m
m
m
m
NO
Resultados
)(-.,/#9(OO/(O*&. ),'OO
m
m
(&30 T DMB[m -1'!'A,m "#m *-1m 1m ,*'8"-1m #,m *m *>,#m LHjGGIm #,m !"m %03.-m "#m *'%+'#,2-m "#*m +.m
%#,<2'!-m"#m+#*A,mm^#0,9,"#8j'*4m7m!-*YZmHFFN_Ymm
DMBMBMCTTTTTTTTT
,-m "#m *-1m - (#2'4-1m +91m '+.-02,2#1m .0m "#100-**0m 3,m .- *!'A,m "#m m
#$#!2'4m#1m/3#m2#,%m3,m#*#4"m21m"#m+32!'A,Ym0m#**-Zm&!#m$*2m#12 *#!#0m!39*m
#1m#*m0,%-m"#m"-1'1m"#m+329%#,-m+91m"#!3"-m.0m- 2#,#0m3,m*2m21m"#m+32!'A,m
OF
)(-.,/#9(OO/(O*&. ),'OO
Resultados
!-+.2' *#m!-,m3,m 3#,m21m"#m13.#04'4#,!'m7m$#02'*'""m"#m*1m.*,21m^',)*#0m7m
!-*YZm HFFK_Ym ,m ,3#120-m * -02-0'-m 1#m 0#*'8Am 3,m #6.#0'+#,2-m .0#4'-m *m "#100-**-m "#m *m
.- *!'A,m "#m m .0m .-"#0m #12 *#!#0m #*m 0,%-m "#m "-1'1m +91m "#!3"-Ym 12#m
!-,1'12'Am #,m +320m 2,"1m "#m GFFm 1#+'**1m !-,m "'12',21m !-,!#,20!'-,#1m "#m m m ^FYGZm
FYKZmGYFZmGZKm7mHYFmP_m7m- 1#040m*m21m"#m%#0+',!'A,m*m! -m"#m"-1m1#+,1m^'%30m
IYH_Ym *m .0'+#0m #$#!2-m "#12! *#m .3"-m - 1#0401#m #,m *1m .*,21m +32"1m m 3,m
!-,!#,20!'A,m"#mFYKmPm"#mm7m/3#m3,mHHPm"#m*1m1#+'**1m,-m1#m"#100-**0-,m!-,m
,-0+*'""Ym 12#m 4*-0m 3+#,2 m &12m 3,m KFPm #,m *1m .*,21m 202"1m !-,m "-1'1m m
.02'0m"#*mGPm"#mYm-0m*-m2,2-Zm#12#m0#13*2"-m',"'! m/3#m!-,m0,%-1m13.#0'-0#1mm
FYKPm"#mm*m%#0+',!'A,m#,m*mFm"'1+',3>m!-,1'"#0 *#+#,2#Ym
,m!3,2-mm*m%#0+',!'A,m"#m*m%#,#0!'A,mHm1#m- 1#04Am3,m.#/3#@m"'1+',3!'A,m
#,m*m"-1'1m"#mFYKPZm#,m*m!3*m%#0+',0-,m#*mOIYKGP "#m*1m1#+'**1Zm+'#,201m/3#m#,m*1m
"-1'1m *m GPm 7m GYKPm *m "'1+',3!'A,m "#m *m %#0+',!'A,m $3#m *%-m +7-0m - 2#,'#,"-m
%#0+',!'-,#1m"#*mNHYMIm7mMKYOJPZm0#1.#!2'4+#,2#Ymm
m
m
m
(&30 T DMC[m -0!#,2(#1m "#m %#0+',!'A,m #,m 2,"1m "#m GFFm 1#+'**1m Fm 202"1m !-,m "'12',2-1m .-0!#,2(#1m "#m
m ^"#m FPm m HP_Zm "-1m 1#+,1m "#1.3<1m "#*m 202+'#,2-m 1#%B,m *m - 1#04!'A,m "#*m !0#!'+'#,2-m "#m *m
%#,#0!'A,m GYm ^Sm 1#+'**1m ,-m %#0+',"1Zm Sm 1A*-m "#100-**-m 0>8Zm Sm !-2'*#"-,#1m ,-0+*#1m 7m
"#100-**-m 0>8Zm Sm "#100-**-m ,-0+*_Ym 123"'-m 0#*'8"-m .-0m ,2-,'-m -,$-02#Zm ,m "30"-m 7m -0"'m
0!'m1m"30,2#m#*m@-mHFFJm^_Ym
m
',*+#,2#m 1#m "#!'"'Am 310m *1m "-1'1m "#m FZKPZm GPm 7m GZKFPm ^ *m IYH_m .0m +320m *1m
1#+'**1m 7m /3#m <121m .0-"3!>,m 3,-1m ,'4#*#1m !#.2 *#1m "#m 13.#04'4#,!'m #,m *m Gm
OG
)(-.,/#9(OO/(O*&. ),'OO
Resultados
^%#0+',!'A,m13.#0'-0m*mJFPm^'%30mIYH__Zm$#02'*'""m#,m*mGm7m4'%-0m#,m*m%#0+',!'A,m
7m "#100-**-m "#m *m %#,#0!'A,m HYm *m 202+'#,2-m 1#m &'8-m #,m GMYFFFm 1#+'**1Zm .*'!,"-m
"'12',21m "-1'1m "#m Ym 1m 1#+'**1m 202"1m 1#m 0#.02'#0-,m #,20#m *-1m "-1m !#,20-1m
!-* -0"-0#1m^'#&&-O#.9Zm#,m 0#0m"#m0m^0!#*-,_m7mjm^*#,!'__m
.0m 13m !0#!'+'#,2-Ym ,m !"m *3%0m 1#m !0#!'#0-,m *1m .*,21m - 2#,'#,"-m *m %#,#0!'A,m
GZm3,/3#m#,m#*m!1-m"#*mm&3 -m3,m"0912'!m.<0"'"m"#m.*,21m"# '"-mm3,m
#*#4"m ',!'"#,!'m "#m !-*.1-m "#*m +#*A,m #,m #*m @-m HFFKYm 1m .*,21m Gm 1#m
32-$#!3,"0-,m7m1#m- 234'#0-,mIGJFm$032-1Zm*1m1#+'**1m"#m*1m!3*#1Zm1#m.*,20-,m.0m
- 2#,#0m *m %#,#0!'A,m HYm #m !"m 3,-m "#m *-1m $032-1m 1#m .*,20-,m GFm 1#+'**1Zm
- 2#,'#,"-m 1>m *m %#,#0!'A,m HYm ',*+#,2#Zm .0m !"m $+'*'m 1#m !-%'Am 3,m "'1!-m "#m
&-(m "#m !"m 3,m "#m *1m "'#8m .*9,23*1m HZm /3#m 1#m +#8!*0-,m .0m #620#0m #*m Ym *m
,B+#0-m "#m $+'*'1m Hm #,m *1m /3#m 1#m - 234-m m $3#m HILNYm 12-1m 1m $3#0-,m
#,4'"-1m m ,3#120-m * -02-0'-m #,m 0'*1m "-,"#m 1#m 20,1$'0'#0-,m m .*!1m .0m
.-12#0'-0+#,2#m-0%,'80m*1m+#8!*1m/3#m$-0+0>,m*m.- *!'A,m"#mYmm
T
!+ T DMC[m -1'1m "#m m 31"1m .0m +320m *1m 1#+'**1Zm ,B+#0-m "#m .*,21m !-,m $032-1m - 2#,'"-1m #,m *m
%#,#0!'A,mGm7m,B+#0-m"#m$+'*'1mHm!-,mm"'1.-,' *#Ym
m
Dosis EMS
Número de frutos recogidos en las
plantas M1
Número de familias M2 incluidas
en la población de TILLING
0,50%
204
177
1%
2544
1922
1.5%
392
269
TOTAL
3140
2368
m
DMBMBMDTTTTTTTTTTTT
m .- *!'A,m "#m m#129m 0#.0#1#,2"m #,m Mm .*!1m .--*m ^-m +#8!*_m "-,"#m 1#m &,m
+#8!*"-m *-1m 1m "#m *1m HILNm $+'*'1m #,m +#8!*1m "#m !320-m $+'*'1Ym m .- *!'A,m
!-,12m "#m 20#1m 2'.-1m "#m .*!1[m 3,1m !-,m *-1m 1m -0'%',*#1m "#m *1m #620!!'-,#1m .0m
!"m$+'*'mHm^.*!1me2-!)e_Zm-201m!-,m#*mm"'*3'"-mGiGFm^.*!1me'*32'-,e_mm3,m
!-,!#,20!'A,m"#m^Km,%ix*_m7m$',*+#,2#m*1m.*!1m"#m20 (-m/3#m',!*37#,m*m+#8!*m"#m
!320-m $+'*'1Ym 1m +#8!*1m 1#m &,m #* -0"-m !-,m 3,m !0'2#0'-m "#m -0"#,m +37m #120'!2-m
.3#12-m /3#m .-12#0'-0+#,2#m m *m "#2#!!'A,m "#m *-1m +32,2#1m "# #m .-"#01#m '1*0m #m
'"#,2'$'!0m$9!'*+#,2#m*m$+'*'m/3#m!-,2'#,#m*m+32!'A,Ym
OH
)(-.,/#9(OO/(O*&. ),'OO
Resultados
*m-0"#,m"#*mm#,m*1m.*!1m!-00#1.-,"#m*m-0"#,m"#m*1m20#1m2,"1m"#m+32!'A,m"#m
*1m1#+'**1Ymm,-+#,!*230m31"m.0m*1m$+'*'1mHm*m#12 *#!'#0-,m*-1m"-1m!#,20-1m
!-* -0"-0#1m#,m$3,!'A,m"#*m-0"#,m"#m0#!-%'"m"#m*m.*,2m#,m!"m!#,20-Zm1>m2-"1m*1m
$+'*'1m/3#m1#m!0#!'#0-,m#,m'#&&-O#.9m^ 0#0m"#m0_m!-+'#,8,m.-0mbcZm+'#,201m
/3#m*1m/3#m1#m!0#!'#0-,m#,m#*mjm^*#,!'_m!-+'#,8,m.-0mbcYm1m1#+'**1m
"#m2-"1m*1m$+'*'1m#129,m!-00#!2+#,2#m%30""1m7m#2'/3#2"1m#,m#*mjYm
*m*'12"-m7m*m.-1'!'A,m.0m!"m$+'*'mHm#,m*1m.*!1m"#m*m.- *!'A,Zm1#m#,!3#,20,m
"#2**"-1m#,m#*m,#6-mYYm
m
DMBMBMETTTTTT$
"-m /3#m #*m +<2-"-m m ,-m & >m 1'"-m 31"-m .0m "#100-**0m .- *!'-,#1m "#m
+32,2#1m#,m+#*A,Zm#0m,#!#10'm*m-.2'+'8!'A,m"#m*m2<!,'!m#,m,3#120-m* -02-0'-Ymm
0m .-"#0m "#100-**0m A.2'++#,2#m *m .- *!'A,m "#m +32,2#1m 1#m "#!'"'Am 0#*'80m 3,m
#12,!'m #,m #*m %03.-m "#*m 0Ym "#*m #,"&+,#m "#*m jm ^407Zm 0,!'_Ym ,m
3,m .0'+#0m $1#m #,m #*m @-m HFFKm 1#m .0#,"'Am *m 2<!,'!m .0m .-"#0m "#100-**0*m #,m
,3#120-m * -02-0'-Ym m 1#%3,"m $1#m #,m #*m HFFNm 1'04'Am .0m ,*'80m *-1m %#,#1m #,m *1m
"'12',21m .- *!'-,#1m "#m +32,2#1m /3#m 1#m & >,m "#100-**"-m #,m #*m * -02-0'-m "#*m
Ym #m #1#m +-"-m 1#m .0#2#,">m !-+.00*1m 7m .-"#0m "#2#0+',0m *m #$'!'#,!'m "#m *m
.- *!'A,m Em -0m #12#m +-2'4-Zm #,m *-1m .02"-1m 1'%3'#,2#1m 1#m '0,m "#1!0' '#,"-m
+32,2#1m!0!2#0'8"-1mm.02'0m"#m*m.- *!'A,m"#*mm7m"#*mYmm
m
IYGYGYJYGmmmmmm
m .- *!'A,m "#m m "#*m m 2'#,#m *%3,1m "'$#0#,!'1m 0#1.#!2-m m *m .- *!'A,m
"#*m m 7m /3#m !-,12m "#m 40'1m 13 .- *!'-,#1m - 2#,'"1m .-0m "'12',21m #+.0#11m
.0'4"1m "#m 1#+'**1m ^)''Zm m 7m 32'#0_Ym m "'$#0#,!'m "#m *m ,3#120Zm #129m 0#*'8"m
!-,m3,m40'#""m2'.-m",(.#-Em*m,B+#0-m"#m$+'*'1m/3#m*m!-+.-,#,m#1m"#mHJNIm7m
<121m #129,m +32"1m 31,"-m "-1'1m !-+.0#,"'"1m #,20#m Gm 7m IPm "#m Zm .-0m *-m 2,2-m *m
!-,!#,20!'A,m "#m m 1#m 3+#,2Am !-,1'"#0 *#+#,2#m 0#1.#!2-m m *m "-1'1m 31"m #,m *m
.- *!'A,mYm"#+91Zm*1m+#8!*1m#129,m!-+.3#121m.-0mNm$+'*'1mHm+'#,201m/3#m
*1m ,3#1201m !-,2'#,#,m Jm $+'*'1m Hm ^ *m IYI_Ym + '<,m #1m '+.-02,2#m "#12!0m /3#m
.0m+320m*m.- *!'A,m"#*mm1#m310-,m20#1m.0-2-!-*-1m"'12',2-1m#,m*-1m!3*#1m,-m1#m
.0#&'"020-,m *1m 1#+'**1m ,2#1m "#m .*'!0m #*m Zm 3,/3#m *-1m .0-2-!-*-1m 1-,m
!-,$'"#,!'*#1m7m.#02#,#!#,mm!"m3,m"#m*1m#+.0#11m.0'4"1m/3#m*-1m"#100-**AYm1m
'+.-02,2#m 2#,#0m #,m !3#,2m #12#m .3,2-m .3#12-m /3#m .3#"#m ',$*3'0m "#m 3,m +-"-m
"#2#0+',,2#m#,m*m#$'!'#,!'m$',*m"#m+32!'A,m"#m*m.- *!'A,Ym
OI
)(-.,/#9(OO/(O*&. ),'OO
Resultados
!+ TDMD[m *m!-+.02'4m"#m*1m"-1m.- *!'-,#1m,*'8"1m#,m#12#m20 (-m+#"',2#mYmm
m
@T
% ,(+( 1T
CT
KTT
/+(" "(>-T#$+TT
0($# #T
,(+( 1R,$8"+ T
.!+ "(>-T
T
HILNm
FYKZmGZmGYKPm
!-,m.0#&'"02!'A,m"#m
*1m1#+'**1m
'#*m"#m.-m
Jm
.!+ "(>-T
T
HJNIm
GZmGYKZmHPm
1',m.0#&'"02!'A,m"#m*1m
1#+'**1m
&0#,2'1m
Nm
m
IYGYGYJYHmmmmmm
-1m .0-2-!-*-1m "#m m 0#!-+'#,",m #*m 31-m "#m +m -*'+#01m !-,m !2'4'""m
!-00#!2-0m-m"#m*2m$'"#*'""Ym129m"#1!0'2-m/3#m*m+m2'#,#m3,m21m"#m#00-0m"#mGYG6GFjJm
#00-0#1i. m ^0,#1Zm GOOJ_m .-0m #**-m 1#m ','!'0-,m *-1m #6.#0'+#,2-1m !-,m +m .-*'+#011m
!-+#0!'*#1m /3#m !-,2#,>,m !2'4'""m !-00#!2-0m !-+-m *m /m ^-!&#_m - 2#,'"m "#m
3,))/-O /,#)-/-Fm!-,m3,m21m"#m#00-0mGYLm6mGFjLm#00-0#1i. m^3," #0%m7m!-*YZmGOOG_m-m*m
2*(m+O*)&3',-m^-!&#_m!-,m3,m21m"#m#00-0m"#mJYNm6mGFjLZm3,/3#m#*m!-12#mm"#m*1m
0#!!'-,#1m #0m #6!#1'4-Ym #m .0- 0-,m *1m +'1+1m +.*'$'!!'-,#1m !-,m +m 1',m !2'4'""m
!-00#!2-0m 7m 1#m "#2#0+',Am ^+#"',2#m m 7m 1#!3#,!'!'A,_m 1'm 1#m & >,m @"'"-m
#00-0#1m"30,2#m*m+.*'$'!!'A,Zm.3"'#,"-m.0- 0m/3#m,-m#0m1>Ym',*+#,2#Zm1#m"#!'"'Am
/3#m 2-"1m *1m +.*'$'!!'-,#1m 1#m &0>,m !-,m +O 1',m !2'4'""m !-00#!2-0Zm 4#0'$'!,"-m
.-12#0'-0+#,2#m *m +32!'A,m +#"',2#m 1#!3#,!'!'A,Ym 12#m .3,2-m #1m '+.-02,2#m .3#12-m
/3#m0#"3!#m#*m!-12#m"#m*m2<!,'!m!-,1'"#0 *#+#,2#Ym
m
IYGYGYJYImmmmmO
,m &#00+'#,2m ',"'1.#,1 *#m .0m .-,#0m m .3,2-m #*m m #1m "'1.-,#0m "#m 3,m
!-,20-*Ym #m "#!'"'Am 310m 3,m m .0#4'+#,2#m '"#,2'$'!"-m #,m ,3#120-m * -02-0'-Ym ,m
HFFLm'#2-m7m!-*Ym'"#,2'$'!0-,m#*m%#,m (-0O^!-00#1.-,"'#,2#m*m%#,m >m"#m+#*A,_m/3#m
!-,$'#0#m0#1'12#,!'m*m4'031mm^&)(O,).#O*).O#,/-_Ym*m%#,m(-0m!-"'$'!m.0m
3,m $!2-0m "#m ','!'!'A,m "#m *m 20"3!!'A,m "#m .0-2#>,1m 7m 3,m +32!'A,m #,m #12#m %#,m
!-,$'#0#m 0#1'12#,!'m m !'#02-1m 4'031m ^- %*'m 7m 0,2Zm HFFL\m '#2-m 7m !-*YZm HFFK_Ym m
+32!'A,m/3#m"m0#1'12#,!'mmm#1m3,m!+ '-m"#m+',-9!'"-m"#m3,m*#3!',m#,m#*m
%#,-2'.-m0#1'12#,2#mmGLGIMKm^_mm3,m&'12'"',m#,m#*m%#,-2'.-m131!#.2' *#m^GGG_m#,m*m
.-1'!'A,mHHNm"#m*m.0-2#>,Ym12m.-1'!'A,m1#m*-!*'8m#,m#*m#6A,mKZm+91m!-,!0#2+#,2#m#,m
*m .-1'!'A,m LNIm "#*m %#,m ^'#2-m 7m !-*YZm HFFL_Ym #!'"'+-1m 310m #12#m m !-+-m !-,20-*m
.-1'2'4-m .0m .-,#0m m .3,2-m #*m +<2-"-Ym 0m #**-Zm 1#m 1#!3#,!'Am #*m $0%+#,2-m "#*m %#,m
OJ
)(-.,/#9(OO/(O*&. ),'OO
Resultados
>m"-,"#m#129m"#1!0'2m*m+32!'A,m#,m*m*>,#mLHjGGIm7m1#m!-+.0Am!-,m*m1#!3#,!'m
"#m *m *>,#1m GGGm 7m Ym m 1#!3#,!'m "#m LHjGGIm 7m GGGm #,m #12m 0#%'A,m #1m '"<,2'!Ym
1>+'1+-m1#m*-!*'80-,m-20-1m.-*'+-0$'1+-1m#,20#mLHjGGIiGGGm7mm^'%30mIYIm_Ymm
m
T
T
(&30 T DMD[m _m 1/3#+m "#*m $0%+#,2-m 1#!3#,!'"-m "#*m %#,m >m !-,m *1m .-1'!'-,#1m "#m *-1m .-*'+-0$'1+-1m
#,!-,20"-1m#,20#m*1m40'#""#1mLHjGGIm7mYm _m#*m"#mjm3,m4#8m.*'!"-m#*m+<2-"-m"#mmm
"'12',21m+3#1201m"#m1Ym"m.,#*m0#.0#1#,2m*-1m!,*#1mMFFm7mNFFZm0#1.#!2'4+#,2#Ymgmm*',#m
#,m %0'1m "#2#0+',m #*m 2+@-m m .02'0m "#*m !3*m #*m %#*m .#0">m 0#1-*3!'A,m ^7m .-0m *-m 2,2-m *-1m 2+@-1m "#m *-1m
&#2#0-"B.*#6m4'13*'8"-1m#,m#*m%#*m,-m!-',!'"#,m!-,m#*m2+@-m0#*_m!-+-m!-,1#!3#,!'m"#*m3+#,2-m#6!#1'4-m
"#m*m2#+.#0230m"#*m%#*m"#m!0'*+'"Ym#m!-+#,209m#,m"#2**#m#,m#*m2#62-Ymm
m
OK
Resultados
)(-.,/#9(OO/(O*&. ),'OO
m .02#m "#*m m 0#1.-,1 *#m "#m (-0m m NKOiIGFm . m "#m "'12,!'m "#m *-1m !# "-0#1m
;M /&&=Km#,!-,20+-1m3,m',1#0!'A,mm#,m#*m%#,-2'.-mm#,m*m.-1'!'A,mILGiNFNYm
*m2#0!#0m.-*'+-0$'1+-m#,!-,20"-m#0m3,mm/3#m#,m#*m%#,-2'.-mLHjGGIm!-,2#,>m3,m
m+'#,201m/3#m#,m#*m%#,-2'.-mm!-,2#,>m3,mm#,m*m.-1'!'A,mNONiHMGYm#m+-"-m/3#m*m
.*'!0m #*m .0-2-!-*-m "#m m #,m *m +.*'$'!!'A,m "#m #12#m $0%+#,2-m 31,"-m !-+-m
m +-*"#m *m +#8!*m "#m + 1m 40'#""#1m - 2#,>+-1m 3,m .20A,m "#m "'%#12'A,m
!0!2#0>12'!-m#,m*-1m%#*#1m"#m!0'*+'"m^'%30mIYIm _Ymmm
#m310-,m!# "-0#1m7m"'1#@"-1m,2#0'-0+#,2#m.0m+.*'$'!0m*m8-,m"#*mm^'#2-m
7m!-*YZmHFFM_m^(.,)(O;O3O /&&O=K_m7m.-12#0'-0+#,2#m1#m0#*'8Am#*m.0-2-!-*-m40',"-m
"'4#01-1m .09+#20-1m .0m .-"#0m "#2#0+',0m *1m !-,"'!'-,#1m A.2'+1m 7m .-"#0m
#12,"0'80*1Ym12-1m.09+#20-1m$3#0-,m'_m*m!,2'""m"#mm/3#m"# >+-1m@"'0m#,m
*m "'%#12'A,Zm ''_m #*m .-0!#,2(#m "#m "'*3!'A,m "#m *m #,"-,3!*#1Zm '''_m #*m 1'12#+m "#m
.30'$'!!'A,m"#m*m0#!!'A,m"#m"'%#12'A,Zm'4_m*m!-,!#,20!'A,m"#m*m+3#120m^#*m2'#+.-m"#m
!#,20'$3%!'A,m7m*m2#+.#0230m.*'!"m#,m#*m*_m7m4_m#*m+-"-m"#m!0%m#,m#*mj
m ^!-,m .#',#m -m +,3*_Ym ',*+#,2#Zm "#1.3<1m "#m 40'1m .03# 1Zm 1#m #12,"0'80-,m
*1m!-,"'!'-,#1m#,m*1m/3#m#*mm!-,20-*m1#m4#>m!-,m+7-0m0#1-*3!'A,Ym#m"#+-120Am/3#m
*m .30'$'!!'A,m "#m *m 0#!!'A,m "#m "'%#12'A,m #0m ,#!#10'm .3#12-m /3#m 1'm ,-m 1#m 0#*'8 m
.0#!>,m 3,1m ,"1m #,m #*m %#*m "#m jm /3#m ',2#0$#0>,m #,m *m 4'13*'8!'A,m "#m *1m
"'%#12'-,#1Ym 1m !-,"'!'-,#1m A.2'+1m "#m !-,!#,20!'A,m "#m *m +3#120m #,m #*m *O
$3#0-,m "#m JKm +',32-1m m LKEZm .3#12-m /3#m m +#,-0m 2'#+.-m 7m +#,-0m 2#+.#0230m *m
+3#120m /3#" m "#+1'"-m "'*3'"m .0m - 2#,#0m 3,m 4'13*'8!'A,m !-00#!2m #,m #*m j
Ym',*+#,2#Zm1#m!-+.0- Am/3#m#*m+<2-"-m"#m!0%m31,"-m*-1m.#',#1m"#1#!& *#1m
^
OL_m.0-.-0!'-, m3,m4'13*'8!'A,m!-,m+#(-0m0#1-*3!'A,m.0m*m'"#,2'$'!!'A,m"#m
*-1m!00'*#1m/3#m!-,2'#,#,m+32,2#1m1>m!-+-m*m!.!'""m"#m!0%m^OLm+3#1201m.-0m%#*m
+'#,201m/3#m*m!0%m+,3*m1A*-m.#0+'2#m*m!0%m"#mLJm+3#1201_Ym
,m *m '%30mIYIY m.3#"#m4#01#m#*m0#13*2"-m "#m 3,m "#m *1m .03# 1m .0m "#2#0+',0m *-1m
.09+#20-1mA.2'+-1m"#*m.0-2-!-*-m7m!-+.0- 0m*m1#,1' '*'""m"#*m+<2-"-Ym,m#*m!,*m
MFFm 1#m 4'13*'8m *m "'%#12'A,m "#1"#m #*m !# "-0m "'0#!2-m ^;_m &12m #*m .3,2-m "#m *m
"'%#12'A,Zm +'#,201m /3#m #,m #*m !,*m NFFm 1#m 4'13*'8m #*m $0%+#,2-m "#1"#m #*m !# "-0m
0#4#01-m ^ /&&=K_m &12m #*m +'1+-m .3,2-m "#m *m "'%#12'A,m ^'%30m IYI_Ym ,m
!-,1#!3#,!'Zm *-1m 2+@-1m "#m *-1m $0%+#,2-1m "'%#0'"-1m /3#m 1#m 4'13*'8,m #,m + -1m
!,*#1m "# #,m 13+0m *-m +'1+-m /3#m *m 2-2*'""m "#*m 2+@-m "#*m $0%+#,2-m 1',m "'%#0'0Ym
#*m !00'*m Gm *m Nm .3#"#,m 4#01#m *1m "'%#12'-,#1m 0#13*2,2#1m 1#%B,m *-1m "'12',2-1m 1m
#+.*#"-1Ym 1m +#8!*1m /3#m !-,2#,%,m 1m !-,m 1#!3#,!'1m "'12',21m +-1200,m *m
OL
)(-.,/#9(OO/(O*&. ),'OO
Resultados
"'%#12'A,m .0-"3!'"m .-0m ()O ^!00'*#1m "#*m GjJm 7m L_m +'#,201m /3#m *1m +3#1201m /3#m
!-,2#,%,m 1m '%3*#1m #,m 1#!3#,!'m ^M_m -m ,-m 1#,m +#8!*1m "#m 1m ^Km 7m N_m ,-m
+-1200,m"'%#12'A,Ym3#"#m- 1#0401#m/3#m#,m!3*/3'#0m"#m*1m"'*3!'-,#1m&#!&1m#,m*1m
+#8!*1m"#m*-1m+'1+-1m1m^!00'*#1mGjJ_m*m"'%#12'A,m.-">m"#2#!201#m!-,m*m+'1+m
',2#,1'""Ym-0m*-m2,2-m1#m!-+.0- Am/3#m#,m*1m"'12',21m"'*3!'-,#1m"#mmGiGLZmGiNZm
GiJm 7m GiGm ^!00'*#1m GZm HZm Im 7m J_m *-1m &#2#0-"B.*#6m 1#m 4'13*'8 ,m .#0$#!2+#,2#m 7m
.09!2'!+#,2#m!-,m*m+'1+m',2#,1'""Ym1m+3#1201m1-,m+#8!*1m"#m"'12',2-1m1m7m
"#m "'12',2-1m &#2#0-"B.*#6Ym 1m #,"-,3!*#11m ,-m !-02,m 2-2*+#,2#m 2-"-1m *-1m
&#2#0-"B.*#6m 1'+3*29,#+#,2#Ym -0m #12#m +-2'4-m #,m *-1m %#*#1m 1#m .3#"#,m 4'13*'80m *-1m
$0%+#,2-1m"#m*-1m"'12',2-1m2+@-1m"#.#,"'#,"-m"#*m&#2#0-"B.*#6m/3#m&7m"'%#0'"-m
*m#,"-,3!*#1Ym+ '<,m.-0m#**-Zm#,m*-1m%#*#1m1#m.3#"#,m4'13*'80m#*m2+@-m2-2*m"#*m
$0%+#,2-m ,*'8"-m ^.#02#,#!'#,2#m m /3#**1m +3#1201m #,m *1m /3#m ,-m 1#m &m "'%#0'"-m
,',%B,m &#2#0-"B.*#6_m 7m *-1m 2+@-1m "#m !"m &#2#0-"B.*#6m ^.#02#,#!'#,2#1m m /3#**1m
+3#1201m#,m*1m/3#m1#m&m"'%#0'"-m!"m3,-m"#m#**-1_Ymm
20m-.2'+'8!'A,m.-12#0'-0m$3#m!+ '0m#*m+-"#*-m"#mjYm*m,3#4-m+-"#*-mJIFFZm
,-1m.#0+'2'Am+#(-01m'+.-02,2#1[m0#"3!'0m#*m2'#+.-m.0m!-00#0m*1m+3#1201Zm3+#,20m
*m 0#1-*3!'A,m 7m ,*'80m jMFFm 7m NFFm !-,(3,2+#,2#Zm *-m /3#m .#0+'2#m *m '"#,2'$'!!'A,m
.0#!'1m"#m*1m"'%#12'-,#1m7mm*m4#8Zm"#1!020m*-1m02#$!2-1Ym
'#,201m /3#m #,m #*m +-"#*-m ,2'%3-m ^'jm H_m #*m 2'#+.-m "#m #*#!20-$-0#1'1m "30 m
.0-6'+"+#,2#m 1'#2#m &-01Zm !-,m #*m jm JIFFm !-,1#%3'+-1m 0#"3!'0*-m m IjJm &-01m
"#.#,"'#,"-m "#*m $0%+#,2-m m ,*'80Ym "#+91Zm #12-m .#0+'2>m /3#m *-1m %#*#1m 234'#0,m
+91m 0#1-*3!'A,m 7m /3#m !-,m Mm &-01m "#m +'%0!'A,m *m !0'*+'"m 3+#,2 m
#6!#1'4+#,2#m "#m 2#+.#0230m 7Zm #,m !-,1#!3#,!'Zm *m .02#m 13.#0'-0m "#*m %#*m .#0">m
+3!&m0#1-*3!'A,m^'%30mIYI Zm2+@-1m13.#0'-0#1mmJLFm ._Ymm
m
DMBMCT$
01m#*m"#100-**-m7m-.2'+'8!'A,m"#m*m.*2$-0+m"#mm#,m+#*A,m! #m"#12!0m
/3#m #1m '+.-02,2#m 3,m #123"'-m .0#4'-m "#m +32%<,#1'1m .0m - 2#,#0m 3,m #$'!'#,!'m "#m
+32!'A,m"#!3"Ym0m#**-Zm#*m#123"'-m"#*m"#100-**-m"#m*m.*,2m201m*m%#0+',!'A,m
1>m !-+-m #*m "#m *m !.!'""m "#m .0-"3!'0m 1#+'**m Hm 4' *#m #1m +37m '+.-02,2#Ym *m
.0-2-!-*-m "#m +32!'A,m "#m *1m 1#+'**1m "# #m 1#0m -.2'+'8"-m .0#4'+#,2#m ,-m 1A*-m .0m
$'(0m*m"-1'1m"#m+329%#,-m+91m#$'!'#,2#m1',-m2+ '<,m.0m.*'!0*-m"#m3,m+-"-m#$'!8Ym
#%B,m,3#120-1m"2-1m#*m.-0!#,2(#m"#m+329%#,-mA.2'+-m#,m+#*A,m#129m.0- *#+#,2#m
"#,20-m"#*m0,%-m"#*mGZKjHZKPYmm
OM
Resultados
)(-.,/#9(OO/(O*&. ),'OO
,m 4#8m "#100-**"m *m .- *!'A,m #1m ,#!#10'm *m .3#12m m .3,2-m "#*m +<2-"-m "#m
"#2#!!'A,m 1'#,"-m ',"'1.#,1 *#m 3,m !-,20-*m .-1'2'4-m /3#m .#0+'2m -.2'+'80m 2-"-1m *-1m
.1-1m "#*m .0-2-!-*-Zm 1>m !-+-m "'1.-,#0m "#m 3,m 2#!,-*-%>m /3#m .#0+'2m *m "#2#!!'A,m
09.'"m 7m #$'!8Ym ,m #*m !1-m "#m #12#m 20 (-m 1#m "#!'"'Am 310m !-+-m !-,20-*m #*m %#,m >m
.3#12-m /3#m 7m 1#m 1 >m /3#m !-,2#,>m 3,m .-*'+-0$'1+-m #,20#m *-1m %#,-2'.-1m "#*m +#*A,m
GGGm7m#*m+#*A,m!-0#,-mYm3"'+-1m"#2#0+',0m/3#m,-m.0#!#m,#!#10'-m#*m31-m"#m+m
!-,m !2'4'""m !-00#!2-0m 7m /3#m #*m +<2-"-m #1m .0#!'1-m !-,m *m +3#120m "#m m +32,2#m
"'*3'"m&12mGLm4#!#1Ym*m+'1+-m2'#+.-Zm"#+-120+-1m/3#m#*m.1-m"#m*m.30'$'!!'A,m"#m
*m"'%#12'A,m!-,m()m#1m',"'1.#,1 *#m.0m3,m4'13*'8!'A,mA.2'+m"#m*-1m$0%+#,2-1m
#,m*-1m%#*#1m7m/3#m#*mjmJIFFm0',"#m3,m+37m*2m0#1-*3!'A,m7m0#"3!#m#*m2'#+.-m"#m
+'%0!'A,m"#m*1m+3#1201Ymm
m
ON
.#9(OO'/.#)(-O
Resultados
T
m
DMCT T T T T T T T
TTTTTTT
m
*m +<2-"-m m #129m "'1#@"-m .0m 310*-m %#,#0*+#,2#m !A+-m &#00+'#,2m "#m
%#,<2'!m',4#01[m.0'+#0-m1#m#*'%#m3,m%#,m"',m"#*m!3*m/3#0#+-1m#,!-,200m+32,2#1m7m
3,m4#8m#,!-,20"-1m7m#123"'"-1m131m$#,-2'.-1m1#m.3#"#m$-0+3*0m3,m&'.A2#1'1m1- 0#m
#*m#$#!2-m-m*m$3,!'-,*'""m"#*m%#,m,*'8"-Ym-1m%#,#1m"',m#,m#12#m20 (-m&,m1'"-m
1#*#!!'-,"-1m 91'!+#,2#m.-0m13m.-1' *#m',2#0<1m%0-,A+'!-Zm#6!#.2-m#,m#*m!1-m"#*m%#,m
"#m *m m ^'2-#,-m "#12301_m /3#m $3#m #*#%'"-m !-+-m !-,20-*Ym -1m #$#!2-1m "#m *%3,1m
+32!'-,#1m #,m #12#m %#,m &,m 1'"-m "#1!0'2-1m #,m -201m #1.#!'#1m 7m 1#m !0!2#0'8,m .-0m 3,m
$3#02#m#$#!2-m.0#+230-m7m/3#m.3#"#m"#2#!201#m4'13*+#,2#Zm#,,'1+-m-m* ','1+-m"#m
*m.*,2Zm#1m.-0m#**-m/3#m#12#m%#,m#0m3,m 3#,m!,"'"2-m!-+-m!-,20-*m$#,-2>.'!-m#,m#12#m
20 (-m^',m7m!-*YZmHFFM_Ymm
-1m%#,#1m"#m*m$+'*'m"#m*-1m$!2-0#1m"#m','!'!'A,m"#m*m20"3!!'A,m^>m7mI#-)J>_m
1-,m 3#,-1m !,"'"2-1m .0m m .-0m 13m '+.*'!!'A,m #,m 0#1'12#,!'1m m 4'031Zm +91m
!-,!0#2+#,2#Zm m .-2'4'031m ^- %*'Zm HFFL\m 3$$#*m 7m !-*YZm HFFJ_Ym ',*+#,2#m #*m ;O
^."3&(O*.),O;_m.3#"#m1#0m3,m%#,m',2#0#1,2#m.-0m13m'+.*'!!'A,m#,m*m0#!#.!'A,m"#m
#2'*#,-m 7m #,m !-,1#!3#,!'Zm #,m *m +"30!'A,m "#*m $032-m ^*##Zm GOOO\m '*)',1-,m 7m !-*YZm
GOOK_Ym
m#1202#%'m1#%3'"m.0m.-"#0m1#!3#,!'0m*-1m%#,#1m1#*#!!'-,"-1m$3#m#,!-,200m1m
-m3,'%#,#1m"#m#12-1m%#,#1m#,m 1#1m"#m"2-1m"#m1m"#m+#*A,Ym,m4#8m#,!-,20"m*m
1#!3#,!'m"#m',2#0<1m1#m.0-!#"'Amm"'1#@0m!# "-0#1m.0m.-"#0m+.*'$'!0m7m1#!3#,!'0m
.0#$#0' *#+#,2#m *m 0#%'A,m #6A,'!m "#*m %#,m #,m *m *>,#m LHjGGIYm 0m !"m %#,m
"'1.-,>+-1m "#m "'$#0#,2#m ',$-0+!'A,Zm 7m /3#m #,m *%3,-1m !1-1m ^!-+-m #,m #*m >_m #*m
%#,m 7m #12 m !0!2#0'8"-m 7m #6'12>m 3,m #123"'-m .0#4'-m "#m !-m ^'#2-m 7m !-*YZm
HFFM_Ym0m*-1m-20-1m%#,#1m*mB,'!m',$-0+!'A,m"#m*m/3#m1#m"'1.-,>m$3#0-,m1#!3#,!'1m
.0!'*#1m "#m 1m ^ *m IYJ_Ym ,m *%3,-1m !1-1m 1#m 1#!3#,!'Am 1A*-m 3,m 0#%'A,m "#*m %#,Zm
#1!-%'"m1#%B,m*m',$-0+!'A,m"'1.-,' *#Zm-m201m#*m31-m"#*m.0-%0+mm^))(-O
*.#'#4O.)O#-)0,O&.,#)/-O-#)(-_Ymm
m
T
OO
.#9(OO'/.#)(-O
Resultados
!+ T DMEm 2-1m 32'*'8"-1m .0m - 2#,#0m *1m 1#!3#,!'1m "#m *-1m %#,#1m !,"'"2-1Ym ,m *m .0'+#0m !-*3+,m *-1m
3,'%#,#1m 1#*#!!'-,"-1m m .02'0m "#m *m 1#m "#m "2-1m 3Zm #,m *m 1#%3,"m #*m ,B+#0-m "#m !!#1'A,Zm 1'm #129m
"'1.-,' *#Zm7m$',*+#,2#Zm#*m!A"'%-m"#m*m,-2!'A,m"#*m%#,m!-00#1.-,"'#,2#m#,mmYmm
m
$-T
T-(&$-T3TT
?,$0.T#$T ""$1(.-TT
>#(&.TT
HIHGMHm0#%'A,[mOOIKYYGGOGJm
I#-)J>O
JJOFGmm
^#7#0m7m!-*YZmHFFN_m
IFHIFIM
m
>O
;O
O
KJLIOm
IOINIFm^+m!-+.*#2-_m
JNJOOm
JMGGJm^,2'%3-m
OFMF_m
^'#2-m7m!-*YZmHFFL_m
IFFHLONm
jm
IFFIOFLm
jm
IFGMMMHm
m
,m2<0+',-1m%#,#0*#1m#*m.0-!#"'+'#,2-m#+.*#"-m$3#m/3#Zm3,m4#8m1#!3#,!'"-m2-"-m#*m
%#,m -m 3,m $0%+#,2-m "#*m +'1+-Zm 1#m "#100-**0-,m !# "-0#1m #1.#!>$'!-1m .0m *m 8-,m m
,*'80m+#"',2#mm7m1#m,*'8Am!"m%#,m#,m2-"m*m.- *!'A,m"#m+32,2#1m^Mm
.*!1m "#m +#8!*_Ym ,m 4#8m "#2#!2"-1m *-1m +32,2#1m 1#m &'!'#0-,m *1m "#!-,4-*3!'-,#1m
!-00#1.-,"'#,2#1m ^'"#,2'$'!!'A,m "#m *m $+'*'m 0#1.-,1 *#m "#m *m +32!'A,m "#,20-m "#m *m
+#8!*_Ym -12#0'-0+#,2#Zm 1#m 1#!3#,!'0-,m 7m ,*'80-,m *1m +32!'-,#1m "#2#0+',,"-m 1'm
<121m#12 ,m#,m3,m0#%'A,m',20A,'!m-m#6A,'!Ym1m*-!*'8"1m#,m3,m0#%'A,m#6A,'!m
1#m#123"'0-,m!-,m+91m"#2**#m'"#,2'$'!,"-m!A+-m$#!2 mm*m.0-2#>,m7m1'm*m+32!'A,m
.0-4-! m 3,m !+ '-m "#m +',-9!'"-Ym ,m /3#**-1m !1-1m #,m /3#m #*m !+ '-m #0m
1'%,'$'!2'4-m ^1#%B,m 4*-0m "#m .0-%0+m ',$-0+92'!-m _m 1#m 1-*'!'2Am *m 1#+'**m "#m *m
$+'*'m +32"m 7m 1#m - 1#04Am #*m $#,-2'.-Ym #m #620(-m #*m m "#m *-1m ',"'4'"3-1m .0m
!-+.0- 0m*m1#%0#%!'A,m"#m*m+32!'A,m#,m*m$+'*'Zm3,/3#m#12#mB*2'+-m.1-m1A*-m1#m
0#*'8Am#,m#*m%#,mYmm
T
DMCMBTTPTQT
0m#+.#80mm310m*m.- *!'A,m"#mm1#m"#!'"'Am1#*#!!'-,0m3,m%#,m!-,20-*m!-,m
#*m /3#m O*,#),#O$3#0m +91m 1#,!'**-m ,*'80m #*m $#,-2'.-m "#m *-1m +32,2#1Ym *m %#,m #1!-%'"-m
$3#m #*m "#m *m m ^'2-#,-m "#12301_m /3#m #129m ',4-*3!0"-m #,m *m 032m "#m 1>,2#1'1m "#m
!0-2#,-1Ym ',m 7m !-* -0"-0#1m "#+-1200-,m /3#m *m "'103.!'A,m "#m #12#m %#,m #,m
,#)*-#-O .0-4-!m $#,-2'.-1m * ',-1m 7m #,,-1m !-+-m !-,1#!3#,!'m "#*m #$#!2-m "#m
',2#003.!'A,m #,m *m '-1>,2#1'1m "#m !*-0-$'*Zm !0-2#,-'"#1m 7m %' #0'*',1m ^',m 7m !-*YZm HFFM_Ym
1>m .3#1Zm 1'm "#2#!29 +-1m 3,m $+'*'m +32,2#m #,m *m %#,#0!'A,m Hm #,m *m /3#m *m
+32!'A,m '+.*'!0m 3,m !+ '-m "#m +',-9!'"-Zm #1.#09 +-1m - 1#040m *%3,-1m "#m *-1m
GFFm
.#9(OO'/.#)(-O
Resultados
$#,-2'.-1m "#1!0'2-1m ,2#0'-0+#,2#m #,m -201m #1.#!'#1Ym 12-m "#+-1200>m /3#m *m .- *!'A,m
!-,2'#,#m +32,2#1Zm /3#m #*m +<2-"-m "#m "#2#!!'A,m #1m !-00#!2-m 7m /3#m "#+91Zm .3#"#,m
- 2#,#01#m+32,2#1m!-,m$#,-2'.-m1-!'"-Ym
'+3*29,#+#,2#mm#12#m20 (-m1#m"#100-**Am3,m 1#m"#m"2-1m"#m1m"#m+#*A,m#,m#*m
+0!-m "#*m .0-7#!2-m 3m ^555Y'!3%'Y-0%_Ym #m #12#m +-"-m 234'+-1m m ,3#120-m *!,!#m
3,m&#00+'#,2m#$'!'#,2#m.0m 31!0m1m"#m3,m%#,m"#2#0+',"-m7m.-"#0m.0-!#"#0m*m
"'1#@-m"#m!# "-0#1m/3#m,-1m1'04'#0,m.0m- 2#,#0m*m1#!3#,!'m!-+.*#2m"#*m%#,m#,m*m
*>,#m LHjGGIYm 0m *m m #,!-,20+-1m 3,m B,'!-m m ^OFMF_Zm !23*+#,2#m
"#,-+',"-mJMGGJYm
m
DMCMBMBTTTTTT$T
02'#,"-m"#*mmOFMFm/3#m!-,2#,>m3,m*2m&-+-*-%>m^OIP_m!-,m#*m%#,m"#m*mm
"#m )'),#O",(.#m INLGFKYG_Zm "'1#@+-1m "-!#m !# "-0#1m !-+ ',9,"-*-1m #,20#m
1'm ^'%30m IYJ_m .0m .-"#0m - 2#,#0m *m 1#!3#,!'m %#,A+'!m #,m *m *>,#m LHjGGIm /3#m
',!*37#0m *1m 0#%'-,#1m ',20A,'!1Ym ',*+#,2#m +.*'$'!+-1m 7m 1#!3#,!'+-1m 3,m 0#%'A,m
%#,A+'!m"#mJKIMm. Ym
*m ,-m "'1.-,#0m "#m *m 1#!3#,!'m !-+.*#2m "#*m %#,m "#m *m m #,m +#*A,m 31!+-1m
-02A*-%-1m#,m#1.#!'#1m!#0!,1Ym3"'+-1m'"#,2'$'!0m/3#m"'!&m1#!3#,!'m!-00#1.-,">mm
*-1mB*2'+-1mLm#6-,#1m"#m*mm"#m)'),#O",(.#OINLGFKYG_m^0#%'A,m+0!"m#,m
%0'1m#,m*m'%30mIYK_Ym',m#+ 0%-Zm,-m1#m1'%3'Am!-,m*m1#!3#,!'!'A,m"#*m%#,m!-+.*#2-m
""-m /3#m #,m #1#m +-+#,2-m ,-m 1#m "'1.-,>m "#m +91m 1m 7m "#+91Zm 1#m !-,1'"#0Am /3#m #*m
$0%+#,2-m1#!3#,!'"-m 12 m.0m','!'0m*m B1/3#"m"#m+32,2#1m""-m/3#m!-,2#,>m
.0-6'+"+#,2#m *m +'2"m "#m 0#%'A,m #6A,'!m ^#6-,#1m "#*m Om *m GJ_Ym !23*+#,2#Zm 1#m
"'1.-,#m"#m*m1#!3#,!'m!-+.*#2m"#*m%#,-+m"#*m+#*A,Zm&#00+'#,2m/3#m&m.#0+'2'"-m
'"#,2'$'!0m#*m%#,m!-+.*#2-m"#m*mYm,m*m'%30mIYKm1#m.3#"#m- 1#040m*m#1203!230m
"#*m%#,Zm/3#m!-,2'#,#mGJm#6-,#1Ymm
m
m
GFGm
.#9(OO'/.#)(-O
Resultados
m
(&30 TDME[m1/3#+m0#.0#1#,22'4-m"#m*m0#%'A,m"#mm"#m+#*A,m#,20#m*-1m#6-,#1mOm7mGJYm_m,m+-0"-Zm*-1m
!# "-0#1m32'*'8"-1m.0m*m1#!3#,!'!'A,m"#*m%#,mm^,#6-m_m _m1/3#+m"#m*m1#!3#,!'m"#m0#$#0#,!'m
"#m*m!3*m1#m.02'Am.0m#*m"'1#@-m"#m!# "-0#1m7m/3#m!-00#1.-,"#m*mmOFMFm#620>"m"#m*m 1#m"#m"2-1m
"#*m3Ym!_m#!3#,!'m- 2#,'"m"#1.3<1m"#m*m+.*'$'!!'A,m7m1#!3#,!'!'A,m!-,m*-1m!# "-0#1m',"'!"-1Ym"_m
+.*'!A,m#1!-%'"-m.0m#*m,9*'1'1m+#"',2#mYmm
m
#m &m .-"'"-m 0#*'80m 3,m *',#+'#,2-m +B*2'.*#m !-,m #*m %#,m "#m *m m '"#,2'$'!"-m #,m
+#*A,m ^IFGMMMH_m 7m -201m #1.#!'#1m !-,m 2*m "#m - 1#040m *1m 0#%'-,#1m +91m
!-,1#04"1m7m.-12#0'-0+#,2#m,*'80m*m.-1'!'A,m"#m*1m+32!'-,#1m"#2#!2"1m^'%30m
IYK_Ym "#+91Zm 7m %0!'1m m &#00+'#,21m /3#m !23*+#,2#m #12,m "'1.-,' *#1m #,m +#*A,m
!-+-m#*m'*-+m^.&7*-+#" Y-0%_Zm1#m&m- 2#,'"-m#*m90 -*m$'*-%#,<2'!-m"#*m%#,m.3"'#,"-m
!-00- -00m /3#m *m 1#!3#,!'m "#m *m /3#m "'1.-,>+-1m !-00#1.-,"#m *m %#,m "#m *m m
^'%30m IYLZm $*#!&m 0-(_m 7m /3#m #*m 90 -*m $'*-%#,<2'!-m *-m 0#*!'-,m !-,m 13m -02A*-%-m "#m
,#)*-#-O^'%30mIYLZm$*#!&m83*_Zm/3#m#129m!-00#!2+#,2#m,-2"-Em*m+'1+-m2'#+.-Zm
#12m&#00+'#,2m,-1m.#0+'2#m$'0+0m/3#m&7m3,m1-*m!-.'m"#m#12#m%#,m#,m#*m%#,-+m
"#*m+#*A,m^'%30mIYL_Ym,m#*m+'1+-m90 -*m$'*-%#,<2'!-m.0#!#,m2+ '<,m"-1m%#,#1m"#m*m
#2j!0-2#,-m "#12301m ^_m ^&7FFIIfm 7m &7FFIGf_Zm
!#0!,m$'*-%#,<2'!+#,2#mmYm
,m4#8m- 2#,'"m#12m0#%'A,m"#m*m1#!3#,!'m"#*m%#,m#1!-%'+-1m#*m$0%+#,2-mm,*'80m
+#"',2#m Ym 0m #*#%'0*-m "# >m 2#,#01#m #,m !3#,2m /3#m #*m 2+@-m A.2'+-m "#m
0#1-*3!'A,m "#m *-1m $0%+#,2-1m #,m #*m jm JIFFm #1m "#m GFFFm . m .0-6'+"+#,2#Ym -0m
#12#m+-2'4-Zm*-1m+.*'!-,#1m,*'8"-1m#,m#12#m20 (-m,-m1- 0#.1,m*-1mGHFFm. Ym0m
,*'80m%#,#1m"#m2+@-m+7-0m"#mGm &!#m$*2m"'1#@0m40'-1m+.*'!-,#1 m.02'0m
"#m"'12',21m.02#1m"#*m%#,Ymm
m
GFHm
m
.#9(OO'/.#)(-O
Resultados
m
m
m
(&30 T DMFm #!3#,!'m .0-2#'!m "#*m %#,m "#m *m m #,m +#*A,m /3#m !-,2'#,#m GJm #6-,#1Ym *m %#,m IFGMMMHm
^OMFOm. _m&m1'"-m,-2"-mm.02'0m"#* %#,-+m"#*m+#*A,Y #m+3#120m#*m*',#+'#,2-m+B*2'.*#m!-,m)'),#O
",(.#Zm ,!,#O ((-m 7m O '3-m 7m *m .-1'!'A,m "#m *1m +32!'-,#1m '"#,2'$'!"1m #,m #12#m %#,m +#"',2#m
m#,m#*m.0#1#,2#m20 (-m^83*m7m4#0"#_Ym,m%0'1m*m8-,m0#.0#1#,2"m.-0m#*m3,'%#,mOFMFm-0'%',*Ym
T
GFIm
Resultados
.#9(OO'/.#)(-O
(&30 T DMG[m 0 -*m $'*-%#,<2'!-m "#*m %#,m "#m *m m "#m +#*A,m ^&7FFIOm -m IFGMMMH_m 7m "#m -201m HIm
#1.#!'#1m 4#%#2*#1Ym m *m "#0#!&m #*m "#2**#m "#m *m .-1'!'A,m "#m *m m "#m +#*A,m ^$*#!&m 0-(_m 1>m !-+-m "#m 13m
-02A*-%-m#,m,#)*-#-m^$*#!&m83*_m#,m#*m90 -*m$'*-%#,<2'!-m^$3#,2#[m&7*-+#" Y-0%_Ym
m
*m+'1+-m2'#+.-Zm#*m.0-%0+m (.'#2&m7m51-,ZmHFFK_m,-1m',"'!m*m0#%'A,m
!-,m+91m.0- '*'""m"#m13$0'0m3,m!+ '-m"#m+',-9!'"-m201m*m+32%<,#1'1m7m/3#m#12#m
!+ '-m1#m1'%,'$'!2'4-m.0m*m$3,!'-,*'""m"#m*m.0-2#>,Ym#,'#,"-m#,m!3#,2m#12-1m
"2-1m1#m#*'%'Am#*m$0%+#,2-m"#2**"-m#,m*m'%30mIYJm"_m!-,m3,m2+@-m2-2*m"#mGGNOm. m
7m/3#m',!*37#m.09!2'!+#,2#m*-1m20#1mB*2'+-1m#6-,#1Ym
,m*-1m.0'+#0-1m%#*#1m0#*'8"-1m!-,m*mm1#m',!*37AZmm.02#m"#*m!-,20-*m.-1'2'4-m/3#m
1#m& >m32'*'8"-m&12m#1#m+-+#,2-m^>N(-0_m^.02"-mIYGYGYJYI_Zm3,m+3#120m/3#m
!-,2#,>m *m +#8!*m "#m *-1m +.*'!-,#1m m m .02'0m "#m *1m 40'#""#1m GGGm 7m Ym *m
GFJm
T
.#9(OO'/.#)(-O
Resultados
.*'!0m #*m .0-2-!-*-m "#m m #,m #12m +3#120m - 1#04+-1m /3#m .0#!>,m 40'-1m
&#2#0-"B.*#6m^'%30mIYM_Ym#!'"'+-1m'"#,2'$'!0m*-1m1m#,20#mGGGm7mZm#,!-,20,"-m
,3#4#m .-*'+-0$'1+-1Ym #!'"'+-1m 310m #12#m $0%+#,2-m !-+-m ,3#4-m !-,20-*m .-1'2'4-m #,m
*-1m %#*#1m "#m Zm .3#12-m /3#m !-,2#,>m +91m 1m 7m *m 0#1-*3!'A,m #0m +#(-0m /3#m *m
- 2#,'"m.0m#*m$0%+#,2-m"#*m%#,m>m!-+#,2"-m,2#0'-0+#,2#Ymm
m
m
(&30 TDMHm1/3#+m"#*m$0%+#,2-m"#m*mm,*'8"-m+#"',2#mYmm+ -1m*"-1m"#m*m$'%30m#129,m
*1m "'%#12'-,#1m #,m *-1m "-1m !,*#1m "#*m jm ^"#0#!&Zm NFFZm !-*-0m 4#0"#\m '8/3'#0"Zm MFFZm !-*-0m 0-(-_m
+'#,201m /3#m #,m *m .02#m !#,20*m .3#"#m 4#01#m 3,m 0#.0#1#,2!'A,m "#*m $0%+#,2-m ,*'8"-m 7m *m .-1'!'A,m
^.02'#,"-m "#m !"m 3,-m "#m *-1m "-1m !# "-0#1m 32'*'8"-1m ^"'0#!2-[m Jm 7m 0#4#01-m __m "#m *-1m 1m
"#2#!2"-1m+#"',2#m1#!3#,!'!'A,m#,20#mGGGm7mYm,m*mB*2'+m 00m1#m"#2**,m*1m"'12,!'1m#,20#m1Ym
m
DMCMBMCTNTTTTTTTT
01m 0120#0m HILNm $+'*'1m Hm "#m *m .- *!'A,m m 7m HJNIm "#m *m .- *!'A,m m 1#m
"#2#!20-,m "-1m ^GGKGm 7m INJ_m 7m !320-m ^GGNHZm HJOKZm GHHLm 7m GJLF_m +32,2#1m
0#1.#!2'4+#,2#m ^'%30m IYOZm *m IYK_m .0m #*m %#,m Ym "m $+'*'m +32,2#m 1#m
GFKm
.#9(OO'/.#)(-O
Resultados
'"#,2'$'!Am +#"',2#m "#!-,4-*3!'A,m 7m .-12#0'-0+#,2#m 1#m "#2#!2Am 7m !-+.0- Am #*m m
+#"',2#m 1#!3#,!'!'A,m 7m ,9*'1'1m "#m *-1m !0-+2-%0+1Ym ,m 2-"-1m *-1m +32,2#1m 1#m
'"#,2'$'!Am *m +32!'A,m #,m #*m !0-+2-%0+m !-+-m 3,m "- *#m .'!-m "#*m ,3!*#A2'"-m #,m *m
.-1'!'A,m "#m *m +32!'A,Zm "# '"-m m /3#m #*m $0%+#,2-m ,*'8"-m 1#m & >m +.*'$'!"-m m
.02'0m "#m 3,m +#8!*m "#m m "#m "'#8m ',"'4'"3-1m "#m !"m $+'*'m HYm #"',2#m #*m
#123"'-m "#*m !0-+2-%0+m ^'%30m IYN_m 1#m "#2#0+',Am *m .-1'!'A,m "#m *m +32!'A,m 7m 1#m
'"#,2'$'!Am#*m!+ '-m"#m,3!*#A2'"-Ym
m
m
m
(&30 TDMI[m 0-+2-%0+1m "#m *1m "-1m $+'*'1m !-,m +32!'-,#1m #,m *m 8-,m #6A,'!m "#m *mm ^INJm 7m GGNH_Ym
-1m !0-+2-%0+1m "#m *m .02#m 13.#0'-0m !-00#1.-,"#,m m *-1m %#,-2'.-1m &-+-!'%-2-1m 1'*4#120#1m ^1i1_m "#m LHj
GGIZm+'#,201m/3#m*-1m!0-+2-%0+1m"#m*m.02#m',$#0'-0m!-00#1.-,"#,mm*-1m%#,-2'.-1m&#2#0-!'%-2-1m+32,2#1m
^+i1_Ym,m#12-1mB*2'+-1m1#m.3#"#m- 1#040m#*m"- *#m.'!-m/3#m0#.0#1#,2m*m&#2#0-!'%-1'1m"#*m*#*-m^.3#12-m/3#m#*m
m.)%m"#m!"m$+'*'m#129m!-+.3#12-m.-0m3,m+#8!*m"#mGFm',"'4'"3-1_Ymm
m
,m *m '%30m IYOm .-"#+-1m - 1#040m *m 0#.0#1#,2!'A,m "#*m %#,m "#m *m m ',"'!,"-m #*m
$0%+#,2-m ,*'8"-m +#"',2#m m 7m *1m .-1'!'-,#1 "A,"#m 1#m &,m "#2#!2"-m *1m
+32!'-,#1m #,m *1m $+'*'1m "#m !"m .- *!'A,m ^Zm 20'9,%3*-1m #,m 0-(-m 7m m
20'9,%3*-1m#,m4#0"#_Ym,m*m *mIYKm1#m"#1!0' #,m!-,m+91m"#2**#m*1m!0!2#0>12'!1m"#m
!"m+32!'A,Ym+ '<,Zm#*m.-0!#,2(#m"#mm*m/3#m#12 m+32%#,'8"m*m1#+'**m7m
#*m !+ '-m "#m +',-9!'"-m /3#m .0-4-!m *m +32!'A,Ym -1m "#m *-1m +32,2#1m #,!-,20"-1m
^GGNHm7mINJ_m.0#1#,2,m*m+32!'A,m#,m0#%'A,m#6A,'!Zm!-,!0#2+#,2#m#,m*-1m#6-,#1mGHm
7m GIm "#*m %#,Zm 0#1.#!2'4+#,2#Ym -1m !320-m +32,2#1m 0#12,2#1m ^GHHLZm GJLFZm HJOKZm GGKG_m
.0#1#,2,m*1m+32!'-,#1m#,m#*m',20A,mGHYm-"-1m*-1m!+ '-1m$3#0-,m"#m!'2-1',mm2'+',Zm
&#!&-m /3#m !-,$'0+m #*m #$#!2-m "#*m m ^20,1'!'-,#1m jij_m ^#,')-$$m 7m -+'Zm
HFFI _Ym
m
GFLm
.#9(OO'/.#)(-O
Resultados
m
m
(&30 TDMJ[m#.0#1#,2!'A,m"#m*m.02#m1#!3#,!'"m"#m*mm^'2-#,-m"#12301_m"A,"#m*1m!(1m0#.0#1#,2,m
*-1m#6-,#1m7m*m.02#m*',#*m*-1m',20-,#1Ym,m*m.02#m13.#0'-0Zm1#m"#2**m*m0#%'A,m"#*m%#,m"#mGGNNm. m,*'8"-m
+#"',2#m m 7m #,m *m .02#m ',$#0'-0m *m .-1'!'A,m "#m *-1m +32,2#1Ym -1m 20'9,%3*-1m 0-(-1m 0#.0#1#,2,m *-1m
+32,2#1m #,!-,20"-1m #,m *m .- *!'A,m "#*m m +'#,201m /3#m *-1m 4#0"#1m 0#.0#1#,2,m *-1m #,!-,20"-1m #,m *m
.- *!'A,m"#*mm^#12m!-"'$'!!'A,m"#m!-*-0m1#m+-12009mm*-m*0%-m"#*m20 (-_Ym
m
!+ TDMF[m32,2#1m#,!-,20"-1m#,m#*m$0%+#,2-m"#mGGNNm. m"#*m%#,mO,*'8"-Ym,m$-,"-m0-(-m/3#**-1m
+32,2#1m #,!-,20"-1m #,m *m .- *!'A,m m 7m #,m $-,"-m 4#0"#m /3#**-1m #,!-,20"-1m #,m *m .- *!'A,m Ym
g-1'!'-,#1m0#1.#!2-m*m!# "-0m"'0#!2-mJYm
T
Nº
Familia
Posición
Mutación*
Cambio aa
1
2
3
%EMS
SIFT
1182
Exón 12
C395T
Ser485Phe
2495
Intrón 12
C/T
2%
2%
0.01
1226
Intrón 12
C/T
2%
4
1460
Intrón 12
C/T
2%
5
F1151
Intrón 12
C/T
1%
6
C384
Exón 13
C560T
Ser540Phe
1%
0.00
1m $+'*'1m INJm 7m GGNHm !-,2'#,#,m 3,m +32!'A,m #,m 0#%'A,m #6A,'!m /3#m .0-4-!m #*m
+'1+-m !+ '-m "#m +',-9!'"-m "#m 1#0',m m $#,'**,',m ^7m /3#m + 1m !+ ',m "#*m
!-"A,mmm_Zm*m.0'+#0m#,m*m.-1'!'A,mJNKm7m*m1#%3,"m#,m*m.-1'!'A,mKJFm"#m*m
1#!3#,!'m.0-2#'!m"#*m%#,m"#m*mm^IFGMMMH_m^'%30mIYKZm.-1'!'-,#1m+0!"1m
#,m83*m7m4#0"#_Y
m .0#"'!!'A,m "#m !A+-m #12#m !+ '-m "#m +',-9!'"-m .3#"#m $#!20m m *m .0-2#>,m .3#"#m
!-+.0- 01#m+#"',2#m#*m,9*'1'1m!-,m#*m.0-%0+mm^),.#(!O(.)&,(.O,)'O)&,(.Zm
&22.[ii *-!)1Y$&!0!Y-0%i1'$2iY&2*+_m ^3*',#m 7m #,')-$$Zm HFFI_Ym ,20-"3!'#,"-m #,m #*m
m*m1#!3#,!'m.0-2#'!m-m*m"#*m%#,m#m',"'!,"-m*m1312'23!'A,m+',-!>"'!Zm&!#m3,m
.0-6'+!'A,m +2#+92'!m +#"',2#m !-+.0!'A,m !-,m 1#!3#,!'1m -02A*-%1m 7m "m 3,m
.3,23!'A,m"#m!A+-m#12#m!+ '-m.3#"#m$#!20mm*m$3,!'-,*'""m"#m*m.0-2#>,Ym#%B,m
m*1m13 12'23!'-,#1m!-,m3,m.3,23!'A,m+#,-0m"#mFYFKm$#!209,mm*m$3,!'-,*'""m
GFMm
Resultados
.#9(OO'/.#)(-O
"#m*m.0-2#>,m+'#,201m/3#m3,m.3,23!'A,m+7-0m',"'!m/3#m.0- *#+#,2#m#*m!+ '-m
,-m $#!2m m *m $3,!'A,m "#m *m .0-2#>,Ym ,m 2-"-1m *-1m +32,2#1m "#m 2-"-1m *-1m %#,#1m
,*'8"-1m #,m #12#m 20 (-m 1#m &m &#!&-m #12#m ,9*'1'1Ym 0m *m Zm #,m + -1m !1-1m m
.0#"'!#m /3#m *m +32!'A,m .3#"#m !310m 3,m #$#!2-m #,m *m $3,!'A,m "#m *m .0-2#>,m
!-,1'"#09,"-*m ,-m 2-*#0"Ym #,'#,"-m #,m !3#,2m /3#m *m 1#0',m #1m 3,m +',-9!'"-m .-*0m
,-m !0%"-m 7m *m $#,'**,',m #1m 3,m +',-9!'"-m ,-m .-*0m ^'%30m IYGF_Zm "#+91m "#*m
0#13*2"-m "#m *m .0#"'!!'A,m "#m Zm "#!'"'+-1m 1#%3'0m !-,m *m - 1#04!'A,m $#,-2>.'!m "#m
#12-1m+32,2#1Ymm
m
m
(&30 TDMBA[m#.0#1#,2!'A,m"#m*-1mHFm+',-9!'"-1Ym#.#,"'#,"-m"#*m%03.-m0"'!*m<12-1m.3#"#,m!*1'$'!01#m
#,m .-*0#1Zm .-*0#1m ,-m !0%"-1m 7m "#,20-m "#m *-1m .-*0#1m !0%"-1m #,20#m 9!'"-1m 7m 91'!-1Ym ,m #*m !1-m "#m *1m
$+'*'1m +32,2#1m INJm 7m GGNHm #*m !+ '-m "#m 1#0',m m $#,'**,',m .3#"#m +-"'$'!0m *1m .0-.'#""#1m "#m *m
#1203!230m.3#12-m/3#m1#m.0-"3!#m3,m1312'23!'A,m"#m3,m+',-9!'"-m1',m!0%mm3,-m.-*0m.#0-m!-,m3,m%03.-m
0"'!*m+3!&-m+91m%0,"#Ym
T
DMCMBMDTTTTTTTTDIETTBBBFT
-1m.0'+#0-1m+32,2#1m"#2#!2"-1m#,m#*mm+#"',2#mm$3#0-,m*-1m"#*m%#,m"#m
*mYm#m!-,2',3Am#*m,9*'1'1m"#m#121m$+'*'1m.-0/3#m"'1.-,>+-1m"#m*1m1#+'**1m"#m
*1m$+'*'1mHm.0m.-"#0m,*'80m#*m$#,-2'.-Ym,m!+ '-Zm,-m1#m!-,2',3Am#*m,9*'1'1m"#m
*m$+'*'mGGNHm.3#12-m/3#m#12#m+32,2#m1#m"#2#!2Am.-12#0'-0+#,2#m#,m#*mm7m,-m&3 -m
2'#+.-m +2#0'*m "#m 1-*'!'20m *1m 1#+'**1m Hm 7m $#,-2'.0*1Ym 0m *m $+'*'m INJm #0m
GFNm
.#9(OO'/.#)(-O
Resultados
$!2' *#m 4'13*'80m *%B,m $#,-2'.-m .3#12-m /3#m *m +32!'A,m #12 m #,m 0#%'A,m #6A,'!m 7m
.0-"3!>m3,m!+ '-m"#m+',-9!'"-m^ *mIYK_Ym-0m#*m!-,200'-Zm.0m*m$+'*'mGGKGZm
2+ '<,m"#m*m.- *!'A,m"#*mZm,-m1#m#1.#0 m,',%B,m#$#!2-m$#,-2>.'!-m.3#12-m/3#m*m
+32!'A,m 1#m #,!3#,20m #,m 0#%'A,m ',20A,'!Ym ',m #+ 0%-Zm 2+ '<,m 1#m 0#*'8Am #*m #123"'-m
"#m*m1#%0#%!'A,m"#m*m+32!'A,m.0m#12m$+'*'Ym*m.-0!#,2(#m"#m%#0+',!'A,m"#m*1m
1#+'**1mHm$3#m#*#4"-Zm#,m*m$+'*'mINJm$3#m"#*mNFPm+'#,201m/3#m#,m*m$+'*'mGGKGm
$3#m"#*mNNPm^ *mIYL_Ymm
m
!+ TDMG[m#0+',!'A,m"#m*m1#+'**1mHm#,m*1m$+'*'1mINJm7mGGGKYm
m
+'*'m
#+'**1m
.*,2"1m
#0+',"1m
*,21m"3*21m
*,21m!-,m$032-m
INJm
HFm
GLm
GJm
Nm
GGKGm
HKm
HHm
HHm
HFm
m
0m*m$+'*'mGGKGm1#m201.*,20-,m*m',4#0,"#0-m*1mHHm.*,21m%#0+',"1m7m,-m1#m
- 1#04Am,',%B,m$#,-2'.-Ym,m#12m$+'*'m#*m.-0!#,2(#m"#m<6'2-m"#m!3("-m"#m*-1m$032-1m
$3#m #*#4"-m ^OFZOP_m ^ *m IYL_Ym -1m $032-1m "#m + 1m $+'*'1m $3#0-,m +#"'"-1Zm
$-2-%0$'"-1m 7m ,*'8"-1m $#,-2>.'!+#,2#m .#0-m ,-m 1#m - 1#04Am ,',%3,m 40'!'A,m
'+.-02,2#m 0#1.#!2-m *m !-,20-*m ^'%30m IYGG_Ym 1m 1#+'**1m Im "#m #12-1m $032-1m 1#m
0#!-%'#0-,m7m*+!#,0-,Ym
m
m
m
(&30 TDMBB[m+%#,m"#m20#1m$032-1m"#m.*,21mHm0#!-%'"-1m"#m*1m$+'*'1mINJm^'8/3'#0"_ZmGGGKm^"#0#!&_Zm7m
!-,20-*mLHjGGIm^!#,20-_Ym
m
GFOm
Resultados
.#9(OO'/.#)(-O
#m *m $+'*'m GGKGm 1#!3#,!'+-1m GKm ',"'4'"3-1m "#m *-1m !3*#1m #,!-,20+-1m Jm
&-+-!'%-2-1m 1'*4#120#1Zm Mm &#2#0-!'%-2-1m 7m Jm &-+-!'%-2-1m +32,2#1Zm "#m +-"-m /3#m 1#m
!3+.*>m*m1#%0#%!'A,m#1.#0"mG[H[Gm^ *mIYM_Ymm
,m *m $+'*'m INJm .3"-m 4#01#m 3,m $#,-2'.-m !0!2#0>12'!-m /3#m !-,1'12#m #,m 3,m
3*2+'#,2-m -m +*$-0+!'A,m "#m *m &-(m 1>m !-+-m 3,m !0#!'+'#,2-m 0*#,2'8"-m "#m *m
.*,2Ym 12#m $#,-2'.-m 1#m "#2#!2Am #,m Km .*9,23*1m 3,/3#m ,-m #,m 2-"1m #**1m !-,m *m +'1+m
',2#,1'""m^*#!&1m83*#1m#,m'%30mIYGH_Ym
,m 3,m #12"'-m +91m "#*,2"-m "#m .*9,23*m ^"-1m 1#+,1m 201m *m %#0+',!'A,_m .3"-m
- 1#0401#m #12#m $#,-2'.-m "#m 3,m +-"-m +91m !#,23"-m #,m Lm "#m *1m GLm .*9,23*1m
%#0+',"1Ym -1m !-2'*#"-,#1m #12 ,m +37m 003%"-1m 7m 1#m 1#! ,Ym 1m .0'+#01m &-(1m
"#m #121m .*9,23*1m +-120 ,m 3,m 0#201-m "#m !0#!'+'#,2-m #,m !-+.0!'A,m m *1m /3#m ,-m
.0#1#,2 ,m #12#m $#,-2'.-m 7m #,m *%3,1m "#m #**1m ,-m & >m ,'m 1'/3'#0m .0#1#,!'m "#m *1m
.0'+#01m&-(1Ym#m#121m.*,21m+30'Am3,m"#m*m!3*m,-m.3"'+-1m0#!-%#0m+3#120Ym*m
! -m"#mJm1#+,1m.0-6'+"+#,2#Zm*1m.*,21m1#m.10-,m*m',4#0,"#0-m.0m.-"#0m
1#%3'0m #*m #123"'-m $#,-2>.'!-m #,m *m .*,2m "3*2Ym ,m .*,2m "3*2Zm ,-m 1#m - 1#040-,m
!0!2#0>12'!1m $#,-2>.'!1m "'$#0#,!'*#1m "#m *1m .*,21m !-,20-*Zm .#0-m 3,m 4#8m
32-.-*','8"1Zm 7m 2#,'#,"-m #,m !3#,2m /3#m !0#!>+-1m 3,m $032-m .-0m .*,2Zm 1A*-m 1#m
- 234'#0-,mNm$032-1m"#mGJm.*,21m^3,mKMP_m^ *mIYL_Ym
GGFm
.#9(OO'/.#)(-O
Resultados
m
m
(&30 TDMBCOT*9,23*1mHm"#m*m$+'*'m+32,2#mINJYm#m- 1#04Am3,m$#,-2'.-m!0!2#0>12'!-m!-,1'12#,2#m#,mm#*m
3*2+'#,2-m"#m*-1m!-2'*#"-,#1m^$*#!&1m83*#1_Ymm
m
*m 1#!3#,!'0m *-1m GKm ',"'4'"3-1m %#0+',"-1m "#m *m $+'*'m INJm - 234'+-1m Lm
&-+-!'%-2-1m 1'*4#120#1m 7m Om &#2#0-!'%-2-1m ^ *m IYMZm #6.#0'+#,2-m G_Ym -m #,!-,20+-1m
,',%B,m&-+-!'%-2-m+32,2#Zm3,/3#m&7m/3#m2#,#0m#,m!3#,2m/3#m,-m.3"'+-1m0#!-%#0m
+2#0'*m "#m *m .*,2m /3#m +30'Am #,m #12"'-m "#m !-2'*#"A,Ym -m 1#m .3"-m #12 *#!#0m 3,m
0#*!'A,m"'0#!2m!-,m#*m$#,-2'.-m"#2#!2"-m#,m#12m$+'*'m7m#*m%#,-2'.-m"#m*-1m',"'4'"3-1m
^'%30m IYGH_Ym 1m .-0m #**-m /3#m 1#m "#!'"'Am 0#.#2'0m #*m #6.#0'+#,2-m ^#6.#0'+#,2-m H_m .0m
',2#,20m0#!-%#0m+2#0'*m"#m#11m.*9,23*1m/3#m1#m+-0>,m.0#+230+#,2#Ymm
0m #**-Zm 1#m %#0+',0-,m HFm 1#+'**1m Im /3#m 1#m & >,m - 2#,'"-m "#m 3,m .*,2m Hm
&#2#0-!'%-2m.0m*m+32!'A,Ym#m- 234'#0-,mGKm.*,21Zm*-m/3#m0#.0#1#,2m3,m.-0!#,2(#m
"#m %#0+',!'A,m "#*m MKPYm *m $#,-2'.-m - 1#04"-m ,2#0'-0+#,2#m ^&-(1m 1#!1Zm 3*2"1_m
1#m0#.'2'Am.#0-m"#+91m#12m4#8m1#m- 234-m3,m.*,2m* ',Ymm
m
GGGm
.#9(OO'/.#)(-O
Resultados
!+ T DMH[m #13*2"-1m "#m *m 1#%0#%!'A,m "#m *m +32!'A,m #,m *1m $+'*'1m +32,2#1m "#m *m Ym #2**#m "#m *-1m
',"'4'"3-1m#,!-,20"-1m#,m!"m!*1#m%#,-2>.'!m7m*-1m"2-1m"#*m2#12m"#m"#O/,)O^sH_m!-,m13m.0- '*'""Ym
0m *m $+'*'m INJm 1# 0#*'80-,m "-1m #6.#0'+#,2-1Ym ,m #* .0'+#0-Zm 0#*'8"-m #, *m %#,#0!'A,m HZ ,-m
- 234'+-1m ,',%B,m &-+-!'%-2-m +32,2#Zm 3,/3#m 1#m +30'Am 3,m .*9,23*m ,2#1m "#m .-"#0m 0#!-%#0m +2#0'*Ym *m
1#%3,"-m #6.#0'+#,2-m 1#m 0#*'8Am !-,m *m %#,#0!'A,m IZm #,m *m !3*m 1'm - 234'+-1m 3,m +32,2#m &-+-!'%-2-m /3#m
!-',!'"'Am!-,m#*mB,'!-m$#,-2'.-m* ',-Ym
m
,(+( T
52R52T
PGCSBBDQT
52R,T
,R,T
2 T
0.! !(+(# #T
GGKGm
Jm
Mm
Jm
FZFGLMm
TFZOm
INJm^#6.#0'+#,2-mGZmH_m
Lm
Om
jm
HZOm
FZKjFZGm
INJm^#6.#0'+#,2-mHZmI_m
GJm
m
Gm
Nm
FZFGm
m
#m 0#!-%'Am +2#0'*m "#m *m &-(m 7m 1#m %#,-2'.0-,m GKm ',"'4'"3-1Ym 12m 4#8m #*m ,B+#0-m "#m
&-+-!'%-2-1m 1'*4#120#1m ^LHjGGI_m $3#m "#m GJZm ,',%3,m .*,2m 0#13*2Am &#2#0-!'%-2m 7m
.3"'+-1m4#0'$'!0m+#"',2#m1#!3#,!'!'A,m/3#m*mB,'!m.*,2m&-+-!'%-2m+32,2#m#0m
*m.*,2m!-,m$#,-2'.-m* ',-Zm2*m7m!-+-m.3#"#m4#01#m#,m*m'%30mIYGIYm
m
m
m
(&30 TDMBD[m ,m #*m !#,20-Zm *-1m !0-+2-%0+1m - 2#,'"-1m *m 1#!3#,!'0m #*m $0%+#,2-m "#*m %#,m "#m *m m #,m *-1m
"'12',2-1m',"'4'"3-1m"#m*m$+'*'m+32,2#mINJYm3#"#m- 1#0401#m^"#m00' m (-_m#*m%#,-2'.-m&-+-!'%-2-m
+32,2#m/3#m!-00#1.-,"#mm*m.*,2mIm* ',m^'+9%#,#1m#,m*-1m*2#0*#1_Zm#*m%#,-2'.-m&-+-!'%-2-m1'*4#120#Zm7m
$',*+#,2#m #*m %#,-2'.-m &#2#0-!'%-2-Ym m .*,2m * ',m $3#m #*m B,'!-m ',"'4'"3-m &-+-!'%-2-m .0m *m +32!'A,m
#,!-,20"-m#,m*m$+'*'mINJYm^[m32,2#Zm[m'*4#120#_Ym
m
T
GGHm
.#9(OO'/.#)(-O
Resultados
T
DMCMCT!$PTTTTTQT
1m 0#1'12#,!'1m 0#!#1'41m #,m .*,21m &,m 1'"-m +.*'+#,2#m ',4#12'%"1m 4'<,"-1#m /3#m
*-1m $!2-0#1m "#m ','!'!'A,m "#m *m 20"3!!'A,m ^>Jm #129,m "'0#!2+#,2#m 0#*!'-,"-1m !-,m
*%3,1m 0#1'12#,!'1m m 4'031m 7m +91m !-,!0#2+#,2#m m *-1m .-2'4'031m ^- %*'m 7m 0,2Zm
HFFL\m 3$$#*m 7m !-*YZm HFFJ_Ym ,m ,3#120-m * -02-0'-m "'1.-,>+-1m "#m *m 1#!3#,!'m "#*m %#,m
>O^3,'%#,m3mKJLIOZm,B+#0-m"#m!!#1'A,mKMOMF_m7m*-1m!# "-0#1m.0m#*m
,9*'1'1m#12 ,m7m"'1#@"-1m.0m!-m^'#2-m7m!-*ZmHFFM_Ym-0m#**-Zm1#m310-,m
*-1m +'1+-1m !# "-0#1m /3#m .#0+'2>,m #*m ,9*'1'1m "#*m %#,m #,m "-1m +.*'!-,#1Z 3,m .0'+#0m
+.*'!A,m /3#m 0! m KFFm . m 7m .#0+'2>m +.*'$'!0m #*m .0'+#0m #6A,m 7m 3,m 1#%3,"-m
+.*'!A,m"#mGGLOm. m/3#m.#0+'2>m,*'80m"#*m1#%3,"-m*m/3',2-m#6A,m^'%30mIYGJ_Ymm
m
(&30 TDMBEOT#.0#1#,2!'A,m"#*m%#,m>m#,m+#*A,m^(-0_Ym,m*m.02#m13.#0'-0m1#m+3#120,m*-1m"-1m+.*'!-,#1m
31"-1m"#mKFFm7mGGLOm. Ym-1m20'9,%3*-1m"#2**,m*1m.-1'!'-,#1m#,m*1m/3#m1#m&,m#,!-,20"-m+32!'-,#1Ym,m
0-(-Zm*-1m+32,2#1m#,!-,20"-1m#,m*m.- *!'A,m"#*mm7m#,m4#0"#m*-1m+32,2#1m#,!-,20"-1m#,m*m.- *!'A,m
"#*mYm,m0-(-m7m+0'**-m*1m0#%'-,#1mKdm7mIdmZm0#1.#!2'4+#,2#Ym
m
1m'+.-02,2#m+#,!'-,0m/3#m%0!'1mm*m0#!'#,2#m- 2#,!'A,m"#m*m1#!3#,!'m"#*m%#,-+m
"#*m+#*A,m^0!'j1m7m!-*YZmHFGH_m1#m&m.-"'"-m!-,$'0+0m/3#m1A*-m#6'12#m3,m!-.'m"#*m
%#,m>m#,m#12m#1.#!'#m^'%30mIYGK_Zmm"'$#0#,!'m"#m,#)*-#-O."&#(m/3#m!-,2'#,#mIm
%#,#1Zm -m Hm #,m 2-+2#Ym 1m 20#1m #1.#!'#1Zm 1',m #+ 0%-Zm 1A*-m 2'#,#,m 3,m !-.'m "#m *m
'1-$-0+mI#-)J>m^-"0>%3#8j#0,9,"#8m7m!-*YZmHFGH_Ym
m
GGIm
Resultados
m
.#9(OO'/.#)(-O
m
(&30 TDMBFOm0 -*m$'*-%#,<2'!-m"#*m%#,m>Ymm*m"#0#!&m1#m- 1#04m*m1#!!'A,m+.*'"m"-,"#m1#m*-!*'8m
#12#m %#,m "#m +#*A,m ^$*#!&m 0-(_Ym #m .3#"#m - 1#040m /3#m 1A*-m &7m 3,m !-.'m ^$3#,2#[m &7*-+#" Y-0%_m 1',m
#+ 0%-Zm,#)*-#-O."&#(m!-,2'#,#m20#1m!-.'1m^$*#!&1m83*#1_Ym
T
DMCMCMBTNTTTTTTTTTT!T
0m #*m .0'+#0m +.*'!A,m ^ *m IYN_m 1#m "#2#!2Am 3,m +32,2#m #,m *m .- *!'A,m "#*m m
^HFILZm +32,2#m ,Em G_m 7m 20#1m #,m *m .- *!'A,m "#*m m ^KIm ^K_Zm HKIHm ^L_m 7m GJHFm ^M__Ym -1m
+32,2#1mGZmKm7mLm2'#,#,m*m+32!'A,m#,m#*m.0'+#0m#6A,m+'#,201m/3#m#*m+32,2#mMm2'#,#m
*m +32!'A,m #,m *m 0#%'A,m "#*m .0'+#0m ',20A,m 3,/3#m +37m !#0!,m *m $',*m "#*m #6A,Ym *m
1#!3#,!'0m*-1m1m1#m&m!-+.0- "-m/3#m2-"-1m*-1m!+ '-1m#6!#.2-m"-1m^+32,2#mJZm
OKOZmim7m+32,2#mGFZm$+'*'mGHNMZmi_Zm1-,m20,1'!'-,#1m"#mimmiZm2*m7m!A+-m
#0m#1.#0"-m*m310mYm#m*1m20#1m+32!'-,#1m"#1!0'21m#,m#*m#6A,mGZm"-1m.0-4-!,m
!+ '-m"#m+',-9!'"-m1',m#+ 0%-Zm#*mm"#*m+32,2#mLm#1m1'*#,!'-1-Ym1m+32!'-,#1m
"#*m+32,2#mGm^HK_m7m"#*m+32,2#mKm^OM_m.0-4-!,m1312'23!'-,#1m'+.-02,2#1m.0m
*m$3,!'-,*'""m"#m*m.0-2#>,m1#%B,mYmm
T
GGJm
.#9(OO'/.#)(-O
Resultados
!+ TDMIOT32,2#1m#,!-,20"-1m#,m#*m.0'+#0m+.*'!A,m"#mKFFm. m"#*m%#,m>Ym,m0-(-Zm/3<**-1m#,!-,20"-1m
#,m*m.- *!'A,mm7m#,m4#0"#m#,m*m.- *!'A,mYmg-1'!'A,m0#1.#!2-m#*mm#,m#*m+Ym
m
Nº
Familia
Posición
CDS*
Cambio aa
%EMS
SIFT
1
F2036
Exón 1
G 74 A
Arg25Lys
1%
0.00
0.02
5
53
Exón 1
G 289 A
Glu97Lys
1%
6
2532
Exón 1
C 294 T
Phe98Phe
2%
7
1420
Intrón 1
G/A
2%
0m #*m 1#%3,"-m +.*'!A,m ^ *m IYO_m 1#m '"#,2'$'!0-,m 3,m 2-2*m "#m GJm +32,2#1m "#m *-1m
!3*#1mGGm1#m&,m"#2#!2"-m#,m*m.- *!'A,m"#*mm7mIm#,m*m.- *!'A,m"#*mYm#m
#12-1Zm Km 2'#,#,m *m +32!'A,m #,m 0#%'A,m #6A,'!Zm Mm #,m 0#%'A,m ',20A,'!m 7m Hm #,m #*m #620#+-m
IaYm 320-m "#m *-1m +32,2#1m /3#m 2'#,#,m *m +32!'A,m #,m *m 0#%'A,m #6A,'!Zm .0-4-!,m
3,m !+ '-m "#m +',-9!'"-m ^KMZm GHHOZm JJJZm HFLF_m 7m *m $+'*'m GNNFm !-,2'#,#m 3,m
+32!'A,m1'*#,!'-1Ym
m$+'*'mGLLMm!-,2'#,#m#*mm#,m#*m.0'+#0m,3!*#A2'"-m"-,"#m#+.'#8m#*m',20A,mIZm.-0m
*-m2,2-Zm.-"0>m4#01#m$#!2"m*m$3,!'A,m"#m-*&##(!O7m#,m!-,1#!3#,!'m*m$3,!'-,*'""m
"#m *m .0-2#>,Ym -"1m *1m +32!'-,#1m '"#,2'$'!"1m #,m #12#m +.*'!A,m 1-,m 20,1'!'-,#1m
ijim#6!#.2-m#,mOKOZm#,m#*m/3#m#*m!+ '-m#1m3,m20,14#01'A,m"#m%3,',mm!'2-1',m
7mGILNZm/3#m!-,2'#,#m3,m!+ '-m"#m2'+',mm!'2-1',Ym
m
!+ T DMJOT 32,2#1m '"#,2'$'!"-1m #,m #*m 1#%3,"-m +.*'!A,m "#m GGLOm . m "#*m %#,m >Ym ,m 0-(-Zm /3<**-1m
#,!-,20"-1m#,m*m.- *!'A,mm7m#,m4#0"#m#,m*m.- *!'A,mYmm
m
Nº
Familia
Posición
CDS
Cambio aa
%EMS
SIFT
2
F57
Exón 2
G 385 A
Glu129Lys
1%
0.00
3
F1880
Exón 2
G 465 A
Thr155Thr
1%
4
F959
Intrón 2
G/C
8
1229
Exón 2
C 401 T
Ala134Val
2%
0.10
9
444
Exón 2
G 407 A
Gly136Glu
2%
0.00
10
1287
Intrón 2
C/A
2%
11
1115
Intrón 2
C/T
2%
12
1667
Intrón 3
G/A primer
nucleótido del
intrón
2%
13
2646
Intrón 3
C/T
2%
14
1233
Intrón 3
C/T
15
2060
Exón 4
G 616 A
1%
2%
Glu206Lys
2%
0.09
GGKm
.#9(OO'/.#)(-O
Resultados
16
2035
Intrón 4
C/T
2%
17
1368
3’UTR
T/C
2%
18
2163
3’UTR
G/A
2%
m
,m 0#13+#,Zm #,m #*m %#,m >m 1#m &,m #,!-,20"-m 3,m 2-2*m "#m GNm +32,2#1m 2#,'#,"-m #,m
!3#,2m*-1m0#13*2"-1m"#m*-1m"-1m+.*'!-,#1Ym!&-m"#m#**-1m^JJZJP_m#,m0#%'A,m#6A,'!ZmNm
#,m0#%'A,m',20A,'!m^JJZJP_m7mHm#,m#*m#620#+-mIam^GGZGGP_Ym#m*-1m+32,2#1m#,m0#%'A,m
#6A,'!Zm Im ^IMZKP_m .#02#,#!#,m m *m .- *!'A,m "#*m m +'#,201m /3#m Km ^LHZKP_m m *m
.- *!'A,m"#*mYm,m*m'%30mIYGLm1#m.0#1#,2m#*m*',#+'#,2-m#,20#m#*m%#,m>m7m
13m '1-$-0+Zm I#-)J>Zm "#m +-"-m /3#m .3#"#m - 1#0401#m *m &-+-*-%>m #,20#m + -1m 7m
"#+91m *m *-!*'8!'A,m "#m *1m +32!'-,#1m '"#,2'$'!"1m #,m #*m .0'+#0-m "#m #**-1m #,m
.-1'!'-,#1m #6A,'!1Ym *%3,1m "#m #121m +32!'-,#1m #129,m *-!*'8"1m #,m 0#%'-,#1m +37m
!-,1#04"1m.0m+ -1m%#,#1Ym
m
m
(&30 T DMBGOT *',#+'#,2-m #,20#m *1m .0-2#>,1m "#m >m 7m #*m I#-)J>m "#m +#*A,m ',"'!,"-m *1m +32!'-,#1m
"#2#!2"1m#,m>m^*1m4#0"#1m!-00#1.-,"'#,2#1mm+32!'-,#1m"#2#!2"1m#,m$+'*'1m"#m*m.- *!'A,mm7m
*1m0-(1m#,m*m.- *!'A,m_Ym
m
1m'+.-02,2#m"#12!0m/3#m"#m*1m!320-m$+'*'1m+32,2#1m"#2#!2"1m#,m*m.- *!'A,m
"#*m Zm Im 2'#,#,m *m +32!'A,m #,m 0#%'A,m #6A,'!m ^MKP_Ym ',m #+ 0%-Zm #,m *m .- *!'A,m
"#*m Zm "#m 3,m 2-2*m "#m GJm $+'*'1m +32,2#1m 1A*-m Km 1#m *-!*'8,m #,m 0#%'A,m #6A,'!m
^IKZMP_Ymm
#%B,m *-1m 4*-0#1m "#m Zm *1m +32!'-,#1m /3#m $#!20,m m *m $3,!'-,*'""m "#m *m
.0-2#>,m1#09,m*1m"#m*1m$+'*'1mHFILZmKIZmKMm7mJJJZm+'#,201m/3#m*1m+32!'-,#1m"#m
GGLm
.#9(OO'/.#)(-O
Resultados
*1m$+'*'1mGHHOm7mHFLFm1#09,m2-*#0"1m1#%B,mm^4*-0TFZFK_m7m*m$3,!'-,*'""m"#m*m
.0-2#>,m ,-m 1#m 4#09m $#!2"Ym #1.#!2-m m *m "'120' 3!'A,m "#m *1m +32!'-,#1m "# #+-1m
"#12!0m/3#m1#m&,m#,!-,20"-m+32,2#1m#,m2-"-1m*-1m#6-,#1m#6!#.2-m#,m#*m#6A,mIYm-0m
-20-m *"-Zm *m "'120' 3!'A,m "#m *1m +32!'-,#1m #,m *1m .02#1m ',20A,'!1m ,*'8"1m .0#!#m
1#0m&-+-%<,#Ym
m
DMCMCMCTTTTTTDTTTT!T
0m !-+.*#+#,20m *-1m #123"'-1m "#m .0#"'!!'A,m !-,m m "#m !A+-m .3#"#,m $#!20m *1m
1312'23!'-,#1m +',-!>"'!1m m *m $3,!'-,*'""m "#m *m .0-2#>,m "#*m %#,m >Zm 1#m 0#*'8Am
3,m#123"'-m"#2**"-m"#m*m#1203!230m#,mIm"#m>Em*m%#,m>m2'#,#m3,m%0,m',2#0<1m
.3#12-m /3#m *m - 2#,!'A,m "#m 3,m 40'#""m 0#1'12#,2#m m "#2#0+',"-1m 4'031m 1#0>m 3,m
&**8%-m'+.-02,2#m2,2-mm,'4#*m!'#,2>$'!-m!A+-m!-+#0!'*Ym-0m#12#m+-2'4-m0#*'8+-1m
3,m #123"'-m +91m #6&312'4-m "#m .0#"'!!'A,m "#m !A+-m .-">,m $#!20m *1m +32!'-,#1m
"#2#!2"1m m *m #1203!230m "#m >Ym *m +-"#*(#m #,m Im 1#m 0#*'8Am !-,m #*m .0-%0+m
'-',$-0+92'!-m 7Ym ,m *1m $'%301m /3#m 1#m .0#1#,2,m m !-,2',3!'A,m 1#m .3#"#,m
4'13*'80m *1m *-!*'8!'-,#1m "#m *1m +32!'-,#1m '"#,2'$'!"1m /3#m .-"0>,m $#!20m m *m
$3,!'-,*'""m"#m>Em
*m+32,2#m,B+#0-mHm^$+'*'mKMZm+32!'A,m*3GHO71_m^1#%B,m *mIYO_m^'%30mIYGM_m
.0#!#m !-,2#,#0m *m +32!'A,m +91m '+.-02,2#m #,m !3,2-m *m .-1' *#m #$#!2-m 1- 0#m *1m
',2#0!!'-,#1m#,20#m*m.0-2#>,m7m13m*'%,"-Ym30,2#m#*m+-"#*(#m1#m#12 *#!'Am*m.-1' *#m
8-,m "#m ',2#0!!'A,m #,20#m >m 7m #*m KaZm 7m /3#m 7m -$0#!>m *m .-1' '*'""m "#m
"#$','0m4'023*+#,2#m^m204<1m"#m!-+.0!'-,#1m!-,m*1m 1#1m"#m"2-1m"#m!0'12*'8!'A,m
"#m3,'-,#1m"#m.0-2#>,1m!-,m13m131202-m-m*'%,"-_m#*m!#,20-m!2'4-m7m#*m*'%,"-m^'%30m
IYGN_Ym,m#12#m!1-m#*m*'%,"-m#1m*mMj+#2'*m%3,-1',m20'$-1$2-m^GGGNKGGHQ_m-mKaZm
+-*<!3*m /3#m 1#m @"#m *m +m 7m *-m .0-2#%#m "#m 13m "#%0"!'A,Ym 12m +-*<!3*m #1m #*m
*3%0m"#m0#!-,-!'+'#,2-m.0m#*m','!'-m"#m*m20"3!!'A,m7m.-0m*-m2,2-m*m/3#m',2#0!!'-,m
"'0#!2+#,2#m!-,m*m.0-2#>,m>m#,m#*m+-+#,2-m"#*m','!'-m"#m*m20"3!!'A,m^- %*'m
7m 0,2Zm HFFL_Ym 12m +32!'A,m #1m #1.#!'*+#,2#m ',2#0#1,2#m .-0/3<m .0#!#m /3#m
- *'%0>m m #12 *#!#0m #,*!#1m "'12',2-1m !-,m #*m *'%,"-Zm *-m /3#m .-"0>m 2#,#0m #$#!2-m #,m *m
$3,!'-,*'""m"#m>Ymm
m
GGMm
.#9(OO'/.#)(-O
Resultados
T
T
(&30 T DMBH[m -"#*(#m "#m *m .0-2#>,m >m #,m Im +#"',2#m #*m /.*/.O "#*m .0-%0+m 7m "#*m +32,2#m Hm
^$+'*'mKM_Ym,m0-(-m7m+0!"m+#"',2#m*m$*#!&Zm*m.-1'!'A,m"#m*m+32!'A,m*3GHO71Ym
m
m
m
(&30 T DMBI[m -"#*(#m "#m *m .0-2#>,m >m #,m Im +#"',2#m #*m /.*/.O "#*m .0-%0+m 7m !-,m .-1' *#m
*'%,"-m ^+-*<!3*m Kdm "#*m +_Ym ,m +0'**-Zm "#2**#m "#m *1m .-1' *#1m ',2#0!!'-,#1m #,20#m #*m *'%,"-m ^#,m
0-(-_m7m#*m+',-9!'"-m*3GHOm^#,m0-(-m7m$*#!&_Ym
m
*m +32,2#m ,B+#0-m Km ^$+'*'m KIZm +32!'A,m *3OM71_m ^1#%B,m *m IYN_m ^'%30m IYGO_m
!-,2'#,#m 3,m +32!'A,m /3#m .3#"#m .0-4-!0m 3,m #$#!2-m !-+.0-+#2'"-m .0m *m
$3,!'-,*'""m "#m *m .0-2#>,Ym *m 9!'"-m %*329+'!-m "#m *m .-1'!'A,m OMm #,m #*m %#,-2'.-m
1'*4#120#m !0#m 3,m #,*!#m "#m &'"0A%#,-m !-,m *m 1#0',m OIm ^'%30m IYHF_Ym ,m !+ '-Zm #,m #*m
+32,2#m #*m 9!'"-m %*329+'!-m /3#"m 1312'23'"-m .-0m 3,m *'1',m 7m <12m .10>m m
GGNm
.#9(OO'/.#)(-O
Resultados
',2#0!!'-,0m !-,m *m 20#-,',m *-!*'8"m #,m *m .-1'!'A,m OGYm -0m *-m 2,2-Zm *m +32!'A,m
.0-"3!'0>m 3,m !+ '-m #,m *1m ',2#0!!'-,#1m #,20#m +',-9!'"-1m 7m .-"0>m $#!20m *m
#1203!230m-m*m#12 '*'""m"#m>Ymm
m
m
m
(&30 T DMBJ[m -"#*(#m "#m *m .0-2#>,m >m #,m Im +#"',2#m #*m /.*/.O "#*m .0-%0+m 7m "#*m +32,2#m KZm
$+'*'mKIYm,m0-(-m7m+0!"m+#"',2#m*m$*#!&Zmm*m.-1'!'A,m"#m*m+32!'A,m*3OM71Ym
m
m
T
(&30 TDMCAOm/.*/.O"#*m.0-%0+m7m"#*m+32,2#mKZm$+'*'mKIYm#2**#m"#m!A+-m!+ '0>m#*m#,*!#m#,m#*m
+32,2#Ym ,m #*m %#,-2'.-m 1'*4#120#m ',2#0!!'-,m #*m 9!'"-m %*329+'!-jOMm !-,m *m 1#0',jOIm 7m #,m !+ '-Zm #,m #*m
+32,2#m',2#0!!'-,0>m*m*'1',jOMm!-,m*m20#-,',jOGYm
*m +32,2#m ,B+#0-m Nm ^$+'*'m GHHOZm +32!'A,m *GIJ*_m ^1#%B,m *m IYO_m 2'#,#m *m
+32!'A,m*-!*'8"m#,m*m13.#0$'!'#m"#m>m^'%30mIYHG_m.-0m*-m/3#m13m#$#!2-m"# #0>m
1#0m +#,-0m .3#12-m /3#m #*m !#,20-m !2'4-m "#m >m 1#m *-!*'8m #,m 3,m .-1'!'A,m ',2#0,Ym
GGOm
.#9(OO'/.#)(-O
Resultados
"#+91Zm*m4*',m7m*m*,',m,-m.-1##,m!0%Zm7m.-0m*-m2,2-m*m1312'23!'A,m,-m2#,"0>m
.0- *#+#,2#m,',%B,m#$#!2-mm,'4#*m#1203!230*Ym
m
(&30 T DMCB -"#*(#m "#m *m .0-2#>,m > #,m Im +#"',2#m #*m 32.32m "#*m .0-%0+m 7m "#*m +32,2#m NZ
$+'*'mGHHOYm,m0-(-m7m+0!"m+#"',2#m*m$*#!&Zm*m.-1'!'A,m"#m*m+32!'A,m*GIJ*YT
m
*m+32,2#m,B+#0-mOm^$+'*'mJJJZm+32!'A,m*7GIL*3_m^1#%B,m *mIYO_m',20-"3!#m3,m
!+ '-m"#m%*'!',mm9!'"-m%*329+'!-Zmm3,m0#1'"3-m"#m!"#,m*0%m*2#0*m!-,m!0%Ymm
.-1'!'A,m"#m#12#m0#1'"3-m,-m#1m13.#0$'!'*m1',-m',2#0,m^'%30mIYHH_Ymm
m
T
T
(&30 T DMCC[m -"#*(#m "#m *m .0-2#>,m >m #,m Im +#"',2#m #*m /.*/.O "#*m .0-%0+m 7m "#*m +32,2#m OZm
$+'*'mJJJYm,m$3!1'm7m+0!"m+#"',2#m*m$*#!&Zm*m+32!'A,m*7GIL*3Ym
GHFm
.#9(OO'/.#)(-O
Resultados
m
#m&,m',!*3'"-m#,m*m'%30mIYHIm2-"1m*1m+32!'-,#1m!-+#,2"1m,2#0'-0+#,2#m.0m
#*m%#,m>Ym1m+32!'-,#1mGm7mGKm,-m1#m,*'80-,Ymmm
m
T
(&30 TDMCDOm-"#*(#m"#m*m.0-2#>,m#,mIm"#*m%#,m>Ym,m0-(-m*1m+32!'-,#1m"#2**"1m,2#0'-0+#,2#m.-0m
13m.-1' *#m'+.*'!!'A,m#,m#*m!+ '-m"#m$3,!'-,*'""m"#m>Em
T
DMCMDT "#!T PT T T T T T T
TEQT
,m #*m * -02-0'-m 7m 1#m & >m ','!'"-m *m 1#!3#,!'!'A,m "#*m %#,m I#-)J>m ^3,'%#,m
JJOFGm "#m *m 1#m "#m "2-1m "#m 3_m 7m .-0m *-m 2,2-m "'1.-,>+-1m "#m 3,m .02#m
'+.-02,2#m "#m *m 1#!3#,!'Ym 3,m 7m 1>Zm 1#m "'1#@0-,m !320-m ,3#4-1m !# "-0#1m ^'%30m
IYHJm _m .0m .-"#0m 2#0+',0m "#m 1#!3#,!'0m *-1m $0%+#,2-1m /3#m $*2 ,m .0m - 2#,#0m *m
1#!3#,!'m!-+.*#2Ym,m4#8m- 2#,'"Zm0#1#!3#,!'+-1m#,m*m40'#""mLHjGGIYm,m#1#m
+-+#,2-m ,-m 1#m & >m .3 *'!"-m 2-"4>m *m 1#!3#,!'m !-+.*#2m "#*m %#,Zm 1',m #+ 0%-Zm
!23*+#,2#m 1'm #129m "'1.-,' *#(#,#,)[m HIHGMHm ^1#!3#,!'m "#*m m /3#m !-,2'#,#m
I#-)J>m#,m*m0#%'A,[mOOIKjGGOGJ__m^#7#0m7m!-*YZmHFFN_Ym#!'"'+-1m','!'0m*m"#2#!!'A,m
"#m*-1m+32,2#1m31,"-m3,m+.*'!A,m#,m*m0#%'A,m"#*m%#,m"#2**"m#,m*m'%30mIYHJZm"#m
GFGJm. m7m/3#m!-,2#,>m20#1m#6-,#1Y
m
GHGm
Resultados
.#9(OO'/.#)(-O
T
T
(&30 T DMCEOT ^ _T 1/3#+m "#*m %#,m I#-)J>m !-+.3#12-m "#m !',!-m #6-,#1m ^!(1m 83*#1_Ym + '<,m .3#"#m
- 1#0401#m ^!_m #*m $0%+#,2-m ,*'8"-m +#"',2#m m "#m GFGJm . m 1>m !-+-m ^_m *-1m !# "-0#1m 32'*'8"-1m
2,2-m .0m 1#!3#,!'0m *m 0#%'A,m !-+-m .0m "#2#!20m *-1m +32,2#1m ^,#6-m Y(_Ym -1m 20'9,%3*-1m "#2**,m *1m
.-1'!'-,#1m#,m*1m/3#m1#m&,m#,!-,20"-m+32!'-,#1Ym,m4#0"#m*-1m+32,2#1m#,!-,20"-1m#,m*m.- *!'A,m"#*m
Ym
T
!23*+#,2#m #12#m %#,m &m 1'"-m '"#,2'$'!"-m #,m *m 1#!3#,!'m "#*m %#,-+m !-+-m
IFHIFIMm!-,m3,m*-,%'23"m2-2*m"#mGOOGm. Ym*m7m!-+-m1#m&m.-"'"-m- 1#040m#,m
*m '%30m IYGLZm *-1m %#,#1m >m 7m 13m '1-$-0+m !-,2'#,#,m 0#%'-,#1m +37m !-,1#04"1Ym
+ '<,m %0!'1m m *m - 2#,!'A,m "#m *m 1#!3#,!'m "#*m %#,-+m "#*m +#*A,m ^0!'j1m 7m
!-*YZm HFGH_m 1#m &m .-"'"-m !-,$'0+0m /3#m 1A*-m #6'12#m 3,m !-.'m "#*m %#,m I#-)J>m ^'%30m
IYHK_Ym
m
(&30 T DMCFOm 0 -*m $'*-%#,<2'!-m "#*m %#,m I#-)J>Ym m *m "#0#!&m 1#m - 1#04m *m 1#!!'A,m +.*'"m "-,"#m 1#m
*-!*'8m #12#m %#,m "#m +#*A,m ^$*#!&m 0-(_Ym #m .3#"#m - 1#040m /3#m 1A*-m &7m 3,m !-.'m ,-2"m #,m #*m %#,-+m
^$3#,2#[m&7*-+#" Y-0%_Ym
GHHm
.#9(OO'/.#)(-O
Resultados
m
DMCMDMBTNTTTTTTT
,m #*m %#,m I#-)J>m 1#m &m '"#,2'$'!"-m 3,m B,'!-m +32,2#m #,m *m .- *!'A,m m 7m
,',%3,-m#,m*m.- *!'A,mm^ *mIYGF_Ym*m+32,2#m"#2#!2"-m.#02#,#!#mm*m$+'*'m
GKNMm 7m 2'#,#m 3,m m #,m #*m .0'+#0m #6A,m "#*m %#,Ym 12m +32!'A,m .0-4-!m 3,m !+ '-m "#m
+',-9!'"-m "#m 0%',',m m *'1',Zm 1',m #+ 0%-Zm 1#%B,m *-1m 4*-0#1m - 2#,'"-1m #,m Zm
.-"0> 1#0m2-*#0"-m7m,-m$#!20mm*m$3,!'A,m"#m*m.0-2#>,Ym
m
!+ TDMBAOT0!2#0>12'!1m"#*m+32,2#m#,!-,20"-m#,m#*m%#,mI#-)J> #,m*m.- *!'A,m"#mm!-,m#*m,-+ 0#m
"#m*m$+'*'Zm.-1'!'A,Zm2'.-m"#m+32!'A,m7mPm"#mm32'*'8"-Ymm+32!'A,m1#%B,mm,-m#1m1'%,'$'!2'4m7m
,-m$#!20>mm*m$3,!'A,m"#m*m.0-2#>,Yg-1'!'A,m0#1.#!2-m*m!# "-0m'1-JYm
m
Nº
Familia
Posición
CDS *
Cambio aa
%EMS
SIFT
1
M1587
Exón1
G128A
Arg43Lys
2%
1.00
T
m
DMCMET
TPTTTBQT
*m ; ^#!#.2-0m "#m 2'*#,-m G_m .#02#,#!#m m 3,m $+'*'m +3*2'%<,'!m ^2-j0m 7m !-*Zm
GOOO_m7m#129m'+.*'!"-m#,m*m1#@*m"#m20,1"3!!'A,m"#*m#2'*#,-m^*##!)#0m7m#,"#ZmHFFF_Ym
129,m "#1!0'21m +32!'-,#1m #,m #12#m %#,m #,m ,#)*-#-O /3#m .0-4-!,m ',1#,1' '*'""m *m
#2'*#,-m ^'*)',1-,m 7m !-*YZm GOOK_m 7m 2+ '<,m 1#m &m "#+-120"-m /3#m &7m 3,m +7-0m
#6.0#1'A,m"#m#12#m%#,m#,m1#+'**1m7m.*!#,2m^2-j0m7m!-*YZmGOOO_Ym,m*m 1#m"#m"2-1m
^&22.[ii555Y'!3%'Y-0%_m "'1.-,>+-1m "#*m 3,'%#,m JNJOOm "#1!0'2-m !-+-m .02#m "#*m %#,m
;Zm.-0m#**-m1#m','!'Am*m1#!3#,!'!'A,mm.02'0m"#m#12#mYm',*+#,2#Zm1#!3#,!'+-1m
3,m%0,m.02#m"#*m%#,m32'*'8,"-mGIm!# "-0#1m#,m"'12',21m!-+ ',!'-,#1Zm"#2**"-1m
#,m*m'%30mIYHLYm
m
(&30 TDMCGOT1/3#+m"#*m%#,m;m^ _Zm"#m*-1m!# "-0#1m"'1#@"-1m.0m13m1#!3#,!'!'A,m^_m^,#6-mY(_m7m"#*m
$0%+#,2-m"#*m%#,m"#mLGKm. m,*'8"-m+#"',2#mm^!_Ym
m
!23*+#,2#m1#m&m.-"'"-m- 2#,#0m*m1#!3#,!'m!-+.*#2m"#*m%#,m;O#,m+#*A,Ym*m%#,m
'"#,2'$'!"-m!-+-mIFFIOFLm2'#,#m3,m*-,%'23"m2-2*m"#mJONNm. m7m#129m!-+.3#12-m
GHIm
T
Resultados
.#9(OO'/.#)(-O
.-0m 1#'1m #6-,#1m 2*m 7m !-+-m 1#m & >m '"#,2'$'!"-m +#"',2#m *m 1#!3#,!'!'A,m #,m *m
40'#""mLHjGGIYm-+-m#,m*-1m%#,#1m.0#1#,2"-1m,2#0'-0+#,2#Zm2+ '<,m1#m&m.-"'"-m
- 2#,#0m#*m90 -*m$'*-%#,<2'!-m"#*m%#,m;m!-+.0- ,"-m/3#m$-0+m.02#m"#m3,m$+'*'m
+3*2'%<,'!m7m!-+-m2*Zm#,!-,20+-1m"-1m%#,#1m.09*-%-1m#,m+#*A,m^'%30mIYHM_Ym
m
m
T
(&30 TDMCHOm0 -*m$'*-%#,<2'!-m"#*m%#,m;Ymm*m"#0#!&m1#m- 1#04m*m1#!!'A,m+.*'"m"-,"#m1#m*-!*'8m
#12#m%#,m"#m+#*A,m^$*#!&m0-(_Ym#m.3#"#,m- 1#040mm*m'8/3'#0"m^1#@*"-1m!-,m$*#!&1m83*#1_m*-1m.09*-%-1m
*m%#,m;m^$*#!&m0-(_m^$3#,2#[m&7*-+#" Y-0%_Ym
T
DMCMEMBTNTTTTTTT
01m #*m ,9*'1'1m +#"',2#m m "#*m %#,m ;m ,-m 1#m "#2#!20-,m +32,2#1m #,m *m
.- *!'A,m "#*m Ym -0m -20-m *"-Zm #,m *m .- *!'A,m "#*m m ,-m 1#m .3"-m ,*'80m #12#m
%#,m .3#12-m /3#m *-1m !# "-0#1m "'1#@"-1m m .02'0m "#m *m 40'#""m 31"m #,m *m .- *!'A,m
Zm ,-m $3,!'-,0-,m !-00#!2+#,2#m 7m ,-m +.*'$'!0-,m #,m *m 40'#""m ",(.#-Ym #m
"'1#@0-,m ,3#4-1m !# "-0#1m 3,/3#m 1'#+.0#m m .02'0m "#m *m 1#!3#,!'m "#m 0#$#0#,!'m
GHJm
.#9(OO'/.#)(-O
Resultados
- 2#,'"m"#m*m40'#""mLHjGGIYm12#m1#%3,"-m"'1#@-m"#m!# "-0#1m2+.-!-m+.*'$'!Am
*m40'#""m",(.#-Ym',*+#,2#Zm,-m&3 -m+91m2'#+.-m.0m,*'80m!39*m#0m#*m+-2'4-m
.-0m#*m!39*m*-1m!# "-0#1m,-m&' 0'" ,m!-00#!2+#,2#m7m.-0m#**-m1#m"#1!02Am#12#m%#,m#,m
#*m ,9*'1'1m "#m m #,m *m .- *!'A,m Ym -0m #*m !-,200'-Zm #*m %#,m ;m 1'm 0#13*2Am
',2#0#1,2#m.0m#*m,9*'1'1m"#m!-m/3#m1#m.0#1#,209m.-12#0'-0+#,2#m
m
DMCMFTTTTTTTTTTT
,m *m ' *'-%0$>m 1#m #,!3#,20,m "'12',2-1m +-"-1m "#m #6.0#10m *m 21m "#m #$'!'#,!'m "#m
+32!'A,m"#m3,m.- *!'A,m"#mYm*m!9*!3*-m 91'!+#,2#m"# #m2#,#0m#,m!3#,2m#*m
,B+#0-m"#m+.*'!-,#1m,*'8"-1Zm#*m,B+#0-m"#m$+'*'1mHm"#m*1m/3#m#129m!-+.3#12m
*m.- *!'A,m7m#*m,B+#0-m"#m+32,2#1m#,!-,20"-1Ym,m*m *mIYGGm1#m.3#"#,m4#0m*1m
211m"#m+32!'A,m"#1!0'21m#,m"'4#011m.- *!'-,#1m"#mm.0m"'12',21m#1.#!'#1Ym
7m /3#m 2#,#0m #,m !3#,2m /3#m *m 21m - 2#,'"m ,-m 1#m &m !*!3*"-m "#m '%3*m +,#0m #,m
!"m.- *!'A,Zm.-0m*-m/3#m!3*/3'#0m!-+.0!'A,m#1m.0-6'+2'4Ymm
,m #12#m 20 (-Zm #*m !9*!3*-m 1#m &m 0#*'8"-m 2#,'#,"-m #,m !3#,2m #*m ,B+#0-m "#m .0#1m "#m
1#1m,*'8"-1m#,mIm"#m*-1m%#,#1m/3#m1#m&,m#123"'"-m^Zm>m7mI#-)J>_Ym*m,-m
.-"#0m,*'80m#*m%#,m;m#,m*m.- *!'A,m"#*mm1#m"#!'"'Am"#1!020m#12#m%#,m7m,-m
2#,#0*-m#,m!3#,2m.0m#*m!9*!3*-m"#m*m21m"#m+32!'A,Ym1>Zm#*m!9*!3*-m1#m&m&#!&-m"#*m
1'%3'#,2#m+-"-[m
m
Gj Em "#m 1#1m ,*'8"1m 6m Em "#m $+'*'1m /3#m 1#m &,m ,*'8"-m Sm Em 1#1m
,*'8"1m#,m2-2*m
m
Hj mEm 1#1m,*'8"1m#,m2-2*imEm"#m+32,2#1m#,!-,20"-1m#,m!"m.- *!'A,mSmmmm
. im32,2#m
T
!+ TDMBBOT$'!'#,!'m"#m+32!'A,m"#m"'12',21m.- *!'-,#1m"#mm#,m"'12',21m#1.#!'#1Ym1m"-1mB*2'+1Zm
1-+ 0#"1Zm!-00#1.-,"#,mm*1m"#100-**"1m#,m+#*A,Ym
m
1/$"($T
%("($-"( T#$T
,32 "(>-T
$%$0$-"( T
# 0$'1&m
GiHIK jGiKHMm m
'#,&-*"1m7m!-*YZmHFFIm
0.#0m7m!-*YZmHFFJ\mm
,)-)*"#&O'&()!-.,O
GiGKLm m
',)*#0m7m!-*YZmHFFKm
,#)*-#-O."&#(O
GiIFFm m
0##,#m7m!-*YZmHFFIm
O'3-O
GiKFFm m
'**m7m!-*YZmHFFJm
GHKm
.#9(OO'/.#)(-O
Resultados
,34O-.#0O
GiKFFm m
3m7m!-*YZmHFFKm
),/'O0/&!,O
GiGFFFm m
*"5#**m7m!-*YZmHFFJm
,#.#/'O-.#0/'OI@2JO
GiHJm m
*"#m7m!-*YZmHFFKm
,#.#/'O./,!#/'O-/-*EO
/,/'OI>2JO
GiJFm m
*"#m7m!-*YZmHFFKm
#!)O.,/(./&O
GiJNKm m
#m'%,-0m7m!-*YZmHFFOm
GiKMIm m
&+,'j0"1m7m!-*YZm
HFGFm
GiGYKm m-m
FYIIiKFFm m
-,8*#8j-m7m!-*YZmHFGGm
$$P' 0$-2 (1QT
PTQT
$$P($+T#$T
/.Q$"#$
m
,m*m.- *!'A,mm1#m&,m,*'8"-mIYNMGmm. m^GYGNNm#,mZmGYLLOm#,m>m7mGYFGJm#,m
I#-)J>_m#,m3,m2-2*m"#mHYILNm$+'*'1Ym*m,B+#0-m2-2*m"#m. m,*'8"-1m#1m"#mOZGMm Ym
#,'#,"-m #,m !3#,2m /3#m .0m *-1m 20#1m %#,#1m 1#m &,m #,!-,20"-m Lm +32,2#1Zm .-"#+-1m
#6.0#10m*m21m"#m+32!'A,m!-+-m/3#m!"mGYKHNm m.-"0>+-1m#,!-,200m3,m+32,2#Ym
m*-m/3#m#1m*-m+'1+-ZmFZIIm+32,2#1imKFFm m.0m.-"#0m!-+.00m+#(-0m!-,m*1m211m
- 2#,'"1m#,m-201m.- *!'-,#1m"#m-201m#1.#!'#1m^ *mIYGG_Ym
#m "#!'0m /3#m .0m /3#m 3,m 21m "#m +32!'A,m 1#m +91m $' *#m &!#m $*2m ,*'80m 3,m
+7-0m,B+#0-m"#m%#,#1mm*-1m,*'8"-1m#,m#12#m20 (-Ym*m,B+#0-m"#m%#,#1m,*'8"-1m
"30,2#m#*m"#100-**-m"#m-201m.- *!'-,#1m#1m%#,#0*+#,2#mGFm^3m7m!-*YZmHFFK\m*"5#**m
7m!-*YZmHFFJ\m0##,#m7m!-*YZmHFFI\m&+,'j0"1m7m!-*YZmHFGF_Ym
129m"#1!0'2-m/3#m.0m/3#m3,m.- *!'A,m"#mm1#mB2'*m13m21m"#m#$'!'#,!'m"# #m
"#m 1#0m TGm +32,2#iKFFm m ^'**Zm HFFM_Ym ',m #+ 0%-Zm "# #+-1m 2#,#0m #,m !3#,2m /3#m #12m
21m "#m +32!'A,m 1#m &m !*!3*"-m #,m 00-8m 7m #,m 3,m .- *!'A,m "#m MLNm ',"'4'"3-1m ^'**Zm
HFFM_Ymm
m21m- 2#,'"m#,m*m.- *!'A,m"#*mZm201m,*'80mGGm%#,#1m&m1'"-m"#mGiKMIm m
^&+,'j0"1m 7m !-*YZm HFGF_Ym #0-m .0m .-"#0m #12 *#!#0m 3,m +#(-0m !-+.0!'A,m
#,20#m *1m .- *!'-,#1m m #m Zm 1#m &m 2#,'"-m #,m !3#,2m .0m #12m B*2'+m 1A*-m #*m
,9*'1'1m !-,m *-1m %#,#1m /3#m 1#m &,m ,*'8"-m #,m + 1m .- *!'-,#1m ^>FO I#-)J>Zm
_Ym,m4#8m0#*'8"-m#*m!9*!3*-m"#m*m21m"#m+32!'A,m"#m*m.- *!'A,mm!-,m*-1m
!0'2#0'-1m "#1!0'2-1m ,2#0'-0+#,2#m 1#m &m - 2#,'"-m 3,m 21m "#m +32!'A,m "#m Gm +32!'A,m
!"mKFFm Zmm.02'0m"#mGOm+32,2#1m#,mHYJNIm$+'*'1Ym
m
T
T
T
T
GHLm
.#9(OO'/.#)(-O
Resultados
!+ TDMBCOT1m"#m+32!'A,m#,m*1m.- *!'-,#1m"#mm"#*mm7m#*mYm
m
T
32 -2$1T
$-$1T
- +(8 #.1T
/!T - +(8 #.1T
,(+( 1T
1 T
Lm
Im
INMHm
HILNm
GiGZK m
GOm
Im
INMHm
HJNIm
GiKFF m
.!+ "(>-T
T
.!+ "(>-T
T
m
',*+#,2#Zm2#,'#,"-m#,m!3#,2m*m#$'!'#,!'m0#/3#0'"m1#%B,m'**ZmHFFMm1#m&m!*!3*"-m#*m
0#,"'+'#,2-m"#m*m.- *!'A,[m
m
$'!'#,!'m0#*mmmmmm6mGFFmSmPm"#m0#,"'+'#,2-m#$'!'#,!'m#1.#0"m
$'!'#,!'m2#A0'!m
m
-0m*-m2,2-m#,m*m.- *!'A,mm1#m&m- 2#,'"-m3,mIIPm^^GiGKFFm iGiKFFm _m6GFF_m"#m
0#,"'+'#,2-m "#m +32!'A,m +'#,201m /3#m #,m *m .- *!'A,m "#*m Zm 3,m GFFPm ^^GiKFFm
iGiKFFm _m 6m GFF_Ym m #$'!'#,!'m "#m +32!'A,m #,m *m .- *!'A,m "#*m m &m 1'"-m
!*0+#,2#m13.#0'-0m^20#1m4#!#1m+91Zm.0-6'+"+#,2#_mm*m#,!-,20"m#,m*m.- *!'A,m
"#*mYmm
m
GHMm
)
Resultados
T
DMDT".T
m
,m#12#m20 (-m1#m&,m,*'8"-m+#"',2#m!-m*-1m%#,#1m>m7m;Ym-1m
%#,#1mm7mI#-)J>m$3#0-,m,*'8"-1m#,m#*mZm#,m#*m+0!-m"#*m.0-7#!2-m
Ym 1m GGIm 40'#""#1m "#m +#*A,m ,*'8"1m !-00#1.-,"#,m m .02#m "#m *m
!-*#!!'A,m "#*m
,!-m "#m %#0+-.*1+m "#*m m 7m 0#.0#1#,2,m 3,m %0,m
40' '*'""m m ,'4#*m $#,-2>.'!-m 7m /3#m 1-,m -0'%',0'1m "#m IOm .>1#1m "'12',2-1m 7m
.#02#,#!'#,2#1m m "'12',2-1m %03.-1Ym 91m !-,!0#2+#,2#m #,!-,20+-1m Gm 40'#""m
.#02#,#!'#,2#m#*m%03.-m"3"'+ZmHLm+#*-ZmIm!-,-+-,ZmGHm!,2*3.#,1'1ZmJJm',-"-031Zm
Mm $*#63-1-1Zm Hm +-+-0"'!Zm Gm "8&30j!&2#Zm GFm %0#12'1Zm Jm !&,"*)Zm Gm 22')m 7m Gm
+#0'm ^,#6-m Y)_Ym #m &m - 1#04"-m /3#m #6'12#m 40' '*'""m ,230*m #,20#m *1m GGIm
40'#""#1m,*'8"1m7m*m*>,#m"#m0#$#0#,!'mLHjGGIm.0m#12-1m"-1m%#,#1Ym
m
DMDMBTTT".TTTT!T
0m#*m,9*'1'1m"#m!-m1#m31Am*m+'1+m#1202#%'m31"m.0m#*mm
"#m+-"-m/3#m1#m,*'80-,m*-1m"-1m+.*'!-,#1m"#*m%#,m>m#,m*1mGGIm!!#1'-,#1Ym
*m.0'+#0m+.*'!A,m',!*37#m#*m.0'+#0m#6A,m+'#,201m/3#m#,m#*m1#%3,"-m.0#1#,2m*-1m
!320-m#6-,#1m0#12,2#1m/3#m!-,2'#,#m#*m%#,m>m^'%30mIYGJ_Ym*m'%3*m/3#m.0m#*m
+<2-"-m"#mZm+#"',2#m*m1#!3#,!'!'A,m"#m*1m40'#""#1m^1#m#1!-('#0-,m"-1m
40'#""#1m.-0m!"mm4'13*'8"-m#,m#*m%#*_m.3"'+-1m"#2#0+',0m*m.-1'!'A,m"#m
*-1m 1m 7Zm *m +'1+-m 2'#+.-Zm !-+.0- 0m 1'm <12-1m .0-4-! ,m !+ '-1m #,m *m
1#!3#,!'m+',-!>"'!Ymm
,m#*m.0'+#0m+.*'!A,m1#m"#2#!20-,mMm1m^GYGmmGYM_m^'%30mIYHNm7m *mIYGI_Zm"#m
*-1m/3#m1A*-m#*mmGYKm.0-4-!m3,m!+ '-m"#m+',-9!'"-[m1.MG*7Ym*m.0-%0+m
m 1'%,m 3,m 4*-0m "#m FYFFm m #12#m !+ '-m 7m .-0m *-m 2,2-Zm "#2#0+',m /3#m .3#"#m
$#!20m m *m $3,!'-,*'""m "#m *m .0-2#>,Ym A*-m *m !!#1'A,m m KFKLFHm !-,2'#,#m #12#m
Zm#,m&#2#0-!'%-1'1Ymm
m
GHNm
)
Resultados
m
m
!_m
T
(&30 T DMCIOT #*m "#m !-m .0m #*m .0'+#0m +.*'!A,m "#m >Ym _m !,*m MFFZm _m !,*m NFFYm
"m!00'*m!-00#1.-,"#mm*m+#8!*m"#m3,m"#m*1mGGIm40'#""#1m!-,m*m0#$#0#,!'mLHjGGIYm,m0-(-m7m
4#0"#m 1#m &,m "#2**"-m *-1m &#2#0-"B.*#6m "#2#!2"-1m ^GYGjGYM_Ym ,m +0'**-m 1#m "#2**,m *%3,-1m
&#2#0-"B.*#6m $*1-1m -m 02#$!2-1m "#1!02"-1m +#"',2#m 1#!3#,!'!'A,Ym *%3,-1m &#2#0-"B.*#6m ,-m 1#m
4'13*'80-,m !-00#!2+#,2#m #,m + -1m !,*#1m 1',-m 1A*-m #,m 3,-m "#m #**-1Zm !_m *-!*'8!'A,m "#m *-1m 1m
"#2#!2"-1m+#"',2#m!-m#,m#*m.0'+#0m+.*'!A,Ym
m
GHOm
)
Resultados
*m 0#12-m "#m 1m "#2#!2"-1m #,m #*m .0'+#0m +.*'!A,m "#m >m 1-,m 1'*#,!'-1-1m -m ,-m
#129,m #,m 0#%'A,m #6A,'!Ym *m m GYLm ^'%30m IYHO_m #1m 3,m .-*'+-0$'1+-m !-+.*#(-Zm
$-0+"-m .-0m 3,m ',1#0!'A,m "#m "-1m 2'+',1m 7m 3,m +'!0-12<*'2#Ym -0m *-m 2,2-Zm #*m
&#2#0-"B.*#6m $-0+"-m #,m #12#m !1-m .3#"#m 1#0m "# '"-m m *m ',1#0!'A,m "#m *1m "-1m
2'+',1m-m*m+'!0-12<*'2#m/3#m#129m*-!*'8"-m(312-mm!-,2',3!'A,Ym,m*m'%30mIYHOm
.3#"#m 4#01#m !-+-m 3,m "#m *1m 40'#""#1m /3#m !-,2'#,#,m #12#m .-*'+-0$'1+-m
^JMJ_m !-,2'#,#m *1m ',1#0!'A,m "#m "-1m 2'+',1m 1#%3'"1m "#*m +'!0-12<*'2#m ^_MZm
+'#,201m /3#m *m 40'#""m "#m 0#$#0#,!'m LHjGGIm ,-m !-,2'#,#m *m ',1#0!'A,m 7m #*m
,B+#0-m"#m0#.#2'!'-,#1m"#*m+'!0-12<*'2#m#1m^_LYmm
m
(&30 TDMCJOT0-+2-%0+m"#m*1m40'#""#1mJMJm7mLHjGGIm#,m*m0#%'A,m/3#m!-,2'#,#m#*mmGYLmYm
*mmGYLm#129m!-+.3#12-m.-0m3,m',1#0!'A,m"#m"-1m2'+',1m7m3,m0#.#2'!'A,m"'!'-,*mm#,mJMJYm
m
m
GIFm
m
)
Resultados
!+ TDMBDOT-1'!'A,Zm!+ '-m"#m,3!*#A2'"-m7m"#m+',-9!'"-m"#m!"mm"#2#!2"-Zm40'#""#1m/3#m*-m
!-,2'#,#,m7m#*m2+@-m"#*m$0%+#,2-m"#m"'%#12'A,m1#%B,mMFFm7mNFFm.0m#*m.0'+#0m+.*'!A,m"#*m
%#,m >Ym ,m 1-+ 0#"-m %0'1Zm #*m m GYKm /3#m .0-4-!m 3,m !+ '-m ,-m 1',A,'+-m #,m *m .-1'!'A,m MGm "#m *m
1#!3#,!'m.0-2#'!Ymg[m'#*m"#m.-Zm![m!!#1'A,Ym
m
m
GYGm
-1'!'A,m
Kdm
m
^j!_gm
^jJH_m
m
jm
m
jm
-+ 0#m0'#""#1m
mKFKLFHZmmHNHJJNm
mHLLOJMZmmGHJGGHm
+@-m
+@-m
$0%+#,2-m
$0%+#,2-m
!,*mm
!,*mm
mMFFm
mNFFm
KFm. m
JKFm. m
GMHm. m
IHNm. m
HGJm. m
HNLm. m
HMMm. m
HHIm. m
DADT/!T
BJHT/!T
IIJm. m
GLLm. m
JGOm. m
NGm. m
HFLZmGONm
GYHm
6A,Gm
NGm
0%HM0%m
jm
mJILKIJZmmGHJGGHm
mHLLOJMZmmHNHJJNm
mLGNNKJZmjjGFm
GYIm
6A,Gm
GHIm
*JG*m
jm
mGNKGGGZmmmmJILKIJm
mHNMm
HOJZmHKOm
mGJFLIHZmILIm
HHKZmIMHm
mJILKIJZmjHNMm
GYJm
6A,Gm
GNLm
#3KK#3m
jm
mGNKGGGZmmGLJIHFm
jjHZmjjGFOm
jKKFZmjLHm
JGLZmJLNm
JMJZmKFFm
,1GIFFIHZmmHNHJJNm
BMFT
6>-BT
CBCT
1/HB+7T
AMAAT
TFAFGACT
HOJZmHKOm
mGJFLIHZmHHKm
m^_mMm
GYLm
,20A,mGm
HJIm
&0NG&0m
ST
jj^_mLm
mHNHJJNZmIJJm
KIZmjjHm
jKKFZmjLHm
JGLZmJMJm
GYMm
,20A,mGm
IHNm
jm
ST
mGJFLIHZmjjHm
jjNZmJMJm
m
GIGm
Resultados
)
0m#*m1#%3,"-m+.*'!A,m1#m"#2#!20-,mKm1m^'%30mIYIFZm *mIYGJ_m.#0-m1A*-m
#*mmHYKm#129m*-!*'8"-m#,m0#%'A,m#6A,'!Ym',m#+ 0%-Zm#*m!+ '-m"#m+',-9!'"-m
/3#m.0-4-!m#1m1',A,'+-m^71HIF71_m7m.-0m*-m2,2-m,-m$#!20>mm*m$3,!'-,*'""m
"#m *m .0-2#>,Ym ,m *m '%30m IYIFm .3#"#m 4#01#m #*m %#*m !-,m *-1m &#2#0-"B.*#6m
'"#,2'$'!"-1m.0m#12#m+.*'!A,Ym#1"#m#*m.3,2-m"#m4'12m$#,-2>.'!-m*-1m&.*-2'.-1m
"#2#!2"-1m #,m #12#m 1#%3,"-m +.*'!A,m ,-m 2'#,#,m ,',%B,m ',2#0<1m 7m /3#Zm -m #129,m
*-!*'8"-1m#,m0#%'A,m',20A,'!Zm-m.0-4-!,m!+ '-1m1',A,'+-1Ym
1#04,"-m *1m "'12',21m !-+ ',!'-,#1m #,20#m *-1m 1m '"#,2'$'!"-1m #,m *1m GGIm
!!#1'-,#1m1#m&,m1'%,"-m3,m2-2*m"#mGOm&.*-2'.-1Ym#m*1mGGIm!!#1'-,#1ZmLNm,-m
.0#1#,2,m ,',%B,m .-*'+-0$'1+-m 0#1.#!2-m *m 40'#""m "#m 0#$#0#,!'m 7m .-0m *-m 2,2-m
2'#,#,m#*m+'1+-m%#,-2'.-m#,m*-1m$0%+#,2-1m,*'8"-1m"#*m%#,m>O1'%,9,"-*#1m
1>m #*m &.*-2'.-m !#0-m ^F_Ym ,m !+ '-Zm *1m JKm !!#1'-,#1m 0#12,2#1m .0#1#,2,m 1m
/3#m*1m"'$#0#,!',m"#m*m40'#""mLHjGGIYm
,m *m *m IYGKm 1#m .0#1#,2,m *-1m .-*'+-0$'1+-1m /3#m .0#1#,2m !"m !!#1'A,Zm *1m
!3*#1m &,m 1'"-m -0"#,"1m 1#%B,m *m !,2'""m "#m 1m /3#m !-,2'#,#,Zm #+.#8,"-m
.-0m *1m /3#m !-,2'#,#,m 1A*-m 3,m m 7m !-,2',3,"-m .-0m *1m /3#m !-,2'#,#,m
!-+ ',!'-,#1m"#m"-1Zm20#1Zm!320-Zm1#'1m7m1'#2#m1Ym
1#04,"-m *m *m IYGKm 1#m .3#"#m $'0+0m /3#m *-1m 1m /3#m .0#!#,m !-,m +91m
$0#!3#,!'m1-,m#*mmGYJm.0m#*m.0'+#0m+.*'!A,m7m#*mmHYGm.0m#*m1#%3,"-Zm/3#m
.0#!#,m #,m HFiGGIm 7m GLiGGIm !!#1'-,#1Zm 0#1.#!2'4+#,2#Ym m *m ',4#01Zm *-1m +#,-1m
$0#!3#,2#1m7m.-0m*-m2,2-m*-1m+91m#1.#!>$'!-1m"#m!!#1'A,m1-,m*-1m1mGYGZmGYIZmGYKm7m
#*m m GYMm .0m #*m .0'+#0m +.*'!A,Zm .0#!'#,"-m #,m 2,m 1A*-m JiGGIZm IiGGIZm GiGGIm 7m
JiGGIm!!#1'-,#1Zm0#1.#!2'4+#,2#Ym0m#*m1#%3,"-m+.*'!A,m#*mmHYKm#1m#*m+#,-1m
$0#!3#,2#m.0#!'#,"-mB,'!+#,2#m#,m*m!!#1'A,mmKFKLFHZm/3#m2+ '<,m#1m*mB,'!m
/3#m!-,2'#,#m#*mmGYKYmm
,m 2-2*Zm 1#m &,m "#2#!2"-m GHm .-*'+-0$'1+-1m 0#1.#!2-m m *m 40'#""m LHjGGIm #,m JKm
!!#1'-,#1m"#m*1mGGIm,*'8"1Zm#,m3,m0#%'A,m"#mGLLOm. m^KFFQGGLOm. _Ymm
*m&.*-2'.-m+91m$0#!3#,2#m#1m#*mKm^mHYG_Zm#,!-,20"-m#,mGFm!!#1'-,#1Zm1#%3'"-m
"#*m Hm ^m GYJ_m #,!-,20"-m #,m Mm !!#1'-,#1Ym 6'12#,m GGm &.*-2'.-1m /3#m 1#m
#,!3#,20,m1A*-m#,m3,m!!#1'A,m^JZmMZmNZmGFZmGGZmGIZmGJZmGKZmGLZmGMm7m
GN_Ym*m+91m0#*#4,2#m#1m#*mGIZm7m/3#m#1m#*mB,'!-m/3#m!-,2'#,#m*-1m1mGYKm7mHYKm
^ *m IYGK_Zm 1'#,"-m #*m GYKm ^'%30m IYIG_m #*m B,'!-m /3#m .0-"3!#m 3,m !+ '-m "#m
+',-9!'"-m 7m 2'#,#m 3,m 4*-0m FYFFm #,m Ym m !!#1'A,m /3#m *-m !-,2'#,#m #1m *m m
KFKLFHZm .#02#,#!'#,2#m *m %03.-m %0#12'1m 7m .0-4#,'#,2#m "#m + 'Ym ',m #+ 0%-Zm m
GIHm
)
Resultados
#12#m!+ '-m"#m+',-9!'"-m,-m1#m*#m.3#"#m20' 3'0m3,m$#,-2'.-m!-,!0#2-m0#*!'-,"-m
!-,m0#1'12#,!'m.3#12-m/3#m,-m&7m',$-0+!'A,m ' *'-%09$'!m,'m#6.#0'+#,2*m!-,m*m
/3#m1#m.3#"m0#*!'-,0m#12#m%#,-2'.-m!-,m3,m0#1'12#,!'m$0#,2#m*%B,m4'031Ymm
m
m
m
!_m
m
m
(&30 TDMDAOT#*m"#m!-m.0m#*m1#%3,"-m+.*'!A,m"#m>Ym_m!,*mMFFm _m!,*mNFFYm
"m!00'*m!-00#1.-,"#mm*m+#8!*m"#m3,m"#m*1mGGIm40'#""#1m!-,m*m0#$#0#,!'mLHjGGIYm,m0-(-m7m
4#0"#m 1#m &,m "#2**"-m *-1m &#2#0-"B.*#6m "#2#!2"-1m ^m HYGjHYK_Ym ,m +0'**-m 1#m "#2**,m *%3,-1m
GIIm
)
Resultados
&#2#0-"B.*#6m $*1-1m -m 02#$!2-1m "#1!02"-1m +#"',2#m 1#!3#,!'!'A,Zm !_m *-!*'8!'A,m "#m *-1m 1m
"#2#!2"-1m+#"',2#m!-m#,m#*m1#%3,"-m+.*'!A,Ym
m
!+ TDMBEOT-1'!'A,Zm!+ '-m"#m,3!*#A2'"-m7m"#m+',-9!'"-m"#m!"mm"#2#!2"-Zm40'#""#1m/3#m*-m
!-,2'#,#,m7m#*m2+@-m"#*m$0%+#,2-m"#m"'%#12'A,m1#%B,mMFFm7mNFFm.0m#*m1#%3,"-m+.*'!A,m"#*m
%#,m>Emg[m'#*m"#m.-Zm![m!!#1'A,Ym
m
m
-1'!'A,m
^j!_gm
m
m
-+ 0#m0'#""#1m
+@-m
+@-m
$0%+#,2-m
$0%+#,2-m
!,*mm
!,*mm
mMFFm
mNFFm
ILGm. m
NFNm. m
KOGm. m
KMNm. m
MOIm. m
IMLm. m
NHJm. m
IJKm. m
NLJm. m
IFKm. m
JINZmmGJFLIHm
mJOFINNZmILIm
IINZmmHNHJJNm
HYGm
,20A,Hm
HHNKiQm
jm
jm
mHLLOJMZmmGHJGGHm
jjOZmmGLOIHOm
mGLOILNZmjHJGm
YmNHGKZmKFFm
1'!jLZm7HNKm
JINZmGONm
HYHm
,20A,Im
HKGKm
jm
jm
mHNHJJNZmHNMm
mHLLOJMZmmGHJGGHm
mJOFINNZmmHMLLLFm
HYIm
,20A,mJm
HMGMm
jm
jm
mHLLOJMZmmGHJGGHm
GONZmmGLMFKMm
mHNHJJNZmmHLLOJMm
HYJm
,20A,Jm
HMJNm
jm
jm
mGHJGGHZmGONm
JINm
HYKm
T
m
m
m
m
m
m
GIJm
6A,Km
HMNNm
71HIF71m
jm
mKFKLFHm
)
Resultados
!+ T DMBFOT .*-2'.-1m #,!-,20"-1m +#"',2#m #*m ,9*'1'1m .-0m !-m "#m *-1m "-1m +.*'!-,#1m "#m
#JYm ,m 1-+ 0#"-m %0'1m 1#m &m "#2**"-m #*m m GYKm /3#m .0-4-!m 3,m !+ '-m "#m +',-9!'"-m #,m *m
!!#1'A,mKFKLFHYmm
Tm
-+ 0#m
GYGm
GYHm
GYIm
GYJm
BMFT
GYLm
GYMm
HYGm
HYHm
HYIm
HYJm
HYKm
HFLm
m
6m
m
m
T
m
m
m
m
m
m
m
mLGNNKJm
m
6m
m
m
T
m
m
m
m
m
m
m
jjGFm
m
6m
m
m
T
m
m
m
m
m
m
m
ILIm
m
m
m
6m
T
m
m
m
m
m
m
m
HHKm
m
m
m
6m
T
m
m
m
m
m
m
m
IMHm
m
m
m
6m
T
m
m
m
m
m
m
m
mGLJIHFm
m
m
m
6m
T
m
m
m
m
m
m
m
40'#""m
jjGFOm
m
m
m
6m
T
m
m
m
m
m
m
m
JLNm
m
m
m
6m
T
m
m
m
m
m
m
m
1GIFFIHm
m
m
m
6m
T
m
m
m
m
m
m
m
HHKm
m
m
m
m
T
6m
m
m
m
m
m
m
IJJm
m
m
m
m
T
6m
m
m
m
m
m
m
KIm
m
m
m
m
T
6m
m
m
m
m
m
m
jNm
m
m
m
m
T
m
6m
m
m
m
m
m
mGJFLIHm
m
m
m
m
T
m
m
6m
m
m
m
m
ILIm
m
m
m
m
T
m
m
6m
m
m
m
m
IINm
m
m
m
m
T
m
m
6m
m
m
m
m
jjOm
m
m
m
m
T
m
m
6m
m
m
m
m
mGLOIHOm
m
m
m
m
T
m
m
6m
m
m
m
m
mGLOILNm
m
m
m
m
T
m
m
6m
m
m
m
m
.*-2'.-[m1m
G[GYHm
H[mGYJm
I[mGYLm
J[GYMm
K[mHYGm
jHJGm
m
m
m
m
T
m
m
6m
m
m
m
m
YmNHGKm
m
m
m
m
T
m
m
6m
m
m
m
m
1'!jLm
m
m
m
m
T
m
m
6m
m
m
m
m
HNKm
m
m
m
m
T
m
m
6m
m
m
m
m
mJOFINNm
m
m
m
m
T
m
m
m
6m
m
m
m
mHMLLLFm
m
m
m
m
T
m
m
m
6m
m
m
m
mGLMFKMm
m
m
m
m
T
m
m
m
m
6m
m
m
M[HYIm
mGNKGGGm
m
m
6m
6m
T
m
m
m
m
m
m
m
N[mGYIQGYJm
HOJm
m
m
m
6m
T
6m
m
m
m
m
m
m
HKOm
m
m
m
6m
T
6m
m
m
m
m
m
m
jKKFm
m
m
m
6m
T
6m
m
m
m
m
m
m
L[HYHm
O[mGYJQGYLm
GIKm
)
Resultados
jLHm
m
m
m
6m
T
6m
m
m
m
m
m
m
JGLm
m
m
m
6m
T
6m
m
m
m
m
m
m
KFFm
m
m
m
6m
T
m
m
6m
m
m
m
m
GF[mGYJQHYGm
mJILKIJm
m
6m
6m
6m
T
m
m
m
m
m
m
m
GG[mGYHQGYIQGYJm
jjHm
m
m
m
6m
T
6m
6m
m
m
m
m
m
JMJT
m
m
m
6m
T
6m
6m
m
m
m
m
m
TFAFGACm
6m
m
m
m
6T
m
m
m
m
m
m
6m
BDOBMBLBMFLCMFT
HNMT
m
m
6m
6m
T
m
m
m
6m
m
m
m
GJ[mGYIQGYJQHYHT
JINm
m
m
m
m
T
m
m
6m
6m
m
6m
m
GK[mHYGQHYHQHYJT
GONm
m
6m
m
m
T
m
m
m
6m
6m
6m
m
mGJFLIHm
m
m
m
6m
T
6m
6m
6m
m
m
m
m
mHLLOJMm
6m
6m
m
m
T
m
m
6m
6m
6m
6m
m
mGHJGGHm
6m
6m
m
m
T
m
m
6m
6m
6m
6m
m
mHNHJJNm
6m
6m
m
6m
T
6m
m
6m
6m
m
6m
m
GH[mGYJQGYLQGYMm
GL[m
GYHQHYHQHYIQHYJm
GM[m
GYJQGYLQGYMQHYGm
GO[m
GYGQGYHQHYGQHYHQHYI
QHYJm
GN[GYGQGYHQGYJQGY
LQHYGQHYHQHYJm
m
m
m
(&30 TDMDB[m0-+2-%0+1m"#m*m40'#""m"#m0#$#0#,!'mLHjGGIm7m*m!!#1'A,mmKFKLFHZm/3#m!-,2'#,#m
#*mmGYKm0#1.-,1 *#m"#m3,m!+ '-m"#m+',-9!'"-m^9!'"-m1.902'!-mm%*'!',_Ym
T
DMDMCTTT".TTTT
T
*m+.*'!A,m0120#"-m+#"',2#m!-m!-00#1.-,"#m*m.0'+#0m#6A,m"#m;Zm
"#mLGKm .m^'%30mIYHL_Ym#m&,m"#2#!2"-m"-1m.-*'+-0$'1+-1m#,20#m*m40'#""mLHj
GGIm7m*1mGGIm!!#1'-,#1m,*'8"1Ym,m*m'%30mIYIHm1#m.3#"#m- 1#040m#*m%#*m!-,m*m
.-1'!'A,m "#m *-1m "-1m 1m "#2#!2"-1Ym 0m !"m %#,m ,*'8"-m +#"',2#m
GILm
)
Resultados
!-m234'#0-,m/3#m0#*'801#m"-1m%#*#1m""-m/3#m#*m,B+#0-m"#m40'#""#1mm
,*'80m #0m 13.#0'-0m *m +B+#0-m "#m +3#1201m /3#m .3#"#,m !0%01#m #,m 3,m %#*m "#m
Ym 12#m #1m #*m +-2'4-m .-0m #*m !39*m #,m *m '%30m IYIHm ,-m #129,m 0#.0#1#,2"1m
2-"1m*1m40'#""#1m7m.-0m*-m2,2-m,-m#129,m"#2**"-1m2-"-1m*-1m&#2#0-"B.*#6Ym
m
m
m
!_m
m
(&30 TDMDCOT#*m"#m!-m.0m#*m+.*'!A,m"#*m%#,m;Ym_m!,*mMFFZm _m!,*mNFFYm"m
!00'*m!-00#1.-,"#mm*m+#8!*m"#m3,m"#m*1mGGIm40'#""#1m^3,/3#m#,m!"m%#*m1A*-m1#m!0% ,mOLiGGI_m
!-,m*m0#$#0#,!'mLHjGGIYm,m0-(-m7m4#0"#m1#m&,m"#2**"-m*-1m&#2#0-"B.*#6m"#2#!2"-1m^mGm7mH_Ym,m
+0'**-m 1#m "#2**,m *%3,-1m &#2#0-"B.*#6m $*1-1m -m 02#$!2-1m "#1!02"-1m +#"',2#m 1#!3#,!'!'A,Zm !_m
*-!*'8!'A,m"#m*-1m1m"#2#!2"-1m+#"',2#m!-m#,m#*m.0'+#0m#6A,m"#*m%#,mGYm
T
GIMm
)
Resultados
*m m Gm ^ *m IYGLZm '%30m IYII_m .0-4-!m 3,m !+ '-m "#m +',-9!'"-m "#m *#3!',m m
4*',m 3,/3#m 1#%B,m *-1m 4*-0#1m - 2#,'"-1m +#"',2#m m ^4*-0m GYFF_m #1m .-!-m
.0- *#m/3#m$#!2#mm*m$3,!'-,*'""m"#m*m.0-2#>,Ym1m40'#""#1m/3#m!-,2'#,#,m
#*mmGm1-,mmGHJGGHZmmJNHJHOZmIJOZmmJILKIJm7mmHNHJJNYmm
m
m
(&30 T DMDDOT 0-+2-%0+m "#*m m Gm "#*m .0'+#0m #6A,m "#*m %#,m ;Zm /3#m .0-4-!m 3,m !+ '-m "#m
+',-9!'"-m"#m*#3!',mm4*',m#,m*m40'#""mmHNHJJNYm
m
-"1m#121m40'#""#1m.#02#,#!#,mm%3.-1m"'12',2-1m^+-+-0"'!Zm%0#12'1Zm+#*-m7m
$*#63-131_m7m1-,m"#m-0>%#,#1m%#-%09$'!-1m"'12',2-1m^,"'Zm'+ 5#Zm0)Zm#,#%*m7m
3"9$0'!_Ym
*m m Hm ,-m .0-4-!m ,',%B,m !+ '-m "#m +',-9!'"-Ym #m .3#"#m "#12!0m /3#m <12#m
.0-4-!m 3,m !+ '-m #,m *m 40'#""m m GHJGGHm "#m LOIm 7Zm 1',m #+ 0%-Zm #,m *1m
40'#""#1mmHNHJJNm7mmJNHJHOm#*m!+ '-m#1m"#mLOIYmm
#"',2#m *m !-+ ',!'A,m "#m *-1m "-1m 1m 1#m &,m '"#,2'$'!"-m "-1m &.*-2'.-1m
"'12',2-1m ^ *m IYGM_Ym #%B,m *-1m 0#13*2"-1m - 2#,'"-1m 1#m .3#"#m - 1#040m !-+-m #*m
.0'+#0m#6A,m"#*m%#,m;m.0#1#,2m.-!m40' '*'""m.3#12-m/3#m1A*-mKm!!#1'-,#1m
"#m *1m GGIm ,*'8"1m "'$'#0#,m #,m *%3,-m "#m *-1m "-1m .-*'+-0$'1+-1m 0#1.#!2-m m *m
40'#""m#129,"0Ymm
T
!+ TDMBGOm -*'+-0$'1+-1m "#2#!2"-1m #,m #*m .0'+#0m #6A,m "#*m %#,m;m #,20#m *m 40'#""m LHjGGIm 7m *1m
m
-1'!'A,m
m
^j!_gm
m
m
Gm
6A,Gm
LMFm
#3HHJ*m
GYFFm
Hm
6A,Gm
LOIm
LOIm
0-HIG0-m
jm
GINm
-+ 0#m0'#""m
mGHJGGHZmmJNHJHOm
IJOZmmJILKIJm
mHNHJJNm
mGHJGGHm^_ZmmHNHJJNm^_m
mJNHJHOm^_m
m
+@-m
+@-m
$0%+#,2-m
$0%+#,2-m
!,*m
!,*m
mMFFm
mNFFm
IMNm. m
HMHm. m
JFGm. m
m
HJOm. m
m
)
Resultados
GGIm!!#1'-,#1m,*'8"1Ym#m',!*37#,m2+ '<,m*-1m"2-1m"#m*1m40'#""#1m/3#m!-,2'#,#,m!"mm1>m
!-+-m#*m2+@-m"#*m$0%+#,2-m"#m"'%#12'A,m4'13*'8"-m#,m+ -1m!,*#1m^MFFm7mNFF_Ymg[m'#*m
"#m.-Zm![m!!#1'A,Ym
T
!+ TDMBHOT *m!-,m*-1m&.*-2'.-1m"#2#!2"-1m.0m#*m%#,m;Ym,m1-+ 0#"-m%0'1m1#m"#2**m#*mmGm
/3#m.0-4-!m3,m!+ '-m"#m+',-9!'"-m^*#3!',mm4*',_m#,m*m.-1'!'A,mHHJm"#m*m1#!3#,!'m.0-2#'!Ym
m
-+ 0#m40'#""m
BT
Hm
m
IJOm
6T
m
T
mJILKIJm
6T
m
mGHJGGHm
6T
6m
mJNHJHOm
6T
6m
mHNHJJNm
6T
6m
m
.*-2'.-im
G[mGm
H[GQHm
GIOm
Resultados
.5&)!)OO'/.(.-O ().7*#)-OO
m
m
DMETTTT$
T
30,2#m#*m1#%3,"-m@-m"#m2#1'1Zm1#m- 234'#0-,m*1m1#+'**1mHm"#m*1m$+'*'1m/3#m
0#.0#1#,20>,m *m .- *!'A,m "#m Ym -,(3,2+#,2#m !-,m *m #+.0#1m '#&&-O
#.9m 1#m "#!'"'Am - 1#040m *m .- *!'A,m Hm m ,'4#*m $#,-2>.'!-Ym 1m .- *!'-,#1m "#m
m1#m!0#,m+7-0+#,2#m.0m.*'!!'-,#1m"#m%#,<2'!m',4#01Ym1>Zm1#m','!'m
*m "#2#!!'A,m "#m +32,2#1m #,m 3,m %#,m "#m ',2#0<1m 7m .-12#0'-0+#,2#m 1#m ,*'8m 1'm #*m
%#,-2'.-m"#2#!2"-m#1m#*m0#1.-,1 *#m"#m3,m$#,-2'.-m- 1#04"-Ym#0-m2+ '<,m#6'12#m
*m .-1' '*'""m "#m #4*30m $#,-2>.'!+#,2#m 2-"m *m .- *!'A,Zm -m .02#m "#m #**Zm .0m
"#2#!20m $#,-2'.-1m ',2#0#1,2#1m 7m .-12#0'-0+#,2#m !0!2#0'80*-1m !-,m $',#1m
!-+#0!'*#1m -m .-0m 13m ',2#0<1m '-*A%'!-Zm m .#10m "#m ,-m !-,-!#0m *m +32!'A,m
0#1.-,1 *#m"#m"'!&-m$#,-2'.-Ymm
*m+<2-"-mA.2'+-m#1m"#100-**0m3,m 1#m"#m"2-1m"-,"#m*+!#,0m*-1m$#,-2'.-1m
- 1#04"-1m "#m 3,m +-"-m -0"#,"-m .0m .-"#0m &!#0m B1/3#"1m #6&312'41m #,m
$3,!'A,m"#m*m+-0$-*-%>m-m#*m2'.-m"#m#$#!2-m/3#m.0-"3!#m*m+32!'A,m"#1!-,-!'"Ym
20-m +-"-m "#m 310m *-1m "2-1m $#,-2>.'!-1m #1m #* -0,"-m 3,m .9%',m 5# m "A,"#m #*m
3130'-m .3#"m #*#%'0m 3,m +32,2#m $#,-2>.'!-m /3#m *#m ',2#0#1#Ym ,m 3#,m #(#+.*-m #1m
" m &22.[ii30%4Y#407Y',0Y$0i" m "-,"#m 1#m .3#"#,m 1-*'!'20m 1#+'**1m
+32,2#1m"#m%3'1,2#m^#-/'O-.#0/'m_m7m"#m2-+2#m^)&(/'O&3)*,-#/'_Ym#m.3#"#m
#1!-%#0m #*m +32,2#m #,m $3,!'A,m B,'!+#,2#m "#m 3,m $#,-2'.-m ,-0+*+#,2#m
+-0$-*A%'!-Zm#,m3,m%#,m"#m',2#0<1Zm-m&!'#,"-m&-.m!-,m#*m$0%+#,2-m"#m1#!3#,!'m
/3#m,-1m.3#"m',2#0#10m#,m*m 1#m"#m"2-1Ym,m#12#m20 (-m,-m1#m&m#* -0"-m3,m
1#m "#m "2-1m .#0-m 1'm 3,m !29*-%-m $#,-2>.'!-m #,m $3,!'A,m "#m *1m "'12',21m #2.1m "#m
!0#!'+'#,2-m "#m *m .*,2Ym 0m *-1m $#,-2'.-1m '"#,2'$'!"-1Zm 1#m &m &#!&-m 3,m
"#1!0'.!'A,m"#m*m+32!'A,m!-,(3,2+#,2#m!-,m*m'"#,2'$'!!'A,m"#m*m$+'*'m/3#m*m
!-,2'#,#m7m*m'+%#,m"#*m$#,-2'.-Ym
0m *m #+.0#1m '#&&-O #.9Zm *m .02#m +91m ',2#0#1,2#m 1#m !#,20 m #,m *1m
!0!2#0>12'!1m "#*m $032-m 7m #,m 2020m "#m #,!-,200m $#,-2'.-1m ',2#0#1,2#1m m ,'4#*m
!-+#0!'*Ym0m#**-Zm#,m#*m@-mHFFMm1#m.*,20-,mJFFm$+'*'1mHZm!-,!0#2+#,2#mGFm
',"'4'"3-1m .0m !"m $+'*'m ^3,m 2-2*m "#m JFFFm .*,21m .0-6'+"+#,2#_Ym
*,2,"-m"'#8m',"'4'"3-1m.-0m$+'*'m1#m.0#2#,">m.-"#0m"'$#0#,!'0m*-1m$#,-2'.-1m
"# '"-1m m #$#!2-1m + '#,2*#1Zm "#m *-1m #$#!2-1m .0-"3!'"-1m .-0m *%3,m +32!'A,m
GJFm
Resultados O
.5&)!)OO'/.(.-O ().7*#)-O
',"3!'"m .-0m Ym + '<,Zm #,m 2-"-1m *-1m #12"'-1m #1234'#0-,m .0#1#,2#1m .*,21m
!-,20-*m ^.*,21m "#m *m 40'#""m LHjGGI_m .0m .-"#0m !-+.00m 1'm #*m $#,-2'.-m
- 1#04"-m#0m"# '"-mm3,m+32!'A,m-m*m+ '#,2#mm
#1"#m*m%#0+',!'A,m"#m*1m1#+'**1mHm1#m&'8-m3,m1#%3'+'#,2-m#6&312'4-m"#m*1m
.*,21m - 1#04,"-m 1'm .0#!>,m $#,-2'.-1m +32,2#1Ym ,m $3,!'A,m "#*m #12"'-m "#m *m
.*,2m 1#m - 1#040-,m "'12',2-1m !0!2#0#1m ^.*9,23*Zm .*,2m "3*2m 7m $032-_Ym # #m
"4#02'01#m/3#m#,m*%3,1m"#m*1m$+'*'1m.0#1#,2"1m#,m#*m!29*-%-Zm1A*-m3,-m"#m*-1m
"'#8m',"'4'"3-1m.0#1#,2 m#*m$#,-2'.-m+32,2#Ym,m#12-1m!1-1m"# #0>,m.*,201#m
+91m ',"'4'"3-1m Hm .0m .-"#0m 1#%300m /3#m #*m #$#!2-m $#,-2>.'!-m - 1#04"-m $3#m
.0-4-!"-m .-0m 3,m +32!'A,m 7m ,-m !-+-m !-,1#!3#,!'m "#m *1m !-,"'!'-,#1m
+ '#,2*#1Ym*m20 (-m"#m!0#!'+'#,2-m7m+,2#,'+'#,2-m"#m*1m.*,21m$3#m0#*'8"-m
.-0m*m#+.0#1m'#&&-O#.9Ymm
T
DMEMBTTTTTT
m ,'4#*m "#m .*9,23*m 7m ,2#1m "#m 201.*,20m *m ',4#0,"#0-m 1#m &'8-m 3,m 1#%3'+'#,2-m
"#2**"-m "#m !0!2#0>12'!1m 2*#1m !-+-m #*m ,B+#0-m "#m 1#+'**1m %#0+',"1m .-0m
$+'*'m7m#*m,B+#0-m"#m.*,21m4' *#1m.-0m$+'*'Ym-,2 '*'8,"-m#12-1m0#13*2"-1m
1#m- 234'#0-,m*-1m.-0!#,2(#1m.0#1#,2"-1m#,m*m *mIYGNYmm
m
!+ TDMBIOT-0!#,2(#1m"#T%#0+',!'A,m#,m*1mJFFm$+'*'1mHm- 1#04"1Ym
m
+'*'1m,-m%#0+',"1m
GZHKPm
+'*'1m!-,m (m%#0+',!'A,m
+'*'1m%#0+',"1m.#0-m!-,m
^+#,-1m"#mJjLmiGF_m
*#2*'""m
GGZNFPm
IZHKPm
mm
#m- 1#040-,m40'-1m$#,-2'.-1m,-0+*#1m#,m*-1m/3#m*1m.*9,23*1Zm1',m**#%0mm1#0m
"3*21Zm .0#1#,2 ,m +*$-0+!'-,#1Zm "#100-**-m ,-0+*m #,m #*m !0#!'+'#,2-Zm
!*-0-1'1m3m-20-1m2'.-1m"#m*2#0!'-,#1m/3#m1#m.3#"#,m4#0m"#2**"1m#,m*m *mIYGOm7m
#,m*1m'+9%#,#1m!-00#1.-,"'#,2#1m^IYJYJm29*-%-m"#m'+9%#,#1_Ym
m
m
m
m
m
m
GJGm
.5&)!)OO'/.(.-O ().7*#)-OO
Resultados
!+ T DMBJT #,-2'.-1m #,!-,20"-1m #,m #12"'-m "#m .*9,23*Yg29*-%-m "#m '+9%#,#1m IYJYJYm ggm *%3,1m
.*,21m &,m +-120"-m #*m $#,-2'.-m +32,2#m #,m "'$#0#,2#1m #12"'-1m "#m !0#!'+'#,2-Zm .-0m *-m 2,2-Zm *1m
#,!-,200#+-1m2,2-m#,m#*m!29*-%-m0#*'8"-m.0m#*m#12"'-m"#m.*9,23*m!-+-m#,m#*m"#m.*,2m"3*2Ym
m
*,21m!-,m
#,-2'.-m
+'*'m
$#,-2'.-m
+%#,gm
+32,2#i+'*'m
04'20-.'1+-Ym
#100-**-m
,-0+*m
,!&1m
!*-0A2'!1Ym
*-0-1'1m.0!'*m
HHm
IiGFm
Gm
KKm
KiGFm
Hm
MONm
GiGFm
Mm
GIHLm
KiOm
GFm
GKFKm
GiHm
GGm
GKHKm
HiOm
GHm
GLJLm
IiGFm
GJm
GMOFm
NiOm
GKm
GNMLm
IiMm
GLm
GOFHm
GiNm
Om
HGOIm
GiOm
GNm
HIGKm
JiLm
GOm
KKm
KiGFm
Hm
KNggm
HiGFm
Im
NGm
MiOm
Km
GIMm
MiGFm
Lm
MONm
GiGFm
Mm
OMOm
JiGFm
Nm
GKHKm
HiOm
GHm
GKKFm
GiHm
GIm
GIHLm
KiOm
GFm
GOFHm
GiNm
Om
GOIJm
KiLm
GMm
HIGKm
JiLm
GOm
"&#1'A,m"#m*1m
MJm
LiGFm
Jm
.0'+#01m&-(1m
NGm
MiGFm
Km
m
T
DMEMCTTTTTTT
,m #*m #12"'-m "#m .*,2m "3*2m 1#m - 1#040-,m !+ '-1m #,m #*m "#100-**-Zm 1>m !-+-m
!+ '-1m#,m*m.'%+#,2!'A,Zm!+ '-1m#,m*m$-0+m"#m*m&-(m-m#,m*m$*-0Zm#,,'1+-Zm7m
-20-1m $#,-2'.-1m ^ *m IYHF_Ym -+-m #*m !0' "-m #0m *#2-0'-m ,-m #1.#09 +-1m 3,m
GJHm
Resultados O
.5&)!)OO'/.(.-O ().7*#)-O
$#,-2'.-m !-,!0#2-Zm 1',-m "#1!3 0'0m ,3#4-1m $#,-2'.-1Zm /3'891m ,-m - 1#04"-1m
,2#0'-0+#,2#m#,m*m#1.#!'#Ym
m
!+ TDMCAT#,-2'.-1m#,!-,20"-1m#,m#12"'-m"#m.*,2m"3*2Ymg29*-%-m"#m'+9%#,#1mIYJYJYm
m
#,-2'.-m
+'*'m
04'20-.'1+-Ym
#100-**-m,-0+*m
*,21m!-,m$#,-2'.-m
+32,2#i+'*'m
+%#,gm
ONKm
KiGFm
HGm
GFNLm
GiOm
HHm
HHLGm
HiOm
HNm
HHNJm
HiLm
HOm
HIGMm
JiOm
IGm
#$-0+!'-,#1m#,m
GFNLm
GiOm
HHm
&-(1m
GOFHm
IiNm
HMm
KNm
HiGFm
HFm
*-0-1'1m
ONKm
KiGFm
HGm
GFOLm
KiGFm
HIm
GJKKm
HiOm
HJm
GOFHm
IiNm
HMm
GJKKm
HiOm
HJm
GKKFm
HiLm
HKm
GLIHm
JiMm
HLm
HIGKm
HiGFm
IFm
12#0'*'""m-m
+*$-0+!'A,m#,m
$*-0m
1.#!2-m"#m
',$#!!'A,m4'0*m
#2*'""m
T
DMEMDTTTT
#m "#"'!Am 3,m +7-0m #1$3#08-m #,m #*m $#,-2'."-m "#*m $032-Zm 7m /3#m #*m - (#2'4-m "#m
'#&&-O#.9m#0m#,!-,200m*%B,m+32,2#m/3#m.0-.-0!'-,0m3,-m-m40'-1m!0!2#0#1m
20!2'4-1m"#1"#m#*m.3,2-m"#m4'12m!-+#0!'*Ym-1m+#(-0"-0#1m#,m#12#m!1-m 31! ,m
!0!2#0#1m /3#m !+ '0,m #*m 1.#!2-m 2>.'!-m "#*m +#*A,m b'#*m "#m 1.-cm 7m /3#m
0#13*20,mm13m4#8m20!2'4-1m.0m#*m!-,13+'"-0Ym,m$-0+m+91m0#"-,"#"m"#m
$032-Zm -m 3,m .'#*m 1',m +,!&1m 7m .-!-m #1!0'230"m .-"0>,m 1#0m *%3,-1m "#m *-1m
!0!2#0#1m/3#m.-"0>,m1#0m"#m',2#0<1Ym
#m0#!-%'#0-,m*-1m$032-1m"#m.0-6'+"+#,2#mIMFm$+'*'1Ym0m!"m3,m"#m#**1m1#m
!0#!'#0-,m"'#8m.*,21m7m.0m!"m.*,2m1#m0#!-%'Am3,m+#*A,Ym #m!-+.00-,m*-1m
.09+#20-1m- 1#04"-1m.0m2-"-1m*-1m+#*-,#1m"#m*m+'1+m$+'*'m"#m+-"-m/3#m1#m
1#*#!!'-, ,m /3#**-1m /3#m .0#1#,20,m 3,m 01%-m "'$#0#,!'*m 0#1.#!2-m *-1m "#+91Ym
-"-1m *-1m .09+#20-1m 1#m ',20-"3(#0-,m #,m 3,m 1#m "#m "2-1m "#100-**"m .-0m *-1m
GJIm
.5&)!)OO'/.(.-O ().7*#)-OO
Resultados
+#(-0"-0#1m "#m '#&&-O #.9m #,m *m /3#m 1#m ',"'! m .0m !"m +#*A,Zm #*m 4'%-0Zm *m
0#1'12#,!'mm->"'-Zm#*m.#1-Zm#*m!-*-0m#62#0'-0Zm#*m#1!0'230"-Zm*m03%-1'""Zm#*m!'#00#Zm*m
!4'""Zm#*m!-*-0m"#m*m!0,#Zm#*m 0'6m^!,2'""m#,m8B!0#1_Zm*m"30#8Zm#*m4',"-Zm#*m
1 -0m 7m *1m +#"'"1m "#m $-0+Ym /3#**-1m $032-1m /3#m .0#1#,2 ,m 3,m !09!2#0m
"'$#0#,!'"-m7m/3#m.3"'#0m0#13*20m',2#0#1,2#m.0m*m+#(-0m1#m1#*#!!'-, ,m7m1#m
*+!#, ,m 131m 1#+'**1Ym 12-1m 0#13*2"-1m ,-m 1#m +3#120,m #,m #12#m 20 (-m 7m /3#m
1-,m"2-1m"#m*m#+.0#1Ym
m
,m*m'%30mIYIJm1#m+3#120m3,m#(#+.*-m"#m!A+-m1#m!-+.0 ,m2-"-1m*-1m$032-1m"#m
*1m .*,21m "#m 3,m +'1+m $+'*'Ym ,m 4#8m ,*'8"-1m 2-"-1m *-1m !0!2#0#1m 1#m
1#.0 m /3<*m -m /3#**-1m $032-1m /3#m 1#m 1*>,m "#m *-1m .09+#20-1m #129,"0#1Zm .0m
.0-"3!'0m +91m 1#+'**m #m ',2#,20m $'(0m #*m $#,-2'.-Ym ,m #12#m #(#+.*-Zm 1#m 1#*#!!'-,Am #*m
.0'+#0-m .-0m *m '8/3'#0"m .-0m 1#0m +91m 0#"-,"-m /3#m #*m 0#12-m 7m .-0m *-m 2,2-Zm 3,m
$#,-2'.-m',2#0#1,2#m.0m.-"#0m$'(0m#12#m!09!2#0m#,m#*m+#*A,m2'.-mb'#*m"#m1.-cYm
m
m
m
m
(&30 TDMDEOT0-!#"'+'#,2-m .0m *m 1#*#!!'A,m "#m +#*-,#1m !-,m !0!2#0#1m $#,-2>.'!-1m ',2#0#1,2#1m m ,'4#*m
!-+#0!'*Ym,m#12#m#(#+.*-Zm#,m*m'+%#,m13.#0'-0m4#+-1m*-1m+#*-,#1m0#!-%'"-1m"#m#12m$+'*'m^HHNL_m7m
GJJm
Resultados O
.5&)!)OO'/.(.-O ().7*#)-O
#,m*m'+%#,m',$#0'-0m#*m+#*A,m1#*#!!'-,"-m.-0m13m!#,23"m$-0+m0#"-,"m#,m!-+.0!'A,mm*m$-0+m
#*>.2'!m2>.'!m"#*m+#*A,mb'#*m"#m1.-cYm
m
#m - 1#040-,m *1m "'$#0#,!'1m $#,-2>.'!1m #,m *-1m $032-1Zm *m '%3*m /3#m .0m *-1m -20-1m
"-1m #12"'-1Zm .0m .-"#0m !2*-%0m *%3,-1m $#,-2'.-1m +32,2#1Ym 1m "'12',21m
+32!'-,#1m$#,-2>.'!1m1>m!-+-m*1m$+'*'1mHm!-00#1.-,"'#,2#1m1#m"#2**,m#,m*m
*mIYHGYm
m
!+ TDMCBOT#,-2'.-1m#123"'"-1m#,m$032-1Yg29*-%-m"#m'+9%#,#1mIYJYJYm
m
B+#0-m"#m
#,-2'.-m
+'*'m
$032-1m!-,m#*m
$#,-2'.-m
+%#,gm
+32,2#i+'*'m
GFOJm
Hm
IHm
0'!'-,#1m#,m
GGJOm
Hm
ILm
!-*-0i#1!0'230"-m
GKFKm
Gm
INm
GOFHm
Gm
JHm
GFOOm
Gm
IIm
#$-0+!'-,#1m#,m
GIJJm
Gm
IMm
!'#00#m7m
GKINm
Gm
JFm
+*$-0+!'A,m
GMKIm
Gm
JGm
GOFHm
Gm
JHm
GGFKm
Hm
IJm
GGIIm
Gm
IKm
0'!'A,m#,m*m
GGJOm
Hm
ILm
$-0+m
GKFLm
Gm
IOm
GOFHm
Gm
JHm
HHNLm
Gm
JIm
m
DMEMETTTTTTTT
,m#*m1'%3'#,2#m.02"-m1#m&,m0#3,'"-m2-"1m*1m'+9%#,#1m!-00#1.-,"'#,2#1mm*-1m
$#,-2'.-1m"#2#!2"-1m#,m*-1m#12"'-1m"#m.*9,23*Zm"#m.*,2m"3*2m7m"#m$032-Ym
j32,2#1m#,m#12"'-m"#m.*9,23*[m+9%#,#1m"#m*mGmm*mGOYm
j32,2#1m#,m.*,2m"3*2[m+9%#,#1m"#m*mHFmm*mIGYm
j32,2#1m#,m$032-[m+9%#,#1m"#m*mIHmm*mJIYm
GJKm
.5&)!)OO'/.(.-O ().7*#)-OO
Resultados
12"'-m"#m.*9,23*m
m
GJLm
.5&)!)OO'/.(.-O ().7*#)-O
Resultados O
m
GJMm
.5&)!)OO'/.(.-O ().7*#)-OO
Resultados
$#"(
)
&
)
%())
)
))
)
)
&)
)&
)
m
m
GJNm
% ())
))
))&
)
!())
'))
)
)
) &) ) ) ) ) ) )
))
))
)
))&
&
)
Resultados O
.5&)!)OO'/.(.-O ().7*#)-O
12"'-m"#m.*,2m"3*2m
m
GJOm
Resultados
T
GKFm
.5&)!)OO'/.(.-O ().7*#)-OO
m
.5&)!)OO'/.(.-O ().7*#)-O
Resultados O
T
GKGm
Resultados
T
GKHm
.5&)!)OO'/.(.-O ().7*#)-OO
T
Resultados O
.5&)!)OO'/.(.-O ().7*#)-O
12"'-m"#m032-m
T
GKIm
Resultados
.5&)!)OO'/.(.-O ().7*#)-OO
T
m
GKJm
)&#9(O'/.!(#4OO'&9(O
#-/-#9(m
m
m
EMTT
T
EMBTTTTTTTTT
TTT
m
m#$'!'#,!'m"#m+32!'A,m"#m*m.- *!'A,m"#mm- 2#,'"m#,m#12#m20 (-m&m
1'"-m "#m Gm +32,2#iGZKm Ym 12m #1m 3,m #$'!'#,!'m +#,-0m /3#m /3#**1m "#1!0'21m #,m
-201m #1.#!'#1m "'.*-'"#1m !-+-m ,#)*-#-O."&#(m ^GiIFFm _m ^0##,#m 7m !-*YZm HFFI_Zm
+>8m ^GiKFFm _m ^'**m 7m !-*YZm HFFJ_m -m !# "m ^Gi _m ^*"5#**m 7m !-*YZm HFFJ_Ym ,m
#1.#!'#1m .-*'.*-'"#1m !-+-m #*m 20'%-m 2#20.*-'"#m -m #*m 20'%-m &#6.*-'"#m *-1m 4*-0#1m
2-"4>m1-,m+91m#*#4"-1mGiJFm m7mGiHJm m0#1.#!2'4+#,2#m^*"#m7m!-*YZmHFFK_Ymm
,m #*m +-+#,2-m "#m ','!'0m #12#m 20 (-m ,-m #6'12>m ,',%3,m .- *!'A,m "#m +32,2#1m
"#100-**"m#,m+#*A,Em*m.0-2-!-*-m"#m+32%<,#1'1m"#m*1m1#+'**1m$3#m".2"-mm
.02'0m"#m-201m#1.#!'#1m7m3,/3#m1#m&'8-m3,m#,17-m.0#4'-m.0m"#2#0+',0m*m"-1'1m
A.2'+m "#m Zm *-1m 0#13*2"-1m - 2#,'"-1m ,-1m ',"'!,m /3#m *m "-1'1m 31"m ,-m $3#m
.0- *#+#,2#m 13$'!'#,2#Ym ,m ,3#120-m !1-m *1m !-,!#,20!'-,#1m 31"1m "#m m
#1234'#0-,m #,m #*m 0,%-m "#m FZKPm &12m GZKPYm ,m !+ '-Zm .0m #*m "#100-**-m "#m *m
.- *!'A,m"#mm"#*mZm.02'#0-,m!-,m*m4#,2(m"#m!-,-!#0m*-1m0#13*2"-1m
"#m ,3#120-m 20 (-Zm 7m .*'!0-,m !-,!#,20!'-,#1m +91m *21m "#m m ^"#*m GPm *m HP_Ym
-0m -20-m *"-Zm "30,2#m #*m .0-2-!-*-m "#m +32!'A,m "#m *1m 1#+'**1m 1#m 0#*'8Am 3,m
&'"02!'A,m "#m *1m +'1+1m .0#4'm m *m .*'!!'A,m "#*m +329%#,-Ym 6'12#m 3,m .0-!#1-m
"#,-+',"-m )-')*,#'#(!m #,m #*m /3#m *m .0#&'"02!'A,m "#m 1#+'**1m "#1#!"1m
.0-.-0!'-,m 3,m %#0+',!'A,m +37m &-+-%<,#m ""-m /3#m !2'4m *-1m 1'12#+1m "#m
0#.0!'A,m "#*m m ^1&0$m 7m 07Zm GOOI_Ym 1m .-0m #**-m /3#m .-1' *#+#,2#m *m
.0#&'"02!'A,m0#*'8"m#,m*1m1#+'**1m.3"-m!2'40m*-1m1'12#+1m"#m0#.0!'A,m"#*m
m 7m /3'891Zm .0-4-!0m *m !-00#!!'A,m "#m *%3,1m +32!'-,#1m .0-"3!'"1m .-0m #*m
m7m#,m!-,1#!3#,!'m (0m*m#$'!'#,!'m"#m*m+32%<,#1'1Ym
#,'#,"-m #,m !3#,2m #*m #$#!2-m "#*m +329%#,-m 31"-m ^_m #*m .-0!#,2(#m "#m 1m
.0#1#,2#m #,m 3,m %#,m #1m 3,m "#m *1m 40' *#1m /3#m .3#"#m !-,"'!'-,0m *m #$'!'#,!'m "#m
+32!'A,Ym #m &,m !-+.0"-m *-1m .-0!#,2(#1m #,m 1m #,m *1m 8-,1m !-"'$'!,2#1m "#m
*-1m%#,#1m,*'8"-1Ym0m#*m%#,m#Jm#*m!-,2#,'"-m"#m1m#1m"#m3,mILZMmPZm7m13m
'1-$-0+mI#-)J>m2'#,#m3,m!-,2#,'"-m*'%#0+#,2#m13.#0'-0m^IMZJGmP_Ym*m%#,m"#m*m
GKKm
#-/-#9(
O)&#9(O'/.!(#4OO'&9(
m 2'#,#m 3,m !-,2#,'"-m "#m 1m "#*m IJZOIm Pm ^ *m JYG_Ym #,'#,"-m #,m !3#,2m #*m
#$#!2-m "#*m m #1.#09 +-1m .-"#0m #12 *#!#0m 3,m 0#*!'A,m "'0#!2+#,2#m
.0-.-0!'-,*m #,20#m #*m .-0!#,2(#m "#m 1m 7m #*m ,B+#0-m "#m +32,2#1m "#2#!2"-1m #,m
!"m%#,Zm.#0-m,-m&m1'"-m1>Ym*m%#,mI#-)J>m#1m#*m/3#m2'#,#m+7-0m.-0!#,2(#m"#m
1#1m131!#.2' *#1mm1#0m+32"1m7m1',m#+ 0%-Zm.0m#12#m%#,m,-m&#+-1m#,!-,20"-m
,',%B,m +32,2#m #,m *m .- *!'A,m m 7m 2,m 1A*-m 3,-m #,m *m .- *!'A,m Ym ,m
!+ '-Zm .0m #*m %#,m m #*m .-0!#,2(#m "#m 1m #1m +#,-0m 7m 1#m &,m "#2#!2"-m "-1m
+32,2#1m#,m*m.- *!'A,mm7m!320-m#,m*m"#*mYm-0m*-m2,2-Zm#,m#12#m20 (-m
.0#!#m/3#m*m.0-.-0!'A,m"#m1m"#*m%#,m,-m1#m&m!-00#*!'-,"-m!-,m*m- 2#,!'A,m
"#m +32,2#1m ^ *m JYG_Ym 7m "-1m 08-,#1m /3#m .-"0>,m #6.*'!0m #12#m 0#13*2"-Ym m
.0'+#0Zm !-+-m 7m 1#m &m !-+#,2"-m ,2#0'-0+#,2#Zm #1m /3#m ,-m 1#m &m ,*'8"-m 3,m
,B+#0-m 13$'!'#,2#m "#m %#,#1m .0m .-"#0m #12 *#!#0m 3,m 21m "#m +32!'A,m +91m
1'%,'$'!2'4m m ,'4#*m #12">12'!-Ym m 1#%3,"Zm #1m /3#m "# #m 2#,#01#m #,m !3#,2m *m
'+.-02,!'m "#*m %#,m #,m !3,2-m m *m $3,!'-,*'""m /3#m 0#*'8Ym 'm 3,m %#,m 2'#,#m 3,m
$3,!'A,m 4'2*m .0m *m #1.#!'#Zm #,2-,!#1m *m .0- '*'""m "#m #,!-,200m +32,2#1m
&-+-!'%-2-1m#,m#1#m%#,m"'1+',37#m!-,1'"#0 *#+#,2#m.3#12-m/3#m#*m',"'4'"3-m!-,m
"'!&m +32!'A,m ,-m 1#0>m 4' *#Zm +30'<,"-m ,2#1m "#m *m .-1' *#m "#2#!!'A,m "#m *m
+32!'A,Ymm
0m .-"#0m "#100-**0m 3,m .- *!'A,m B,m +91m #$#!2'4m #,m !3,2-m m 21m "#m
+32!'A,m 1#0>m !-,4#,'#,2#m !*!3*0m *1m .*,21m ,#!#10'1m .0m 1#%300m *m
- 2#,!'A,m "#m 3,m +32,2#m .0m !"m ,3!*#A2'"-m ,*'8"-Ym #%B,m *+'1m 7m !-*YZm
^HFFM_m !-,-!'#,"-m *m $0#!3#,!'m "#m +32!'A,m - 2#,'"m #,m 3,m .- *!'A,m .-"#+-1m
#12'+0m#*m,B+#0-m"#m.*,21m,#!#10'1m.0m!-,1#%3'0m3,m+32!'A,m"#1#"m!-,m
3,m #*#4"m .0- '*'""Ym -0m !-,1'%3'#,2#m 1#m .-"0>m 3+#,20m #*m 2+@-m "#m *m
.- *!'A,m&12m- 2#,#0m3,m+32,2#m.-0m!"m 1#m,*'8"m^*+'1m7m!-*YZmHFFM_Ym
12#m 1#0>m 3,m +-"-m "#m 3+#,20m *m #$'!'#,!'m "#m +32%<,#1'1m "#m *m .- *!'A,Zm
3,/3#m#*m.0-!#1-m"#m!0#!'+'#,2-m"#m3,m,B+#0-m2,m#*#4"-m"#m.*,21m1#0>m+37m
*0%-m7m!-12-1-m#,m3,m#1.#!'#m!-+-m#*m+#*A,Ym
m .02'0m "#m *-1m 0#13*2"-1m "#m #12#m 20 (-Zm #,m m ^.0-7#!2-m HFGFjHFGHm
"-,"#Zm #,20#m -20-1m 0#13*2"-1Zm 1#m &m 1#!3#,!'"-m #*m %#,-+m "#*m +#*A,_m 1#m ',!*37Am
!-+-m- (#2'4-m*m- 2#,!'A,m"#m-20m.- *!'A,m"#mYmm#+.0#1m'#&&-O#.9m
(3,2+#,2#m!-,m#*mjm&,m"#100-**"-m3,m,3#4m.- *!'A,m"#mm
m .02'0m "#m 3,m *>,#m .30m "#m +#*A,m 2'.-m e'#*m "#m .-eYm ,m #12m -!1'A,m 1#m &,m
+32"-m *1m 1#+'**1m !-,m 3,m .-0!#,2(#m "#m GZKPm "#m m 7m 1#m &,m - 2#,'"-m
.0-6'+"+#,2#mIGFFm',"'4'"3-1mHm#,m#*m+-+#,2-m"#m#1!0' '0m#12m2#1'1Zm#,m*-1m
GKLm
)&#9(O'/.!(#4OO'&9(O
#-/-#9(m
!3*#1Zm 7m 1#m &,m "#2#!2"-m Nm ,3#4-1m +32,2#1m #,m #*m %#,m >m 7m Im #,m #*m %#,m
I#-)J>m^#12-1m0#13*2"-1m1#09,m!-+#,2"-1m!-,m+91m"#2**#m.-12#0'-0+#,2#_Ym
m
!+ T EMB[m -0!#,2(#m "#m 1#1m 131!#.2' *#1m *m +329%#,-m Zm -m !-,2#,'"-m #,m 1Zm #,m !"m %#,m
,*'8"-Zm7m*m!-+.0!'A,m"#*m,B+#0-m"#m+32,2#1m#,!-,20"-1m#,m!"m.- *!'A,Ymg# #m2#,#01#m#,m
!3#,2m /3#m *m 40'#""m 32'*'8"m .0m "#100-**0m *m .- *!'A,m "#*m m ^&0#,2'1_m #0m "'12',2m m *m
32'*'8"m .0m *m .- *!'A,m "#*m m ^'#*m "#m .-_m .-0m *-m 2,2-Zm *-1m .-0!#,2(#1m .-"0>,m 40'0m
*'%#0+#,2#Zm7m/3#m*m1#!3#,!'m32'*'8"m.0m*-1m!9*!3*-1m#1m*m"#m*m40'#""mLHjGGIYm
m
#,m
Pm1gm
32,2#1mmm
32,2#1mmm
>O
ILZMmPm
Jm
GJm
I#-)J>O
IMZJGmPm
Fm
Gm
O
IJZOImPm
Hm
Jm
GKMm
#-/-#9(
)'*,#9(O(.,O&-O*)&#)(-OM
,m -201m .- *!'-,#1m 1#m &m "#2#0+',"-m *m #$'!'#,!'m "#m +32!'A,m +#"',2#m #*m
,9*'1'1m#,m3,m,B+#0-m"#m%#,#1m+91m#*#4"-Ym-0m#(#+.*-Zm.0m"#2#0+',0m*m21m
"#m +32!'A,m - 2#,'"m #,m *m .- *!'A,m "#m +#*A,m "#100-**"m #,m #*m m 1#m
,*'80-,m GGm %#,#1m ^&+,'j0"1m 7m !-*YZm HFGF_Zm "#m #12#m +-"-m *m 21m "#m
+32%<,#1'1m!*!3*"m#1m+91m$' *#Ymm
,m#12#m20 (-m1#m&,m,*'8"-m!320-m%#,#1m#,m"-1m.- *!'-,#1m"'12',21Ym12-m&m
.#0+'2'"-m /3#m .-"+-1m #12 *#!#0m *%3,1m !-+.0!'-,#1m #,20#m + 1Ym #%B,m *m
*m JYGm - 1#04+-1m /3#m .0m #*m %#,O >m 1#m &,m "#2#!2"-m Jm +32,2#1m #,m *m
.- *!'A,m m +'#,201m /3#m #,m *m .- *!'A,m m 1#m &,m "#2#!2"-m GJZm *-m /3#m
0#.0#1#,2m3,m"'$#0#,!'m,-2 *#m#,20#m+ 1m.- *!'-,#1Ym*m+'1+-m!1-m1#m"m#,m
#*m %#,m m .3#12-m /3#m #,m *m .- *!'A,m "#*m m 1#m &,m "#2#!2"-m Hm +32,2#1m
+'#,201m/3#mJm#,m*m"#*mYmm
"-m /3#m 1#m !-,-!#,m *-1m "#2**#1m "#*m .0-2-!-*-m #+.*#"-m .0m *m +32%<,#1'1m 7m
"#100-**-m "#m + 1m .- *!'-,#1Zm .-"#+-1m $'0+0m /3#m *m 21m "#m +32!'A,m
- 2#,'"m#,m!"m.- *!'A,m&m1'"-m!-,"'!'-,"m 91'!+#,2#m.-0m*m#$'!'#,!'m!-,m
/3#m $3#m +32"Zm #1m "#!'0Zm *m "-1'1m "#m +329%#,-m 32'*'8"m 7m #*m .0-2-!-*-m "#m
+32%<,#1'1m#+.*#"-Ymm
,m*m *mJYHm1#m0#13+#,m2-"-1m*-1m+32,2#1m#,!-,20"-1m#,m#12#m20 (-Zm2,2-m#,m
*m.- *!'A,mm!-+-m#,m*m"#*mZm1>m!-+-m2-"m*m',$-0+!'A,m"#2**"m"#m
!"m3,-m"#m#**-1Ym,m*m!-*3+,m"-,"#m1#m"#2**,m*1m$0#!3#,!'1m"#m+32!'A,m1#m
#4'"#,!'m /3#m #,m 2-"-1m *-1m %#,#1m *m $0#!3#,!'m "#m +32!'A,m #1m +7-0m #,m *m
.- *!'A,m m ^GiIOKm m #,m m 0#1.#!2-m GiGZNm m #,m m #,m #*m .0'+#0m
+.*'!A,m"#*m%#,m#JZmGiHLJm m0#1.#!2-mGiOHIm m#,m#*m1#%3,"-m+.*'!A,m"#*m%#,m
#JZmGiHZKHm m0#1.#!2-mm,',%B,m+32,2#m#,m#*m%#,mI#-)J>O7mGiMIMm m0#1.#!2-m
GiGZJGm m #,m *m _Ym *m +'1+-m 2'#+.-Zm m .#10m "#m /3#m #,m *m .- *!'A,m m *m
$0#!3#,!'m "#m +32%<,#1'1m #1m +7-0Zm #,m *%3,-1m !1-1m !-+m #*m "#*m %#,m I#-)J>m
1'%3#m 1'#,"-m +37m (Ym 12#m "2-m !-00- -0m *m &'.A2#1'1m "#m /3#m #6'12#,m *%3,-1m
%#,#1m /3#Zm .-0m +-2'4-1m 2-"4>m "#1!-,-!'"-1Zm 0#13*2,m +91m "'$>!'*#1m "#m
+32%#,'80Ymm
m
m
m
m
m
GKNm
)&#9(O'/.!(#4OO'&9(O
#-/-#9(m
!+ TEMC[m *m0#13+#,m"#m*-1m+32,2#1m#,!-,20"-1m#,m!"m.- *!'A,m7m.0m!"m%#,Zm!-,m"#2**#m
"#*m 2'.-m "#m !+ '-m "#m 1#Zm #*m #$#!2-m "#m *m +32!'A,m m ,'4#*m +',-!>"'!-Zm #*m ,-+ 0#m "#m *m $+'*'Zm *m
1#%0#%!'A,m Hm .0m /3#**-1m +32,2#1m /3#m 1#m &,m .-"'"-m #123"'0Zm *m $0#!3#,!'m "#m +32!'A,Zm #*m
$#,-2'.-m#,m!1-m"#m& #0m1'"-m#123"'"-Zm7m$',*+#,2#m#*m4*-0mYm^gm2-1m#6.*'!"-1m#,m#*m.02"-m
JYHYHZm *mJYImgg2-1m#6.*'!"-1m#,m#*m.02"-mIYHYGYIZm *mIYMYm
m
KFFm
Im
Im
&0GKK&0m
GNNFm
jm
,20A,mHm
im
m
OKOm
jm
6A,mGm
HNOm
*3OM71m
KIm
jm
6A,mGm
HOJm
&#ON&#m
HKIHm
jm
,20A,mGm
im
m
GJHFm
jm
GiOHI m
GiIOKm m
*-0m
m
jm
FYFFm
jm
FYFFm
jm
jm
jm
jm
jm
FYFHm
jm
jm
jm
jm
6A,mHm
JFGm
*GIJ*m
GHHOm
jm
jm
FYGFm
6A,mHm
JFMm
*7GIL*m
JJJm
jm
jm
FYFFm
,20A,mHm
im
m
GHNMm
jm
jm
jm
im
m
GGGKm
jm
jm
jm
jm
jm
jm
jm
,20A,mHm
im
>O
.0'+#0
GGLOm
GGm
-*&##(!O
m
m
GiGZN m
#,-2'.-m
JLKm
+32!'A,m
6A,mHm
0#!3#,!'m
FKiFIiFFgm
^ii_m
FOiFMiFNgm
KMm
Hm
HFILm
1#%0#%!'A,m
0%HK71m
*3GHO71m
-1'!'A,m
MJm
INKm
6A,im
6A,mGm
6A,mHm
,20A,m
B+#0-m
+32,2#1m
+@-m
GGLOm
Gm
Hm$+'*'m
m
KFFm
+ '-mm
>O
+.*'!A,m^. _m
#,m
i- *!'A,m
m
GLLMm
,20A,mIm
im
m
HLJLm
jm
jm
,20A,mIm
im
m
GHIIm
jm
jm
jm
6A,mJm
LGLm
*3HFL71m
HFLFm
jm
jm
FYFOm
,20A,mJm
im
m
HFIKm
jm
jm
jm
Iamm
im
m
GILNm
jm
jm
jm
Iamm
im
m
HGLIm
jm
jm
jm
,20A,mIm
m 1#m
"#*m
GiHLJm m
',20A,m
I#-)J
Jm
m
GFGJm
Fm
jm
jm
jm
jm
jm
Fm
jm
jm
GFGJm
Gm
6A,mGm
GHNm
0%JI71m
GKNMm
jm
GiHYKH m
jm
GYFFm
,20A,mGm
im
m
GGKGm
FJiFMiFJggm
jm
jm
m
I#-)J
Jm
m
m
O
m
GGNNm
Hm
6A,mHm
KLFm
#0KJF&#m
INJm
FLiFOiFFggm
GiGYJG m
*,2m
* ',m#,m
FYFFm
Im
O
GGNNm
Jm
6A,mGm
IOKm
#0JNK&#m
GGNHm
jm
GiMIM m
jm
FYFGm
GKOm
#-/-#9(
m
)'*,#9(O(.,O&-O*)&#)(-OM
,20A,mGm
im
jm
HJOKm
jm
jm
jm
jm
,20A,mGm
im
jm
GJLFm
jm
jm
jm
jm
,20A,mGm
im
jm
GHHLm
jm
jm
jm
jm
m
,m #6.*'!!'A,m .0m *-1m 0#13*2"-1m - 2#,'"-1m #,m #*m %#,m I#-)J>Zm .-"0>m 1#0m #*m
+-2'4-m 7m !-+#,2"-m ,2#0'-0+#,2#Zm /3#m *m +32!'A,m .0-4-!0m ',"'4'"3-1m
',4' *#1m7m.-0m*-m2,2-m,3,!m.3"'#0,m"#2#!201#m.-0/3#m,-m**#%,mm"#100-**01#Ym
m *#2*'""m -m *m $*2m "#m +32,2#1m .3#"#m 1#0m .0-4-!"m 2,2-m .-0m #*m #$#!2-m "#m *m
+32!'A,m#,m#*m%#,m,*'8"-m!-+-m.-0m#*m#$#!2-m"#m-201m+32!'-,#1m/3#m!-,2'#,#m
#*m+'1+-m+32,2#m0#.02'"1m*#2-0'+#,2#m.-0m2-"-m#*m%#,-+Ym'(9,"-,-1m#,m*m
$0#!3#,!'m"#m*m.- *!'A,m+91m#$'!'#,2#m^_m#12#m%#,m.0#1#,2m3,m$0#!3#,!'m"#m
Gm+32,2#m!"mHZKHm Zm!*0+#,2#m+37m (m1'm*m!-+.0+-1m!-,m*m"#*m%#,mm
^GiMIMm _m -m !-,m *m "#*m 1#%3,"-m +.*'!A,m "#*m %#,m >O ^GiHLJm _Ym ,m "#m *1m
$3,!'-,#1m"#*m%#,mI#-)J>m#1m*m"#m73"0mm#12 '*'80m#*m!-+.*#(-m.0-2#'!-m.0m
*m ','!'!'A,m "#m *m 20"3!!'A,m "#m *1m .0-2#>,1m ^0-5,',%m 7m !-*YZm GOOH_Zm $3,!'A,m
91'!m .0m *m !<*3*Ym -0m #12#m +-2'4-m .-"0>+-1m .#,10m /3#m +32,2#1m !-,m 3,m
!+ '-m/3#m,-m.#0+'2'#0m*m#12 '*'""m"#m*m.0-2#>,mI#-)J>m#,m#*m!-+.*#(-m"#m
','!'!'A,m "#m *m 20"3!!'A,m 7m .-0m *-m 2,2-Zm ,-m .#0+'2'#0m *m 20"3!!'A,m "#m !'#021m
.0-2#>,1Zm&0>m/3#m,-m$3#0,m4' *#1Ymm
m#$'!'#,!'m"#m+32!'A,m- 2#,'"m#,m3,m.- *!'A,m"#mm"#m2-+2#m"-,"#m
1#m ,*'80-,m GNm %#,#1Zm 3,-m "#m #**-1m #*m %#,m >Zm $3#m "#m GiKMJm m ^'0-,m 7m !-*YZm
HFGF_Ym ',m #+ 0%-Zm .0m #*m %#,m I#-)J>m *m #$'!'#,!'m "#m +32!'A,m $3#m "#m GiGGGFm
Ym 12#m "2-m !-',!'"#m !-,m *-1m 0#13*2"-1m - 2#,'"-1m #,m #12#m 20 (-m 7m "#+3#120m
!-+-m .0m !'#02-1m %#,#1m *m #$'!'#,!'m "#m +32!'A,m .3#"#m 1#0m +91m (m 0#1.#!2-m *m
#$'!'#,!'m +#"'m #,m *m .- *!'A,Zm .0- *#+#,2#m "# '"-m m /3#m 13m $3,!'A,m #1m +91m
4'2*m.0m#*m-0%,'1+-m/3#m-20-1m%#,#1m!-,m3,m21m"#m+32!'A,m+3!&-m+91m*2Ymm
$
GLFm
.)-OO&-O'/.#)(-O#(.# #-O
#-/-#9(m
m
m
EMCT T T T T T T T
TT
m
EMCMBTTPTQT
-"#0m 0#*!'-,0m #*m $#,-2'.-m "#m 3,m +32,2#m !-,m 3,m +32!'A,m !-,!0#2m .#0+'2#m
!-,$'0+0m*m$3,!'A,m"#*m%#,Ym0m#**-Zm&!#m$*2m!0!2#0'80m '#,m*-1m+32,2#1m7m
1#m m ,'4#*m +-0$-*A%'!-Zm +#2 A*'!-Zm '-/3>+'!-m -m +#"',2#m -20-1m ,9*'1'1m /3#m
!0!2#0'!#,m!-,m#6!2'23"m#*m#$#!2-m"#*m%#,m+32"-Ym
*m%#,mm0#*'8m3,m$3,!'A,m"#2#0+',,2#m.0m*m '-1>,2#1'1m"#m!0-2#,-'"#1m7m
#,m!-,1#!3#,!'Zm.0m/3#m1#m!-+3*#,m.'%+#,2-1m#,m*m.*,2Ymm,-m$3,!'-,*'""m
-m#*m1'*#,!'+'#,2-m"#*m%#,mm.0-4-!m$#,-2'.-1m* ',-1Zm!-+-m1#m&m"#+-120"-m
#,m-201m#1.#!'#1m!-+-m,#)*-#-m-m00-8m^0-*m7m3,28ZmHFFG\m02&+,,ZmHFFN_Ym
-0m#**-Zm1#m#1!-%'Am#*m%#,mm!-+-m!-,20-*m$#,-2>.'!-m"#m*m.- *!'A,m.3#12-m/3#m
*m"#2#!!'A,m"#m+32,2#1m.0m#12#m%#,m,-m0#/3#0>m"#m#,17-1m!-+.*#(-1Ym,!-,200m
3,m.*,2m* ',m*m+320mm"#+-1200>m,-m1A*-m*m$3,!'-,*'""m"#*m%#,Zm1',-m
*m #$'!'#,!'m "#m *m .- *!'A,m "#m m 7m #*m +<2-"-m 31"-m .0m "#2#!20m *-1m
+32,2#1Ym
,m *m .3#12m m .3,2-m "#*m +<2-"-m 1#m "#2#!20-,m Hm +32,2#1m #,m #*m %#,m m #,m *m
.- *!'A,m Zm 3,-m "#m #**-1m #,m 8-,m !-"'$'!,2#m ^+32,2#m INJ_m 7m !-,m 3,m 4*-0m
m"#mFYFFm/3#m!-,$'0+ m/3#m*m+32!'A,m.-">m$#!20mm*m$3,!'-,*'""m"#m*m
.0-2#>,Ym 01m %#0+',0m HFm 1#+'**1m Hm "#m *m $+'*'m +32,2#m INJm 1#m - 234'#0-,m
.*9,23*1m!-,m3,m$#,-2'.-m/3#m+-120 m*-1m!-2'*#"-,#1m*2#0"-1m#,m13m+-0$-*-%>Zm
.#0-m ,-m 1#m - 234-m ,',%3,m .*,2m * ',Ym ',m #+ 0%-Zm *m %#0+',0m 1#+'**1m Im
- 2#,'"1mm.02'0m"#m*m32-$#!3,"!'A,m"#m3,m&#2#0-!'%-2-mHm.0m*m+32!'A,Zm1'm
1#m - 234-m 3,m ',"'4'"3-m * ',-m ^'%30m JYG_Ym "#+91Zm 1#m !-+.0- Am +#"',2#m #*m
,9*'1'1m "#m *m 1#!3#,!'m "#m m /3#m #0m *m B,'!m .*,2m &-+-!'%-2m .0m *m
+32!'A,Ym -0m *-m 2,2-Zm *m "#2#!!'A,m "#m #12#m +32,2#m "#+-120Am /3#m #*m +<2-"-m
m 1#m #12 m .*'!,"-m !-00#!2+#,2#Zm 7m /3#m 1#m .-">,m - 2#,#0m $#,-2'.-1m
+32,2#1m#,m,3#120m.- *!'A,Ym
m
GLGm
#-/-#9(
.)-OO&-O'/.#)(-O#(.# #-
m
m
(&30 MEMB[m*9,23*m!-,m$#,-2'.-m* ',-m'"#,2'$'!"m#,m*m$+'*'mINJZm+32,2#m#,m#*m%#,mm^'2-#,-m
"#12301_Ym0'+#0m+32,2#m"#2#!2"-m%#,-2>.'!m7m$#,-2>.'!+#,2#m7m/3#m"#+3#120m*m#$'!'#,!'m"#m*m
.- *!'A,m"#mm"#mLHjGGIYm
m
1m '+.-02,2#m "#12!0m /3#m #,m #*m +-+#,2-m "#m ','!'0m #12#m 20 (-m ,-m 1#m !-,-!>m *m
1#!3#,!'m!-+.*#2m"#*m%#,mm#,m+#*A,m7m.-0m*-m2,2-Zm#*m"'1#@-m"#*m+.*'!A,m"#m
m1#m&'8-mm.02'0m"#m3,m3,'%#,m^OFMF_Ym!23*+#,2#Zm7m%0!'1mm/3#m1#m
"'1.-,#m "#m *m 1#!3#,!'m "#*m %#,-+m "#*m +#*A,Zm 1#m &m .-"'"-m "#2#0+',0m *m
#1203!230m!-+.*#2m"#*m%#,mZm/3#m!-,12m"#mGJm#6-,#1Ymm
m
EMCMCT$ETPTTTTTTEQT
*m%#,m#Jm&m1'"-m#*m%#,m#,m#*m/3#m1#m&,m"#2#!2"-m+91m+32,2#1m#,m#12#m20 (-Ym
,m #12#m %#,Zm 1#m &,m '"#,2'$'!"-m +32!'-,#1m ,230*#1m 0#1.-,1 *#1m "#m '+.#"'0m *m
',2#0!!'A,m #,20#m #*m $!2-0m "#m ','!'!'A,m 7m *m !"#,m ',$#!2'4m "#m m .-1'2'4m "#m
*%3,-1m4'031m^- %*'m7m0,2ZmHFFL_Ym- %*'m7m0,2m#,mHFFLm0#4'10-,m*-1m
$!2-0#1m "#m ','!'!'A,m "#m *m 20"3!!'A,m "#m *%3,1m #1.#!'#1m 7m 13m ',2#0!!'A,m !-,m
4'031m 7m !A+-m *%3,1m +32!'-,#1m .0-.-0!'-, ,m 0#1'12#,!'m ,230*m m "'4#01-1m
.-2'4'031Ym-+.00-,m*m1#!3#,!'m.0-2#'!m"#m*-1m%#,#1m*0,<m"#m.#.',-m^*-#/'O
((/'_Zm *).;m "#m 2-+2#m ^)&(/'O &3)*,-#/'_Zm ');m "#m *#!&3%m ^./.O -.#0_m 7m
-';m "#m %3'1,2#m ^#-/'O -.#0/'_Ym ,m #12#m *',#+'#,2-m 1#@*0-,m *1m "'$#0#,!'1m
+',-!>"'!1m #,20#m *-1m %#,-2'.-1m 0#1'12#,2#1m 7m *-1m 131!#.2' *#1m ^'%30m JYHZm #,m
$-,"-m ,#%0-_m #,m !"m 3,m "#m *1m #1.#!'#1m !'2"1Ym + '<,m '"#,2'$'!0-,m "-1m
GLHm
.)-OO&-O'/.#)(-O#(.# #-O
#-/-#9(m
0#%'-,#1m^'%30mJYHZm8-,1m$3!1'm7m83*m230/3#1_m"A,"#m1#m!-,!#,20,m3,m+7-0m
,B+#0-m "#m .-*'+-0$'1+-1m #,20#m *-1m $#,-2'.-1m 131!#.2' *#1m 7m 0#1'12#,2#1Ym "#+91Zm
+ 1m0#%'-,#1m!-,$-0+,m&))*-O)O*8-1m#62#0,-1m#,m*m#1203!230mIm"#m*m.0-2#>,Ym
,m#12#m+'1+-m*',#+'#,2-m.0-2#'!-m&#+-1m',!*3'"-m#*m%#,m#Jm"#m+#*A,Zm!-,m*m
&'12'"',m #,m *m .-1'!'A,m HHNm +0!"m #,m *,!-m 7m $-,"-m 83*m +0',-m ^'#2-m 7m !-*YZm
HFFL_m ^'%30m JYH_Zm 0#1.-,1 *#m "#m *m 0#1'12#,!'m m m ^(-0_Zm #*m %#,m -02A*-%-m #,m
!# "m ^),/'O 0/&!,_m ,3'm 0#1.-,1 *#m "#m *m 0#1'12#,!'m m m ^'031m "#*m
-1'!-m +0'**-m #,m # "_m 7m *-1m +32,2#1m #,!-,20"-1m +#"',2#m m #,m
#12#m20 (-m^'%30mJYHZm#,m$-,"-m0-(-m7m4#0"#_Ym#m#12#m+-"-m.-"#+-1m!-+.0- 0m
/3#m*1m+32!'-,#1m"#2#!2"1m#,m#12#m20 (-m#129,m*-!*'8"1m#,m"'12',21m0#%'-,#1m
m *1m "#1!0'21m ,2#0'-0+#,2#m #,m -201m #1.#!'#1Ym *m +'1+-m 2'#+.-Zm 1#m &m .-"'"-m
"#2#0+',0m/3#m*1m+32!'-,#1m*3GHO71m^$+'*'mKM_Zm*GIJ*m^$+'*'mGHHO_m7m
*7GIL*3m ^$+'*'m JJJ_m .0-4-!,m 3,m !+ '-m "#m +',-9!'"-m #,m 0#%'-,#1m +37m
!-,1#04"1m"#m*m.0-2#',Zm.-0m*-m2,2-m13m#$#!2-m1#m.0- *#+#,2#m+7-0m/3#m*1m
+32!'-,#1m '"#,2'$'!"1m #,m 8-,1m +#,-1m !-,1#04"1Zm *1m +32!'-,#1m 0%HK71m
^$+'*'mHFIL_Zm*3OM71m^$+'*'mKI_m7m*m*3HFL71m^$+'*'mHFLF_Ym1m+32!'-,#1m
1'*#,!'-11m ,-m .0-4-!,m !+ '-m "#m +',-9!'"-m 7m .-0m *-m 2,2-m ,-m $#!209,m m *m
$3,!'-,*'""m"#m*m.0-2#>,m^&#ON&#m^$+'*'mHKIH_m7m&0GKK&0m^$+'*'mGNNF__Ymm
'(9,"-,-1m #,m #*m *',#+'#,2-m .0#1#,2"-m #,m *m '%30m JYHm #,!-,20+-1m #*m .0'+#0m
+32,2#m "#2#!2"-m #,m *m .- *!'A,m m #,m *m .-1'!'A,m HKm "#m *m 1#!3#,!'m
+',-!>"'!m"#*m%#,m#Jm#,m+#*A,O^*-1m+32,2#1mm#129,m"#2**"-1m#,m$-,"-m
0-(-_Ym12m+32!'A,m.#02#,#!#mm*m$+'*'mHFILm7m#*m!+ '-m/3#m.0-4-!m#1m"#m3,m
0%',',m m 3,m *'1',Ym + -1m +',-9!'"-1m !-,2'#,#,m #*m 0"'!*m "#m !"#,m !0%"-m
.-1'2'4+#,2#m .#0-m *m 0%',',m !-,2'#,#m *m $',*m "#*m 0"'!*m "-1m %03.-1m +',-m
+'#,201m/3#m*m*'1',m,-Ym0m#12m+32!'A,m#*mm.0#"'!#m3,m4*-0mFYFFm7m.-0m*-m
2,2-m3,m.-1' *#m#$#!2-m#,m*m$3,!'-,*'""m"#m*m.0-2#>,Ym,m#12#m!1-m1#m#,!3#,20m
#,m 3,m 0#%'A,m +37m .-!-m !-,1#04"m 7m .-0m *-m 2,2-m /3'891m 3,m 0#%'A,m ,-m 2,m
'+.0#1!',"' *#m.0m*m!-00#!2m$3,!'-,*'""m"#m#JYmm
m
m
m
m
m
m
m
GLIm
#-/-#9(
.)-OO&-O'/.#)(-O#(.# #-
pot1
pvr2
rym
nsv
sbm1
mo1
MAAAEMERTMSFDAAEKLKAAD---GGGGEVDDELEEGEIVEES----NDTASYLGKEIT
MATAEMEKTTTFDEAEKVKLN------ANEADDEVEEGEIVEET----DDTTSYLSKEIA
MAEDTETRPASAGAEER------------------EEGEIADDG----DGSAAAAAGRVS
MVVEDSMKATSAEDLSNSIANQNPRGRGGDEDEELEEGEIVGDDD--LDSSNLSAS-LVH
MVVEETPKSIITDDQITTNPN-----RVIEDDNNLEEGEILDED----DSSATSKP-VVH
MKSEE-QKLIDVNKHRGVRSD-------GEEDEQLEEGEIVGGDADTLSSSSSSRPGTAI
*
:
*****
..:
53
50
38
57
50
52
pot1
pvr2
rym
nsv
sbm1
mo1
VKHPLEHSWTFWFDNPTTKSRQTAWGSSLRNVYTFSTVEDFWGAYNNIHHPSKLIMGADF
TKHPLEHSWTFWFDNPVEKSKQDAWGSSLRNVYTFSTVEDFWGAYNNIHHPSKLVVGADL
-AHPLENAWTFWFDNPQGKSRAVAWGSTIHPIHTFSTVEDFWSLYNNIHHPSKLNVGADF
QPHPLEHSWTFWFDNPSAKSKQATWGASIRPIYTFSTVEEFWSVYNNIHHPSKLAMRADL
QPHLLENSWTFWFDTPAAKSKQDDWGSSMRPIYTFSTVEEFWSIYNNIHHPGKLAVGADF
AQHPLEHSWTFWFDTPSAKSKQVAWGSSMRPIYTFSSVEEFWSLYNNIHRPSKLAQGADF
* **::*** **.* **:
**:::: ::***:**:**. *****:*.**
**:
113
110
97
117
110
112
pot1
pvr2
rym
nsv
sbm1
mo1
HCFKHKIEPKWEDPVCANGGTWKMSFSKGKSDTSWLYTLLAMIGHQFDHGDEICGAVVSV
HCFKHKIEPKWEDPVCANGGTWKMSFSKGKSDTSWLYTLLAMIGHQFDHEDEICGAVVSV
HCFKDKIEPKWEDPICANGGKWTISCGKGKSDTFWLHTLLALIGEQFDFGDEICGAVVSV
YCFKHKIEPKWEDPVCANGGKWTVNFPRGKSDNGWLYTLLAMIGEQFDCGDEICGAVVNV
YCFKHKIEPKWEDPICANGGKWTANYPKGKSDTSWLYTLLAMIGEQFDHGDEICGAVVKV
YCFKNKIEPKWEDPVCANGGKWTMTFTKAKSDTCWLYTLLAMIGEQFDHGDDICGAVVNV
:***.*********:*****.*. . :.***. **:****:**.*** *:******.*
173
170
157
177
170
172
pot1
pvr2
rym
nsv
sbm1
mo1
RAKGEKIALWTKNAANETAQVSIGKQWKQFLDYSDSVGFIFHDDAKRLDRNAKNRYTV
RGKGEKISLWTKNAANETAQVSIGKQWKQFLDYSGSVGFIFHDDAKRLDRNAKNRYTV
RKNQERVAIWTKNAANETAQISIGKQWKEFLDYKDSIGFVVHEDAKRSDKGAKNRYTV
RSGQDKISIWTKNASNEAAQASIGKQWKEFLDYNESIGFIFHDDAKKFDRLAKNKYMV
RGRAEKISIWTKNASNEAAQVSIGKQWKEFLDYNETMGFIFHDDARKLDRNAKNKYVV
RARQEKIALWTKNAANESAQLSIGKQWKEFIDYNDTIGFIFHEDAKTLDRSAKNKYTV
*
:::::*****:**:** *******:*:**. ::**:.*:**: *: ***:* *
231
228
215
235
228
230
(&30 T EMC[m *',#+'#,2-m +#"',2#m &/-.&m "#m *m .0-2#',m #Jm "#m "'12',21m #1.#!'#1Ym #m "#12!m #,m
$-,"-m,#%0-m*m*-!*'8!'A,m"#m*-1m+',-9!'"-1m/3#m"'$'#0#,m#,20#m*-1m%#,-2'.-1m131!#.2' *#1m7m0#1'12#,2#1mm
m .-2'4'031m "#m .'+'#,2-m ^*-#/'O ((/'Fm *0,<_Zm 2-+2#m I)&(/'O &3)*,-#/(Fm *).;_Zm *#!&3%m ^./O
-.#0Zm');_Zm%3'1,2#m^#-/'O-.#0/'Fm-';_Zm+#*A,m^//'#-O'&)Zm(-0_m7m!# "m^),/'O0/&!,Zm,3'_Ym
1m.02#1m!-*-0#"1m#,m$3!1'm7m83*m230/3#1m0#.0#1#,2,m*1m8-,1m!-,m+91m!+ '-1m"#m+',-9!'"-1m7m
m 13m 4#8m 8-,1m +91m #6.3#121m *m #62#0'-0m 2*m 7m !-+-m "#2#0+',m *m !-,$'%30!'A,m #,m Im "#m *m .0-2#>,m
^- %*'m7m0,2ZmHFFL_Ym,m0-(-m*1m+32!'-,#1m"#2#!2"1m+#"',2#mm#,m*m.- *!'A,mm7m
#,m4#0"#m*1m+32!'-,#1m"#2#!2"1m#,m*m.- *!'A,m"#*mYm
m
1m1'%3'#,2#1m+32!'-,#1m/3#m#,!-,20+-1m.#02#,#!#,mm*1m$+'*'1mKIm7mHKIHm^#,m
#12#m!1-m#,m4#0"#m7m/3#m1#m"#2#!20-,m#,m*m.- *!'A,m"#*m_Ym,m*m$+'*'mKIm
*m+32!'A,m1#m#,!3#,20m#,m*m.-1'!'A,mOMm7m.0-4-!m3,m!+ '-m"#m9!'"-m%*329+'!-m
m *'1',Ym *m m #,m #12#m !1-m .0#"'!#m 3,m .3,23!'A,m "#m FYFHm 7m .-0m *-m 2,2-m 3,m
.-1' *#m !+ '-m #,m *m $3,!'-,*'""m "#m *m .0-2#>,Ym "#+91Zm #*m !+ '-m "#m 9!'"-m
%*329+'!-mm*'1',m.0-4-!m2+ '<,m3,m!+ '-m"#m!0%m^"#m,#%2'4-mm.-1'2'4-_m/3#m
.-"0>m $#!20m #,m *m ',2#0!!'A,m !-,m -20-1m +',-9!'"-1m 7m .-0m *-m 2,2-m #,m #*m
.*#%+'#,2-m"#m*m.0-2#>,Ym,m.-1' *#m!+ '-m#,m*m!-,$-0+!'A,m"#m*m.0-2#>,mm
,'4#*m .0'+0'-m 3m -20-1m ,'4#*#1m 13.#0'-0#1m .-"0>m $#!20m m *m $3,!'-,*'""m "#m *m
GLJm
.)-OO&-O'/.#)(-O#(.# #-O
#-/-#9(m
.0-2#>,m-mm13m',2#0!!'A,m!-,m13m*'%,"-Ymm$+'*'mHKIHm2'#,#m*m+32!'A,m(312-m
#,m *m .-1'!'A,m !-,2'%3m m *m ,2#0'-0m #,m #*m +',-9!'"-m ,B+#0-m ONYm -m - 12,2#Zm *m
+32!'A,m #1m 1'*#,!'-1Ym -,m #12#m *',#+'#,2-m &#+-1m .-"'"-m - 1#040m /3#m &7m
!'#021m 0#%'-,#1m "#m *m 1#!3#,!'m .0-2#'!m +91m 131!#.2' *#1m m 1#0m +32"1m -m /3#m
.#0+'2#,m *m +32!'A,Zm .3#12-m /3#m *m .0- '*'""m "#m +320m "-1m +',-9!'"-1m
!-,2'%3-1m-m+37m!#0!,-1m#1m (Ymm
,m *m 0#%'A,m "#*'+'2"m .-0m *-1m +',-9!'"-1m GHOm *m GKKm &#+-1m "#2#!2"-m !320-m
+32,2#1Zm"-1m#,m!"m.- *!'A,Ymm+32!'A,m!-00#1.-,"'#,2#m*m+',-9!'"-mGHOm
$3#m "#2#!2"m #,m *m $+'*'m KMm "#m *m .- *!'A,m Ym 12#m !+ '-m #1m 1'%,'$'!2'4-m
^SFYFF_m .3#12-m /3#m .0-4-!m 3,m !+ '-m "#m 9!'"-m %*329+'!-m m *'1',Ym 1m
+32!'-,#1m"#m*1m$+'*'1mGHHOm7mJJJm"#m*m.- *!'A,mm#129,m*-!*'8"1m!-,m
1A*-m 3,m +',-9!'"-m "#m 1#.0!'A,Ym + 1m .0-"3!#,m !+ '-m "#m +',-9!'"-Zm *m
.0'+#0m "#m *,',m m 4*',m ^+ -1m &'"0-$A '!-1_m !-,m 3,m 4*-0m "#m m "#m FYGFm
+'#,201m/3#m*m1#%3,"m"#m%*'!',mm9!'"-m%*329+'!-m!-,m3,m4*-0m"#mm"#mFYFFYm
12#mB*2'+-m.0#!#m/3#m.-"0>m$#!20m+91mm*m$3,!'-,*'""m"#m*m.0-2#>,m.3#12-m
/3#m #*m !+ '-m #1203!230*m 2+ '<,m .-"0>m 1#0m +91m !#,23"-Ym m %*'!',m #1m 3,m
+',-9!'"-m+37m.#/3#@-m7m13m0"'!*m1#m 1m#,m3,m92-+-m"#m&'"0-%#,-Zm+'#,201m
/3#m#*m9!'"-m%*329+'!-m#1m3,m+',-9!'"-m!0%"-m,#%2'4+#,2#m7m!-,m3,m0"'!*m
+3!&-m+91m%0,"#m/3#m1#m#1203!230m#,m3,m!"#,m"#m20#1m!0 -,-1m!-,m3,m%03.-m
!0 -6'*-Ym ',*+#,2#Zm *m !302m +32!'A,m #,m #12m 0#%'A,m $3#m '"#,2'$'!"m #,m *m
$+'*'m GNNFm "#m *m .- *!'A,m Zm .#0-m #,m #12#m !1-m *m +32!'A,m #1m 1'*#,!'-1Ym
#m "#12!0m /3#m *1m 20#1m +32!'-,#1m ^KMZm GHHOm 7m JJJ_m #129,m *-!*'8"1m #,m 3,m
0#%'A,m +37m !-,1#04"m ^'%30m JYH_Ym m (m 21m "#m .-*'+-0$'1+-1m #,m #12m 0#%'A,m
,-1m.-"0>m',"'!0m/3#m#1m'+.-02,2#m.0m*m$3,!'-,*'""m"#m*m.0-2#>,Ym
,m *m .-1'!'A,m HFLm #,!-,20+-1m #*m B*2'+-m +32,2#m "#2#!2"-m #,m *m $+'*'m HFLFm
.#02#,#!'#,2#mm*m.- *!'A,mYm,m#12#m!1-m#*m!+ '-m.0-"3!'"-m#1m2+ '<,m"#m
9!'"-m %*329+'!-m m *'1',Ym 12m B*2'+m +32!'A,m 1#m #,!3#,20m #,m 3,m 8-,m ,-m 2,m
!-,1#04"m!-+-m.3#"#m4#01#m#,m*m'%30mJYHm7m.-0m#**-m13m4*-0mm#1m2+ '<,m
+7-0m^FYFO_Ym
#m 3,m 2-2*m "#m -!&-m +32,2#1m #,m 0#%'A,m #6A,'!m "#*m %#,m #JZm 1#'1m .0-"3!#,m
!+ '-m "#m +',-9!'"-m 7m 20#1m "#m #**-1m 1-,m "#m 9!'"-m %*329+'!-m m *'1',Ym 3,/3#m
.0#!#m /3#m *m +32!'A,m +#"',2#m m #1m 2-2*+#,2#m *#2-0'Zm #,m ,3#120-m !1-m &m
& '"-m3,m!'#02m2#,"#,!'mm+320m*%3,1m0#%'-,#1m+91m/3#m-201m7mm13m4#8Zm&m
& '"-m20#1m!1-1m"#m1#'1m#,m/3#m#*m!+ '-m&m1'"-m#*m+'1+-m^i_Ymm
GLKm
#-/-#9(
.)-OO&-O'/.#)(-O#(.# #-
,m *m '%30m JYHm 2+ '<,m 1#m .3#"#m - 1#040m !-+-m ,',%3,m "#m *1m +32!'-,#1m
"#2#!2"1m1#m*-!*'8m#,m*1m0#%'-,#1m"#m#Jm!-,m*2-m!-,2#,'"-m#,m.-*'+-0$'1+-1m
#,20#m*1m40'#""#1m0#1'12#,2#1m7m*1m131!#.2' *#1m"#m-201m#1.#!'#1Ym
!23*+#,2#m #6'12#,m +3!&1m &#00+'#,21m ',$-0+92'!1m .0m .-"#0m #123"'0m !-,m
"#2**#m !-+-m $#!2,m *1m +32!'-,#1m m *m $3,!'-,*'""m "#m 3,m .0-2#>,Ym ,m #12#m
20 (-m1#m&m31"-m+7-0'20'+#,2#m#*mYm
,m HFFLm 3*',m 7m #,')-$$m .3 *'!0-,m 3,m #123"'-m "-,"#m .0#1#,20-,m 3,m 1#0'#m "#m
+<2-"-1m7m&#00+'#,21m.0m.-"#0m.0#"#!'0m*-1m#$#!2-1m"#m!+ '-1m"#m+',-9!'"-1m
#,m *m .0-2#>,Zm +3!&-1m "#m #**-1m "'1.-,' *#1m 4'm 5# Ym ,-m "#m #**-1m #1m m
^".)&)!3O '/..#)(-_m ^-12m 7m ,"#0Zm GOOI_m 7m 3,/3#m #12<m "'1#@"-m .0m "#2#!20m
+32!'-,#1m#,m&3+,-1m0#13*2m#1.#!'*+#,2#mB2'*m.0m*m.0#"'!!'A,m"#m#1203!2301m
1#!3,"0'1m"#m.0-2#',1Ym
,m ,3#120-m !1-m 1#m &,m &#!&-m 2+ '<,m 1'+3*!'-,#1m #,m Im !-,m &#00+'#,21m
',$-0+92'!1m /3#m 1#m &,m .0#1#,2"-m #,m #*m .02"-m "#m #13*2"-1Zm 32'*'8,"-m
7-*Ym ',m #+ 0%-Zm 0#13*2m 2+ '<,m ',2#0#1,2#m &!#0m 3,m #123"'-m "#2**"-m "#m *m
.0#"'!!'A,m "#m #1203!2301m 1#!3,"0'1m #,m *m 1#!3#,!'m .0-2#'!Ym 121m #1203!2301m
1-,m*1m.0'+#01m$#!2"1m7m/3#m1#m!-,12037#,mm204<1m"#m*1m',2#0!!'-,#1m#,20#m
+',-9!'"-1Ym -0m #**-m 31+-1m #12m &#00+'#,2m ^_m #,m *m /3#m .-"#+-1m
- 1#040m *m 1#!3#,!'m .0-2#'!m "#m #Jm !-,m *m .0#"'!!'A,m #1203!230*m
!-00#1.-,"'#,2#m.0m!"m+',-9!'"-m^'%30mJYI_Ym
1m +32!'-,#1m !-00#1.-,"'#,2#1m m *1m $+'*'1m GHHOm 7m JJJm !-,m !+ '-1m #,m *1m
.-1'!'-,#1m GIJm 7m GILm #129,m *-!*'8"1m #,m 3,m 0#%'A,m !-,m 3,m *2m .0- '*'""m
^4*-0mL_m"#m13+'0m!-,$-0+!'A,m"#m&))*m^'%30mJYIZm"#2**"-m#,m$-,"-m4#0"#_Ym-1m
&))*-m1-,m%'0-1m#,m*m#1203!230m1#!3,"0'm"#m*m.0-2#>,m7m.-0m*-m2,2-m"#$',#,m#,m
%0,m+#"'"m*m#1203!230m2#0!'0'm7m!32#0,0'Ym1m.-0m#**-m/3#m+ -1m+32,2#1m
.-"0>,m .0-"3!'0m 3,m !+ '-m #1203!230*m "#m *m .0-2#>,Ym 12m !-+.0!'A,m ,-m
!-',!'"#m "#*m 2-"-m !-,m *-1m 4*-0#1m - 2#,'"-1m !-,m #*m m .3#12-m /3#m m *m .0'+#0m
+32!'A,m *#m 1'%,m 3,m 4*-0m "#m FZGFm 7m #,m !+ '-m #*m m *#m !-,$'#0#m 3,m 4*-0m
#*#4"-m #,m !3,2-m m 13m .0- '*'""m #1203!230*Ym ,m !3*/3'#0m !1-m + -1m
0#13*2"-1m 1-,m !-+.*#+#,20'-1m .3#12-m /3#m !"m .0-%0+m 31m 3,1m +20'!#1m
"'12',21m7m1#m 1,m#,m"'12',2-1m.09+#20-1m','!'*#1Ym
m
m
m
m
GLLm
.)-OO&-O'/.#)(-O#(.# #-O
SeqAA
prH
prE
prL
SUBsec
#-/-#9(m
MVVEDSMKATSAEDLSNSIANQNPRGRGGDEDEELEEGEIVGDDDLDSSNLSASLVHQPH
000011111111112344444444322111111111111222211111221111111221
000000011111000000000000111111111233333333321111111122223333
988877777677776654554444556667776554554444456666666666655444
LLLLLLLLLLLLLLL...........LLLLLLL...........LLLLLLLL........
SeqAA PLEHSWTFWFDNPSAKSKQATWGASIRPIYTFSTVEEFWSVYNNIHHPSKLAMRADLYCF
prH
212211222222222221111100112211111112222222111112211100101011
prE
221111222211000000123443210111123334444333322111135676665543
prL
556666544556666677765445676667655443333444566666543212223344
SUBsec....LL......LLLLLLL.....LLLLLLL.............LLL.....EEE.....
SeqAA KHKIEPKWEDPVCANGGKWTVNFPRGKSDNGWLYTLLAMIGEQFDCGDEICGAVVNVRSG
prH
112222212222211111122221222222122222222111111111112111111112
prE
322233343332211123333323333333332222333333211111232333344311
prL
555444444444566655444444444444444444333445667677654444433566
SUBsec.............LLL...........................LLLLL..........LL
SeqAA
prH
prE
prL
SUBsec
QDKISIWTKNASNEAAQASIGKQWKEFLDYNESIGFIFHDDAKKFDRHAKNKYMV
1111111111222233222233334322112211111112357666553311100
1111223332111112343212112333222222355431000000000000100
7776665445665654433444543334555455433356542323346678789
LLLL...................................L..H.......LLLLL
T
(&30 TEMD[m /.*/.O"#*m .0-%0+m m !-,m *m 1#!3#,!'m .0-2#'!m "#*m %#,m #Jm "#m //'#-O'&)m 7m *-1m
+32,2#1m #,!-,20"-1m +0!"-1m #,m 0-(-m ^.- *!'A,m _m 7m #,m 4#0"#m ^.- *!'A,m _Ym 1m *>,#1m
',$#0'-0#1m!-00#1.-,"#,mm*1m.0- '*'""#1m.0m!"m+',-9!'"-m"#m!-,20' 3'0m#,m3,m!-,$-0+!'A,m
1#!3,"0'Ym.0[m.0- '*'""m"#m&<*'!#Zm.0[m.0- '*'""m"#m!"#,m*0%m7m.0[m.0- '*'""m"#O&))*Ym
',*+#,2#Zm *m B*2'+m *>,#m ^1#!_m !-00#1.-,"#m m *m .0#"'!!'A,m "#m #1203!230m 1#!3,"0'm #,m !"m
+',-9!'"-m1#%B,m*1m.0- '*'""#1m,2#0'-0#1Zm"-,"#m[m&<*'!#Zm[m*))*m7m[m!"#,m*0%YmbYcm1'%,'$'!m
/3#m,-m1#m.3#"#m1'%,0m3,m#1203!230m.-0/3<m*m.0- '*'""m"#m!"m!-,$-0+!'A,m2'#,#m.3,23!'A,m
'%3*m-m+#,-0m"#mKYm
m
0!'1m m *1m .0#"'!!'A,m "#m *m #1203!230m Im "#m *m .0-2#>,m #Jm 7m m 13m +-"#*(#m
+#"',2#m #*m .0-%0+m 7-*m 1#m &,m .-"'"-m ,*'80m *%3,1m "#m *1m +32!'-,#1m
"#2#!2"1m !-,m +7-0m "#2**#Ym *m #123"'-m #,m #*m +32,2#m Hm ^$+'*'m KM_m ,-1m &m
.#0+'2'"-m- 1#040m/3#m#*m!+ '-m*3GHO71m.-"0>m$#!20m#,m*m',2#0!!'A,m!-,m#*m
131202-m^*m+-*<!3*m*m"#*m+_Ym#!-0"#+-1m/3#m2091m*m',$#!!'A,m4'0*Zm1'm*m
',2#0!!'A,m #,20#m *m .0-2#>,m #Jm 7m #*m +m "#*m 4'031m ,-m 1#m .0-"3!#m
!-00#!2+#,2#Zm#*m4'031m,-m.-"09m',$#!20m*m.*,2Ym,m#12#m+32,2#m*m1312'23!'A,m#1m
"#m 3,m +',-9!'"-m !0%"-m ,#%2'4+#,2#m ^9!'"-m %*329+'!-_m m -20-m !0%"-m
.-1'2'4+#,2#m^*'1',_Ym12#m!+ '-m.-"0>m.0-4-!0m*m"#1.0'!'A,m-m.0'!'A,m"#m
,3#4-1m #,*!#1m -!1'-,,"-m 3,m 3,'A,m +91m "< '*m #,20#m #*m ','!'-m "#*m +m 7m #*m
!#,20-m!2'4-m-m"#m3,'A,m"#m#JYm-0m#12#m+-2'4-Zm.-"0>m20201#m"#m3,m+32,2#m
+#,-1m#$'!'#,2#m#,m*m',2#0!!'A,m.0-2#>,j131202-Ym"#+91Zm#*m4*-0m"#mm.0m
GLMm
#-/-#9(
.)-OO&-O'/.#)(-O#(.# #-
#12m+32!'A,m#1m"#mFYFFZm7m.-0m*-m2,2-m!-,$'0+m#*m.-1' *#m#$#!2-m"#m*m+32!'A,m#,m
*m$3,!'-,*'""m"#m*m.0-2#>,Ym
*m+'1+-m!+ '-m.#0-m#,m-20m.-1'!'A,m1#m&m.0-"3!'"-m#,m#*m+32,2#mKm^$+'*'mKI_m
/3#m 1312'237#m 2+ '<,m #*m 9!'"-m %*329+'!-m .-0m *m *'1',Ym *m 9!'"-m %*329+'!-m #,m #*m
%#,-2'.-m 1'*4#120#m !0#m 3,m #,*!#m "#m &'"0A%#,-m !-,m *m 1#0',m OIYm 12#m #,*!#m
+,2'#,#m .0- *#+#,2#m *m &<*'!#m *$m 7m *m &-(m #2m 3,'"1m ","-m #12 '*'""m 7m
!'#02m0'%'"#8mm+ 1m!-,$-0+!'-,#1Ym,m!+ '-Zm*m*'1',m/3#m!-,2'#,#m#*m+32,2#m
,-m.3#"#m#12 *#!#0m#12#m#,*!#m.3#12-m/3#m13m0"'!*m#129m!0%"-m.-1'2'4+#,2#m7m
,-m ,#%2'4+#,2#m !-+-m #*m 0"'!*m "#*m 9!'"-m %*329+'!-Ym m $*2m "#m #12#m #,*!#m
.-"0>m .0-.-0!'-,0m +91m $*#6' '*'""m #,m #12m 8-,m "#m *m +-*<!3*m !-,m
!-,1#!3#,!'1m '+.0#"#!' *#1Ym 12m 1312'23!'A,m .0-.-0!'-,m +#,-0m 0'%'"#8m m #12m
.02#m #1203!230*m 7m #,m !-,1#!3#,!'m +#,-0m #12 '*'""Zm "#+91Zm #*m 4*-0m "#m m
.0m#12m+32!'A,m#1m"#mFYFHYm
#"',2#m*m.0#"'!!'A,m#,mIm1#m&m.-"'"-m- 1#040m/3#m*m+32!'A,m"#*m+32,2#mNm
^$+'*'m GHHO_m .09!2'!+#,2#m ,-m .0-4-!0>m !+ '-1m #1203!230*#1Ym *m !+ '-m "#m
*,',m m 4*',m ,-m *2#0m *m #1203!230m .3#12-m /3#m + -1m 1-,m +',-9!'"-1m ,-m
.-*0#1m7m!-,m3,m0"'!*m+37m.#/3#@-Ym*m4*-0m"#mm2+ '<,m!-,$'0+m#12#m"2-m
"9,"-*#m 3,m .3,23!'A,m "#m FYGFYm ',m #+ 0%-Zm 7m !-+-m 1#m &m !-+#,2"-m
,2#0'-0+#,2#Zm #12#m 0#13*2"-m #1m !-,20"'!2-0'-m *m 0#13*2"-m - 2#,'"-m +#"',2#m
Ym12-m#1m"# '"-mm/3#m+ -1m.0-%0+1m1#m 1,m#,m"'12',2-1m.09+#20-1Ym,m
#12#m !1-Zm *-m !-,4#,'#,2#m 1#0>m 2#,#0m #,m !3#,2m + -1m 0#13*2"-1m 7m .-0m *-m 2,2-m
!-,1'"#00m /3#m *m +32!'A,m "#m *m $+'*'m GHHOm .3#"#m $#!20m m *m #1203!230m
1#!3,"0'm7m/3'891mm*m$3,!'-,*'""m"#m*m.0-2#>,Ym
',*+#,2#Zm*m+32!'A,m"#*m+32,2#mOm^$+'*'mJJJ_Zm1#%B,m#*m+-"#*-mIZm,-m.0#!#m
/3#m.0-4-/3#m3,m!+ '-mm,'4#*m"#m%'0-m#,m*m#1203!230m1#!3,"0'm"#m*m.0-2#>,Ym
',m#+ 0%-Zm#*m!+ '-m"#m%*'!',mm9!'"-m%*329+'!-m',20-"3!#m3,m!0%m,#%2'4m7m
3,m 0"'!*m +3!&-m +7-0Ym #,'#,"-m #,m !3#,2m *m .-1'!'A,m ',2#0,m "#m #12#m
+',-9!'"-m7m*m',20-"3!!'A,m"#m*m!0%m,#%2'4Zm#12m+32!'A,m.-"0>m$#!20m#,m
*1m',2#0!!'-,#1m!-,m*-1m*'%,"-1m"#m*m.0-2#>,m#JYm#%B,m#*m,9*'1'1m!-,mm
*m+32!'A,m/3#m!-,2'#,#m#12m$+'*'m#,m*m.-1'!'A,mGILm"#m*m1#!3#,!'m.0-2#'!m#1m
3,m 0#%'A,m !-,m 3,m *2m .0- '*'""m ^4*-0m L_m "#m 13+'0m !-,$-0+!'A,m "#m &))*O -m
%'0-Em
,m#*m.0-2-!-*-m"#mZmm.02#m"#m*m"#2#!!'A,m#m'"#,2'$'!!'A,m"#m+32,2#1Zm1#m
"# #m0#*'80m#*m#123"'-m"#m1#%0#%!'A,m"#m*m+32!'A,m7m"#m13m$#,-2'.-Ym0m#*m%#,m
GLNm
.)-OO&-O'/.#)(-O#(.# #-O
#-/-#9(m
m.3"-m0#*'801#m#12#m#123"'-m""-m/3#m#*m,9*'1'1m$#,-2>.'!-m,-m0#/3#0>m+3!&-m
20 (-Zm 2029,"-1#m "#m - 1#040m 1A*-m #*m !0#!'+'#,2-m "#m *m .*,2m 7m 13m #12"-m "#m
.'%+#,2!'A,Ym 0m #*m 0#12-m "#m *-1m %#,#1m ,*'8"-1m #,m #12#m 20 (-m ^>Fm
I#-)J>_m#*m#123"'-m$#,-2>.'!-m2-"4>m/3#"m.#,"'#,2#m.-0m0#/3#0'0m+91m2'#+.-m7m
0#!301-1Ym',m#+ 0%-Zm.0m#*m%#,m#Jm1#m&'8-m*m0#!-*#!!'A,m"#m*1m1#+'**1mHm7m
*m - 2#,!'A,m "#m *1m 1#+'**1m Im .0m .-"#0m $'(0m *m +32!'A,m .0Zm #,m 3,m $3230-Zm
.-"#0m!-,$'0+0m#*m#$#!2-m$#,-2>.'!-m+#"',2#m',-!3*!'A,m!-,m3,m 2#0>m"#m4'031m
^ *mJYI_Ymm
!+ TEMD[m#13*2"-1m"#*m#123"'-m"#m1#%0#%!'A,m"#m*1m$+'*'1m+32,2#1m"#*m%#,m#Jm#,!-,20"-1m#,m
*m.- *!'A,mYg[m'*"m27.#m-m1'*4#120#Zm[m-+-!'%-2-Zm[m32,2#Ym
m
Amplicón
Exón
Posición
cambio AA
Familia M2
Plantas
secuenciadas
en M2
Segregación en M2
(WT/H/M)*
eIF4E-1
Exón 1
G74A
Arg25Lys
F2036
24
9/7/8
eIF4E-2
Exón 2
G385A
Glu129Lys
F57
8
5/3/0
m
,m4#8m'"#,2'$'!"1m*1m$+'*'1m+32,2#1m#,m#*m%#,m>m"#m*m.- *!'A,mm1#m
!0#!'Am *m %#,#0!'A,m HYm 0m *m $+'*'m HFILm 1#m .3"'#0-,m !0#!#0m 7m 1#!3#,!'0m HJm
.*,21Zm+'#,201m/3#m.0m*m$+'*'mKMm1A*-m1#m- 234'#0-,m-!&-m"# '"-mm3,m (m
21m"#m%#0+',!'A,Ym0!'1mm*m1#!3#,!'!'A,m"#*m$0%+#,2-m"#*m%#,m,*'8"-m1#m
.3"'#0-,m- 2#,#0m*-1m"2-1m"#m1#%0#%!'A,m/3#m.0#!#,m#,m*m *mJYIYmm
1m0#*#4,2#m#*m&#!&-m"#m/3#m.0m*m$+'*'mKMm,-m1#m- 234-m,',%B,m&-+-!'%-2-m
+32,2#Ym 12#m 0#13*2"-m .-"0>m #120m 0#*!'-,"-m !-,m #*m &#!&-m "#m /3#m *m +32!'A,m
#,m &-+-!'%-1'1m !-,"'!'-,#m #*m !-00#!2-m !0#!'+'#,2-m "#m *m .*,2m -m 13m %#0+',!'A,Ym
m +32!'A,m #,m #12m $+'*'m 2'#,#m 3,m 4*-0m #,m m "#m FYFFm 7m .-0m *-m 2,2-m *m
+32!'A,m $#!20>m m *m $3,!'-,*'""m "#m *m .0-2#>,Zm !-+-m 7m 1#m &m !-+#,2"-m
,2#0'-0+#,2#Ym
,m#*m!1-m"#m*m$+'*'mHFILm*m1#%0#%!'A,m$3#m"#m,3#4#m1'*4#120#1m&-+-!'%-2-1Zm
1'#2#m &#2#0-!'%-2-1m 7m -!&-m &-+-!'%-2-1m +32,2#1Ym -1m &-+-!'%-2-1m +32,2#1m 1#m
.-"0>,m $#,-2'.0m .0m !-,-!#0m 7m 1 #0m "#m /3<m +-"-m $#!2m #12m +32!'A,m m *m
$3,!'-,*'""m"#m*m.0-2#>,m7m1'm&,m"/3'0'"-m*%3,m0#1'12#,!'mm*%B,m4'031m/3#m
',$#!2m m *m .*,2m "#*m +#*A,Zm 1- 0#2-"-m .-2'4'031m !-+-m Zm m -m Zm
#,20#m-20-1Ym
m
GLOm
#-/-#9(
.)-OO&-O'/.#)(-O#(.# #-
EMCMDT"#!TT
,m#*m!1-m"#*m%#,mI#-)J>m1#m- 234-m3,m+32,2#m#,m*m.- *!'A,mYmm$+'*'m
GKNMm 2'#,#m 3,m +32!'A,m /3#m .0-4-!m 3,m !+ '-m "#m +',-9!'"-m "#m 0%',',m m
*'1',Ym *%3,-1m 20 (-1m 0#!'#,2#1m &,m 73""-m m #,2#,"#0m +#(-0m !39*m #1m *m
"'$#0#,!'m"#m$3,!'-,*'""m#,20#m%#,#1m"#*m!-+.*#(-m"#m','!'!'A,m"#m*m20"3!!'A,m
7m 131m '1-$-0+1Ym ,m #(#+.*-m #1m #*m "#m >m 7m I#-)J>m #,m *-1m !3*#1m 1#m &m
"#+-120"-m/3#m!"m3,-m.-1##m+7-0m$','""m.-0m*%B,m2'.-m"#m+m^',%01m
7m !-*YZm GOOO_Ym 20-1m #123"'-1m "#+3#120,m /3#m *-1m %#,#1m >m 7m I#-)J>m .3#"#,m
#6.0#101#m "'$#0#,!'*+#,2#m #,m 1#%B,m /3#m 2#('"-1m 7m .#0'-"-1m "#m "#100-**-m "#m *m
.*,2Ym'#,201m/3#m#,m,#)*-#-m1#m!-+.0- Am/3#m+ -1m%#,#1m1-,m#6.0#1"-1m
.-0m'%3*m#,m2#('"-1m+#0'12#+92'!-1m7m#,m!<*3*1m#,m.#0>-"-m"#m"'4'1'A,m"#mA0%,-1m
',+"30-1Zm1#m#6.0#1,m#,m"'12',2m.0-.-0!'A,m#,m2#('"-m+"30-m^-"0'%3#8m7m!-*YZm
GOON_Ym 0#!#m /3#m *1m 0#1'12#,!'1m m 4'031m "#1!0'21m &12m &-0m ',"'!,m /3#m !"m
4'031m 2'#,#m 3,m !'#02m #1.#!'$'""m !-,m 3,-m "#m *-1m "-1m %#,#1m 7m 2+ '<,m #12m
#1.#!'$'!'""m.0#!#m40'0m#,m$3,!'A,m"#m*m#1.#!'#m"#m.*,2Ym*mm^))O."O
0#,/-_m',2#0!!'-,m!-,m#*m%#,m>m#,m2-+2#Zm*#!&3%m7m.'+'#,2-m+'#,201m/3#m#,m
,#)*-#-m',2#0!!'-,m!-,m13m'1-$-0+m^#**'1m7m!-*YZmHFGH_Ym
1m.-0m#**-m/3#m2,2-m#*m%#,mI#-)J>m!-+-m#*m>m"# #,m2#,#01#m#,m!3#,2m#,m*m
B1/3#"m "#m .-1' *#1m ,3#41m 0#1'12#,!'1m 0#!#1'41Ym "#+91Zm #6'12#,m !1-1m #,m *-1m
/3#m 1A*-m #*m %#,m I#-)J>m #1m #*m 0#1.-,1 *#m "#m !-,$#0'0m *m 0#1'12#,!'m m 3,m 4'031Ym
#!'#,2#+#,2#m 1#m &m "#+-120"-m /3#m #*m 4'031m m ^&/'O *)2O 0#,/-_m 1A*-m
',2#0!!'-,m !-,m #*m %#,m I#-)J>m +'#,201m /3#m ,-m *-m &!#m !-,m #*m %#,m >Ym ,m #*m
1'*#,!'+'#,2-m "#*m %#,m I#-)J>m #,m .*,21m "#m ,/(/-FO <121O 1#m +-1200-,m
0#1'12#,2#1m$0#,2#mm*m',$#!!'A,m!-,m#*m4'031mm+'#,201m/3#m#,m#*m1'*#,!'+'#,2-m
"#*m %#,m >m ,-m 1#m - 234-m 0#1'12#,!'Ym "#+91Zm 1#m !-+.0- Am *m #1.#!'$'!'""m "#m
',2#0!!'A,m #,20#m #*m .%m "#*m 4'031m 7m #*m %#,m I#-)J>m +#"',2#m #,17-m "#m "- *#m
&> 0'"-Zm!-00- -0,"-m#*m+'1+-m0#13*2"-m^,%m7m!-*YZmHFGI_Ym
,m +#*A,m #6'12#,m #123"'-1m /3#m !-,$'0+,m /3#m #*m 1'*#,!'+'#,2-m "#*m %#,m >m
.-02m 0#1'12#,!'m $0#,2#m *%3,-1m 4'031Ym 91m !-,!0#2+#,2#m #*m #6.#0'+#,2-m
0#*'8"-m.-0m-"0>%3#8j#0,9,"#8m7m!-*YZmHFGHm"#+-120Am/3#m#*m1'*#,!'+'#,2-m"#m
#12#m%#,m!-,$#0>m0#1'12#,!'mm!320-m^ZmZmZm_m"#m*-1m-!&-m
4'031m!-,m*-1m/3#m',$#!20-,m*1m.*,21m"#m+#*A,Ym',m#+ 0%-Zm,-m1#m!-+.0- Am#*m
1'*#,!'+'#,2-m"#*m%#,mI#-)J>m7m.-0m*-m2,2-m,-m1#m.3#"#m1 #0m1'm#12#m!-,$#0'0>m
0#1'12#,!'m.0m*%3,-1m"#m#12-1m4'031Ymm
GMFm
.)-OO&-O'/.#)(-O#(.# #-O
#-/-#9(m
m .#10m "#m /3#m #*m +32,2#m "#2#!2"-m #,m #12#m 20 (-m !-,2'#,#m 3,m !+ '-m "#m
+',-9!'"-m!-,m3,m4*-0mGYFFm#,mm&#+-1m/3#0'"-m4*-00m*m'+.-02,!'m"#m*m
0#%'A,m"-,"#m1#m#,!3#,20m#,m$3,!'A,m"#m*m&-+-*-%>m/3#m2'#,#,m#*m%#,m#Jm7m13m
'1-$-0+m^'%30mJYJ_Ymm#m&m- 1#04"-m/3#m*m+32!'A,m#129m*-!*'8"m#,m3,m8-,m
"-,"#m-201m#1.#!'#1m2'#,#,m+32!'-,#1m#,m#*m%#,m>m/3#m!-,$'#0#,m0#1'12#,!'1mm
*%3,-1m 4'031m ^'%301m JYHm 7m JYJ_Ym *m !+ '-m 0%JI71m 1#m .-1'!'-,m #,m *m 8-,m
!-*-0#"m #,m $3!1'm ^'%301m JYHm 7m JYJ_m 7m "-,"#m 1#%B,m - %*'m 7m 0,2Zm #6'12#m
+91m !3+3*!'A,m "#m !+ '-1m "#m +',-9!'"-1m #,m #*m %#,m #Jm /3#m !-,$'#0#,m
0#1'12#,!'m,230*mm4'031Ym*m+'1+-m2'#+.-Zm#1m3,m0#%'A,m+91m#6.3#12m*m#62#0'-0m
1#%B,m*m!-,$'%30!'A,m#,mIm"#m*m.0-2#>,m^- %*'m7m0,2ZmHFFL_Ym-0m*-m2,2-Zm
7m 2#,'#,"-m 1- 0#2-"-m #,m !3#,2m *m &-+-*-%>m #,20#m + -1m %#,#1Zm #12#m +32,2#m
"# #0>m 2#1201#m *m '%3*m /3#m *-1m "#2#!2"-1m #,m #*m %#,m #Jm #m ',$#!201#m !-,m *m
+'1+m 2#0'm"#m4'031m.0m!-+.0- 0m1'm*m+32!'A,m.0-.-0!'-,m-m,-m0#1'12#,!'m
m*%B,m4'031Ym
m
(&30 TEMEMm*',#+'#,2-m+#"',2#m*312*m#,20#m*m1#!3#,!'m.0-2#'!m"#m*-1m%#,#1m>m7mI#-)J>Ym
,m $3!1'm *m 8-,m "#1!0'2m .-0m - %*'m 7m 0,2m /3#m !-,2'#,#m +91m !3+3*!'A,m "#m !+ '-1m
+',-!>"'!-1m/3#m!-,$'#0#,m0#1'12#,!'mm4'031m#,m"'12',21m#1.#!'#1m^'%30mJYH_m7m#,m0-(-m#*m!+ '-m"#m
+',-9!'"-m/3#m.0-4-!m*m+32!'A,m"#2#!2"m+#"',2#mm#,m*m$+'*'mGKNMm"#m*m.- *!'A,m
m.0mI#-)J>Ymm
m
EMCMET
$PTTTQT
0m #*m %#,m ;m ,-m 1#m #,!-,20Am ,',%B,m +32,2#m #,m *m .- *!'A,m m 7m ,-m 1#m
.3"-m&!#0m#*m,9*'1'1m#,m*m.- *!'A,mYmm0#%'A,m,*'8"m#0m0#*2'4+#,2#m
.#/3#@m^LGKm. _m7m!-,m3,m.0-.-0!'A,m"#mm"#*mJJPYm-1m!# "-0#1m!-,m*-1m/3#m
1#m ,*'80-,m *1m .- *!'-,#1m $3#0-,m "'1#@"-1m m .02'0m "#m *m 1#!3#,!'m "#m *m
40'#""m LHjGGIZm #12#m .-"0>m 1#0m #*m +-2'4-m .-0m #*m !3*m *m &!#0m #*m ,9*'1'1m #,m *m
GMGm
#-/-#9(
.)-OO&-O'/.#)(-O#(.# #-
40'#""m&0-,-m"#m*m.- *!'A,mm*-1m!# "-0#1m,-m$3#0-,m13$'!'#,2#+#,2#m
#1.#!>$'!-1Ym 0- *#+#,2#m *-1m !# "-0#1m "'1#@"-1m ,-m +.*'$'!,m #1m 0#%'A,m
.-0/3<m *m 0#%'A,m !-+.*#+#,20'm ,-m #6'12#m -m !-,2'#,#m "#+1'"-1m .-*'+-0$'1+-1m
0#1.#!2-mm*m0#%'A,m-0'%',*m"#mLHjGGIYm-0m#**-m1#m"#!'"'Am,-m1#%3'0m"#*,2#m!-,m
#*m,9*'1'1m"#m#12#m%#,m#,m*m.- *!'A,mYm
T
EMCMFTTT
-,m*-1m0#13*2"-1m- 2#,'"-1m#,m#12#m20 (-m/3#"m.#,"'#,2#m*m$'(!'A,m"#m*%3,-1m
"#m*-1m+32,2#1m#,m&-+-!'%-1'1m7m*m- 2#,!'A,m"#m13$'!'#,2#m1#+'**m"#m*-1m+'1+-1Zm
.0m.-"#0m&!#0m*-1m#6.#0'+#,2-1m"#m$#,-2'."-m"#!3"-1m.0m!"m%#,Ymm
+ '<,m #1m ',"'1.#,1 *#m - 2#,#0m #*m +32,2#m '1*"-m "#m -201m +32!'-,#1Zm .3#12-m
/3#m 3,m "#m *1m "#14#,2(1m "#*m m #1m /3#m *1m .*,21m 1#*#!!'-,"1m ,-m 1A*-m
!-,2'#,#,m*m+32!'A,m"#m',2#0<1m1',-m2+ '<,m+3!&1m-201m/3#m.-"0>,m$#!20m*m
$#,-2'.-m#,m#123"'-Ym
,m#12#m20 (-m#12m* -0m"# #0>m&!#01#m!-,m*-1m+32,2#1m- 2#,'"-1m"#*m%#,m#Jm
^HFILm 7m 1#m &m $'("-m 7m $*20>m $'(0m *1m $+'*'1m GHHOZm HFLFZm JJJZm KMm 7m KI_Ym m
!-,2',3!'A,m1#m"# #0>,m',$#!20m!-,m3,m 2#0>m"#m4'031m.0m.-"#0m!-+.0- 0m1'm
*%3,m "#m *1m +32!'-,#1m !-,$'#0#m 0#1'12#,!'m m *m .*,2Ym ',m #+ 0%-Zm #1m .-1' *#m
/3#m .0m *%B,m +32,2#m ,-m 1#m .-1' *#m - 2#,#0m #*m &-+-!'%-2-Ym ,m #*m !1-m "#m *m
$+'*'m KMm 7m 1#m &m ',2#,2"-m 40'1m 4#!#1m m .02'0m "#m *m 32-$#!3,"!'A,m "#m 3,m
&#2#0-!'%-2-mHm7m,-m&m1'"-m.-1' *#Ymm
#1.#!2-mm*m 2#0>m"#m4'031m!-,m*m!3*m"# #0>,m',$#!201#m*-1m+32,2#1Zm7m#6'12#m
3,m #123"'-m 0#!'#,2#m ^-"0>%3#8k#0,9,"#8m 7m !-*YZm HFGH_m /3#m &m "#+-120"-m !-+-m
+#"',2#m #*m 1'*#,!'+'#,2-m ^'_m "#*m +m "#m +j#Jm *1m .*,21m "#m +#*A,m
20,1$-0+"1m 0#13*2 ,m 0#1'12#,2#1m m Jm 4'031m "'12',2-1m ^//',O 0#(O 3&&)1#(!O
0#,/-O^_Zm&)(O(,).#O-*).O0#,/-O^_Zm/"#(#O3&&)1O')-#O0#,/-O^_m
7m ),)(O 1.,'&)(O ')-#O 0#,/-m ^__Ym 30,2#m #12#m 20 (-m 1#m .0- 0-,m
2+ '<,m -20-1m 4'031Zm !-+-m //',O ')-#O 0#,/-O ^_m ^%<,#0-m //')0#,/-_Zm
.,'&)(O')-#O0#,/-O^_m^%<,#0-m).30#,#JFO//,#.O3&&)1O-./(.#(!O#-),,O
0#,/-O ^_m ^%<,#0-m ,#'#0#,/-_Zm //,#.O *"#N),(O 3&&)1-O 0#,/-m ^_m
^%<,#0-m )&,)0#,/-_Zm *3O ,#(!-*).O 0#,/-m ^_m ^%<,#0-m ).30#,#_m 7m .O
*-/)3&&)1-O 0#,/-m ^_m ^%<,#0-m &)-.,)0#,#_Zm .#0-m #,m #12-1m !1-1m #*m
1'*#,!'+'#,2-m"#m#Jm,-m!-,$'0'Am0#1'12#,!'m^-"0>%3#8k#0,9,"#8m7m!-*YZmHFGH_Ymm
0!'1m m *-1m 0#13*2"-1m .0#1#,2"-1m #,m #12#m 20 (-m 1#m &,m .-"'"-m "#2#0+',0m *1m
!-,"'!'-,#1m.0m"#100-**0m.-12#0'-0#1m.*2$-0+1m"#mZm3,m#(#+.*-m#1m*m
GMHm
.)-OO&-O'/.#)(-O#(.# #-O
#-/-#9(m
"#100-**"m .-0m *m #+.0#1m '#&&-O #.9m (3,2+#,2#m !-,m #*m jm #,m #*m
+0!-m "#*m .0-7#!2-m Ym *m 7m !-+-m 1#m &m !-+#,2"-m ,2#0'-0+#,2#Zm
#12m.*2$-0+m1#m&m"#100-**"-mm.02'0m"#m3,m*>,#m.30m"#m+#*A,m2'.-me'#*m"#m
.-eYm #m &,m +32"-m *1m 1#+'**1m !-,m 3,m .-0!#,2(#m "#m GZKPm "#m m 7m 1#m &,m
- 2#,'"-m .0-6'+"+#,2#m IGFFm ',"'4'"3-1m Hm ^#,m #*m +-+#,2-m "#m #1!0' '0m #12m
2#1'1_Zm #,m *-1m !3*#1m 7m 1#m &,m "#2#!2"-m Nm +32,2#1m #,m #*m %#,m >m 7m Im #,m #*m %#,m
I#-)J>Ym#m*-1mNm+32,2#1m#,m#*m%#,m#JZmLm2'#,#,m*m+32!'A,m#,m0#%'A,m#6A,'!m
+'#,201m /3#m Hm #,m 0#%'A,m ',20A,'!Ym #m *-1m +32,2#1m #6A,'!-1Zm Hm 2'#,#,m 3,m 4*-0m
m +#,-0m "#m FYFKm 7m .-0m *-m 2,2-m ,-m 1#0>,m 2-*#0"-1Ym 0m #*m %#,m I#-)J>Zm #*m
,B+#0-m"#m+32,2#1m#,!-,20"-1m1'%3#m1'#,"-m+3!&-m+#,-0m7m"#m*-1m20#1m+32,2#1m
"#2#!2"-1m 1A*-m 3,-m 1#m *-!*'8m #,m 0#%'A,m #6A,'!m !-,m 4*-0m FYFHm #,m Ym 12m
.0-.-0!'A,m!-,$'0+0>m*m&'.A2#1'1m"#m/3#m#^'1-_Jm#1m+91m',"'1.#,1 *#m.0m*m
!<*3*m^,%m7m!-*YZmHFGI_Ym
-"-1m#12-1m+32,2#1Zm(3,2+#,2#m!-,m*-1m- 2#,'"-1m#,m#12m2#1'1m1#09,m#,m3,m$3230-m
',$#!2"-1m!-,m*%3,-1m"#m*-1m4'031m+#,!'-,"-1m,2#0'-0+#,2#m7m"#m#12#m+-"-m1#m
.0- 09m1'm*m+32!'A,m/3#m&m"/3'0'"-m!"m3,-m!-,$'#0#m *%3,m0#1'12#,!'mm*m
.*,2Zm"#m+-"-m/3#m.3"'#0m1#0m32'*'8 *#m#,m.0-%0+1m"#m+#(-0m%#,<2'!m"#m*m
#1.#!'#Ym
# #m 2#,#01#m #,m !3#,2m /3#m .0m .-"#0m !-+#0!'*'80m ,3#41m *>,#1m - 2#,'"1m m
.02'0m"#m*m+#(-0m%#,<2'!Zm*m2#!,-*-%>mm32'*'80m#,m30-.m#,m#12-1m+-+#,2-1m
,-m .3#"#m 1#0m *m 20,1$-0+!'A,m %#,<2'!Ym -0m #(#+.*-Zm *m 2#!,-*-%>m 'm ,-m 1#m
.-"0>m 32'*'80m #,m .0-"3!2-1m !-+#0!'*#1Zm #,m !+ '-Zm *m 2<!,'!m "#m m ,-m
.0#1#,2m#12m*'+'2!'A,m7m/3#m1#m- 2'#,#,m,3#41m*>,#1m+#"',2#m3,m2#!,-*-%>m
/3#m,-m#+.*#m20,1%#,#1'1Ymm
T
m
$
GMIm
#-/-#9(
)
m
m
EMDT".T
m
*m !-m -$0#!#m *m .-1' '*'""m "#m '"#,2'$'!0m #$'!'#,2#+#,2#m +32!'-,#1m
,230*#1m #,m *1m 40'#""#1m ,*'8"1Ym -0m #**-m 0#13*2m +37m ',2#0#1,2#m ,*'80m
"'12',2-m%#0+-.*1+m.0m*m"#2#!!'A,m"#m&.*-2'.-1m.0#1#,2#1m#,m*m,230*#8m7m
/3#m .3#",m .-020m 40' '*'""m ,230*m #m ',$-0+!'A,m $3,!'-,*m #,m %#,#1m "#m
',2#0<1Ym ,m #*m +0!-m "#*m .0-7#!2-m m 1#m "#!'"'Am &!#0m #*m ,9*'1'1m "#m
!-m #,m *-1m %#,#1m 7m ,*'8"-1m +#"',2#m Ym ,m #12m 2#1'1m 1#m &m
0#*'8"-m#*m,9*'1'1m"#m*-1m%#,#1m>m7m;m7m#,m#*mm1#m0#*'8Am#*m,9*'1'1m
#,m*-1m%#,#1mI#-)J>m7mYm
*m ,9*'1'1m 1#m 0#*'8Am !-+.0,"-m *m +'1+m 40'#""m !-,m *m /3#m 1#m & >m
"#100-**"-m*m.- *!'A,m"#mm^LHjGGI_m7mGGIm!!#1'-,#1m.0-.-0!'-,"1m
.-0m#*m ,!-m"#m%#0+-.*1+m"#*mYm1mGGIm!!#1'-,#1m#129,m!0!2#0'8"1m
.0m!'#02-1m!0!2#0#1Zm#,20#m#**-1m!0!2#0#1m"#m$032-m!-+-m#*m!-*-0m"#m*m.3*.Zm#*m
0-+m 7Zm "#m *%3,1m "#m #**1Zm 1#m "'1.-,#m "#m '+9%#,#1m "#*m $032-m ^#12m ',$-0+!'A,m
#129m "'1.-,' *#m #,m #*m _Ym ',m #+ 0%-Zm "#1$-023,"+#,2#m ,-m #129,m
!0!2#0'8"1m.0m0#1'12#,!'mm.2A%#,-1Ym-0m#**-Zm#*m,9*'1'1m"#m!-m!-,m
#*m %#,m #Jm %#,#0m 3,m .0'+#0m !0' "-m .0m 1#*#!!'-,0m /3#**1m 40'#""#1m /3#m
.3"'#0,m2#,#0m3,m&.*-2'.-m/3#m!-,$'0'#0m0#1'12#,!'mm.-2'4'031m7m#,m3,m1#%3,"m
#2.Zm1#m"# #0>,m4*-00m$#,-2>.'!+#,2#m*1m!!#1'-,#1m"#m',2#0<1Ym
-,m#*m,9*'1'1m"#*m%#,m;m1#m.0#2#,">m0#*!'-,0m*%B,m&.*-2'.-m"#2#!2"-m!-,m
*%B,m !09!2#0m "#m +"30!'A,m "#m $032-m 2#,'#,"-m #,m !3#,2m /3#m *m 40'#""m "#m
0#$#0#,!'mLHjGGIm#1m3,m40'#""m,-m!*'+2<0'!Ym
m
EMDMBT$ETPTTTTTQT
-+-m1#m&m!-+#,2"-mm*-m*0%-m"#m#12#m20 (-Zm1#m&m"#1!0'2-m/3#m#*m%#,m#Jm#1m
0#1.-,1 *#m"#m*%3,1m0#1'12#,!'1mm.-2'4'031m7mm-20-1m4'031Zm7m/3#m*m.0#1#,!'m
"#m+32!'-,#1m#1.#!>$'!1m#,m#12#m%#,m.0-4-!,m/3#m#*m4'031m,-m.3#"m',2#0!!'-,0m
!-,m #*m !-+.*#(-m "#m ','!'!'A,m "#m *m 20"3!!'A,m 7m .-0m *-m 2,2-Zm 20"3!'01#m !-+-m
.0'+#0m .1-m "#m *m ',$#!!'A,m "#m *m .*,2Ym -0m #12#m +-2'4-m #,!-,200m 40' '*'""m
,230*m#,m#12#m%#,m.-"0>m'+.*'!0m'"#,2'$'!0m*%B,m,3#4-m&.*-2'.-m/3#m!-,$'0'#0m
0#1'12#,!'mm*%B,m4'031mm*m.*,2Ym
GMJm
)OO
#-/-#9(m
#m&,m'"#,2'$'!"-m3,m2-2*m"#mGHm.-*'+-0$'1+-1m#,m#*m,9*'1'1m"#m!-m!-,m
#*m %#,m #JYm #m *-1m GHZm 1#'1m 1m 1#m #,!3#,20,m #,m 8-,1m #6A,'!1m 7m 3,-m #,m #*m Kdm
m "#*m %#,Ym A*-m 3,-m "#m #**-1m .0-4-!m 3,m !+ '-m "#m +',-9!'"-m ^m GYKZm
1.MG*7_m^ *mJYJ_m!-,m3,m4*-0mFYFFm#,mm7m/3#m.-0m*-m2,2-Zm.-"0>m$#!20m
#,m*m$3,!'-,*'""m"#m*m.0-2#>,Ymm
m
!+ TEME[m *m!-+.02'4m"#m*-1m.-*'+-0$'1+-1m"#2#!2"-1m.-0m!-m#,m#*m#123"'-m"#m'#2-m7m
!-*Ym^HFFM_m7m*-1m'"#,2'$'!"-1m#,m#12#m20 (-Ymgm-1'!'A,m#,m*m1#!3#,!'m,3!*#-2>"'!m0#1.#2-m*mm!-"A,m"#m
','!'-m"#m*m20"3!!'A,m7m,3+#0!'A,m1'%,"m#,m#12#m20 (-Ym
m
123"'-m
6A,igm
GiNGm^GYH_m
+ '-m
+',-9!'"-m
'*#,!'-1-m
!!#1'A,m
#1'12#,!'m
m4'031m
m
m
m
!-m
m
GiGHImm^GYI_m
'*#,!'-1-m
m
GiGNLm^GYJ_m
'*#,!'-1-m
m
m
TFAFGACT
-m&7m
"2-1T
BRCBCTPBMFQT
!-m
1/HB+7T
GiHJIm^GYL_m
'*#,!'-1-m
m
m
KiLOFm^HYK_m
'*#,!'-1-m
m
m
GiNGm^GYH_m
'*#,!'-1-m
m
m
GiGHImm^GYI_m
'*#,!'-1-m
m
m
GiGNLm^GYJ_m
'*#,!'-1-m
m
m
jFJLm
m
jGMNm
m
jKGHm
m
mGLGIMKm
m
m
m
'#2-m7m!-*YZm
HFFMm
FRGIDT
$3CCI(1T
KiLOFm^HYK_m
'*#,!'-1-m
m
*m ,9*'1'1m +#"',2#m !-m "#*m %#,m #Jm #,m +#*A,m 7m 1#m & >m 0#*'8"-m
,2#0'-0+#,2#m ^'#2-m 7m !-*YZm HFFM_m !-,m #*m +#*A,m !*'+2<0'!-m #"0,2'1m !-+-m
0#$#0#,!'m 7m -201m GJMm 40'#""#1m "#m +#*A,m - 2#,'"1m "#m *m !-*#!!'A,m "#m
%#0+-.*1+m"#m*m12!'A,m6.#0'+#,2*m"#mem7-0ejmm^9*%_Ym,m#12#m
20 (-m.0#4'-m1#m"#2#!20-,m!',!-m.-*'+-0$'1+-1Zm20#1m"#m#**-1m#,m#*m.0'+#0m#6A,m7m
"-1m #,m #*m /3',2-m ^ *m JYJ_Ym -"-1m *-1m 1m /3#m 1#m "#2#!20-,m 0#13*20-,m 1#0m
GMKm
#-/-#9(
)
1'*#,!'-1-1m#6!#.2-m#*m!+ '-mLNIm"#*m/3',2-m#6A,m/3#m!-00#1.-,"#m*m!+ '-m"#m
*#3!',m m &'12'"',m #,m *m .-1'!'A,m HHNm 7m #*m !39*m !-,$'#0#m 0#1'12#,!'m m m #,m m
GLGIMKYm1m!!#1'-,#1m.0- "1m#,m#12#m20 (-m$3#0-,m1-+#2'"1mm#123"'-1m.0#4'-1m
"#m 0#1'12#,!'m $0#,2#m m 3,m 2#0>m "#m 4'031m .3"'#,"-m '"#,2'$'!0m 40'#""#1m
0#1'12#,2#1m .0m *%3,-1m "#m #**-1Zm !-+-m *1m Jm !!#1'-,#1m 0#1'12#,2#1m m m
^',"'!"1m #,m *m *m JYJ_Ym -1m +.*'!-,#1m 32'*'8"-1m #,m + -1m 20 (-1m 1-,m *-1m
+'1+-1Ym
,m*m'%30mJYJm1#m+3#120,m2-"-1m*-1m1m"#2#!2"-1m#,m#12#m20 (-m7m.-0m'#2-m7m
!-*YZm^HFFM_m.0m#*m%#,m#JYm
m
_m
_m
GMLm
T
)OO
T
#-/-#9(m
T
(&30 T EMF[m _m 0"3!!'A,m m .0-2#>,m "#*m +m "#*m %#,m #JYm ,m 0-(-m 1#m 1#@*'8m #*m B*2'+-m !-"A,m "#m
!"m#6A,m7m#,m4#0"#m#*m','!'-m"#m!"m#6A,Ym _m6-,#1m"#*m%#,m#Jm1#.0"-1m',"'!,"-m#*m2+@-m"#m
!"m3,-m^#,m. _m7m*1m40'!'-,#1m#,!-,20"1[m#,m$3!1'm*1m"#2#!2"1m#,m#12#m20 (-m7m#,m#*m20 (-m"#m
'#2-m7m!-*YZm^HFFM_Ym,m83*Zm#*mGHIm^mGYI_m7m#*mHGHm^mGYK_m"#2#!2"-1m#,m#12#m20 (-m.#0-m,-m
#,m #*m "#m '#2-m 7m !-*YZm ^HFFM_Ym ,m %0'1Zm #*m m "#2#!2"-m #,m #*m 20 (-m "#m '#2-m 7m !-*Ym ^HFFM_m .#0-m ,-m
"#2#!2"-m#,m#12#m20 (-Ym
m
320-m "#m *-1m 1m "#2#!2"-1m #,m #12#m 20 (-m !-',!'"#,m !-,m *-1m /3#m $3#0-,m
"#2#!2"-1m +#"',2#m !-m #,m #*m #123"'-m "#m '#2-m 7m !-*Ym HFFMm ^ *m JYJm 7m
'%30m JYK Zm #,m $3!1'_Ym 12-m "#+3#120m /3#m "#,20-m "#m *m 40'#""m ,230*m
#,!-,20+-1m *%3,-1m 1m !-,m +91m $0#!3#,!'m 7m .-0m *-m 2,2-Zm /3#m 1#m "#2#!2,m #,m
"'12',2-m %#0+-.*1+Ym ,m !+ '-Zm -20-1m 1m 1#m .3#"#,m #,!-,200m !-,m +#,-0m
$0#!3#,!'m !-+-m #1m #*m !1-m "#*m m GYIm ^GHI_m 7m #*m m GYKm ^HGH_m /3#m 1#m &,m
"#2#!2"-m 1A*-m #,m #*m %#0+-.*1+m ,*'8"-m #,m #12#m 20 (-m 7m ,-m 1#m "#2#!20-,m #,m
*1m40'#""#1m,*'8"1m#,m#*m#123"'-m"#m'#2-m#2m*Ym*mmGYKm#129m.0#1#,2#m1A*-m
#,m *m 40'#""m m KFKLFHm ^ *m JYJ_m 7m .0-4-!m 3,m !+ '-m "#m 9!'"-m 1.902'!-m m
%*'!',Ym 0m !-+.0- 0m 1'm #12#m m .0-4-!m *%3,m +-"'$'!!'A,m #,m *m
!-,$-0+!'A,m "#m *m .0-2#>,m 7m #,m !-,1#!3#,!'Zm !-,$'#0#m 0#1'12#,!'m $0#,2#m *%B,m
.2A%#,-m "# #0>m ',$#!201#m #12m 40'#""m ^ *m JYJ_m !-,m 3,m 2#0>m "#m 4'031Zm
!-+-m7m1#m&m!-+#,2"-m,2#0'-0+#,2#Ym#m2-"-1m+-"-1Zm"# #m2#,#01#m#,m!3#,2m
/3#m #12m 40'#""m .0#1#,2m #*m .-*'+-0$'1+-m #,m &#2#0-!'%-1'1m 7m "# #0>m .0'+#0-m
$'(01#m*m+32!'A,Ymm
-0m-20-m*"-Zm#*mm"#2#!2"-m#,m#*m20 (-m"#m'#2-m*-!*'8"-m#,m#*m#6A,mKm7m/3#m
!-,$'#0#m 0#1'12#,!'m m m ,-m 1#m &m '"#,2'$'!"-m #,m ,',%3,m "#m *1m 40'#""#1m
,*'8"1m #,m #12#m 20 (-m ^ *m JYJ_Ym ,m #12#m !1-m #*m m 1#m .0#1#,2 m #,m
&-+-!'%-1'1m#,m*1m40'#""#1m/3#m0#13*20-,m1#0m0#1'12#,2#1mmYm
GMMm
#-/-#9(
)
,m + -1m #123"'-1m #*m #6A,m /3#m .0#1#,2m +91m 40' '*'""m &m 1'"-m #*m .0'+#0-m
+'#,201m/3#m#,m#*m1#%3,"-Zm2#0!#0-m7m!302-m,-m1#m&,m'"#,2'$'!"-m1m#,20#m*1m
40'#""#1m!-+.0"1Ymm
m
EMDMCT
TPTTTBQT
,m #*m %#,m ;m 1#m &,m '"#,2'$'!"-m "-1m .-*'+-0$'1+-1m +#"',2#m *m 2<!,'!m "#*m
!-m#,20#m*1mGGIm40'#""#1m,*'8"1m7m*m40'#""m"#m0#$#0#,!'mLHj
GGIYm*mmG .0-4-!m3,m!+ '-m"#m+',-9!'"-m"#m*#3!',mm4*',m#,m*m.-1'!'A,m
HHJm"#m*m1#!3#,!'m+',-!>"'!m!-,m3,m4*-0mm"#mGYFFm+'#,201m/3#m#*mmHm
#1m1'*#,!'-1-Ym
m .0-2#>,m ;m #,m ,#)*-#-m #129m '#,m "#1!0'2Ym 12m .0-2#>,m !-,2'#,#m 20#1m
"-+','-1m^'%30mJYL_Ym*m"-+','-m+',-m2#0+',*m^+',-9!'"-1mGjIHK_m!-,2'#,#m20#1m
1#%+#,2-1m 20,1+#+ 0,Ym 6.0#1,"-m *m .0-2#>,m ;m #,m *#4"30m 1#m &m
"#+-120"-m /3#m #12#m .0'+#0m "-+','-m #1m #*m 0#1.-,1 *#m "#m *m 3,'A,m !-,m #*m #2'*#,-m
^!&**#0m7m*##!)#0ZmGOOK_Ym"#+91Zm*m+32!'A,m#20GjGm*-!*'8"m#,m#12#m"-+','-m
.'#0"#m*m!.!'""m"#m3,'A,m!-,m*m&-0+-,m^!&**#0m7m*##!)#0ZmGOOK_Ym
*m 1#%3,"-m "-+','-m ^+',-9!'"-1m IHLjLFO_m 1#m #,!3#,20m #,m 3,m 0#%'A,m /3#m
.0#1#,2m &-+-*-%>m !-,m "-+','-1m 2'.-m &'12'"',m /3',1m 7m "#m *m !3* 1#m &m
"#+-120"-m 13m !2'4'""m /3',1m #(O 0#.,)m ^+ *#m 7m !-*YZm GOONZm HFFH_Ym *m B*2'+-m
"-+','-m ^+',-9!'"-1m LGFjMIN_m #1m #*m 0#1.-,1 *#m "#m 0#!' '0m #*m $-1$2-m .0-4#,'#,2#m
"#m *m !2'4'""m "#m *m &'12'"',m "#*m "-+','-m ,2#0'-0m ^&,%m 7m !-*YZm GOOI_m 7m .-0m *-m
2,2-m 0#1.-,1 *#m "#m *m 0#%3*!'A,m "#m *m 0#1.3#12m &!'m #*m ',2#0'-0m !#*3*0m "#m *m
.0-2#>,m;Ymm
m
(&30 TEMG[m1203!230m"#m*-1m"-+','-1m'"#,2'$'!"-1m#,m#*m%#,m;m"#m,#)*-#-Em"m"-+','-m2'#,#m
3,m$3,!'A,m"'12',2m7m#,m$3!1'm1#m"#2**m*m.-1'!'A,m"#m*m&'12'"',mIILm"#m*m1#!3#,!'m+',-!>"'!Zm
0#1.-,1 *#m"#m*m$-1$-0'*!'A,m"#*m"-+','-m0#%3*"-0Ym
m
GMNm
m
)OO
#-/-#9(m
,m,#)*-#-m1#m&,m'"#,2'$'!"-m+32!'-,#1m#,m#*m.0'+#0m"-+','-m/3#m.0-"3!#,m
',1#,1' '*'""m *m #2'*#,-m #,m *1m .-1'!'-,#1m *IG*Zm *#LH&#Zm 71LK70m 7m
*GFH&0m^'%30mJYMm#,m+0'**-_m^'*)',1-,m7m!-*YZmGOOK_Ym
,m2-+2#m1#m&,m.-"'"-m'1*0m*-1m%#,#1m;FO<FO=OIJFO>m
7m?Ym*m#123"'-m"#m#12-1m%#,#1m&m1'"-m+37mB2'*m.0m.-"#0m!-+.0#,"#0m+#(-0m
*m !2'4'""m "#*m %#,m ;m 7m 13m $+'*'m %<,'!Ym *m !-+.00m *m 1#!3#,!'m "#m #12-1m
!320-m%#,#1m1#m&m"#+-120"-m/3#m*m0#%'A,m+91m!-,1#04"m#1m*m0#1.-,1 *#m"#m
*m3,'A,m!-,m#*m#2'*#,-m^'#+,m7m!-*YZmHFFF_Ym"#+91Zm#*m%#,m?m&m.#0"'"-m*m
&'12'"',mIILm"#*m"-+','-m&'12'"',m/3',1m^'%30mJYL_m.0#,2#+#,2#m0#1.-,1 *#m
"#m*m$-1$-0'*!'A,m"#*m2#0!#0m"-+','-Zm"#+-120,"-m/3#m*m!2'4'""m"#m*m&'12'"',m
,-m.0#!#m',"'1.#,1 *#m.0m*m0#1.3#12m*m#2'*#,-m^'#+,m7m!-*YZmHFFF_Ymm
6'12#,m -201m +32!'-,#1m 0#*#4,2#1m /3#m &,m 73""-m #,m *m !-+.0#,1'A,m "#m *m
$3,!'-,*'""m"#*m%#,m;Ym-0m#(#+.*-Zm#,m2-+2#m1#m'"#,2'$'!Am#*m+32,2#m0,O
,#*m^Zm_Ym12#m+32,2#m.0-"3!#m$032-1m/3#m,-m+"30,m^'!)m7m3*2#0ZmGOKL_Zm
7m /3#m #1m ',1#,1' *#m *m #2'*#,-m #,m 2-"-1m *-1m 2#('"-1m ^,&,m 7m !-*YZm GOOJ_Ym m
+32!'A,m 0#1.-,1 *#m "#m #12#m $#,-2'.-m #1m 3,m !+ '-m "#m .0-*',m m *#3!',m #,m *m
.-1'!'A,mILm^'*)',1-,m7m!-*YZmGOOK_m^'%30mJYM_Ymm
,m 4#8m '"#,2'$'!"1m *1m +32!'-,#1m "#1!0'21m #,m ,#)*-#-m 7m #,m 2-+2#m /3#m
$#!2,mm*m$3,!'-,*'""m"#*m%#,m.0-"3!'#,"-m',1#,1' '*'""mm#2'*#,-Zm.0-!#"'+-1m
m !-+.00*1m !-,m *-1m .-*'+-0$'1+-1m /3#m 1#m & >,m '"#,2'$'!"-m +#"',2#m
!-m #,m +#*A,Ym -,m 3,m *',#+'#,2-m +B*2'.*#m ^'%30m JYM_m "#m *-1m %#,#1m
;m "#m ,#)*-#-m ^.;_Zm #*m ;O "#m 2-+2#m ^;_Zm Jm 7m Km "#m 2-+2#m
^>FO ?_Zm #*m m "#m 2-+2#m ^_m 7m #*m ;m "#m +#*A,m ^';_m &#+-1m
.-"'"-m '"#,2'$'!0m 7m !-+.00m *1m .-1'!'-,#1m "#m *-1m .-*'+-0$'1+-1m 7m "'1!32'0m 13m
#$#!2-Ymm
,m 4#0"#m #,m *m '%30m JYMm 1#m "#2**m #*m .-*'+-0$'1+-m #,!-,20"-m +#"',2#m
!-m #,m +#*A,Ym 12#m m .0-4-!m 3,m 1312'23!'A,m "#m #3HHJ*m ^'%30m
JYM_Ym 12#m !+ '-m 1#m *-!*'8m #,m *m 0#%'A,m "#*m "-+','-m 20,1+#+ 0,m ^1#,1-0j
0#!#.2-0m "#*m #2'*#,-_m 7m .-0m *-m 2,2-m !-',!'"#m #,m *m 0#%'A,m #,m /3#m 1#m &,m "#1!0'2-m
+32!'-,#1m/3#m$#!2,mm*m$3,!'-,*'""m"#m*m.0-2#>,m#,m,#)*-#-m7m#,m2-+2#m
^'*)',1-,m7m!-*YZmGOOK_Ym12m1312'23!'A,m^#3HHJ*_m1#09m.0- *#+#,2#m2-*#0"m
7m/3#m1'm,-1m$'(+-1m#,m*m1#!3#,!'Zm2,2-m#,m2-+2#m!-+-m#,m,#)*-#-m#6'12#,m
0#!#.2-0#1m/3#m!-,2'#,#,m3,m4*',m#,m#12m.-1'!'A,Zm*-m/3#m$4-0#!#m.#,10m/3#m#*m
+32,2#m"#m+#*A,m,-m.0-4-!0>m,',%B,m!+ '-m#,m*m$3,!'-,*'""m"#m*m.0-2#>,m
^'%30mJYM_Zm"2-m!-00- -0"-m+#"',2#mYm
GMOm
#-/-#9(
)
m
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
---------------------------MEVCNC-IEPQWPADELLMKYQYISDFFIAIAY
---------------------------MENCYC-IEPQWPADELLMKYQYISDFFIALAY
-------------MGSLLRMNRLLSSIVESCNCIIDPQLPADDLLMKYQYISDFFIALAY
---------------------------MESCDC-IEALLPTGDLLVKYQYLSDFFIAVAY
MLAMLRLLFLVLLISLVIISVSANDGEFFNC-CDEDGFWSIHT-ILDCQKVSDFFIAVAY
---MLRTLASALLVLSFFVSLSAADNGFPRCNCDDEGFWSIES-ILECQKISDLFIAIAY
. * * :
.
::. * :**:***:**
32
53
47
32
58
56
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
FSIPLELIYFVKKSAVFPYRWVLVQFGAFIVLCGATHLINLWTFT-THSRTVALVMTTAK
FSIPLELIYFVKKSAVFPYRWVLVQFGAFIVLCGATHLINLWTFT-MHSRTVAVVMTTAK
FSIPVELIYFVKKSAVFPYRWVLVQFGAFIVLCGATHLINLWTFN-MHTRNVAIVMTTAK
FSIPLELIYFVHKSACFPYRWVLMQFGAFIVLCGATHFISLWTFF-MHSKTVAVVMTISK
FSIPLELLYFISRSN-LPFKWVLVQFIAFIVLCGLTHLLNGWTYNPHPSFQLILSLTVAK
FSIPIELLYFVSCSN-FPFKWVLFQFIAFIVLCGMTHLLNFWTYYGQHPFQLMLALTIFK
****:**:**: * :*::***.** ******* **::. **:
. : : :* *
91
112
106
91
117
115
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
VLTAVVSCATALMLVHIIPDLLSVKTRELFLKNKAAELDREMGLIRTQEETGRHVRMLTH
VLTAVVSCATALMLVHIIPDLLSVKTRELFLKNKAAELDREMGLIRTQEETGRHVRMLTH
ALTALVSCITALMLVHIIPDLLSVKTRELFLKKKAAQLDREMGIIRTQEETGRHVRMLTH
MLTAAVSCITALMLVHIIPDLLSVKTRELFLKTRAEELDKEMGLIIRQEETGRHVRMLTH
ILTALVSCATAITLLTLIPLLLKIKVRELFLAQNVLELDQEVGMMKKQTEASMHVRMLTH
VLTALVSFATAITLITLFPMLLKVKVREFMLKKKTWDLGREVGLIKMQKEAGWHVRMLTQ
*** ** **: *: ::* **.:*.**::* .. :*.:*:*:: * *:. ******:
151
172
166
151
177
175
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
EIRSTLDRHTILKTTLVELGRTLALEECALWMPTRTGLELQLSYTLRHQ---HPVEYTVP
EIRSTLDRHTILKTTLVELGRTLALEECALWMPTRTGLELQLSYTLHQQ---NPVGYTVP
EIRSTLDRHTILKTTLVELGRTLALEECALWMPTRTGLELQLSYTLRHQ---NPVGLTVP
EIRSTLDRHTILKTTLVELGRTLDLAECALWMPCQGGLTLQLSHNLNNL---IPLGSTVP
EIRKSLDKHTILYTTLVELSKTLKLQNCAVWMPNESRSQMNLTHELSPSSAAESHRS-LS
EIRKSLDRHTILYTTLVELSKTLDLHNCAVWKPNENKTEMNLIHELRDSSFNSAYNLPIP
***.:**:**** ******.:** * :**:* * .
::* : *
.
:.
208
229
223
208
236
235
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
IQLPVINQVFGTSRAVKISPNSPVARLRPVSGKYMLGEVVAVRVPLLHLSNFQINDWPEL
INLPVISQVFSSNRALKISPNSPVASLRPRAGRYVAGEVVAVRVPLLHLSNFQINDWPEL
IQLPVINQVFGTNHVVKISPNSPVARLRP-AGKYMPGEVVAVRVPLLHLSNFQINDWPEL
INLPIINEIFSSPEAIQIPHTNPLARMRNTVGRYIPPEVVAVRVPLLHLSNFTN-DWAEL
INDPDVLEIT-KNKGVRILRQDSVLAASSSGGSGEPCAVAAIRMPLLRASDFKG-GTPEL
RSDPDVIQVK-ESDGVKILDADSPLAVASSGGSREPGAVAAIRMPMLKVSNFKG-GTPEL
. * : ::
::*
..
*
*.*:*:*:*: *:*
. .**
268
289
282
267
294
293
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
STKRYALMVLMLPSDSARQWHVHELELVEVVADQVAVALSHAAILEESMRARDLLMEQNV
STKRYALMVLMLPSDSARQWRVHELELVEVVADQVAVALSHAAILEESMRARDLLMEQNV
STKRYALMVLMLPSDSARQWHVHELELVEVVADQVAVALSHAAILEESMRARDLLMEQNV
STRSYAVMVLVLPMNGLRKWREHELELVQVVADQVAVALSHAAILEDSMRAHDQLMEQNI
VDTRYAILVLVLSSVDERVWSYDEMEIVEVVADQVAVALSHATVLEESQTMREKLEMRNR
VPECYAILVLVLPSEQGRSWCSQEIEIVRVVADQVAVALSHAAILEESQHMRETLEEQNR
**::**:*.
* * .*:*:*.*************::**:*
:: * :*
328
349
342
327
354
353
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
ALDLARREAETAIRARNDFLAVMNHEMRTPMHAIIALSSLLQETELTPEQRLMVETILKS
ALDLARREAETAIRARNDFLAVMNHEMRTPMHAIIALSSLLQETELTPEQRLMVETILKS
ALDLARREAEMAVRARNDFLAVMNHEMRTPMHAIIALSSLLQETDLTPEQRLMVETILKS
ALDVARQEAEMAIRARNDFLAVMNHEMRTPMHAVIALCSLLLETDLTPEQRVMIETILKS
VLQQAQENAMKASQARTSFQKVMNNGMRRPMHSILGLLSIFQDEKASSDQRMIVDTMVKT
ALEQAKQDALRASQARNAFQMVMSHGLRRPMHSILGLLSLLQDEKLGNEQRLLVDSMVKT
.*: *:.:* * :**. * **.: :* ***:::.* *:: : .
:**:::::::*:
388
409
402
387
414
413
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
SNLLATLMNDVLDLSRLEDGSLQLELGTFNLHTLFREVLNLIKPIAVVKKLPITLNLAPD
SNLLATLINDVLDLSRLEDGSLQLDIGTFNLHAVFKEVLNLIKPVTLVKKLSLTLHLGPD
SNLLATLINDVLDLSRLEDGSLQLDIGTFNLHALFREVHSLIKPIASVKKLFVTLSLSSD
SNLLATLINDVLDLSRLEDGILELENGTFNLHGILREAVNLIKPIASLKKLSITLALALD
STVLSTLINDAMEISAKDDGRFPVEMKPFQLHLLVREASCLVKCLCVYKGFGFSTDVPTS
SNVVSTLIDDVMDTSTKDNGRFPLEMRYFQLHSMIKEAACLAKCLCAYRGYNISIEVDKS
*.:::**::*.:: * ::* : ::
*:** :.:*. * * :
:
.: : .
448
469
462
447
474
473
AtETR1
CmETR1
LeETR1
LPEFVVGDEKRLMQIILNIVGNAVKFSKQGSISVTALVTKSDT----RAADFFVVPTGSH 504
LPVFAVGDEKRLMQAILNVVGNAVKFSKEGSISISAIVAKSETFREIRVPDFHPVPSDSH 529
LPEYVIGDEKRLMQILLNVVGNAVKFSKEGNVSISAFVAKSDSLRDPRAPEFFAVPSENH 522
GNFm
)OO
#-/-#9(m
LeNR
LeETR5
LeETR4
LPILAVGDAKRLIQTLLNVAGNAVKFTKEGHISIEASVAKPEYARDCHPPEMFPMPSDGQ 507
LPNQVMGDEKRTFQVLLHMVGHLLNVSIGKGSVIFRVVLETGAETGNDKVWGTRRPSTTD 534
LPNHVLGDERRVFQVILHMVGNLLKDPNG-GLLTFRVLPESVSREGIGGAWRTRRSNSSR 532
** .:** :* :* :*::.*: :: .
: :.
..
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
FYLRVKVKDSGAGINPQDIPKIFTKFAQTQSLATRSSGGSGLGLAISKRFVNLMEGNIWI
FYLRVQVKDTGSGISPQDIPKLFTKFAQTT-VGPRNSGGSGLGLAICKRFVNLMEGHIWL
FYLRVQIKDTGIGITPQDIPNLFSKFTQSQALATTNSGGTGLGLAICKRFVNLMEGHIWI
FYLRVQVRDTGCGISPQDIPLVFTKFAESRPTSNRSTGGEGLGLAICRRFIQLMKGNIWI
EYVTIKFEIEVSLEGSQSDSSISTIHFGGRRHNSKEVT-EGLSFNMCKKLVQMMQGNIWM
DNAYIRFEVGTSNNHSQPEGTMLPHYRPKR--CSKEMD-EGLSFTVCRKLVQLMQGDIWV
::..
.*
: . .
.
**.: :.::::::*:*.**:
564
588
582
567
593
589
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
ESDGLGKGCTAIFDVKLGISERSNES-KQSGIPKVPAIPRHSNFTGLKVLVMDENGVSRM
ESEGLGKGCTATFIVKLGIADQSNES-KLPYTSKIHENSIHTSFPGLKVLVMDDNG---ESEGLGKGSTAIFIIKLGIPGRANES-KLPFVTKLPANHTQMSFQGLKVLVMDENGVSRM
ESEGPGKGTTVTFVVKLGICHHPNALPLLPMPPRGRLNKGSDDLFRYRQFRGDDGGMS-SSNAQGHAQGMTLILRFQKQSSFRKR-MFEYRNPLEQPISSTMFRGLHVLLTDDDDVNRL
IPNPEGFDQSMAVVLGLQLRPSIAIG-IPEYGESSDHSHPHSLLQGVKVLLADYDDVNRA
.: *
. : :
:
: : * ..
623
643
641
625
652
648
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
VTKGLLVHLGCEVTTVSSNEECLRVVSHEHKVVFMDVCMPGVENYQIALRIHEKFTKQRH
-----------------------------------------------------------VTKGLLTHLGCDVTTVGSRDECLRVVTHEHKVVIMDVSMQGIDCYEVAVVIHERFG-KRH
------------------------------------------------------VNAQRY
VTRKLLEKLGCQVTAVSTGFQCLSALGPSLTTFQVLILDLQMPEMDGYEVALRVRKFRSR
VTSKLLEKLGCSVSAVSSGRDCIGVLSPAVSSFQIVLLDLHLPDLDGFEVTMRIRKFGSH
683
AtETR1
CmETR1
LeETR1
LeNR
LeETR5
LeETR4
QRPLLVALSGNTDKSTKEKCMSFGLDGVLLKPVSLDNIRDVLSDLLEPRVLYEGM
------------------------------------------------------GRPLIVALTGNTDRVTKENCMRVGMDGVILKPVSVYKMRSVLSELLEHGVVLESQRSL--------------------------------------------------SWPLIIALTASSEEQVWEKCLQVGMNGLIRKPVLLQGLADELQRLLQRGGGGDGL
NWPLIVGLTATADENVTGRCLQIGMNGLIRKPVLLPGIADELQRVLLRGSRMM--
700
631
712
708
738
754
635
767
761
m
(&30 TEMH[m*',#+'#,2-m+B*2'.*#m"#m*-1m%#,#1m;m#,m,#)*-#-Zm2-+2#m7m+#*A,m7m*-1m%#,#1m>Fm
?m7mm"#m2-+2#Ym,m!-*-0m+0'**-m*1m+32!'-,#1m#,m,#)*-#-m/3#m.0-4-!,m',1#,1' '*'""m#,m
#2'*#,-Zm#,m0-(-m*m+32!'A,m"#*m$#,-2'.-m,m^0,O#*_Zm#,m$3!1'm*m&'12'"',m!-,m!2'4'""m$-1$-0'*,2#m
#,m#*m%#,m;m"#m,#)*-#-O7m#,m4#0"#m#*m!+ '-m"#2#!2"-m+#"',2#m!-m#,m+#*A,Ym
m
*mm"'1.-,#m"#m',$-0+!'A,m$#,-2>.'!m"#m!0!2#0#1m+-0$-*A%'!-1m!-+-m#*m
!-*-0m"#m*m.3*.Zm#*m0-+m7m#*m1 -0m.0m*%3,1m"#m*1m40'#""#1Ym,m*m *m
JYKm 1#m "#2**,m *%3,-1m "#m *-1m !0!2#0#1m /3#m .0#!#,m #120m 0#*!'-,"-1m !-,m *m
!2'4'""m "#*m #2'*#,-m "30,2#m #*m .0-!#1-m "#m *m +"30!'A,m "#*m $032-Zm !-+-m *m
!-,1'12#,!'m"#m*m.3*.Zm*m',2#,1'""m"#*m0-+m-m*m+"30!'A,m!*'+2<0'!Ym1mJm
!!#1'-,#1m"#1!0'21m#,m*m *mJYKm.0#1#,2,m#*mmGm/3#m!31m*m1312'23!'A,m"#m
#3HHJ*m 7m 1#m !0!2#0'8,m .-0m 2#,#0m *m !0,#m *,!m ^'%30m JYN_m 7m "#+91Zm 3,m
0-+m 7m "3*830m +#,-1m ',2#,1-1m /3#m #,m -201m 40'#""#1Ym ,m "#m #**1m .0#1#,2m
+"30!'A,m2'.-m!*'+2<0'!-Zm"-1m"#m#**1m,-Zm7m.0m"-1m+91m,-m#61'2#,m"2-1m1- 0#m
2'.-m"#m+"30!'A,Ym
m
GNGm
#-/-#9(
)
!+ TEMFOT0!2#0>12'!1m$#,-2>.'!1m"#m*1m!!#1'-,#1m/3#m.0#1#,2,m#*mmGm^"#m*m40'#""mmHNHJJNZm
/3#m2+ '<,m.0#1#,2m#*mmGZm,-m1#m"'1.-,#m"#m"2-1_Ym[m-m&7m"2-1m"'1.-,' *#1Ym
m
.+.0T7T
".-1(12$-"( T
#$T+ TT
/3+/ T
*,!i+#"'-j
*,"m
-2$-1(# #T
#$+T
0., T
3+830 T
#30 "(>-T
"+(, 2:0(" T
!'"m
,-m"3*!#m
'mm
m
m
m
m
&)-,"m
^'+ 5#_m
*,!i"30m
,-m0-+m
,-m"3*!#m
-m
'%3',!&-0m
^#,#%*_m
*,!i+#"'-j
*,"m
.-!-m
0-+92'!-m
.-!-m"3*!#m
-m
.,!0$T
4 0($# #T
1//T
0(&$-T
IJOm
+#*-m
0)m
mGHJGGHm
%0#12'1m
,"'m
mJNHJHOm
%0#12'1m
mJILKIJm
%0#12'1m
m
(&30 TEMIOT+%#,m "#m *1m !!#1'-,#1m #,m *1m /3#m 1#m &,m "#2#!2"-m #*m m Gm /3#m .0-4-!m 3,m !+ '-m "#m
+',-9!'"-m#,m#*m.0'+#0m+.*'!A,m"#*m%#,m;m^"#mmHNHJJNm,-m1#m"'1.-,#m"#m'+%#,_Ym
T
-0m 2,2-Zm *m ',$-0+!'A,m "#m /3#m 1#m "'1.-,#m ,-m .#0+'2#m $'0+0m /3#m *m +32!'A,m
"#2#!2"m#,m#*m%#,m;m#12<m'+.*'!"m#,m!+ '-1m#,m#*m2'.-m"#m+"30!'A,m"#*m
$032-Ymmm
GNHm
-./#)O ().7*#)O
#-/-#9(m
m
EMETTTTTTTT
m
,'!'*+#,2#m#*m- (#2'4-m"#*m#123"'-m$#,-2>.'!-m"#mJFFm$+'*'1mHm"#m*m.- *!'A,m"#m
m !0#"m #,m #12#m 20 (-m #0m #,!-,200m +32,2#1m !-,m !0!2#0>12'!1m "#m
',2#0<1m !-+#0!'*Ym - 0#2-"-Zm #,m !0!2#0#1m "#*m $032-m !A+-m *m $-0+Zm #*m !-*-0Zm #*m
#1!0'230"-m7m2+ '<,m#,m!0!2#0#1m"#m*m.*,2m!-+-m*%B,m2'.-m"#m0#1'12#,!'Zm.-0m
#(#+.*-Zm 0#1'12#,!'m *m ->"'-m ^))-*",O 2(."##_Ym #m !0#!'#0-,m "'#8m .*,21m .-0m
$+'*'m.0m.-"#0m- 1#040m1'm& >m+91m"#m3,m.*,2m.-0m$+'*'m/3#m+-120 m#*m
$#,-2'.-m +32,2#m 7m "#m #12#m +-"-m !-,$'0+0m /3#m #0m 3,m $#,-2'.-m .0-"3!2-m "#m 3,m
+32!'A,m&#0#""m7m,-m3,m#$#!2-m+ '#,2*Ym
201m .*2$-0+1m "#m m &,m "#100-**"-m 1#1m "#m "2-1m "-,"#m 1#m -$0#!#m
"#+91m3,m1#04'!'-m/3#m.0-.-0!'-,m*1m1#+'**1m"#*m+32,2#m/3#m1#m"#1##m#123"'0Ym
1m 1#1m"#m"2-1m$!'*'2,m+<2-"-1m"#m B1/3#"m 1"-1m#,m#*m%#,m"#m',2#0<1m.0m
#*m !3*m /3#0#+-1m #,!-,200m +32,2#1m -m 1"1m #,m *m B1/3#"m "#m 3,m $#,-2'.-m
!-,!0#2-Ym-0m#(#+.*-Zm#,m#*m!1-m"#m*m 1#m"#m"2-1m"#mm^OO
.-_m^&22.[ii30%4Y#407Y',0Y$0i!%'j ',i.0-(#!21i'**',%i',"#6Y.*_m1#m.3#"#m!!#"#0m
m*m',$-0+!'A,m0#!-.'*"m"#m20#1m.- *!'-,#1m"'12',21m"#m[m3)*,-#/'O
-/&(./'FO #-/'O -.#0/'O 7O ,"3*)#/'O #-."3)(O ^'%30m JYOZm G_YO 0m !"m 3,m
"#m #**1Zm 1#m .3#"#m 0#*'80m 3,m B1/3#"m #,m $3,!'A,m "#*m %#,m #,m #*m /3<m .-"+-1m
#120m ',2#0#1"-1m O ^'%30m JYOZm H_m -m '#,m 3,m $#,-2'.-m 7m !0!2#0'8"-O ^'%30m JYOZm
H _Ym
,m #12m 1#m "#m "2-1m 2'#,#,m !0!2#0'8"-1m IGm %#,#1m !-,m KKOm +32!'-,#1m 7m HYJFKm
$#,-2'.-1Zm13+,"-m*-1m0#13*2"-1m- 2#,'"-1m.0m*1m20#1m#1.#!'#1Ym'm1#m1#*#!!'-,m
*%B,m %#,Zm *m 1#m "#m "2-1m .0-.-0!'-,m ',$-0+!'A,m 1- 0#m *1m $+'*'1m +32,2#1m
/3#m1#m&,m"#2#!2"-m.0m<12#m^'%30mJYOZmIZm!-+-m#(#+.*-m1#m&m1#*#!!'-,"-m#*m
%#,m I#-)J>_Ym -12#0'-0+#,2#Zm 2+ '<,m 1#m .3#"#m - 2#,#0m ',$-0+!'A,m +91m
"#2**"m ^'%30m JYOZm J_m #"-,"#m 1#m +3#120m *m 1#!3#,!'m "#*m %#,m 7m *m .-1'!'A,m
!-,!0#2m "#m *m +32!'A,m 2,2-m m ,'4#*m ,3!*#-2>"'!-m !-+-m +',-!>"'!-YO -0m -20-m
*"-Zm 1#m .3#"#m 1#*#!!'-,0m 3,m $#,-2'.-m ^'%30m JYOZm I _m 7m m .02'0m "#m <12#m 4#0m
'+9%#,#1m7Zm#,m*%3,-1m!1-1Zm1'm1#m&m"#2#!2"-m*%3,m+32!'A,m#,m*m$+'*'m"#m*m
'+%#,m1#*#!!'-,"m^'%30mJYOZmJ _Ym
,m "#$','2'4Zm #12m 1#m "#m "2-1m #1m 3,m #(#+.*-m "#m !-+-m 1#m .3#"#m #6.*-20m *m
',$-0+!'A,m- 2#,'"m#,m3,m-m40'1m.*2$-0+1m"#mYm#m#12#m+-"-Zm*1m
GNIm
#-/-#9(
-./#)O ().7*#)
+32!'-,#1m #129,m '#,m '"#,2'$'!"1m 7m 1-,m "#m 31-m .B *'!-m "#m +-"-m /3#m 2-"m *m
!-+3,'""m!'#,2>$'!m.3#"#m!!#"#0mm#**1Ymm
mm
(&30 TEMJTOm,$-0+!'A,m"'1.-,' *#m#,m*m 1#m"#m"2-1m" Ym#m.3#"#m0#*'80m*m B1/3#"m.0m3,m
%#,m!-,!0#2-m^4>mZm#,m4#0"#_m-m#,m$3,!'A,m"#m3,m$#,-2'.-m^4>mFm#,m83*_Ym
m
,m ,3#120-m !1-m ,-m 1#m &m 0#*'8"-m 3,m 1#m "#m "2-1m .#0-m #,m 3,m $3230-m "# #0>m
"'1#@01#m /3'891m !-,(3,2+#,2#Zm !-,m *1m -201m .*2$-0+1m "# m
"#100-**"1m#,m#12m+'1+m#1.#!'#Ym-0m#*m+-+#,2-Zm#,m#12#m20 (-m1'm1#m&m&#!&-m
3,m .0'+#0m !29*-%-m $#,-2>.'!-m 7m /3#m "30,2#m #*m @-m HFFMZm 1#m 1'%3'Am !-,m "#2**#m #*m
!0#!'+'#,2-m "#m JFFm $+'*'1m Hm "#1"#m *m %#0+',!'A,m &12m *m - 2#,!'A,m "#m $032-Ym
,m #12#m .#0>-"-m 1#m 2-+0-,m '+9%#,#1m .0m .-"#0m 0#%'1200m /3#**-1m +32,2#1m !-,m
,3#4-1m$#,-2'.-1Ym*m- (#2'4-m#0m4'13*'80m$#,-2'.-1m7m"#2#0+',0m#*m,B+#0-m7m*m
"'4#01'""m "#m *-1m +'1+-1Zm 2+ '<,m !-+-m "2-m !-,$'0+2'4-m "#m /3#m *m .- *!'A,m
#12 m +32"m !-,m 3,m !'#02m #$'!'#,!'Ym 1#04+-1m $#,-2'.-1m m ,'4#*m "#m
.'%+#,2!'A,m !-,m .*,21m !*-0A2'!1m -m .0!'*+#,2#m !*-0A2'!1Ym + '<,m m ,'4#*m "#m
!0#!'+'#,2-m "#m *m .*,2m - 1#04,"-m $#,-2'.-1m #,,-1Ym m ,'4#*m "#m "#100-**-m
- 1#04+-1 #,m *%3,1m $+'*'1m !0#!'+'#,2-m 1',m "-+',,!'m .'!*m 7m !-,m 3,m
"#100-**-m ',2#0,-"*m +3!&-m +7-0Ym ',*+#,2#m 1#m - 1#040-,m $+'*'1m +32,2#1m
GNJm
-./#)O ().7*#)O
#-/-#9(m
#,m*m$-0+m7m!0!2#0>12'!1m"#*m$032-Ym-1m$#,-2'.-1m"#1!0'2-1m#,m#*m!29*-%-m.3#"#,m
1#0m3,m','!'-m.0m*m!0#!'A,m"#m3,m 1#m"#m"2-1m+91m!-+.*#2Ymm
-0m #(#+.*-Zm *-1m $#,-2'.-1m "#1!0'2-1m !-,m .<0"'"m "#m *m "-+',,!'m .'!*m .-"0>,m
.-1##0m3,m"#$'!'#,!'m#,m*m '-1>,2#1'1m-m 20,1.-02#m "#m *1m 36',1m -m !'2-/3',',1Zm
7m /3#m #121m &-0+-,1m 1-,m *1m /3#m 0#%3*,m #*m !0#!'+'#,2-m !-,m "-+',,!'m .'!*m
^-&,j-301#3Zm HFGF_Ym 0m -20-1m $#,-2'.-1m #1m +91m !-+.*'!"-m "#2#0+',0m /3#m
%#,#1m .3#"#,m 1#0m 0#1.-,1 *#1m "#m *-1m !0!2#0#1m +32,2#1Ym ',m #+ 0%-Zm *%3,-1m
$#,-2'.-1m.3#"#,m1#0m',2#0#1,2#1mm,'4#*m%0-,A+'!-m7m!-+#0!'*m1',m,#!#1'""m"#m
1 #0m !39*#1m 1-,m *-1m %#,#1m 0#1.-,1 *#1m "#m #1#m #$#!2-Ym -0m #(#+.*-Zm 1#m &,m
#,!-,20"-m$+'*'1m!-,m!0!2#0>12'!1m"'$#0#,2#1m!-+-m*m$-0+m7m#*m!-*-0m"#m$032-m
0#1.#!2-m *m 40'#""m LHjGGIYm ,m *%3,m $+'*'m *-1m $032-1m 1-,m +3!&-m +91m
0#"-,"#"-1m^'%30mJYGF_m7m""-m/3#m#*m+#*A,m2>.'!-m"#mc'#*m"#m1.-cm#1m#*>.2'!-m
#12#m ,3#4-m !09!2#0m .-"0>m 1#0m ',2#0#1,2#m m ,'4#*m !-+#0!'*Ym #1.#!2-m *m !-*-0Zm 1#m
&,m #,!-,20"-m $+'*'1m !-,m 3,m !-*-0m *'1-Zm 1',m #1!0'230"-m ^'%30m JYGF_m 7m 2+ '<,m
#,m#12#m!1-m.-"0>m1#0m3,m!09!2#0m',2#0#1,2#mm,'4#*m!-+#0!'*m.3#12-m/3#m.-"0>m
0#13*20m 20!2'4-m .0m #*m !-,13+'"-0Ym m #*#!!'A,m "#m #12-1m $#,-2'.-1m .0m 13m
',!*31'A,m#,m.0-%0+1m"#m+#(-0m%#,<2'!m"# #m1#0m4*-0"m.-0m*1m#+.0#11m"#m
1#+'**1m1'#+.0#m/3#m1#m!-,$'0+#,m*-1m$#,-2'.-1m#,m%#,#0!'-,#1m+91m4,8"1Ym
m
m
m
(&30 TEMBA[m8/3'#0"[m+%#,m"#*m$032-m"#m*m$+'*'mGOFHm!-,m*m.'#*m*'1Zm1',m+,!&1m,'m#1!0'230"-Ym
#0#!&[m +%#,m "#*m $032-m "#m *m $+'*'m HHNLm !-,m 3,m $-0+m 0#"-,"#"m 2-2*+#,2#m "'12',2m m *m
#*>.2'!Zm2>.'!m"#*m+#*A,mb'#*m"#m1.-cYm
m
,m 4#8m !0!2#0'8"-1m *-1m $#,-2'.-1m #,!-,20"-1m #,m *m .- *!'A,m "#m m "#m
+#*A,m .-"#+-1m !-+.00*-1m !-,m *-1m $#,-2'.-1m "#2#!2"-1m #,m *1m .- *!'-,#1m
GNKm
#-/-#9(
-./#)O ().7*#)
"#100-**"1m #,m -201m #1.#!'#1Ym ,m *m '%30m JYGGm 1#m +3#120,m $#,-2'.-1m
#,!-,20"-1m #,m -201m .- *!'-,#1m "#m m "#m -201m #1.#!'#1[m )&(/'O
&3)*,-#/'m^',-'m7m!-*YZmHFGF_Zm#-/'O-.#0/'m^*+'1m7m!-*YZmHFFN_m-m#,m+#*A,m
#,m *m 40'#""m 2'.-m &0#,2'1Zm &0-,-m ^&+,'j0"1m 7m !-*YZm HFGF_m 7m /3#m
+3#120,m !'#02m 1#+#(,8m !-,m $#,-2'.-1m #,!-,20"-1m #,m *m .- *!'A,m +32,2#m
"#100-**"m#,m#12#m20 (-Ymm"#$-0+!'A,m"#m!-2'*#"-,#1m^'%30mJYGGZm_Zm*m$*2m
"#m .'%+#,2!'A,m -m .0#1#,!'m "#m !*-0-1'1m #,m &-(1m ^'%30m JYGGZm _Zm 1>m !-+-m *m
+*$-0+!'A,m #,m #*m "#100-**-m .'!*m "#m *m .*,2m ^'%30m JYGGZm _Zm 1-,m *%3,-1m "#m
*-1m$#,-2'.-1m/3#m+91m$0#!3#,2#+#,2#m1#m0#.'2#,m#,m*1m.- *!'-,#1m"#mYmm
m "#$-0+!'A,m #,m !-2'*#"-,#1m /3#m - 1#04+-1m #,m *m $+'*'m GNMLm #1m
.09!2'!+#,2#m'%3*m*m$#,-2'.-m/3#m+-120 m#*m+32,2#m#,m*m40'#""m&0-,-m
/3#m .0#1#,2 m !'#02m "#$-0+!'A,m 7m #,0-1!+'#,2-m #,m *-1m !-2'*#"-,#1Ym *m 2'.-m "#m
+32!'A,m !-,m !-*-0!'A,m * ',m "#2#!2"m #,m *m $+'*'m ^INJ_m 2+ '<,m 1#m &m
"#2#!2"-m#,m*1m"-1m.- *!'-,#1m!-,m*1m/3#m1#m!-+.0,m*-1m$#,-2'.-1Zm1>m!-+-m*m
+*$-0+!'A,m%#,#0*'8"m,-m1A*-m#,m*m+-0$-*-%>m"#m*1m&-(1m1',-m2+ '<,m#,m#*m
!0#!'+'#,2-m.'!*m"#m*m$+'*'mGKFKm^'%30mJYGG_Ymm
GNLm
-./#)O ().7*#)O
T
#-/-#9(m
m
(&30 TEMBBOm(#+.*-1m"#m$#,-2'.-1m#,!-,20"-1m#,m-201m.- *!'-,#1m"#mYm_m*$-0+!'A,m"#m
!-2'*#"-,#1m^GNML_m_m* ','1+-Zm$*2m"#m.'%+#,2!'A,m^INJ_m_m*$-0+!'A,m#,m#*m"#100-**-m.'!*m
^GKFK_Ym1m'+9%#,#1m1#m&,m- 2#,'"-m"#m*1m.- *!'-,#1m"#m)&(/'O&3)*,-#/'m^',-'m7m!-*YZmHFGF_Zm
#-/'O-.#0/'m^*+'1m7m!-*YZmHFFN_m7m+#*A,m40'#""m2'.-m&0#,2'1m&0-,-m^&+,'j0"1m7m
!-*YZmHFGF_Ym',*+#,2#m1#m&,m@"'"-m*%3,1m'+9%#,#1m"#m+32,2#1m"#m*m.- *!'A,m"#m+#*A,m^LHj
GGI_m- 2#,'"1m#,m#12#m20 (-m.0m.-"#0m#12 *#!#0m*m!-+.0!'A,m^-,8*#8j-m7m!-*YZmHFGF_Ym
m
+ '<,m.-"#+-1m#,!-,200m.0*#*'1+-1m#,20#m*-1m$#,-2'.-1m#,!-,20"-1m#,m$032-m
#,m #121m .- *!'-,#1m "#m m "#100-**"1m #,m -201m #1.#!'#1Ym ,m *m .- *!'A,m
"#m+#*A,m"#100-**"m.-0m&+,'m7m!-* -0"-0#1m$#,-2'.0-,m$032-1m"#*m+#*A,m
&0-,-m !-,m 3,m 2+@-m +#,-0m ^'%30m JYGHZm I_m Zm -m !-,m 3,m ,B+#0-m +7-0m "#m
!0.#*-1m ^'%30m JYGHZm H_m 0#1.#!2-m *m !-,20-*m ^'%30m JYGHZm G_Ym + '<,m #,m *m
.- *!'A,m "#m m "#m 2-+2#m ^',-'m 7m !-*YZm HFGF_m 1#m $#,-2'.0-,m $032-1m /3#m
.0#1#,2 ,m3,m$-0+m+91m*0%"m^'%30mJYGHZmG_m-m3,m.'%+#,2!'A,m*2#0"m
^'%30mJYGHZmH_Ym*%3,-1m"#m#12-1m$#,-2'.-1m+32,2#1m2+ '<,m1#m&,m#,!-,20"-m
#,m*m.- *!'A,m"#100-**"m#,m#12#m20 (-m^'%30mJYLZmGjH_Ym
GNMm
#-/-#9(
T
-./#)O ().7*#)
m
(&30 T EMBC[m #,-2'.-1m '"#,2'$'!"-1m #,m $032-m #,m -201m .- *!'-,#1m "#m m #* -0"1m #,m +#*A,m
^&+,'m0"1m7m!-*YZmHFGF_m7m#,m2-+2#m^',-'m7m!-*YZmHFGF_Ym#m+3#120,m"-1m#(#+.*-1m"#m+32,2#1m
#,!-,20"-1m#,m*m.- *!'A,m"#mm"#100-**"m#,m#12#m20 (-m^-,89*#8j-m7m!-*YZmHFGG_\m3,-m"#m
$032-m +37m *0%"-m ^G_m 7m -20-m /3#m ,-m !-,2'#,#m *m .'%+#,2!'A,m -m +,!&1m !0!2#0>12'!1m "#*m '#*m "#m
.-m^H_Ymm
m
-0m*-m2,2-Zm*m!-+.0!'A,m0#1.#!2-mm*-1m$#,-2'.-1m+32,2#1m#,!-,20"-1m#,m-201m
.- *!'-,#1m !-,$'0+m *m #$'!'#,!'m "#*m +<2-"-m #+.*#"-m .0m +32%#,'80m #,m
+#*A,m7mm13m4#8Zm!-,$'0+ *m2#,"#,!'mm#,!-,200m*%3,1m+32!'-,#1 #1.#!>$'!1m
/3#m7m1#m&,m#,!-,20"-m#,m-201m.- *!'-,#1Zm!-+-m.3#"#,m1#0m*m+*$-0+!'A,m
"#m !-2'*#"-,#1Zm *m *2#0!'A,m "#m .'%+#,2!'A,m 2,2-m #,m &-(1m !-+-m #,m $032-m 7m *1m
+*$-0+!'-,#1m#,m#*m"#100-**-m.'!*m-m6'*m"#m*m.*,2Ym
m
m
GNNm
O
)(&/-#)(-m
m
T
T
1- #m &m "#100-**"-m 3,m .- *!'A,m "#m m #,m //'#-O '&)m Ym !-,m 3,m 21m "#m
+32!'A,m"#mGiGYKm Ym12m,3#4m&#00+'#,2m&m"#+-120"-m1#0mB2'*m.0m*m"#2#!!'A,m
"#m+32,2#1m#,m%#,#1m"#m',2#0<1m%0-,A+'!-Ym
2-m.0#&'"02!'A,m"#m*1m1#+'**1m1>m!-+-m*m32'*'8!'A,m"#m (1m"-1'1m"#m+329%#,-m
^FZKPm *m GZKP_m "30,2#m #*m .0-2-!-*-m "#m +32%#,'8!'A,m &,m "#2#0+',"-Zm
.0- *#+#,2#Zm *m (m 21m "#m +32!'A,m #,!-,20"Ym 0m "#100-**0m *m .- *!'A,m "#*m
m,-m1#m.0#&'"020-,m*1m1#+'**1m7m1#m310-,m"-1'1m+91m*21m"#*m%#,2#m+329%#,-m
Zm- 2#,'#,"-m3,m21m"#m+32!'A,m+91m#*#4"m/3#m#,m*m.- *!'A,mYm
3-,m *m ,3#4m .- *!'A,m "#100-**"m 1#m &,m ,*'8"-m !320-m %#,#1[m m 7m *-1m %#,#1m
>FO I#-)J>m 7m ;Ym -1m +'1+-1m %#,#1m &,m 1'"-m ,*'8"-1m #,m *m .- *!'A,m "#m
m"#*mm.0m.-"#0m#12 *#!#0m3,m!-+.0!'A,m#,20#m+ 1Ym
m
4-,m*m.- *!'A,mm1#m&,m"#2#!2"-m"-1m+32,2#1m#,m#*m%#,m"#m*mZm!320-m#,m#*m
#JZm ,',%3,-m #,m 13m '1-$-0+m ^I#-)J>Jm 7m ,',%3,-m #,m #*m %#,m GYm 320-m "#m #12-1m
+32,2#1m 1#m #,!3#,20,m #,m 8-,m !-"'$'!,2#m ^JiL_m 7m 20#1m "#m #**-1m .-"0>,m $#!20m m *m
$3,!'-,*'""m"#m*m.0-2#>,m1#%B,m*-1m4*-0#1m- 2#,'"-1m#,mYm
5-,m*m.- *!'A,mm1#m&,m"#2#!2"-m!320-m+32,2#1m#,m#*m%#,m"#m*mZm!2-0!#m
#,m #*m #Jm 7m 3,-m #,m I#-)J>Ym '#2#m "#m #12-1m +32,2#1m 1#m #,!3#,20,m #,m 8-,m
!-"'$'!,2#m^MiGO_m7m20#1m"#m#**-1m.-"0>,m$#!20mm*m$3,!'-,*'""m"#m*m.0-2#>,m1#%B,m
*-1m4*-0#1m- 2#,'"-1m#,mYm
m
6-m #m &m 0#*'8"-m #*m #123"'-m "#m 1#%0#%!'A,m 7m "#m $#,-2'."-m .0m *-1m +32,2#1m
"#2#!2"-1m #,m #*m %#,m "#m *m m #,m *m .- *!'A,m Ym ,m *m $+'*'m INJm ,-m 1#m &,m
- 2#,'"-m &-+-!'%-2-1m +32,2#1m #,m Hm 7m 1A*-m 1#m &m .-"'"-m - 2#,#0m 3,m &-+-!'%-2-m
+32,2#m #,m *m %#,#0!'A,m IZm /3#m .0#1#,23,m $#,-2'.-m * ',-Ym -0m *-m 2,2-Zm &#+-1m
0#*!'-,"-m #12#m $#,-2'.-m !-,m *m +32!'A,m "#*m %#,m Zm /3#m 0#.0#1#,20>m 3,m
',#$'!'#,!'m#,m*m4>m"#m '-1>,2#1'1m"#m!0-2#,-'"#1Ym
GNOm
)(&/-#)(-O
m
7-0m*-1m+32,2#1m#,m#Jm1#m&,m- 2#,'"-m#123"'-1m"#m1#%0#%!'A,m#,mHm.#0-m,-m1#m
&m .-"'"-m &!#0m 3,m #123"'-m $#,-2>.'!-Ym ',m #+ 0%-Zm 1'm 1#m &m &#!&-m 3,m #123"'-m "#m
.0#"'!!'A,m "#m #1203!230m IZm 13%'0'#,"-m /3#m *%3,1m +32!'-,#1m .3#"#,m $#!20m *m
$3,!'-,*'""m"#m*m.0-2#>,Zm!-+-m.-0m#(#+.*-m*m$+'*'mKMm!-,m#*m!+ '-m*3GHO71Ym
8-30,2#m #12#m 20 (-m 2+ '<,m 1#m 0#*'8Am 3,m #123"'-m "#m !-m !-+.0,"-m GGIm
40'#""#1m"#m+#*A,m!-,m*m40'#""m"#m0#$#0#,!'mLHjGGIYm12#m#,17-m1#m&'8-m!-,m*-1m
%#,#1m>m7m;Ym,m+ -1m%#,#1m1#m#,!-,200-,m"'$#0#,2#1m&.*-2'.-1m!-,m1m/3#m
.0-4-!,m 3,m !+ '-m "#m +',-9!'"-m 7m .3#"#,m $#!20m *m $3,!'-,*'""m "#m + 1m
.0-2#>,1m 1#%B,m *-1m 4*-0#1m - 2#,'"-1m #,m Ym #"',2#m !-m 1#m &,m "#$','"-m
,3#4-1m &.*-2'.-1m -m 40'!'-,#1m ,230*#1m /3#m ,-m & >,m 1'"-m "#1!0'21m !-,m
,2#0'-0'""Ym
9-m #m &m #* -0"-m 3,m !29*-%-m "#m +32,2#1m $#,-2>.'!-1m #,m 20#1m #12"'-1m "'12',2-1m "#m
!0#!'+'#,2-mm.02'0m"#mJFFm$+'*'1mH[m#12"'-m"#m.*9,23*Zm"#m.*,2m"3*2m7m"#m$032-Ym
,m *1m 20#1m #2.1m 1#m &,m "#1!0'2-m 7m !2*-%"-m 40'!'-,#1m $#,-2>.'!1m /3#m ,-m & >,m
1'"-m "#1!0'21m !-,m ,2#0'-0'""m #,m #12m #1.#!'#Ym 12#m .3#"#m 1#0m 3,m ','!'-m .0m "'1#@0m
3,m 1#m "#m "2-1m +91m !-+.*#(m 7m #1.#!>$'!m !-,(3,2+#,2#m !-,m *1m .- *!'-,#1m "#m
m"#m+#*A,m"#100-**"1m.-12#0'-0+#,2#mm#12#m20 (-Ym
m
GOFm
GOGm
O
#&#)!, 7m
(!+(.&0 %; T
m
%3"#*-j-+#0-Zm YZm 0 -,#**Zm YZm "#m *m %*#1'Zm YZm 00#0Zm YZm -"0'%-Zm Ym 7m
!-*Ym ^HFFN_Ym e&,%#1m ',m 2&#m %#,#m #6.0#11'-,m .0-$'*#m -$m 0 '"-.1'1m 2&*',m $2#0m
',$#!2'-,m5'2&m- !!-m#2!&m4'031Yem'0-*m
mK[mOHYm
m
)1)j#,,#"7ZmYZm-+'2mZmYZ830ZmYm^HFFJ_Yme$$'!'#,2m.*,2m0#%#,#02'-,m
,"m %0- !2#0'3+j+#"'2#1m 20,1$-0+2'-,m 4'm 1-+2'!m #+ 07-%#,#1'1m m ',m +#*-,m
^3!3+'1m+#*-mY_Yem*,2m!'#,!#m*1#4'#0mGLL^I_[mMLIjMLOYm
m
*j '*'m YZm 023,%Zm Ym ^GOOO_Ym em -"3*2#1m #4#*1m -$m 0-2#,-%#,'!m
0-2#',1m ,"m 2&#'0m +1m ,"m $$#!21m 0-2#,-'"m ,"m m -,2#,2m 1m 5#**m 1m
&0-+-.*12m203!230#m',m0!'1131m.1#3"-,0!'1131m*-5#01Yem*,2m'-*-%7mG^L_[mLFMj
LGHYm
m
**0"Zm YYm ^GOOO_Ym e0',!'.*#1m -$m *,2m 0##"',%Yem 0',2#"m 7m -&,m '*#7m Xm
-,1Zm,!Ym
m
,ZmYZm0)ZmYZm
#-,%ZmYYZm##ZmYYZm,%ZmYYm7m!-*Ym^HFFI_Yme#,#02'-,m,"m
,*71'1m-$m#,"m1#/3#,!#m"2 1#m$-0mjm2%%',%m*',#1m',m0'!#Yem*,2m&71'-*mGII^J_[m
HFJFjMYm
m
03+3%,2&,Zm Ym ^GOOG_Ym e3!*#0m m !-,2#,2m -$m 1-+#m '+.-02,2m .*,2m
1.#!'#1Yem*,2m-*m'-m#.mO[mHGGjHGKYm
m
1&0$Zm YZm 07Zm Ym ^GOOI_Ym em 17,2&#1'1m ',m -1+-.0'+#"m *##)m ^**'3+m
.-003+mY_m1##"1m,"m#4'"#,!#m$-0m0#.'0m,"m0#.*'!2'-,Yem##"m!'#,!#m#1#0!&mI^GKj
HI_Ym
m
3#0 !&ZmYZm- 1-,Zm
YYm^GOJL_Yme&#+'!*m.0-"3!2'-,m-$m+322'-,1Yem230#m
GKM[mIFHYm
m
-!&Z
Ym 7m -,1Zm Ym ^HFGF_Ym e,2&-+-,1m 401Im $+'*7j27.#m m #$$#!2-01[m
'1!-4#07m,"m$3,!2'-,Ym,,3Y#4m&72-YmJN[mJGOjJILYm
m
*2'+-0#Zm YZm +-*#0Zm Ym ^GOMG_Ym e0'+#0m 0#/3'0#+#,2m ,"m 2#+.*2#m 1.#!'$'!'27m
-$m2&#mm.-*7+#01#m-$mm23+-0m4'031#1Yem0-!m2*m!"m!'mmmmLN^M_[mGKFMj
GGYm
m
0)*#7ZmYYZm,%ZmYYm^HFFN_Yme..*'!2'-,m-$mm,"m!-m1m
#4#01#m#,#2'!m..0-!&#1m2-m*3!'"2#m2&#m3,!2'-,m-$m#,#1m',m*,21m,"m,'+*1Yem
300m#,-+'!1mO^J_[mHGHjHLYm
m
0,#1ZmYYm^GOOJ_Ymem+.*'$'!2'-,m-$m3.m2-mIKj) mm5'2&m&'%&m$'"#*'27m
,"m&'%&m7'#*"m$0-+m*+ "m !2#0'-.&%#m2#+.*2#1Yem0-!m2*m!"m!'mmmmOG^L_[m
HHGLjHHHFYm
m
02*#7ZmYYZm!-*,')ZmYYZm#7#0ZmYm^GOOO_Yme5-m0 '"-.1'1m2&*',m!0-2#,#m
"#12301#1Zm .&72-#,#m "#12301#m ,"m 8#2j!0-2#,#m "#12301#Zm #6.0#11#"m ',m
1!&#0'!&'m !-*'Zm !2*78#m m .-*7j!'1m .2&57m 2-m 7'#*"m .0-j*7!-.#,#Yem 30m m '-!&#+m
HKO^GjH_[mIOLjJFIYm
m
GOHm
#&#)!, 7O
m
m
3"0!!-j0,1ZmYZm'202ZmYm^GOOL_Ymem%#,#2'!m+.m-$m+#*-,m^3!3+'1m+#*-m
Y_m 5'2&m Zm Zm '1-87+#Zm "'1#1#m 0#1'1'2#,!#m ,"m +-0.&-*-%'!*m +0)#1Yem &#-0m
..*m#,#2mOI[mKMjLJYm
m
#!)#0Zm YZm -1Zm YZm 3'.#0Zm YZm *+','Zm YZm #3,Zm Ym ^GOOK_Ym e-+ ',#"m
+..',%m-$mm,"mm+0)#01m',m 0*#7Yem-*m#,m#,#2mHJO^G_[mLKjMIYm
m
#,2Zm YYm ^GOOL_Ym e*,2m '1#1#m #1'12,!#m #,#1[m 3,!2'-,m ##21m 203!230#Yem
*,2m#**mN^GF_[mGMKMjGMMGYm
m
#,2*#7Zm YZm !#,,,Zm ^HFFF_Ym e0%#2#"m 0#!-4#07m -$m +322'-,1m ',m
0-1-.&'*Yem#,#2'!1mGKL^I_[mGGLOjGGMIYm
m
'0, -'+Zm YYZm -*7Zm Ym ^GOMO_Ym em 0.'"m *)*',#m #620!2'-,m .0-!#"30#m $-0m
1!0##,',%m0#!-+ ',,2m.*1+'"mYem3!*#'!m!'"1m#1mM^L_[mGKGIjHIYm
m
*,!Zm YZm ,'80#1Zm YZm -'%Zm YZm '01-*-Zm YZm 3#8Zm Ym 7m !-*Ym ^HFGG_m
e0,1!0'.2-+#m !&0!2#0'82'-,m ,"m &'%&m 2&0-3%&.32m 1m ,"m 1m "'1!-4#07m ',m
3!30 '2m.#.-m^3!30 '2!##_Yemm#,-+'!1mGH[mGFJYm
m
*,!m
Zm@'80#1m
Zm-'%mZm'01-*-mZm3#8mZm'!AmYm^HFGG_Yme0,1!0'.2-+#m
!&0!2#0'82'-,m ,"m &'%&m 2&0-3%&.32m 1m ,"m 1m "'1!-4#07m ',m 3!30 '2m .#.-m
^3!30 '2!##_Yemm#,-+'!1YmGH[GFJYm
m
*,!Zm YZm 12#01Zm YZm '01-*-Zm YZm <0#8Zm YZm #0,:m "#8j#"0-1Zm YZm 7m !-*Ym
^HFGH_Ym e0,1!0'.2-+#m 1#/3#,!',%m $-0m m "'1!-4#07m !0-11m 3!3+'1m +#*-eYm m
#,-+'!1mGI[HNFYm
m
*##!)#0Zm YYm ^GOOO_Ym e2&7*#,#m .#0!#.2'-,m ,"m 1'%,*',%[m ,m #4-*32'-,07m
.#01.#!2'4#Yem0#,"1m*,2m!'mJ^M_[mHLOjHMJYm
m
*##!)#0Zm YYZm #,"#Zm Ym ^HFFF_Ym e2&7*#,#[m m %1#-31m 1'%,*m +-*#!3*#m ',m
.*,21Yem,,3m#4m#**m#4m'-*mGL[mGjGNYm
m
-&,j-301#3ZmYm^HFGF_Yme36',[mm+(-0m0#%3*2-0m-$m-0%,-%#,#1'1Yemmm'-*m
III^J_[mHOFjLYm
m
-,-Zm YZm 388-Zm YZm * #%%','Zm YZm '88-Zm YZm 0,!-$-,2#Zm Ym 7m !-*Ym ^HFGG_Ym
e#,#2'!m1!0##,',%m-$m 07m.2'#,21m5'2&m!-m,"mm2#!&,-*-%7Yemm#1m
-2#1mJ[mIHIYm
m
-0#4'28Zm
YYZm',%ZmYZm*-3$$#ZmYZm&,%ZmYYZm&3ZmYm7m!-*Ym^HFFI_Yme0%#j
1!*#m'"#,2'$'!2'-,m-$m1',%*#j$#230#m.-*7+-0.&'1+1m',m!-+.*#6m%#,-+#1Yem#,-+#m#1m
GI^I_[mKGIjHIYm
m
-3*#+Zm YZm #0%,7Zm YZm #0,,"#8Zm YZm 0-"#!Zm YZm 02',Zm Ym 7m !-*Ym ^HFFN_Ym
em !-,1#04#"m +322'-,m ',m ,m #2&7*#,#m '-17,2&#1'1m #,87+#m *#"1m 2-m ,"0-+-,-#!7m ',m
+#*-,1Yem!'#,!#mIHG^KNOF_[mNILjNYm
m
0,"5%2ZmYYZm,#..#01ZmY
YZm'()+.ZmY
YZm'**#Zm
Ym^HFFH_Yme4#0#6.0#11'-,m
-$m2&#m2-+2-m1!jGm%#,#m+#"'2#1m&'%&m',1#,1'2'4'27m2-mm2-6',1m,"m$3+-,'1',mGm',m
2-+2-m &'07m 0--21m ,"m !-,$#01m 0#1'12,!#m 2-m *2#0,0'm *2#0,2m $Ym 1.Ym *7!-.#01'!'m ',m
'!-2',m3+ 02'!m.*,21Yem-*m*,2m'!0- #m,2#0!2mGK^G_[mIKjJHYm
m
0--)#1ZmY
Ym^GOOO_Yme&#m#11#,!#m-$m1Yem#,#mHIJ^H_[mGMMjGNLYm
GOIm
O
#&#)!, 7m
m
0-2+,Zm YZm '* #012#',Zm YZm -4*1)'Zm YZm #0',Zm YZm -%'+-,2Zm Ym 7m !-*Ym ^HFFH_Ym
e#1'12,!#m%#,#m&-+-*-%3#1m',m+#*-,m0#m*',)#"m2-m%#,#2'!m*-!'m!-,$#00',%m"'1#1#m,"m
.#12m0#1'12,!#Yem&#-0m..*m#,#2mGFJ^LjM_[mGFKKjGFLIYm
m
0-5,',%Zm YYZm # 12#0Zm YZm - #021Zm YYZm 4#*Zm YYm ^GOOH_Ym e"#,2'$'!2'-,m -$m
,m'1-87+#m$-0+m-$m.0-2#',m17,2&#1'1m','2'2'-,m$!2-0mJm',m.*,21Yem
m'-*m&#+mHLM^GJ_[m
GFFOLjGFFYm
m
m
03%%#+,,Zm YZm ,"5#0%#0Zm YZm 11#6Zm YZm 2-08Zm Ym ^GOOL_Ym e,*71'1m -$m $12m
,#320-,j%#,#02#"m+32,21m2m2&#m0 '"-.1'1m2&*',mJm*-!31Yem*,2m
mGF^J_[mMKKjLFYm
m
03++#**ZmYYZm0.12#0ZmYYm^HFFG_Yme#**m5**m+#2 -*'1+m',m$03'2m1-$2#,',%m
,"m/3*'27m,"m'21m+,'.3*2'-,m',m20,1%#,'!m.*,21Yem*,2m-*m'-*mJM^GjH_[mIGGjJFYm
m
3"'+,Zm YYZm -Zm YZm --"Zm YYZm ',%Zm YYm ^HFFF_Ym em "##.j!-4#0%#m
2-+2-m m *' 007m ,"m .0-1.#!21m 2-50"m "#4#*-.+#,2m -$m ,m m $0+#5-0)m $-0m
%#,-+#m1#/3#,!',%Yem#,-+#m#1mGF^G_[mGHOjILYm
m
3*-5Zm YZm !&',"*#0Zm YZm &-'Zm YZm #&*Zm Ym ^HFFJ_Ym e2&-*,2[m m "2 1#m -,m
.*,2j.2&-%#,m',2#0!2'-,1Yem,m'*'!-m'-*mJ^J_[mKHOjILYm
m
31!&%#1ZmYZm-**0'!&#0ZmYZm,1203%ZmYZm'+-,1ZmYZm-*2#0ZmYm7m!-*Ym^GOOM_Ym
e&#m 0*#7m*-m%#,#[mm,-4#*m!-,20-*m#*#+#,2m-$m.*,2m.2&-%#,m0#1'12,!#Yem#**mNN^K_[m
LOKjMFKYm
m
31&Zm YYZm 071,Zm Y
Ym ^HFGF_Ym e'[m m .#01-,*'8#"m ..0-!&m 2-m 2&#m
'"#,2'$'!2'-,m-$m',"3!#"m+322'-,1m',m0 '"-.1'1Yem*,2m&71'-*mGKJ^G_[mHKjIKYm
m
*"5#**ZmYYZm!**3+ZmYZm&5ZmYZm3#&* 3#0ZmY
YZm01&**ZmYYm7m!-*Ym
^HFFJ_Ym em 1203!230#"m +32,2m .-.3*2'-,m $-0m $-050"m ,"m 0#4#01#m %#,#2'!1m ',m 0*#7m
^-0"#3+m43*%0#mY_Yem&#m*,2m
-30,*mJF^G_[mGJIjGKFYm
m
0-*Zm YZm 3,28Zm Ym ^HFFG_Ym em .*12'"m 2#0+',*m -6'"1#m !-+#1m 2-m *'%&2[m
'+.*'!2'-,1m$-0m!0-2#,-'"m '-17,2&#1'1m,"m!&*-0-0#1.'02'-,Yem0#,"1m*,2m!'mL[mIGj
ILYm
m
12%,Zm YZm "#0*'Zm YZm 1/3*','Zm YZm #,13*'j-"'Zm YZm *",Zm Ym 7m !-*Ym
^HFFM_Yme&#m2-+2-m#2&7*#,#m0#!#.2-0mjIm^_m'1m,-2m',4-*4#"m',m+#"'2',%m-8-,#m
1#,1'2'4'27[m !31*m 0#*2'-,1&'.1m +-,%m #2&7*#,#m #+'11'-,Zm -6'"2'4#m 3012m ,"m 2'113#m
"+%#Yem#5m&72-*mGMJ^H_[mIJHjKLYm
m
#!!&','ZmYZm3**'%,ZmY
YZm-4#7ZmYYZm'*,#0Zm
Y
Ym^GOON_Yme&0!2#0'82'-,m-$m
%++m '00"'2'-,j',"3!#"m "#*#2'-,m +322'-,1m 2m m 1#*#!2 *#m *-!31m ',m 0 '"-.1'1Yem
322m#1mJFG^GjH_[mGOOjHFLYm
m
#0+)Zm YZm -7*#Zm YZm &012',Zm m YZm '%Zm YZm &,%Zm YZm !&+'"2Zm YZm 7m !-*Ym
^HFGG_Ym $$'!'#,2m "#1'%,m ,"m 11#+ *7m -$m !312-+m m ,"m -2&#0m m #$$#!2-0j 1#"m
!-,1203!21m$-0mm20%#2',%Ym3!*#'!m!'"1m#1#0!&mIO[NHYm
m
&)04-027Zm YZm 031-4Zm YZm -2#**Zm YYm ^HFGG_Ym e'2#j"'0#!2#"m +32%#,#1'1m -$m
2&#m 0 '"-.1'1m&#2#0-20'+#0'!mm.0-2#',m #2m 13 3,'2m 13%%#121m "'4#0%#,2m +#!&,'1+1m
-$m#$$#!2-0m!2'42'-,m #25##,m.*,2m,"m,'+*mm.0-2#',1Yem*,2mHIK^I_[mLGKjHMYm
GOJm
#&#)!, 7O
m
m
&,%Zm YZm 5-)Zm YYZm *##!)#0Zm YYZm #7#0-5'28Zm YYm ^GOOI_Ym e0 '"-.1'1m
#2&7*#,#j0#1.-,1#m %#,#m G[m 1'+'*0'27m -$m .0-"3!2m 2-m 25-j!-+.-,#,2m 0#%3*2-01Yem
!'#,!#mHLH^KGII_[mKIOjJJYm
m
&5"#Zm YZm ')-0Zm YZm 032'%+Zm YZm 011-,Zm YZm '4#),,"Zm Ym 7m !-*Ym
^HFGF_Ym e#4#*-.+#,2m ,"m !&0!2#0'82'-,m -$m ,m -2m j.-.3*2'-,m ,"m
'"#,2'$'!2'-,m -$m +322'-,1m ',m *'%,',m ,"m #2j%*3!,m '-17,2&#1'1m %#,#1Yem m *,2m
'-*mGF[mNLYm
m
&#,Zm YYZm **'#Zm YYm ^HFGF_Ym e,*71'1m -$m 2&#m $3,!2'-,*m !-,1#042'-,m -$m
#2&7*#,#m0#!#.2-01m #25##,m+'8#m,"m0 '"-.1'1Yem*,2m-*m'-*mMJ^JjK_[mJFKjHGYm
m
&'1&-*+Zm YYZm -)#0Zm YZm 7Zm YZ21)5'!8Zm Y
Ym ^HFFL_Ym e-12j+'!0- #m
',2#0!2'-,1[m1&.',%m2&#m#4-*32'-,m-$m2&#m.*,2m'++3,#m0#1.-,1#Yem#**mGHJ^J_[mNFIjGJYm
m
&-'Zm YYZm 72#,Zm YYZm 23)3+**'Zm YYZm -,%Zm YZm &)7Zm YYm 7m !-*Ym ^HFFM_Ym
em 1-7 #,m 20,1!0'.2m +.[m %#,#m "'120' 32'-,Zm &.*-27.#m ,"m 1',%*#j,3!*#-2'"#m
.-*7+-0.&'1+m,*71'1Yem#,#2'!1mGML^G_[mLNKjOLYm
m
&-4#*-,Zm YZm #12'#0Zm YZm '-4',88-Zm YZm -%'+-,2Zm YZm 00-3$Zm Ym ^HFGG_Ym
e'12-*-%'!*m 123"7m -$m -0%,-%#,#1'1m ',m 3!3+'1m +#*-m Ym $2#0m %#,#2'!m 20,1$-0+2'-,[m
5&7m'1m'2m"'$$'!3*2m2-m- 2',m20,1%#,'!m.*,21]em*,2m#**m#.mIF^GG_[mHFFGjGGYm
m
*0)Zm YYZm 071,Zm Y
Ym ^HFFM_Ym e0-2-!-*[m ,m '+.0-4#"m &'%&j2&0-3%&.32m
+#2&-"m $-0m %#,#02',%m 2'113#m 1+.*#1m ',m OLj5#**m $-0+2m $-0m .*,2m %#,-27.',%m ^!#j.m
HYF_Yem*,2m#2&-"1mI[mNYm
m
-* #02ZmYZm'**ZmY
YZm-+.ZmYZm#7,-*"1ZmYZm2#',#ZmYYm7m!-*Ym^HFFG_Yme'%&j
&0-3%&.32m!0##,',%m$-0m,"3!#"m-',2m322'-,1Yem*,2m&71'-*mGHL[mJNFmjmJNJYm
m
-**',%#Zm YZm -**#0Zm Ym ^HFFG_Ym e'$$#0#,2'*m ',"3!2'-,m -$m 25-m .-22-m %#,#1Zm
2.06Hm ,"m 2Zm ',m 0#1.-,1#m 2-m ',$#!2'-,m 7m &72-.&2&-0m ',$#12,1m ,"m 2-m
5-3,"',%Yem*,2m-*m'-*mJL^K_[mKHGjOYm
m
-+'Zm YZm #,')-$$Zm Ym ^HFFL_Ym e[m .0!2'!*m 1',%*#j,3!*#-2'"#m +322'-,m
"'1!-4#07Yem*,2m
mJK^J_[mLNJjOJYm
m
-+'Zm YZm -3,%Zm YZm '**Zm Y
YZm #7,-*"1Zm YYZm 0##,#Zm YYm 7m !-*Ym ^HFFJ_Ym
e$$'!'#,2m"'1!-4#07m-$mm.-*7+-0.&'1+1m',m,230*m.-.3*2'-,1m 7m!-2'**',%Yem*,2m
mIM^K_[mMMNjNLYm
m
--.#0Zm YYZm '**Zm Y
YZm .-02Zm YYZm 0*-5Zm YYZm *#$$,#0Zm YYm 7m !-*Ym ^HFFN_Ym
e 2-m"#2#!2m',"3!#"m+322'-,1m',m1-7 #,Yemm*,2m'-*mN[mOYm
-0 !&-Zm YZm -+-(0-Zm YZm #!&Zm jYZm 2!&<Zm YZm -+#8j
'+#,#8Zm Ym ^HFGI_Ym
e0,1!0'.2-+'!m 4#,21m ,4-*4#"m ',m #*-,m 230#j03'2m 1!'11'-,m -+.0'1#m 2&#m
#/3#,2'*m ,"3!2'-,m -$m #**j**m #%0"',%m #,#1m -3.*#"m 2-m m 2'+3*2'-,m -$m ,"-m
,"m6-!72-1'1eYm-mmN^I_[m#KNILIYm
mm
&+,'j0"1Zm YZm 0-"#!Zm YZm -3*#+Zm YZm #4#/3#Zm YZm *1"-,Zm YYm 7m
!-*Ym ^HFGF_Ym e,%',##0',%m #*-,m *,21m 5'2&m +.0-4#"m 03'2m &#*$m '$#m 1',%m 2&#m
m..0-!&Yem-m,#mK^GH_[m#GKMMLYm
GOKm
O
#&#)!, 7m
m
*+'1Zm YZm !&+'"2Zm YZm #m '%,-0Zm YZm -3117Zm YZm 3012',Zm Ym 7m !-*Ym ^HFFN_Ym
e" Zm m '13+m 12'43+m ',m 1'*'!-m $-050"m ,"m 0#4#01#m %#,#2'!1m 2--*Yem #,-+#m '-*m
O^H_[mJIYm
m
,',j-*#%Zm YZm #'1Zm YZm 3"0!!-j0,1Zm YZm '202Zm YZm 23 Zm YYm 7m !-*Ym
^HFFF_Ym e'+.*#m 1#/3#,!#m 0#.#21m ',m 3!3+'1m +..',%m ,"m +.m +#0%',%Yem #,-+#m
JI^L_[mOLIjMJYm
m
#*#3ZmYZm-,89*#8ZmYZm-,$-02ZmYZm#,"&+,#ZmYZm3'%"-+#,#!&ZmYm7m!-*Ym
^HFFM_Yme203!230#m-$m25-m+#*-,m0#%'-,1m0#4#*1m&'%&m+'!0-17,2#,7m5'2&m1#/3#,!#"m.*,2m
1.#!'#1Yem-*m#,#2m#,-+'!1mHMN^L_[mLGGjHHYm
m
#*#3Zm YZm 12#01Zm YZm -'%Zm YZm -,89*#8j-Zm YZm #0,9,"#8j'*4Zm Ym 7m !-*Ym
^HFFO_Ym em 1#2m -$m j1m $-0m +.m 12302'-,m ,"m !3*2'40m '"#,2'$'!2'-,m ',m +#*-,Yem
m*,2m'-*mO[mOFYm
m
#,,7Zm YYm ^GOOK_Ym e,4-*4#+#,2m -$m !2#0'*m .-*71!!&0'"#1m ',m .*,2m
.2&-%#,#1'1Yem,,3m#4m&72-.2&-*mII[mGMIjOMYm
m
'8Zm YZm #0%,7Zm YZm -0+'1,-Zm YZm '01-*-Zm YZm *,!Zm Ym 7m !-*Ym ^HFGG_Ym em
!-,1#,131m*',)%#m+.m$-0m+-*#!3*0m+0)#01m,"m/3,2'22'4#m20'2m*-!'m11-!'2#"m5'2&m
#!-,-+'!**7m'+.-02,2m20'21m',m+#*-,m^3!3+'1m+#*-mY_Yemm*,2m'-*mGG[mGGGYm
m
'8Zm YZm 30"7Zm YZm &0'12Zm YZm -0-2j3"07Zm YYZm ',%*#0Zm Ym 7m !-*Ym ^HFFK_Ym
e&0!2#0'82'-,m-$m+0)#01m2-m"#2#0+',#m2&#m#62#,2m,"m40' '*'27m-$m*#$m1#,#1!#,!#m
',m0 '"-.1'1Ymm+#2 -*'!m.0-$'*',%m..0-!&Yem*,2m&71'-*mGIN^H_[mNONjOFNYm
m
-"%1-,Zm
YYZm#**1ZmYYm^GOMM_Yme!2'-,m-$m1',%*#j120,"m1.#!'$'!m,3!*#1#1m-,m
+-"#*mm&#2#0-"3.*#6#1m-$m"#$',#"m1'8#m,"m1#/3#,!#Yem'-!&#+'1207mGL^GG_[mHIMJjOYm
m
-,%Zm YZm ',!#,2Zm YZ&0.Zm Ym ^HFFO_Ym e'+3*2,#-31m +322'-,m "#2#!2'-,m -$m
2&0##m&-+-#-*-%-31m%#,#1m',m5&#2m 7m'%&m#1-*32'-,m#*2',%m,*71'1m,"m322'-,m
304#7-0Yemm*,2m'-*mO[mGJIYm
m
-7*#Zm
YZm-7*#Zm
Ym^GOOF_Yme1-*2'-,m-$m.*,2mm$0-+m$0#1&m2'113#Yem-!31mGH[m
GIjGKYm
m
0.#0Zm YYZm !**3+Zm YYZm 2-32Zm YYZm *"#Zm Y
YZ-#,1Zm YYm ^HFFJ_Ym em
&'%&j2&0-3%&.32m +#2&-"m $-0m '"#,2'$7',%m j#2&7*jj,'20-1-30#m ^_j',"3!#"m .-',2m
+322'-,1m',m8# 0$'1&Yem#2&-"1m#**m'-*mMM[mOGjGGHYm
m
m -,1-02'3+m ^HFFJ_Ym e&#m m ^7!*-.#"'m $m m
*#+#,21_m0-(#!2Yem!'#,!#mIFL^KLOL_[mLILjJFYm
m
830m Zm 3&1&'Zm YZm 132m Ym ^HFFF_Ym e$$#!2m -$m #2&7*#,#m -,m %0- !2#0'3+m
23+#$!'#,1j+#"'2#"m%#,#m20,1$#0m2-m+#*-,Yem*,2m0##"mGGO[mMKjMOYm
m
,%Zm YZm 03+#2Zm Ym ^GOOF_Ym e%0- !2#0'3+m 23+#$!'#,1m +#"'2#"m
20,1$-0+2'-,m,"m0#%#,#02'-,m-$m+31)+#*-,m.*,21Yem*,2m#**m#.mO[mGLFjGLJYm
m
#0,9,"#8j.0'!'-m ^HFGF_Ym e01'2'!m .*,2m ',$#!2'-,m '1m .02'**7m !-,20-**#"m
2&0-3%&m17+ '-2'!m.2&571Yem##"m#1#0!&mKF[mGYm
m
#0,,"#8j'*4ZmYZm"30"-ZmYZm*,!Zm
YZm12#01ZmYZm'!-ZmYm7m!-*Ym^HFFN_Yme',m
+..',%m-$m%#,-+'!m,"mj"#0'4#"m 1m ',m +#*-,m ^3!3+'1m +#*-mY_Yem &#-0m ..*m
#,#2mGGN^G_[mGIOjKFYm
GOLm
#&#)!, 7O
m
m
*-0Zm YYm ^GOMG_Ym e300#,2m 12231m %#,#j$-0j%#,#m !-,!#.2Yem ,,Ym #4Ym
&72-.2&-*YmO[mHMKjHOLYm
m
*-0#1Zm YZm *m &7-3'Zm YZm "#m '**#0 #!)Zm YZm -+-(0-Zm YZm 2!&#Zm Ym 7m !-*Ym
^HFFH_Yme-*#m-$m#2&7*#,#m',m2&#m '-17,2&#2'!m.2&57m-$m*'.&2'!m#12#0m0-+m4-*2'*#1m',m
&0#,2'1m,2*-3.#m+#*-,1Yem
m6.m-2mKI^ILM_[mHFGjLYm
m
*-0#1Zm YYm ^HFFG_Ym '1'-*-%>m 7m +"30!'A,m "#*m +#*A,m ,2*-3.#m &0#,2'1m
!-,m 3,m %#,m ,2'1#,2'"-m "#m *m #,8'+m m -6'"1Ym Ym #1'1Zm ,'4#01'""m "#m 30!'Ym
30!'Ym
m
01#0m^GONL_Yme#,#1m$-0m0#1'12,!#m2-m.*,2m4'031#1Yemm02'Ym#4Ym*,2m!'Y[m
HKMjHOJYm
m
3Zm YYZm &3Zm YYZm &3Zm YYZm ',%Zm YYZm 3-Zm YYm ^HFFK_Ym e'031j',"3!#"m
%#,#m1'*#,!',%m',m2-+2-m$03'2Yem*,2m
mJI^H_[mHOOjIFNYm
m
3&0+,,Zm YZm #02#*Zm YZm #02&-*"Zm YZm #%#+,,Zm Ym ^HFFK_Ym e#+-4*m -$m
+'1+2!&#"m 1#1m $0-+m 17,2&#2'!m %#,#1m 7m #,87+2'!m +'1+2!&m !*#4%#Yem 3!*#'!m
!'"1m#1mII^L_[m#KNYm
m
"7ZmYYZm#0+,1ZmYYZm,m"#m*ZmYYZm,m--ZmYYZm'11#0ZmYYm7m!-*Ym
^HFFO_Ym e+.*#+#,22'-,m -$m 25-m &'%&m 2&0-3%&j.32m 2#!&,'/3#1m ',m m ,-4#*m ..*'!2'-,[m
"#2#!2',%m .-',2m +322'-,1m ',m *0%#m m +322#"m .*,2m .-.3*2'-,1Yem *,2m #2&-"1m K[m
GIYm
m
+ *#ZmYYZm--,$'#*"ZmYYZm!&**#0ZmYYm^GOON_Yme'12'"',#m)',1#m!2'4'27m-$m
2&#mGm#2&7*#,#m0#!#.2-0m$0-+m0 '"-.1'1Yem0-!m2*m!"m!'mmmmOK^GI_[mMNHKjOYm
m
+ *#ZmYYZm3ZmYZ!&**#0ZmYYm^HFFH_Yme322'-,*m,*71'1m-$m2&#m#2&7*#,#m
0#!#.2-0mGYm-*#m-$m2&#m&'12'"',#m)',1#m"-+',m',m"-+',,2m#2&7*#,#m',1#,1'2'4'27Yem
*,2m&71'-*mGHN^J_[mGJHNjINYm
m
-ZmYZm
-&,1#,ZmYZm7#01ZmYZm&-+1ZmYYZm-#*m**'1ZmYYm7m!-*Ym^HFFJ_Yme&#m
.-274'031m 0#!#11'4#m 0#1'12,!#m %#,#Zm 1 +GZm '"#,2'$'#1m m ,-4#*m 0-*#m $-0m 20,1*2'-,m
','2'2'-,m$!2-0m#Jm',m!#**j2-j!#**m20$$'!)',%Yem*,2m
mJF^I_[mIMLjNKYm
m
0!'j1m Zm *'4#0Zm YZm A+#8m m 7m '!#,2#m m ^HFFF_Ym eZm m ,"m
m+0)#01m2-m+#130#m%#,#2'!m"'4#01'27m',m+#*-,Yem&#-0m..*m#,#2mGFG[mNLFjNLJYm
m
0!'j1Zm
YZm#,()ZmYZm,1#4#0',-ZmYZm-30%#-'1ZmYZm'0ZmYm7m!-*Ym^HFGH_Ym
e&#m%#,-+#m-$m+#*-,m^3!3+'1m+#*-mY_Yem0-!m2*m!"m!'mmmmGFO^HO_[mGGNMHjMYm
m
#,0"Zm YZm -3 *#Zm Ym ^HFFK_Ym eYm m 2&#-07m -$m $03'2m !*'+!2#0'!m #2&7*#,#m
#+'11'-,Yem*,2m&71'-*mGIO^G_[mKIGjJKYm
m
#.21m ^HFFH_Ym em !-+.0'1-,m #25##,m 0-.m -+#12'!2'-,Zm *11'!*m *,2m
0##"',%m,"m#,#2'!m,%',##0',%YemmJH^L_[mGMNFjGMOFYm
m
'*!&0'12ZmY
YZm3%&,ZmYYm^HFFK_Ymem5'2&-32mm.*-3%&[mm,#5m+#2&-"m
5'2&m..*'!2'-,1m$-0m0#4#01#m%#,#2'!1Yem300#,2m.','-,m',m*,2m'-*-%7mN[mHGGmjmHGKYm
m
'*!&0'12ZmYZmd#'*ZmYZm-1#ZmYZm#2)ZmYZm3%&,ZmYm^HFFL_Ymem'1m,m
#$$#!2'4#m 0#4#01#m %#,#2'!1m 2#!&,'/3#m $-0m #,-0& "'2'1m #*#%,1Yem m #,-+'!1m M^G_[m
HLHYm
m
GOMm
O
#&#)!, 7m
m
',%01ZmYYZm3%&2ZmYZm-,#, #0%ZmYm^GOOO_Yme#Jm','2'2'-,m$!2-01[m#$$#!2-01m
-$m+m0#!03'2+#,2m2-m0' -1-+#1m,"m0#%3*2-01m-$m20,1*2'-,Yem,,3m#4m'-!&#+m
LN[mOGIjLIYm
m
'-4,,-,'Zm Ym ^HFFG_Ym e-*#!3*0m '-*-%7m -$m 03'2m 2302'-,m ,"m '.#,',%Yem
,,3m#4m*,2m&71'-*m*,2m-*m'-*mKH[mMHKjMJOYm
m
'-4,,-,'Zm Ym ^HFFM_Ym e03'2m 0'.#,',%m +32,21m 7'#*"m ',1'%&21m ',2-m 0'.#,',%m
!-,20-*Yem300m.',m*,2m'-*mGF^I_[mHNIjOYm
m
'3*',-Zm YZm 02*#7Zm YYZm !-*,')Zm YYm ^GOOI_Ym e#%3*2'-,m -$m !0-2#,-'"m
'-17,2&#1'1m"30',%m2-+2-m"#4#*-.+#,2Yem*,2m#**mK^J_[mIMOjNMYm
m
-,89*#8ZmYYZm#,()ZmYZm#,$$ZmYYZm'0ZmYZm1!3 #02Zm
YYm7m!-*Ym^HFGFm
_Ym e#/3#,!',%m -$m LYMm m -$m 2&#m +#*-,m %#,-+#m 31',%m m m .--*',%m 1202#%7Yem m
*,2m'-*mGF[mHJLYm
m
-,89*#8Zm YYZm 0!'j1Zm YZm 0B1Zm YZm 3'%"-+#,#!&Zm Ym ^HFGFm _Ym
e#,#02'-,m -$m m j 1#"m .&71'!*m +.m -$m 2&#m +#*-,m %#,-+#Yem m #,-+'!1m GG[m
IIOYm
m
m
-,89*#8j #1Zm YZm *,!Zm YZm -'%Zm YZm -,8*#8j-Zm YZm '!-Zm Ym 7m !-*Ym ^HFFM_Ym
e[m,mm"2 1#m$-0m+#*-,m$3,!2'-,*m%#,-+'!1Yemm#,-+'!1mN[mIFLYm
m
-,8*#8j #1Zm YZm ,'80#1Zm YZm 0,"Zm YYm ^HFGH_Ym e'!0-007m ,*71'1m
1&-51m2&2m0#!#11'4#m0#1'12,!#m2-m2#0+#*-,m+-1'!m4'031m',m+#*-,m'1m11-!'2#"m5'2&m
2&#m',"3!2'-,m-$m"#$#,1#m0#1.-,1#m%#,#1Yem-*m*,2m'!0- #m,2#0!2mHK^G_[mGFMjGNYm
m
-,89*#8j-Zm YZm 3Zm YZm 12#01Zm YZm -'%Zm YZm -,$-02#Zm Y
Ym 7m !-*Ym ^HFGG_Ym
e-50"1m m m .*2$-0+m $-0m $3,!2'-,*m %#,-+'!1m ',m '#*m "#m .-m +#*-,1Yem m
#1m-2#1mJ[mHNOYm
m
-,8*-ZmY
YZm*'4#0ZmYZm0!'j1Zm
YZm-,$-02ZmYZm-*!#2j,(3,ZmYm7m!-*Ym
^HFFK_Ym e'+.*#j1#/3#,!#m 0#.#2m +0)#01m 31#"m ',m +#0%',%m *',)%#m +.1m -$m +#*-,m
^3!3+'1m+#*-mY_Yem&#-0m..*m#,#2mGGF^K_[mNFHjGGYm
m
0##, #0%Zm YYZ-Zm Ym ^HFFJ_Ym e&#m 0-*#m ,"m 0#%3*2'-,m -$m .0-%0++#"m !#**m
"#2&m',m.*,2j.2&-%#,m',2#0!2'-,1Yem#**m'!0- '-*mL^I_[mHFGjGGYm
m
0##,#ZmYYZm-"-+-ZmYYZm7*-0ZmYYZm#,')-$$Zm
YYZm'**ZmY
Ym7m!-*Ym^HFFI_Ym
e.#!203+m-$m!&#+'!**7m',"3!#"m+322'-,1m$0-+mm*0%#j1!*#m0#4#01#j%#,#2'!m1!0##,m',m
0 '"-.1'1Yem#,#2'!1mGLJ^H_[mMIGjJFYm
m
0-2#5-*"ZmYm^HFFL_Yme&#m%#,#2'!1m,"m '-!&#+'1207m-$m$*-0*m.'%+#,21Yem,,3m
#4m*,2m'-*mKM[mMLGjNFYm
m
03#, 3+Zm YZm 4#&j,7Zm YZm #"0Zm YZ8',Zm Ym ^GONG_Ym e#/3#,!#m
1.#!'$'!'27m-$m+#2&7*2'-,m',m&'%&#0m.*,2mYem230#mHOH^KNHL_[mNLFjHYm
m
3'1m Zm -31,Zm YZm -%'+-,2Zm YZm '202Zm YZm #!&Zm Ym ^GOON_Ym e#*-,m
'-2#!&,-*-%7Yem'-2#!&,-*m#,2m,%m#4mGK[mHNOjIGGYm
m
38+,ZmYZm!)#0Zm
YYm^GOOF_Yme6.*-'2',%m2&#m20'.*#m0#1.-,1#m-$m0 '"-.1'1m2-m
'"#,2'$7m#2&7*#,#j0#*2#"m+32,21Yem*,2m#**mH^L_[mKGIjHIYm
GONm
#&#)!, 7O
m
m
+"m YZm #20',-Zm YYZm )3,%Zm Ym ^GONH_Ym em ,-4#*m 0#.#2#"m #*#+#,2m
5'2&m jjm $-0+',%m .-2#,2'*m '1m 5'"#*7m $-3,"m ',m #4-*323-,07m "'4#01#m #3)0'-2'!m
%#,-+#1Yem0-!m2*m!"m!'mmmmMO[m[mLJLKjLJLOYm
m
,"#*Zm Ym ^GONH_Ym e7,+'!1m -$m %#,#m $*-5m ',m ,m #6.#0'+#,2*m .-.3*2'-,m -$m
3!3+'1m+#*-mem+#0Ym
Ym-2mLO^GF_[mGKINjGKJLYm
m
1#ZmYZm+-)ZmYZm"ZmYZm'0-'ZmYZm)+-2-ZmYm7m!-*Ym^HFFF_Yme0#m3,"#0m
2&#m .*1+m !-,!#,202'-,j2'+#m !304#m -$m ',-0%,'!m $*3-0'"#m $-**-5',%m 1#4-$*30,#m
,#12&#1'm !-00#*2#1m 5'2&m HGm +m *#4#*m ',m +-,-,3!*#0m !#**1Yem ,#12&#1'-*-%7m
OH^L_[mGLLGjLYm
m
#,')-$$ZmYZm-+'ZmYm^HFFI_Yme',%*#j,3!*#-2'"#m+322'-,1m$-0m.*,2m$3,!2'-,*m
%#,-+'!1Yem,,3m#4m*,2m'-*mKJ[mIMKjJFGYm
m
'0-!&')ZmYZm3'"#0"-,'ZmYZm,ZmYZm1',%ZmYYZm3,ZmYYm7m!-*Ym^HFFJ_Yme'!#m
+32,2m0#1-30!#1m$-0m%#,#m"'1!-4#07Yem*,2m-*m'-*mKJ^I_[mIHKjIJYm
m
'01!& #0%Zm Ym ^HFFG_Ym e0-2#,-'"m '-17,2&#1'1m ',m $*-5#0',%m .*,21Yem 300#,2m
.','-,m',m*,2m'-*-%7mJ^I_[mHGFjHGNYm
m
-1.'2*ZmYm^HFFO_Yme&**#,%#1m$-0m#$$#!2'4#m+0)#0j11'12#"m1#*#!2'-,m',m.*,21Yem
#,#2'!mGIL^H_[mIFIjGFYm
m
3Zm YZm &,%Zm YZm 3,Zm YZm #7#0-5'28Zm YYm ^GOOK_Ym e2&7*#,#m ',1#,1'2'4'27m
!-,$#00#"m 7m0 '"-.1'1mm%#,#Yem!'#,!#mHLO^KHIG_[mGMGHjJYm
m
#&'1Zm YYZm &'+'83Zm YZm 2-Zm YZm 13"Zm YZm )3+'Zm Ym ^HFGH_Ym e'1!-4#07m -$m
'%&j-,$'"#,!#m ',%*#m 3!*#-2'"#m -*7+-0.&'1+1m $0-+m 0%#j!*#m #m -4-m
,*71'1m-$m#$m0,1!0'.21m-$m#%'*-.1m231!&''Zmm'*"m&#2m0-%#,'2-0Yemm#1Ym
m
1!1-,Zm YZm -,#,Zm YZm +'0Zm YZm '01!& #0%Zm Ym ^HFFH_Ym e*-,',%m -$m 2,%#0',#m
$0-+m 2-+2-m 0#4#*1m m !0-2#,-'"m '1-+#01#m #11#,2'*m $-0m 2&#m .0-"3!2'-,m -$m #2j
!0-2#,#m,"m6,2&-.&7**1m',m.*,21Yem*,2m#**mGJ^H_[mIIIjJHYm
m
8/3'#0"-Zm Ym ^HFFG_Ym e,%#,'#0>m %#,<2'!m 7m 20,1$#0#,!'m %<,'!eYm "'!'-,#1m
'09+'"#mYYm"0'"Ym
m
#$$0#7Zm Ym ^GOOFY_Ym .>23*-[m ,m -32*',#m !*11'$'!2'-,m -$m 2&#m 3!30 '2!##Ym ,[m
2#1ZmYYZm- ',1-,ZmYYZm
#$$0#7ZmYme'-*-%7m,"m32'*'82'-,m-$m2&#m3!30 '2!##eYm
2&!m,"m-,"-,m-0,#**m,'4#01'27[m.YJJOjJLIYmJNK.Ym
m
-- #30Zm YZm ',%Zm Y
YZm -*',Zm Y
YZm &-+1Zm YYZm #,Zm YYm ^HFFJ_Ym e&#m
310'3+m 5'*2m 0#1'12,!#m *-!31m -+jHm -$m +#*-,m !-,2',1m m 1',%*#m 0#1'12,!#m %#,#m 5'2&m
!-+.*#6m$#230#1Yem*,2m
mIO^I_[mHNIjOMYm
m
28'0ZmYZm,',j-*#%ZmYZm830'ZmYZm0!&'ZmYm^GOOL_Yme#,%2&m.-*7+-0.&'1+m
,"m&-+-*-%'#1m-$m+'!0-12#**'2#1m',m1#4#0*m3!30 '2!#1m1.#!'#1Yem&#-0m..*m#,#2m
OI[mGHNHjGHOFYm
m
##,Zm YYm ^GOOF_Ym e#,#j$-0j%#,#m !-+.*#+#,20'27m ',m .*,2j.2&-%#,m
',2#0!2'-,1Yem,,3m#4m#,#2mHJ[mJJMjLIYm
m
#,"**ZmY
YYm^GONN_Yme#,#2'!j40'2'-,m-$m#2&7*#,#m.0-"3!2'-,m',m&04#12#"mm
+31)+#*-,m$03'21Yem-02!'#,!#mHI[mMKOjMLGYm
GOOm
O
#&#)!, 7m
m
*##ZmYZm'#+,YmYm^GOOO_Yme'$$#0#,2'*m6.0#11'-,m-$m5-m-4#*m#+ #01m-$m
2&#m-+2-m2&7*#,#j#!#.2-0m+'*7Yem*,2m&71'-*mGHF[mGLKjGMHYm
m
,-**Zm YYZm +-1Zm YYZm #,%Zm YZm -* 0--)Zm YYZm &-5Zm Ym 7m !-*Ym ^HFGG_Ym
em$-0m**#0%#,m0#"3!2'-,m,"m'+.0-4#+#,2m-$m/3*'27m20'21m',m.#,32m^0!&'1m
&7.-%#mY_Yemm*,2m'-*mGG[mNGYm
m
-"7+ZmYZm$8ZmYm^HFFI_Yme&71'!*m,"m!&#+'!*m+32%#,#1'1Yem#2&-"1m-*m
'-*mHIL[mGNOjHFJYm
m
-,'#!8,7Zm YZm 313 #*Zm Ym ^GOOI_Ym em .0-!#"30#m $-0m +.',%m 0 '"-.1'1m
+322'-,1m 31',%m !-j"-+',,2m #!-27.#j1.#!'$'!m j 1#"m +0)#01Yem *,2m -30,*m J[m
JFIjJGFYm
m
--0,,##$ZmYZm#**#02ZmYYZm,m"#0m##,Zm
YYm^GONH_Ymejm,"m0"'2'-,j
',"3!#"m +322'-,m $0#/3#,!'#1m 2m ',"'4'"3*m *-!'m ',m 0 '"-.1'1m 2&*',m ^Y_m #7,&Yem
322m#1mOI^G_[mGFOjHIYm
m
071,Zm Ym ^HFFJ_Ym e!#j!.Ym m &'%&j2&0-3%&.32m +#2&-"m $-0m !.230',%m .*,2m
2'113#m1+.*#1m$-0m%#,-27.#m,*71'1Yem*,2m&71'-*mGIK^I_[mGGLHjOYm
m
3,%Zm YZm "+, &,Zm YZm 'Zm YZm +#'Zm YZm &1)0Zm YYm 7m !-*Ym ^HFFO_Ym
e"#,2'$'!2'-,m -$m +','230#m ',4#02#"j0#.#2m 20,1.-1 *#m #*#+#,21m ^1_m ,"m
'-%#,#1'1m -$m 2&#'0m 1'1m ',m 2&#m -*,!##[m ,#5m $3,!2'-,*m '+.*'!2'-,1m $-0m 1Yem
#,-+#m#1mGO^G_[mJHjKLYm
m
3+0ZmYZm+0!&#ZmY
YZm1'ZmYYm^HFFM_Yme1#m-$m"+%#"mm,"m"m
13 1202#1m 7m 2&#m #00-0j.0-,#m m .-*7+#01#m m $0-+m $0'!,m 15',#m $#4#0m 4'031Yem
'-!&#+'1207mJL^GH_[mINGJjHKYm
m
,&,Zm YYZm #,Zm YYZm '-4,,-,'Zm YZm *##Zm Y
Ym ^GOOJ_Ym e&#m ,#4#0m 0'.#m
+322'-,m *-!)1m#2&7*#,#m.#0!#.2'-,m',m2-+2-Yem*,2m#**mL^J_[mKHGjIFYm
m
#m '%,-0Zm YZm 4-'1Zm YZm 3 #02Zm YZm #0"'#0Zm YZm '!-*1Zm Ym 7m !-*Ym ^HFFO_Ym
e.2'+'8',%m m .-.3*2'-,1m $-0m 0#4#01#m %#,#2'!1m ',m #"'!%-m 203,!23*Yem *,2m
'-2#!&,-*m
mM^K_[mJIFjJGYm
m
#&+,Zm YZm *!)Zm YZm !)#0Zm YYm ^GOOL_Ym eGZm ,m #2&7*#,#m 0#1.-,1#m
%#,#Zm '1m 0#/3'0#"m $-0m "'$$#0#,2'*m !#**m #*-,%2'-,m ',m 2&#m 0 '"-.1'1m &7.-!-27*Yem #**m
NK^H_[mGNIjOJYm
m
#**'1Zm YYZm 11!&3Zm YYZm &'2&+Zm YYZm 00',%2-,Zm YYm ^HFFH_Ym e-11j-$j
131!#.2' '*'27m +32,21m -$m 0 '"-.1'1m 2&*',m 0#4#*m ,m #11#,2'*m 0-*#m $-0m #^'1-_Jm
"30',%m.-274'031m',$#!2'-,Yem300m'-*mGH^GH_[mGFJLjKGYm
m
#-,0"ZmYZm*,2#ZmYZm'22+,,ZmYZm'%,#3*2ZmYZm-02',ZmYYm7m!-*Ym^HFFF_Ym
e-+.*#6m$-0+2'-,m #25##,m.-274'031m%m,"m20,1*2'-,m#3)07-2'!m','2'2'-,m$!2-0m
Jm!-00#*2#1m5'2&m4'031m',$#!2'4'27Yem
m'0-*mMJ^GM_[mMMIFjMYm
m
#5'1ZmYZm- #021mYZm* #021m^HFFL_Ymem<*3*Yem"'!'-,#1m+#%Ym"0'"Ym
m
'ZmYZm-,%ZmYZm#,2307ZmYZm20'%&2ZmYZm-,*"ZmYm7m!-*Ym^HFFG_Ymem$12m,#320-,m
"#*#2'-,m+32%#,#1'1j 1#"m0#4#01#m%#,#2'!1m1712#+m$-0m.*,21Yem*,2m
mHM^I_[mHIKjJHYm
m
'ZmYZm&,%ZmYm^HFFH_Yme#4#01#m%#,#2'!1m 7m$12m,#320-,m+32%#,#1'1m',m&'%&#0m
.*,21Yem3,!2m,2#%0m#,-+'!1mH^L_[mHKJjNYm
HFFm
#&#)!, 7O
m
m
'Zm YYZm 22&#51Zm YYZm 300Zm YZm 3028#*Zm YYm ^GOOL_Ym e*-,',%m ,"m
!&0!2#0'82'-,m -$m m +'8#m !m #,!-"',%m .&72-#,#m "#12301#Zm ,m #,87+#m -$m 2&#m
!0-2#,-'"m '-17,2&#2'!m.2&57Yem*,2m-*m'-*mIF^H_[mHLOjMOYm
m
',"#,Zm YZm '15Zm YZm &+-4'28Zm YZm #!)#0Zm YZm '01!& #0%Zm Ym 7m !-*Ym ^GOOG_Ym
e3,!2'-,*m!-+.*#+#,22'-,m',m1!&#0'!&'m!-*'m-$m"'$$#0#,2m.&72-#,#m"#12301#m%#,#1m
,"m,*71'1m-$m!!3+3*2#"m!0-2#,#1Yemm230$-01!&mmJL^GGjGH_[mGFJKjKGYm
m
A.#8j0Zm YYZm -&*#,)+.Zm YZm #'%#0Zm Ym ^HFFL_Ym e&#m .-12j%#,-+'!m #0jj
5&2m!-+#1m,#62]em#4m2',-+m'!0- '-*mJN^G_[mJJjKYm
m
A.#8j#8Zm
YYZm*-01ZmYZm0!'Zm
YYZm-8-ZmY
Ym^HFGG_Ymem17+ '-1'1m*2#01m
.&#,-*'!m!'"m!-,2#,2m',m2-+2-m0--21Yem*,2m'%,*m#&4mK^O_[mGGINjJFYm
m
3ZmYZm02',j#0,,"#8ZmYYZm#02Zm
YYZm*!3'2ZmYZm3*!-+ #ZmYYm^HFFI_Ym
e'031j',"3!#"m%#,#m1'*#,!',%m',m.*,21Yem#2&-"1mIF^J_[mHOLjIFIYm
m
3," #0%Zm YYZm &-#+)#0Zm YYZm "+1Zm YYZm &-02Zm YYZm -0%#Zm YYm 7m !-*Ym
^GOOG_Ym e'%&j$'"#*'27m +.*'$'!2'-,m 31',%m m 2&#0+-12 *#m m .-*7+#01#m '1-*2#"m
$0-+m70-!-!!31m$30'-131Yem#,#mGFN^G_[mGjLYm
m
3-Zm YZm ,%Zm YYZm 0'1!&Zm YZm -- #30Zm YZm ',%Zm YYm 7m !-*Ym ^HFFG_Ym e#*-,m
!2#0'*m 02'$'!'*m !&0-+-1-+#m ^_m *' 007m !-,1203!2'-,m 31',%m '+.0-4#"m +#2&-"1m
,"m'"#,2'$'!2'-,m-$m!*-,#1m*',)#"m2-m2&#m*-!31m!-,$#00',%m0#1'12,!#m2-m+#*-,m310'3+m
5'*2m^-+jH_Yem#,-+#mJJ^H_[mGKJjLHYm
m
,8--0ZmYZm&##+ZmYm^HFFI_Yme"'-1#,1'2'4'27m123"'#1m',m 1+2'm0'!#Yem)Ym
Ym-2mIK^H_[mGOMjHFMYm
m
02',ZmYZm0-"#!ZmYZm-3*#+ZmYZm( ZmYZm#0,,"#8ZmYm7m!-*Ym^HFFO_Ymem
20,1.-1-,j',"3!#"m #.'%#,#2'!m !&,%#m *#"1m 2-m 1#6m "#2#0+',2'-,m ',m +#*-,Yem 230#m
JLG^MHLM_[mGGIKjNYm
m
02',ZmYYZm-%",-4#ZmY
YZm#11ZmYm^HFFI_Yme,"#012,"',%m2&#m$3,!2'-,1m-$m
.*,2m"'1#1#m0#1'12,!#m.0-2#',1Yem,,3m#4m*,2m'-*mKJ[mHIjLGYm
m
1!0#**j0#31ZmYZm,'80#1Zm
YZm'*001j*1'Zm
YZm-0j0!'ZmYZm*,!Zm
Ym
7m!-*Ym^HFFO_Yme,m-*'%-j 1#"m+'!0-007m-$$#01m,-4#*m20,1!0'.2-+'!m..0-!&#1m$-0m2&#m
,*71'1m -$m .2&-%#,m 0#1'12,!#m ,"m $03'2m /3*'27m 20'21m ',m +#*-,m ^3!3+'1m +#*-m Y_Yem
m#,-+'!1mGF[mJLMYm
m
1&*ZmYYZm--,28Zm
YZm)*0Zm
Ym^GOOK_Yme#2#!2'-,m-$m+322'-,1m 7m!*#4%#m-$m
m&#2#0-"3.*#6#1m5'2&m !2#0'-.&%#m0#1-*41#1Yem2m#,#2mO^H_[mGMMjNIYm
m
!**3+Zm YYZm -+'Zm YZm 0##,#Zm YYZm #,')-$$Zm Ym ^HFFFm _Ym e0%#2#"m
1!0##,',%m$-0m',"3!#"m+322'-,1Yem2m'-2#!&,-*mGN^J_[mJKKjMYm
m
!**3+Zm YYZm -+'Zm YZm 0##,#Zm YYZm #,')-$$Zm Ym ^HFFFm _Ym e0%#2',%m
',"3!#"m *-!*m *#1'-,1m m %#,-+#1m ^_m $-0m .*,2m $3,!2'-,*m %#,-+'!1Yem *,2m
&71'-*mGHI^H_[mJIOjJHYm
m
!027Zm YYZm #22*#1Zm YYZm 383)'Zm YZm ,Zm YYZm 21&5Zm Ym 7m !-*Ym ^HFFK_Ym
e2#"7j122#m20,1.-1-,m+32%#,#1'1m',m', 0#"m+'8#Yem*,2m
mJJ^G_[mKHjLGYm
m
!-5#**Zm YYZm -$$#,"#,Zm Y
Ym ^HFFI_Ym e*,2m "'1#1#m 0#1'12,!#m %#,#1[m 0#!#,2m
',1'%&21m,"m.-2#,2'*m..*'!2'-,1Yem0#,"1m'-2#!&,-*mHG^J_[mGMNjNIYm
HFGm
O
#&#)!, 7m
m
!'**',Zm YYZm !,"*'-1Zm YYm ^GONI_Ym e#,#2'!m ,*71'1m -$m 2&#m "3.*'!2#"m
+'2-!&-,"0'*m,"m!72-1-*'!m+*2#m"#&7"0-%#,1#m'1-87+#1m',m+'8#Yem1-87+#1m300m
-.m'-*m#"m#1mN[mLMjOFYm
m
#',)#Zm YYZm &#007Zm YYZm #,Zm YZm -3,1*#7Zm YYZm --0,,##$Zm Ym ^GOON_Ym
e0 '"-.1'1m2&*',[mm+-"#*m.*,2m$-0m%#,-+#m,*71'1Yem!'#,!#mHNH^KINO_[mLLHZmLMOj
NHYm
m
m
m
#7#0Zm YYZm #*#3Zm YZm 0!'j1Zm YZm 4#7Zm Y
Ym ^HFFN_Ym e-,1203!2'-,m -$m m
$-1+'"m *' 007m -$m !3!3+ #0m ^3!3+'1m 12'431_m ,"m !-+.02'4#m ,*71#1m -$m 2&#m #Jm
,"m #^'1-_Jm 0#%'-,1m $0-+m !3!3+ #0m ,"m +#*-,m ^3!3+'1m +#*-_Yem -*m #,#2m
#,-+'!1mHMO^K_[mJMIjNFYm
m
#7#01Zm YYZm '!)#0+,Zm YYZm '!&#*+-0#Zm YYZm '40+)0'1&,,Zm YZm
- 0*ZmYYm7m!-*Ym^GOOO_Yme*,2m"'1#1#m0#1'12,!#m%#,#1m#,!-"#m+#+ #01m-$m,m,!'#,2m
,"m "'4#01#m .0-2#',m $+'*7m 5'2&',m 2&#m ,3!*#-2'"#j ',"',%m 13.#0$+'*7Yem *,2m m HF^I_[m
IGMjIHYm
m
',-'ZmYZm#20-88ZmYZmd,-$0'-ZmYZm'0-,ZmYZm-1!ZmYm7m!-*Ym^HFGF_Ymem,#5m
+32,2m%#,#2'!m0#1-30!#m$-0m2-+2-m!0-.m'+.0-4#+#,2m 7mm2#!&,-*-%7Yem m
#1m-2#1mI[mLOYm
m
-,$-02#ZmY
YZm*'4#0ZmYZm-,8*-ZmY
YZm*40#8Zm
YYZm-*!#2j,(3,ZmYm7m!-*Ym
^HFFJ_Ym e"#,2'$'!2'-,m -$m /3,2'22'4#m 20'2m *-!'m ',4-*4#"m ',m $03'2m /3*'27m 20'21m ',m +#*-,m
^3!3+'1m+#*-mY_Yem&#-0m..*m#,#2mGFN^J_[mMKFjNYm
m
-,2#*-,#YZm YYm ^GOON_Ym e322'-,Zm 32%#,1Zm ,"m m #.'0Yem "'2-0'*[m
3+,m#,#2'1m"'2-0Ym,11m22#m,'4#01'27Ym,11Ym
m
--0#ZmYZm0# *-4Zm
YZm72-,ZmYZ'-4,,-,'Zm
Ym^HFFH_Yme1#m-$m%#,-+'!1m2--*1m
2-m'1-*2#m)#7m0'.#,',%m%#,#1m,"m,*71#m$03'2m+2302'-,m',m2-+2-Yem
m6.m-2mKI^IMM_[m
HFHIjIFYmm
m
-0#,-Zm YZm ,"-Zm YYZm -1j,2-1Zm YZm #0,,"#8j03('**-Zm YYZm -,$-02#Zm
Y
Ym7m!-*Ym^HFFN_Yme,"'"2#m%#,#1m,"m1m$-0m$03'2m0'.#,',%m,"m1-$2#,',%m',m+#*-,Yem
&#-0m..*m#,#2mGGL^J_[mKNOjLFHYm
m
-0%,2#ZmYZm*'4'#0'ZmYYm^GOOI_Ymej.*'$'#"m+'!0-12#**'2#1m1mm+0)#01m
',m.*,2m%#,#2'!1mem*,2mm
mI[mGMKjGNKYm
m
+ 0Zm YZm #'2&Zm YZm !-302Zm YZm '2-Zm Ym ^GOOJ_Ym e1-*2'-,m -$m ,m ',2#0,*m
"#*#2'-,m+32,2m-$m2&#m0 '"-.1'1m2&*',mIm%#,#Yem*,2m#**m&71'-*mIK^I_[mKFOjGIYm
m
'!'1#Zm YZm #0+,j#2,Zm YZm ,(3,Zm YZm 3 0,Zm YYZm 8'#0Zm Ym 7m !-*Ym
^HFFI_Ym e&#m #3)07-2'!m 20,1*2'-,m ','2'2'-,m $!2-0m Jm !-,20-*1m *#223!#m 131!#.2' '*'27m 2-m
2&#m-274'031m#223!#m+-1'!m4'031Yem*,2m&71'-*mGIH^I_[mGHMHjNHYm
m
'#2-Zm YZm -0*#1Zm YZm 0(#"Zm YZm *#.#2Zm YZm -,$-02Zm Ym 7m !-*Ym ^HFFL_Ym e,m
#Jm **#*#m !-,$#01m 0#1'12,!#m 2-m ,m 3,!..#"m ,"m ,-,j.-*7"#,7*2#"m m 4'031m ',m
+#*-,Yem*,2m
mJN^I_[mJKHjLHYm
m
'#2-Zm YZm '0-,Zm YZm *+'1Zm YZm 0!-Zm YYZm -0'-,#1Zm Ym 7m !-*Ym ^HFFM_Ym
e!-m $-0m 2&#m '"#,2'$'!2'-,m -$m **#*'!m 40',21m -$m +#*-,m #JZm m $!2-0m 2&2m
!-,20-*1m4'031m131!#.2' '*'27Yemm*,2m'-*mM[mIJYm
HFHm
#&#)!, 7O
m
m
'1&'7+Zm YZm 3'1Zm YZm -1#Zm YYZm 3 -Zm YZm #,,#22Zm YYm 7m !-*Ym ^HFFM_Ym
e2&7*#,#m0#%3*2'-,m-$m$03'2m1-$2#,',%m,"m!#**m5**m"'111#+ *7m',m&0#,2'1m+#*-,Yem
m
6.m-2mKN^L_[mGHNGjOFYm
m
-,)Zm YZm 3&1&'Zm YZm )"Zm YZm 3%50Zm YZm ',+'15Zm Ym ^HFFN_Ym
e2&7*#,#m .0-"3!2'-,m ',m .*,21m "30',%m %#,#m 20,1$-0+2'-,m 13..0#11#1m 4'0m %#,#m
#6.0#11'-,m',m%0- !2#0'3+m23+#$!'#,1Yem#5m&72-*-%'12mGMN[mLJMjLKLYm
m
3,#8j*#,'31Zm YYZm ,2*'$$#Zm Y
YZm 3 #0Zm Y
YZm '0"'Zm YZm *##Zm Y
Ym ^HFFL_Ym
e0,1$-0+2'-,m -$m m +31)+#*-,m d*'dm &7 0'"m .0#,2*m *',#m ^3!3+'1m +#*-m Ym 40Ym
0#2'!3*231m#0Y_m5'2&m,m,2'1#,1#mm-6'"1#m%#,#Yem*,2m#**m#.mHK^I_[mGONjHFKYm
m
3,#8j*#,'31ZmYYZm-+#8j'+ZmYZm!&-j*#(-ZmYZm03+#2ZmYZm#12#0ZmYm
7m !-*Ym ^HFFN_Ym e#*-,m $03'21[m %#,#2'!m "'4#01'27Zm .&71'-*-%7Zm ,"m '-2#!&,-*-%7m $#230#1Yem
0'2m#4m'-2#!&,-*mHN^G_[mGIjKKYm
m
*#7)-51)'Zm YYZm 0-,1-,m 3**',1Zm YYZm -"5',Zm YYZ#3,%Zm YYm ^GOON_Ym
e322'-,m"#2#!2'-,m31',%mm,-4#*m.*,2m#,"-,3!*#1#Yem3!*#'!m!'"1m#1mHL^HF_[mJKOMj
LFHYm
m
*'4#0ZmYZm0!'j1Zm
YZm0"B1ZmYZm3#7-ZmYZm-.#8j#1#ZmYYm7m!-*Ym^HFFG_Ym
e-,1203!2'-,m-$mm0#$#0#,!#m*',)%#m+.m$-0m+#*-,Yem#,-+#mJJ^K_[mNILjJKYm
m
..#,&#'+#0Zm YYZm #47Zm YYZm ',&Zm YZm #*j"'Zm YZm .388-Zm YYm 7m !-*Ym
^GOOH_Ym e"'-,#!0-1'1m 1#!-,"07m 2-m ',2#012'2'*m 0!&72&#0.7[m !-00#*2'-,m -$m +%,#2'!m
0#1-,,!#m'+%',%m,"m&'12-.2&-*-%7Yem#30-130%#07mIG^H_[mIILjJIYm
m
11-51)'ZmYZm!&,## #0%#0ZmYZm3!1j*#"-Zm
YYZm02&+,,ZmYZm*0)ZmYYm7m
!-*Ym^HFGF_Yme&#m02#m,"m+-*#!3*0m1.#!203+m-$m1.-,2,#-31m+322'-,1m',m0 '"-.1'1m
2&*',Yem!'#,!#mIHM^KOLG_[mOHjJYm
m
0)#0Zm YYZm -*#+,Zm Y
Ym ^GOOM_Ym e-*#!3*0m ',2'+!7m #25##,m .0-2#',1m
1.#!'$7',%m.*,2j.2&-%#,m0#!-%,'2'-,Yem0#,"1m'-!&#+m!'mHH^N_[mHOGjLYm
m
3*',#ZmYZm#,')-$$ZmYm^HFFG_Yme0#"'!2',%m"#*#2#0'-31m+',-m!'"m13 12'232'-,1m
-,m.0-2#',m$3,!2'-,Yem#,-+#m#1mGG^K_[mNLIjMJYm
m
3*',#ZmYmZm#,')-$$ZmYm^HFFI_Yme[m.0#"'!2',%m+',-m!'"m!&,%#1m2&2m$$#!2m
.0-2#',m$3,!2'-,Yem3!*#'!m!'"1m#1mIG^GI_Ym
m
3/3#2Zm YZm 30%3#2Zm YZm %#,Zm Ym ^HFFJ_Ym .j 1#"m !*-,',%m -$m 2&#m 2m %#,#m
$0-+m+#*-,m!-,$#00',%m0#1'12,!#m2-m -2&m.&'"m!-*-,'82'-,m,"m.&'"m20,1+'11'-,m-$m
1#4#0*m4'031#1Ym,[m# #"ZmYZm0'1ZmYm"'2-0'*Ym*!)m,'4#01'27Zm*-+-3!Zm8#!&m
#.3 *'!Ym
m
#!)#0Zm YZm &+-4'28Zm YZm ',"#,Zm YZm ,"+,,Zm YZm '01!& #0%Zm Ym ^GOOH_Ym em
1',%*#m .-*7.#.2'"#m !2*78',%m 2&#m !-,4#01'-,m -$m .&72-#,#m 2-m 8#2j!0-2#,#m '1m
20,1!0'.2'-,**7m 0#%3*2#"m "30',%m 2-+2-m $03'2m 0'.#,',%Yem 0-!m 2*m !"m !'m m m m
NO^GG_[mJOLHjLYm
m
#0',ZmYZmA+#8j
'+<,#8ZmYZm%#,ZmYZm-%'+-,2ZmYZm#!&Zm
YYm7m!-*Ym^HFFH_Ym
e-*#!3*0m ,"m %#,#2'!m !&0!2#0'82'-,m -$m m ,-,j!*'+!2#0'!m .&#,-27.#m ',m +#*-,m
0#4#*1m25-m*-!'m!-,$#00',%m*2#0#"m#2&7*#,#m0#1.-,1#m',m$03'2Yem*,2m&71'-*mGHO^G_[mIFFj
OYm
HFIm
O
#&#)!, 7m
m
#007Zm YYZm ,%Zm YYZm #*&+Zm Y
YZm 0",#0Zm YZm ')#Zm YYm 7m !-*Ym ^HFFI_Ym em
m0#4#01#m%#,#2'!1m2--*m,"mm5# j!!#11' *#m!-**#!2'-,m-$m+32,21m-$m2&#m*#%3+#m
-231m(.-,'!31Yem*,2m&71'-*mGIG[mNLLmjmNMGYm
m
'0-,Zm YZm '!-*'Zm YZm ',-'Zm YZm '#",-'0Zm YZm -0#22'Zm Ym 7m !-*Ym ^HFGF_Ym e,m
',"3!#"m +322'-,m ',m 2-+2-m #Jm *#"1m 2-m '++3,'27m 2-m 25-m .-274'031#1Yem -m ,#m
K^L_[m#GGIGIYm
m
'202Zm YZm ,#*2Zm YZm ++#0Zm ^HFFF_Ym e-+#m !-++#,21m -,m ',$01.#!'$'!m
!*11'$'!2'-,m-$m!3*2'401m-$m+#*-,Yem!2m-02Ym^_mKGF[mHOjIYm
m
-02,-7ZYZ' #0ZYZ-**-!)ZYZ0!&'Zm YZ#4Zm Ym ^HFGG_Ym e1#m -$m ,-,j
,-0+*'8#"Zm -,j+.*'$'#"m !m $-0m JKJj1#"m m #/3#,!',%m -$m *#1&7m #*-,m
03'2eYm*,2m#,Ym^J_[mILjJLYm
m
0-44'"#,2'Zm YZm +.2-,Zm YYm ^GOOH_Ym e-30!#1m -$m 0#1'12,!#m 2-m 4'031#1m ',m 2&#m
-274'0'"#Yem0!&m'0-*m3..*mK[mGNOjHGGYm
m
',m Zm 3Zm YZm Zm YZm #,%Zm YZm #,%Zm YZm 7m !-*Ym ^HFFM_Ym e'103.2'-,m -$m
.&72-#,#m "#12301#m %#,#m 0#13*21m ',m * ',-m ,"m "50$m .&#,-27.#1m ',m 0 '"-.1'1m 7m
'+.'0',%m !&*-0-.&7**Zm !0-2#,-'"Zm ,"m %' #0#**',m '-17,2&#1'1Yem #**m #1#0!&Ym GM^K_[m
JMGjNHYm
m
3"0,ZmYZm-"0>%3#8ZmYYZmA.#8ZmYZm#0+B"#8ZmYZm3,#1j#1'ZmYm7m!-*Ym
^HFGG_Yme-3.*',%m4'031j',"3!#"m%#,#m1'*#,!',%m2-m#6-%#,-31m%0##,m$*3-0#1!#,!#m.0-2#',m
#6.0#11'-,m .0-4'"#1m m &'%&*7m #$$'!'#,2m 1712#+m $-0m $3,!2'-,*m %#,-+'!1m ',m 0 '"-.1'1m
,"m!0-11m**m12%#1m-$m2-+2-m$03'2m"#4#*-.+#,2Yem*,2m&71'-*mGKL^I_[mGHMNjOGYm
m
<"#'ZmYYZm-,!8YZmm^GOOH_Yme*11'!*m+32%#,#1'1Yem#2&-"1m',m0 '"-.1'1m
0#1#0!&YmHI^N_mGIJYm
m
'!)ZmYZm3*2#0ZmYm^GOKL_Yme72-%#,#2'!1m-$m2&#m2-+2-Yem"4m#,#2m[mHLMj
INHYm
m
'%-*ZmYZm,m#4#0#,Zm
YZm
,11#,ZmYZm-,,#ZmYZm!&,#'"#01ZmYm7m!-*Ym^HFFO_Ym
e'%&j2&0-3%&.32m"#2#!2'-,m-$m',"3!#"m+322'-,1m,"m,230*m40'2'-,m31',%m#7-',2m
2#!&,-*-%7Yem-m,#mJ^I_[m#JMLGYm
m
- %*'Zm YZm 0,2Zm Ym ^HFFL_Ym e0,1*2'-,m ','22'-,m $!2-01[m m 5#)m *',)m ',m
.*,2mm4'031m',$#!2'-,Yemm',m*,2m!'#,!#mGG^G_Ym
m
-"0>%3#8Zm YYZm 0#'0#Zm YYZm +'**#0'Zm YZm - %*'Zm Ym ^GOON_Ym e&#m
0 '"-.1'1m 2&*',m !1m !-"',%m $-0m #Jm ,"m #^'1-_Jm 0#m ,-2m $3,!2'-,**7m
#/3'4*#,2m $-0m 7#12m !-+.*#+#,22'-,m ,"m 0#m "'$$#0#,2'**7m #6.0#11#"m "30',%m .*,2m
"#4#*-.+#,2Yem*,2m
mGI^J_[mJLKjMIYm
m
-"0>%3#8j#0,9,"#8Zm YYZm -1*4#8Zm YZm #+.#0#Zm YYZm 30%-1Zm YZm 0,"Zm
YYm7m!-*Ym^HFGH_Yme#*-,mm',2#0$#0#,!#m^'_m*',#1m1'*#,!#"m$-0m+j#Jm1&-5m
0-"m4'031m0#1'12,!#Yem-*m*,2m2&-*mGI^M_[mMKKjLIYm
m
-+#0Zm YZm &,Zm YZm -0",Zm YZm 20311Zm YZm -,1Zm Ym 7m !-*Ym ^HFFM_Ym e*,2m
.2&-%#,m 0#!-%,'2'-,m +#"'2#"m 7m .0-+-2#0m !2'42'-,m -$m 2&#m .#..#0m 1Im 0#1'12,!#m
%#,#Yem!'#,!#mIGN^KNKF_[mLJKjNYm
HFJm
#&#)!, 7O
m
m
-1#m
YYZm"$'#*"ZmYYm^GOON_Yme#+.-0*m1#/3#,!#m-$m!#**m5**m"'111#+ *7m
',m0.'"*7m0'.#,',%m+#*-,m$03'2ZYem*,2m&71'-*mGGM[mIJKjILGYm
m
-12Zm YZm ,"#0Zm Ym ^GOOI_Ym e0#"'!2'-,m -$m .0-2#',m 1#!-,"07m 1203!230#m 2m #22#0m
2&,mMFPm!!30!7Yem
m-*m'-*mHIH^H_[mKNJjOOYm
m
3$$#*ZmYZm3113*2ZmYYZm**-'6ZmYZm-307ZmYZm#,"&+,#ZmYm7m!-*Ym^HFFH_Ym
em,230*m0#!#11'4#m0#1'12,!#m%#,#m%',12m.-22-m4'031mm',m.#..#0m!-00#1.-,"1m2-m2&#m
#3)07-2'!m','2'2'-,m$!2-0mJm^#J_Yem*,2m
mIH^L_[mGFLMjMKYm
m
3$$#*Zm YZm 0,2Zm YZm **-'6Zm YZm #$# 40#Zm YZm -!&#Zm Ym 7m !-*Ym ^HFFJ_Ym
e203!230*m ,*71'1m -$m 2&#m #3)07-2'!m ','2'2'-,m $!2-0m Jm %#,#m !-,20-**',%m .-274'031m
0#1'12,!#m',m.#..#0[m#6.*-'22'-,m-$mmm*' 007Yem#,#mIIN^H_[mHFOjHGLYm
m
3$$#*ZmYZm**-'1Zm
YYZm#1%#ZmYYZm0,2ZmYm^HFFK_Yme&#m0#!#11'4#m.-274'031m
0#1'12,!#m %#,#m .-2jGm '1m 2&#m 2-+2-m -02&-*-%3#m -$m 2&#m .#..#0m .40Hj#Jm %#,#Yem -*m
#,#2m#,-+'!1mHMJ^J_[mIJLjKIYm
m
#22ZmYZm* ZmYZm*,!-ZmYZm-,2#+300-ZmYm^HFGG_Yme3,[mmm
0#1-30!#m$-0m%#,#m$3,!2'-,m,*71'1m',m13,$*-5#0Yem*,2m#2&-"1mM^G_[mHFYm
m
)+-2-Zm YZm ,Zm YYZm 1#Zm YZm &')8-,-Zm YZm 213,%Zm Ym 7m !-*Ym ^HFFI_Ym
e'103.2'-,m -$m 2&#m 2Im %#,#m !31#1m &7.#01#,1'2'4'27m 2-m 3*204'-*#2m m *'%&2m ,"m
%++j071m ',m 0 '"-.1'1[m '+.*'!2'-,m -$m 2&#m .0#1#,!#m -$m m 20,1*#1'-,m 17,2&#1'1m
+#!&,'1+m',m.*,21Yem*,2m#**mGK^O_[mHFJHjKMYm
m
*"'<Zm YZm -1#Zm YZm '!#,2#Zm Ym ^HFFM_Ym e&#m *',)%#m #25##,m !#**m 5**m
+#2 -*'1+m,"m$03'2m1-$2#,',%[m*--)',%m2-m2&#m$3230#Yem
m!'m--"m%0'!mNM[mGJIKjGJJNYm
m
,1#4#0',-Zm YZm -+Zm YZm #m '+-,#Zm YZm ',-Zm YZm #*'2-Zm Ym 7m !-*Ym ^HFFO_Ym
e" [m m '-',$-0+2'!1m .*2$-0+m $-0m .*,2m 0#1'12,!#m %#,#m ,*71'1Yem 3!*#'!m !'"1m
#1mIN^2 1#m'113#_[mNGJjHGYm
m
1)'ZmYZm213+-2-ZmYZm,2-,'-ZmYYZm%+30ZmYm^HFFK_Yme0-+m+..',%m
2-m1#/3#,!',%Zm.-12j1#/3#,!',%m,"m #7-,"Yem*,2m#**m&71'-*mJL^G_[mIjGIYm
m
2-j0m3+'ZmYjYYm^GOOO_Yme2&7*#,#m#!#.2-01m,"m#,#2'!m,%',##0',%m-$m
2&7*#,#m#,1'2'4'27mem*,2m'-2#!&,-*-%7mGL^K_[mIHGjIIJYm
m
!&**#0Zm YYZm *##!)#0Zm YYm ^GOOK_Ym e2&7*#,#j ',"',%m 1'2#1m %#,#02#"m ',m 7#12m
#6.0#11',%m2&#m0 '"-.1'1mGm%#,#Yem!'#,!#mHMF^KHJI_[mGNFOjGGYm
m
!&,#'"#0Zm YYm ^HFFH_Ym e*,2m '++3,'27m ,"m $'*+m -'0[m 5&2m %3+1&-#m
"#2#!2'4#1m!,m2#!&m31m -32m.*,2j.2&-%#,m',2#0!2'-,1Yem#**mGFO^K_[mKIMjJFYm
m
# 12',Zm YYZm #*$-0"Zm YZm ,Zm YZm #,,#0Zm Ym ^HFGF_Ym 3!3+ #0m ^3!3+'1m
12'431_m,"m+#*-,m^Ym+#*-_m&4#m,3+#0-31m5'*"m0#*2'4#1m',m1'm,"m3120*'Zm,"m
2&#m 1'12#0m 1.#!'#1m -$m +#*-,m '1m $0-+m 3120*'Ym 0-!##"',%1m -$m 2&#m 2'-,*m !"#+7m -$m
!'#,!#1m-$m2&#m,'2#"m22#1m-$m+#0'!YmGFM[mGJHLOjMIYm
m
#$$#0,'!)Zm YYZm +,2Zm YYZm -3'#Zm YYZm !)#22Zm YYZm 3 0+,',Zm Ym
^HFFO_Ym e,4#12'%2'4#m +',',%m -$m 1#/3#,!#m "2m $-0m ,-4#*m #,87+#1[m m !1#m 123"7m 5'2&m
,'20'*1#1Yem
m'-2#!&,-*mGJI^G_[mGMjHLYm
m
#%Zm YYm ^GONJ_Ym em 0#4'#5m -$m 2&#m %#,#2'!m #$$#!21m -$m #2&7*m +#2&,#13*$-,2#Yem
322m#1mGIJ^HjI_[mGGIjJHYm
HFKm
O
#&#)!, 7m
m
#7+-30Zm YZm !*11-,Zm Ym ^^GOOI__Ym e#*-,1Ym ',m '-!&#+'1207m -$m 03'2m
'.#,',%Yem"'2-0'*[m#7+-30mZm7*-0m
Zm3!)#0mm^&.+,mXm**_Ym-,"0#1Ym.Ym
HMIjHOFYm
m
&3,Zm YZm -721Zm YZm 23.0Zm YZm ^HFGH_Ym e#,-+#m ,%',##0',%m -$m !0-.1m 5'2&m
"#1'%,#0m,3!*#1#1Ym&#m*,2m#,-+#YmKm^H_YmJHjKFYmm
m
&#**'#ZmYZm')*ZmYm^GOOI_Yme&#m!)m-$mm#1.'02-07m'1#m',m31)+#*-,m03'2m
'.#,',%m -,m 2&#m *,2m &**#,%#1m 2&#m #$','2'-,m -$m *'+!2#0'!m #&4'-30Yem -30,*m -$m
1.#0'+#,2*m-2,7mJJ^N_[mGJFIjGJFLYm
m
&#,Zm YYZm &',Zm YYZm 00-7-j0!'Zm YZm !&-Zm YYZm 4#**#Zm YYm 7m !-*Ym
^HFFH_Ym e+Im '1m -,#m +#+ #0m -$m m *0%#m !-,12'232'4#*7m #6.0#11#"m $+'*7m -$m ,3!*#-2'"#m
',"',%m1'2#j*#3!',#j0'!&m0#.#2m#,!-"',%m%#,#1Yem-*m*,2m'!0- #m,2#0!2mGK^I_[mHKGj
LGYm
m
m
&')8-,-ZmYZm-)-2ZmYZm,)ZmYZm2, #ZmYZ,-ZmYm^GOON_Yme-*#!3*0m
,*71'1m -$m !0 -,m '-,j',"3!#"m +322'-,1m ',m 0 '"-.1'1m 2&*',Yem #,#1m #,#2m 712m
MI^I_[mGMIjOYm
m
&')8-,-Zm YZm ,)Zm YZm 2, #Zm YZm ,-Zm Ym ^HFFG_Ym e#00,%#+#,21m -$m
2&#mm',m!0 -,m'-,j',"3!#"m+32,21m-$m0 '"-.1'1m2&*',Yem#,#2'!1mGKM^G_[mIMOj
NMYm
m
m
&')8-,-ZmYZm-)-2ZmYZm'2+30ZmYZm383)'ZmYZm2, #ZmYm7m!-*Ym^HFFI_Ym
e322'-,m 02#m ,"m ,-4#*m 22m +32,21m -$m 0 '"-.1'1m 2&*',m ',"3!#"m 7m !0 -,m '-,1Yem
#,#2'!1mGLI^J_[mGJJOjKKYm
m
&'0*#7ZmYYZm,*#7ZmYZm--"+,ZmYYm^GOOH_Yme$$#!21m-$m'-,'8',%m0"'2'-,m
-,mm.*,2m%#,-+#[m,*71'1m-$m25-m0 '"-.1'1m20,1.0#,2m2#12m+322'-,1Yem*,2m#**m
J^I_[mIIIjJMYm
m
)#*2-,Zm YYZm 3Zm YZm 0','41,Zm YZm ,1&0"2Zm YZm --0#Zm YYm 7m !-*Ym ^HFFL_Ym
e'113#m"'$$#0#,2'*m#6.0#11'-,m-$m*7!-.#,#m #2j!7!*1#m%#,#m',m..7Yem#**m#1mGL^N_[m
MIGjOYm
m
*"#Zm Y
YZm 3#012#, #0%Zm YYZm -#$$*#0Zm YZm 2#',#Zm YYZ!!'-22'Zm Ym ^HFFK_Ym em
0#4#01#m%#,#2'!Zm,-,20,1%#,'!m..0-!&m2-m5&#2m!0-.m'+.0-4#+#,2m 7mYem2m
'-2#!&,-*mHI^G_[mMKjNGYm
m
+'2&Zm YYZm 3Zm YYZm *40"-m 0#+#0Zm YYm ^HFGH_Ym e#2&-"-*-%'!*m
120#+*',',%m -$m m "'1!-4#07m ,"m %#,-27.',%m 4'm &'%&j0#1-*32'-,m +#*2',%m ,*71'1m
^_m',m,-,j+-"#*m1.#!'#1Yem0m#,-+'!1Ymm
m
- #0A,,m
YZm3'0-%ZmYZm+.'#20-ZmYYm^HFFK_Yme2'*#,-eYm3 *'!"-m.-0m#*m
,12'232-m "#m 123"'-1m #%#2*#1Ym !3*2"m "#m '-/3>+'!m 7m 0+!'Ym ,'4#01'""m
!'-,*m"#m3!3+9,Ym
m
-,%ZmYYZm,%ZmYYZm&#,ZmYYZm'+ZmYYZm'ZmYYm7m!-*Ym^GOOK_Ymem0#!#.2-0m
)',1#j*')#m.0-2#',m#,!-"#"m 7m2&#m0'!#m"'1#1#m0#1'12,!#m%#,#ZmHGYem!'#,!#mHMF^KHJI_[m
GNFJjLYm
m
.'#2&Zm
YZm51-,ZmYm^HFFK_Ymem4#04'#5m-$m%#,#m1203!230#em-0+--)m
-30,*[
&22.[ii555Y5-0+ --)Y-0%iYm
HFLm
#&#)!, 7O
m
m
0##*)1&+'Zm YZm 3.2Zm YZm -",.3Zm YZm &3&,Zm YYZm ,( +Zm Ym 7m !-*Ym
^HFFO_Ym e[m m 1'+.*'$'#"m .0-!#"30#m $-0m ,3!*#'!m !'"m #620!2'-,m $0-+m 007#"m
2'113#m $-0m m ,"m -2&#0m &'%&j2&0-3%&.32m 0#4#01#m %#,#2'!m ..*'!2'-,1Yem *,2m
#2&-"1mL^G_[mIYm
m
2# ',1Zm YYZm --"7Zm YYm ^GONO_Ym e-,!#0,',%m 2&#m &**+0)m -$m m
"'1!0'+',2'-,Yem#&4m,*mGH^H_[mHFIjGGYm
m
2#.,-4Zm YYZm !)#0Zm YYm ^HFFF_Ym e2&7*#,#m 1'%,*',%[m $0-+m +32,21m 2-m
+-*#!3*#1Yem300m.',m*,2m'-*mI^K_[mIKIjLFYm
m
2#.&#,1-,Zm YZm )#0Zm YZm '0',Zm YZm #0#8Zm YZm +-&Zm Ym 7m !-*Ym ^HFGF_Ym em 0'!&m
m0#1-30!#m$-0m123"7',%m%#,#m$3,!2'-,m',m011'!m0.Yemm*,2m'-*mGF[mLHYm
m
20!&,Zm YZm #"Zm YYm ^GOOO_Ym e3+,m -*#!3*0m #,#2'!1Yem "'2-0'*[m
!'#,2'$'!m3 *'1&#01Ym6$-0"Ym
m
20#'1',%#0Zm YZm 5#,Zm Ym ^GONK_Ym e#!&,'1+1m -$m 1.-,2,#-31m ,"m ',"3!#"m
$0+#1&'$2m+322'-,m',m !2#0'-.&%#mJYem#,#2'!1mGFO^J_[mLIIjKOYm
m
3 '7,ZmYYZm2#01ZmYYZm2'70ZmYYZm",,"ZmYYZm"""'ZmYm7m!-*Ym
^HFGH_Ym e#,-+#j5'"#m m .-*7+-0.&'1+1m ',m #*'2#m ',"'!m 0'!#m ', 0#"1m "'1!-4#0#"m 7m
5&-*#j%#,-+#m1#/3#,!',%Yem*,2m'-2#!&,-*m
Ym
m
"+-0Zm YZm 28'0Zm YZm #'0Zm YZm )-4Zm YZm d0Zm Ym ^HFFM_Ym e,"3!#"m
+32%#,#1'1m 2-m 3%+#,2m 2&#m ,230*m 40' '*'27m -$m +#*-,m ^3!3+'1m +#*-m Y_Yem 10#*m m
*,2m!'mKK[mGKOjGLOYm
m
)3+'ZmYZm-7+ZmYZm3('50ZmYZm- 71&'ZmYm^HFGG_Yme"#,2'$'!2'-,m-$mm
*0%#m "#*#2'-,m ',m 2&#m $'012m ',20-,m -$m 2&#m 0,jGm *-!31Zm 11-!'2#"m 5'2&m *-11m -$m
4#0,*'82'-,m 0#/3'0#+#,2m ',m 5'*"m 5&#2m .0-%#,'2-0m #%'*-.1m 231!&''m -11Yem #,#1m
#,#2m712mNL^I_[mGNIjOKYm
m
*+#Zm YZm -4',Zm YZm ,%3',#2'Zm YYZm 3 #0-1Zm YZm 3,"/4'12Zm Ym 7m !-*Ym
^HFFN_Ym e-0#Zm m 0#1-30!#m $-0m 2&#m "'1!-4#07m -$m !&#+'!**7m ',"3!#"m +32,21m ',m
0*#7Yem*,2m'-2#!&,-*m
mL^K_[mJMMjNKYm
m
,)ZmYZm&')8-,-ZmYZm-)-2ZmYZm2, #ZmYZm,-ZmYm^GOOM_Yme$$#!21m-$m
&#47m '-,1m -,m 2&#m %#0+',2'-,m ,"m 1304'4*m -$m 0 '"-.1'1m 2&*',Yem ,2m m "'2m '-*m
MH^G_[mGHGjMYm
m
7*-0Zm YYZm 0##,#Zm YYm ^HFFI_Ym e[m m 2--*m $-0m 2&#m ,*71'1m -$m
,3!*#-2'"#m.-*7+-0.&'1+1Yem3!*m!'"1m#1mIG[mINFNmjmINGGYm
m
&,&Zm YYZm &,Zm YYZm 1&')5Zm YZm 1&''Zm Ym ^HFGF_Ym em ,*71'1m $-0m
$*-5#0',%m 2'+#m 31',%m !)!0-11m .-.3*2'-,m #25##,m 078m 12'4m '..-, 0#m ,"m Ym
03$'.-%-,Yem#,#1m#,#2m712mNK^J_[mHMIjOYm
m
'#+,ZmYYZm7*-0ZmYYZm'0"'Zm
YYZ*##ZmY
Ym^HFFF_Yme&#m2-+2-m#2&7*#,#m
0#!#.2-01m m ,"m #Jm 0#m ,#%2'4#m 0#%3*2-01m -$m #2&7*#,#m 0#1.-,1#m ,"m #6&' '2m
$3,!2'-,*m!-+.#,12'-,m5'2&',mm+3*2'%#,#m$+'*7Yem0-!m2*m!"m!'mmmmOM^GF_[m
KLLIjNYm
m
'**ZmY
YZm-* #02ZmYZm-+.ZmYZm,,1ZmYYZm-"-+-ZmYYm7m!-*Ym^HFFI_Yme'%&j
2&0-3%&.32mm$-0m$3,!2'-,*m%#,-+'!1Yem#2&-"1m-*m'-*mHIL[mHFKjHFYm
HFMm
O
#&#)!, 7m
m
'**Zm Y
YZm #7,-*"1Zm YYZm #'*Zm YZm .0',%#0Zm YZm 302,#0Zm Ym 7m !-*Ym ^HFFJm _Ym
e'1!-4#07m-$m',"3!#"m.-',2m+322'-,1m',m+'8#m%#,#1m 7mYemm*,2m'-*mJ[m
GHYm
m
'**Zm Y
YZm 302,#0Zm YZm -+'Zm YZm #,')-$$Zm Ym ^HFFJm _Ym e'1+2!&m !*#4%#m 7m
1',%*#j120,"m1.#!'$'!m,3!*#1#1Yem3!*m!'"1m#1mIH[mHLIHmjmHLJGYm
m
m
'**ZmY
YZm--.#0Zm
YZm'ZmYYZm-*-5'2ZmYZm0##,#ZmYYm7m!-*Ym^HFFM_Yme'1!-4#07m
-$m!&#+'!**7m',"3!#"m+322'-,1m',m0'!#m 7mYemm*,2m'-*mM[mGOYm
m
0'/3#1Zm YZm 230 -'1Zm YZm **'1Zm YZm *+'1Zm YZm &34',Zm Ym 7m !-*Ym ^HFFM_Ym
e&0!2#0'82'-,m-$m0 '"-.1'1m2&*',m+'1+2!&m1.#!'$'!m#,"-,3!*#1#1[m..*'!2'-,m
2-m+322'-,m"'1!-4#07m 7mm',m.#Yem*,2m
mKG^L_[mGGGLjHKYm
m
0-+1Zm YZm *#,Zm YYm ^HFGF_Ym e&#m 02#m ,"m 1.#!203+m -$m 1.-,2,#-31m
+322'-,1m',mm.*,2mm4'031Yem#,#2'!1mGNK^I_[mONIjOYm
m
03,'%#0Zm YZm '#2-Zm YZm -,89*#8j #1Zm YZm 0,"Zm Ym ^HFFN_Ym e#!&,'1+m -$m
.*,2m #Jj+#"'2#"m 0#1'12,!#m %',12m m 0+-4'031m ^-+ 314'0'"#_[m !.j
',"#.#,"#,2m 20,1*2'-,m -$m m 4'0*m m !-,20-**#"m ',m !'1m 7m ,m ^_4'03*#,!#m
"#2#0+',,2Yem*,2m
mKL^K_[mMGLjHMYm
m
1'Zm YZm -5#**Zm YZm '2!&#0Zm YZm '11'0',Zm YZm 21-,Zm Ym 7m !-*Ym ^HFGG_Ym
e'1!-4#07m -$m 00#m +322'-,1m ',m .-.3*2'-,1[m m 7m 1#/3#,!',%Yem *,2m &71'-*m
GKL^I_[mGHKMjLNYm
m
."&77Zm YYZm &3Zm YYZm &2Zm YYm ^HFGF_Ym e0,1.-1-,m ',1#02'-,*m
+32%#,#1'1m',m0'!#Yem#2&-"1m-*m'-*mLMN[mGJMjMMYm
m
,m "#0m '#8#,Zm YYZm -,#1Zm YYm ^GOON_Ym e*,2m "'1#1#j0#1'12,!#m .0-2#',1m ,"m
2&#m%#,#j$-0j%#,#m!-,!#.2Yem0#,"1m'-!&#+m!'mHI^GH_[mJKJjLYm
m
,m "#0m --0,Zm YYZm #m '2Zm Y
YZm --12#,Zm YYm ^HFFH_Ym e*,!',%m 1#*#!2'-,m
$4-01m%30"',%m0#1'12,!#m.0-2#',1Yem0#,"1m*,2m!'mM^H_[mLMjMGYm
m
,m"#0m*,)Zm
YYm^GOLK_Yme7,+'!1m-$m.'"#+'!1m-$m*,2m'1#1#[m-.3*2'-,m
30121m -$m $3,%'Zm !2#0'Zm -0m 4'031#1m ',m $'#*"m ,"m $-0#12m +)#m ,m ',2#0#12',%m "7,+'!*m
123"7Yem!'#,!#mGJM^ILKJ_[mGHFjJYm
m
,m -'(#,Zm YZm ,m "#,m 30%Zm YYZm -0,#*'11#,Zm Y
YZm ))#,Zm YYm ^HFFM_Ym
e203!230#m ,"m $3,!2'-,m -$m 0#1'12,!#m .0-2#',1m ',m 1-*,!#-31m .*,21Yem ,,3m #4m
&72-.2&-*mJK[mJIjMHYm
m
'%-30-36Zm YZm /3#2&Zm YYZm 213-)Zm YZm +'2&Zm YYZm #4'1Zm YYm 7m !-*Ym
^HFFH_Ym e2#m ,"m .22#0,m -$m +322'-,m 2m +'!0-12#**'2#m *-!'m ',m +'8#Yem -*m '-*m 4-*m
GO^N_[mGHKGjLFYm
m
'**,3#4Zm YZm -,%j-,%Zm YZm --**'+1Zm YYm ^HFFH_Ym e0)#0m 11'12#"m
1#*#!2'-,m 5'2&m -.2'+'1#"m !-,20' 32'-,1m -$m 2&#m !,"'"2#1m 2-m 1#*#!2'-,Yem #,#2m #*m 4-*m
IJ^L_[mLMOjMFIYm
m
-1ZmYZm-%#01ZmYZm*##)#0ZmYZm#'(,1ZmYZm,m"#m##ZmYm7m!-*Ym^GOOK_Yme[m
m,#5m2#!&,'/3#m$-0mm$',%#0.0',2',%Yem3!*#'!m!'"1m#1mHI^HG_[mJJFMjGJYm
HFNm
#&#)!, 7O
m
m
,%Zm YYZm 3,Zm YYZm -Zm YYm ^HFFL_Ym e..*'!2'-,m -$m m ',m .*,2m
'+.0-4#+#,2Yem'm&3,m3#m-mII^GG_[mOKMjLJYm
m
,%Zm
Ym^HFFG_Yme.2'+*m+0)#0j11'12#"m1#*#!2'-,m2-m',!0#1#m2&#m#$$#!2'4#m1'8#m
-$m1+**m.-.3*2'-,1Yem#,#2'!1mGKM^H_[mNLMjMJYm
m
,%Zm YZm -&*+'Zm YYZm 4'0!#4Zm YZm ,%Zm YZm ,$!-,Zm Ym 7m !-*Ym ^HFGI_Ym
e'*#,!',%m-$m2&#m&-12m$!2-0m#^'1-_Jm%#,#m!-,$#01m.*3+m.-6m4'031m0#1'12,!#m',m.*3+Yem
-m,#mN^G_[m#KFLHMYm
m
02&+,,Zm YZm &#,Zm YZm 11-51)'Zm YZm #'%#*Zm YZm #04#Zm Ym ^HFFN_Ym e'%&*7m
1.#!'$'!m%#,#m1'*#,!',%m 7m02'$'!'*m+'1m',m0'!#Yem-m,#mI^I_[m#GNHOYm
m
#'*ZmYZm-,"#ZmYYm^HFFM_Yme#22',%m2-m2&#m.-',2mjm322'-,1m',m+'8#Yem0-.m
!'mJM[mLFjLMYm
m
&'2)#0Zm YYm ^GOML_Ym e3!30 '21Zm -2,7Zm !3*2'42'-,m ,"m 32'*'82'-,Yem
"'2-0'*[m,2#01!'#,!#m3 *'1&#01Ym3#4m-0)Ym
m
'#,&-*"1Zm YZm ,m #"#,Zm YZm -12#01Zm YZm 3""#Zm YZm *12#0)Zm YYm 7m !-*Ym
^HFFI_Yme$$'!'#,2m20%#2j1#*#!2#"m+32%#,#1'1m',m8# 0$'1&Yem#,-+#m#1mGI^GH_[mHMFFjMYm
m
'*)',1-,Zm
YYZm,&,ZmYYZm#,ZmYYZm'-4,,-,'Zm
YZm*##ZmY
Ym^GOOK_Yme,m
#2&7*#,#j',"3!' *#m !-+.-,#,2m -$m 1'%,*m 20,1"3!2'-,m #,!-"#"m 7m ,#4#0j0'.#Yem !'#,!#m
HMF^KHJI_[mGNFMjOYm
m
'**'+1m^GOOF_Ymem.-*7+-0.&'1+m+.*'$'#"m 7m0 '2007m.0'+#01m0#m31#$3*m
1m%#,#2'!m+0)#01Yem3!*m!'"1m#1mGN[mLKIGjLKIKYm
m
',)*#0ZmYZm!&5 #"'11#,ZmYZm!)1!&ZmYZm-)#*ZmYZm#'"#*ZmYm7m!-*Ym^HFFK_Ym
e0%#2j1#*#!2#"m+32,2m1!0##,m 7mm',m0-1-.&'*Yem#,-+#m#1mGK[mMGNmjmMHIYm
m
'22+,,Zm YZm &2#*Zm YZm -02',Zm YYZm *' #02#Zm YYm ^GOOM_Ym e,2#0!2'-,m -$m 2&#m
4'0*m .0-2#',m %#,-+#m *',)#"m -$m 230,'.m +-1'!m .-274'031m 5'2&m 2&#m 20,1*2'-,*m
#3)07-2'!m','2'2'-,m$!2-0m^'1-_mJm-$m0 '"-.1'1m2&*',m31',%m2&#m7#12m25-j&7 0'"m
1712#+Yem'0-*-%7mHIJ^G_[mNJjOHYm
m
-*$1 #0%Zm YYm ^HFGG_Ym e1',%m 2&#m m .m '#5#0m 2-m 0-51#m %#,-+'!m
1#/3#,!#m"2Yem300m0-2-!m3+m#,#2m&.2#0mGN[m,'2GNmKYm
m
0'%&2Zm YYZ-721Zm YYm ^GOON_Ym e-2#,2'*m 0#20-4'031#1m ',m .*,21[m 2Gm '1m
0#*2#"m 2-m m %0-3.m -$m 0 '"-.1'1m 2&*',m 7Ii%7.17m 0#20-20,1.-1-,1m 2&2m #,!-"#m
#,4#*-.#j*')#m.0-2#',1Yem#,#2'!1mGJO^H_[mMFIjGKYm
m
3Zm YYZm 3Zm YZm #'Zm YZm 0-'",Zm YZm -0"#-1Zm Ym 7m !-*Ym ^HFFK_Ym e&#+'!*jm
,"m'00"'2'-,j',"3!#"m+32,21m-$m',"'!m0'!#mLJm$-0m$-050"m,"m0#4#01#m %#,#2'!1Yem
*,2m-*m'-*mKO^G_[mNKjOMYm
m
,%Zm YZm #,Zm YZm -"*'Zm YYZm *#7)-51)'Zm YYZm '**#0Zm YYm 7m !-*Ym ^HFFF_Ym
e30'$'!2'-,Zm!*-,',%Zm,"m!&0!2#0'82'-,m-$m2&#mmm,3!*#1#Yem'-!&#+'1207mIO^GI_[m
IKIIjJGYm
m
#,Zm YYZm ##Zm YZm ,)1*#7Zm YYZm ,&,Zm YYZm *##Zm Y
Ym 7m !-*Ym ^GOOK_Ym e&#m
2-+2-m#4#0j0'.#m*-!31m0#%3*2#1m#2&7*#,#j',"3!' *#m%#,#m#6.0#11'-,m,"m'1m*',)#"m2-mm
&-+-*-%m-$m2&#m0 '"-.1'1mGm%#,#Yem*,2m&71'-*mGFM^J_[mGIJIjKIYm
HFOm
O
#&#)!, 7m
m
#3,%Zm YYZm 22,%"'Zm YZm *)#1*#7Zm YZm '!-*1Zm Ym ^HFFK_Ym e,87+2'!m
+322'-,m"#2#!2'-,m2#!&,-*-%'#1Yem'-2#!&,'/3#1mIN^K_[mMJOjKNYm
m
m
-3'*Zm YZm #+.#0Zm YYZm -22-,Zm YYm ^GOOK_Ym e!0##,',%m $-0m +322'-,1m 7m
#,87+#m+'1+2!&m!*#4%#m5'2&mJm#,"-,3!*#1#mYem0-!m2*m!"m!'mmmmOH^G_[m
NMjOGYm
m
3ZmYYZm&',%ZmYYZm3ZmYYZm'ZmYYZm,%ZmYYm7m!-*Ym^HFGF_Yme#,#02'-,m
-$m 20,1%#,'!m 52#0+#*-,m 0#1'12,2m 2-m 3!!&','m 7#**-5m +-1'!m 4'031m ,"m .7m
0',%1.-2m4'031m27.#mYem*,2m#**m#.mIF^I_[mIKOjMGYm
m
0#+ ',1)'ZmYYZm&#-*-%'1ZmYm^GOOJ_Yme2&7*#,#m '-17,2&#1'1m,"m!2'-,[mm!1#m
-$m!-,1#042'-,Yem*,2m-*m'-*mHL^K_[mGKMOjOMYm
m
&#,%ZmY
YZm,-#0#,ZmYYZm-%#5-,',%ZmYYZm,m--,Zm
Y
YZm '!)#ZmYm^HFGF_Ym
e'103.2'-,m -$m .*,2m !0-2#,-'"m '-17,2&#1'1m 2&0-3%&m 4'031j',"3!#"m %#,#m 1'*#,!',%m
$$#!21m-4'.-1'2'-,m #&4'-30m-$m2&#m 322#0$*7m'#0'1m0.#Yem#5m&72-*mGNL^I_[mMIIjJKYm
m
&-,%Zm YZm ',Zm YZm 0'#01-,Zm Ym ^HFFN_Ym e-+2-m #2&7*#,#m 0#!#.2-0jm
',2#0!2'-,1[m 4'13*'82'-,m -$m ,#4#0j0'.#m ',2#0!2'-,1m 5'2&m +3*2'.*#m 1m 2m 2&#m
#,"-.*1+'!m0#2'!3*3+Yem
m6.m-2mKO^J_[mOLKjMHYm
m
m
HGFm
(2)O
m
TT
T
,m#12#m,#6-m1#m.0#1#,2m#*m02>!3*-m.3 *'!"-m$032-Zm#,m%0,m.02#Zm"#*m20 (-m0#*'8"-m#,m#12m
2#1'1m"-!2-0*Ym#m',!*37#,m2+ '<,m"-1m02>!3*-1m/3#m,-m#129,m0#*!'-,"-1m!-,m*m2#+92'!m"#m*m
2#1'1Zm#,m*-1m/3#m1#m!-* -0Am.0*#*+#,2#m*m"#100-**-m"#m*m.- *!'A,m"#mm"#m+#*A,Ymm
m
T
T
T
HGGm
O
(2)m
T
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
RESEARCH ARTICLE
Open Access
Towards a TILLING platform for functional
genomics in Piel de Sapo melons
Mireia González1, Meihong Xu1,5, Cristina Esteras2, Cristina Roig2, Antonio J Monforte1,3, Christelle Troadec4,
Marta Pujol1, Fernando Nuez2, Abdelhafid Bendahmane4, Jordi Garcia-Mas1 and Belén Picó2*
Abstract
Background: The availability of genetic and genomic resources for melon has increased significantly, but
functional genomics resources are still limited for this crop. TILLING is a powerful reverse genetics approach that
can be utilized to generate novel mutations in candidate genes. A TILLING resource is available for cantalupensis
melons, but not for inodorus melons, the other main commercial group.
Results: A new ethyl methanesulfonate-mutagenized (EMS) melon population was generated for the first time in
an andromonoecious non-climacteric inodorus Piel de Sapo genetic background. Diverse mutant phenotypes in
seedlings, vines and fruits were observed, some of which were of possible commercial interest. The population was
first screened for mutations in three target genes involved in disease resistance and fruit quality (Cm-PDS, Cm-eIF4E
and Cm-eIFI(iso)4E). The same genes were also tilled in the available monoecious and climacteric cantalupensis EMS
melon population. The overall mutation density in this first Piel de Sapo TILLING platform was estimated to be 1
mutation/1.5 Mb by screening four additional genes (Cm-ACO1, Cm-NOR, Cm-DET1 and Cm-DHS). Thirty-three point
mutations were found for the seven gene targets, six of which were predicted to have an impact on the function
of the protein. The genotype/phenotype correlation was demonstrated for a loss-of-function mutation in the
Phytoene desaturase gene, which is involved in carotenoid biosynthesis.
Conclusions: The TILLING approach was successful at providing new mutations in the genetic background of Piel
de Sapo in most of the analyzed genes, even in genes for which natural variation is extremely low. This new
resource will facilitate reverse genetics studies in non-climacteric melons, contributing materially to future genomic
and breeding studies.
Keywords: Cucumis melo, inodorus, TILLING, mutant, disease resistance, fruit quality
Background
Melon (Cucumis melo L.) is an important vegetable
crop. Genetic and genomic information for this crop is
increasing significantly due to several national and international projects [1]. A broad range of genomic tools
are available today [2-7]. An effort is also in progress,
through a Spanish initiative, to obtain the whole genome
sequence of this crop [8]. These tools are generating a
lot of information about genes involved in various biological processes, such as plant resistance and fruit quality
[9,10]. However, the tools necessary for reverse genetic
* Correspondence: [email protected]
2
Institute for the Conservation and Breeding of Agricultural Biodiversity
(COMAV-UPV), Universitat Politècnica de València, Camino de Vera s/n, 46022
Valencia, Spain
Full list of author information is available at the end of the article
studies to conduct the functional validation of candidate
genes in melons are still limited.
The TILLING (Targeting Induced Local Lesions in
Genomes) method may represent an effective means of
addressing limitations in melon research [11]. The application of TILLING has proved to be useful in identifying
novel alleles in genes controlling traits of agronomic
interest in legumes [12-15], cereals [16-23], solanaceous
crops [24-26] and Brassica spp [27,28].
C. melo is a highly variable species divided into two
subspecies, melo and agrestis. Most of the commercial
cultivars belong to the inodorus and cantalupensis
groups of the subspecies melo [29]. Cultivars of these
two commercial groups are quite different in plant,
flowering and fruit traits. Cantalupensis cultivars are
© 2011 Picó et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
T
HGHm
(2)O
m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
early flowering, frequently monoecious, and produce climacteric fruits (aromatic, medium sugar, soft fleshed
and with a short shelf life), whereas inodorus are late
flowering, mostly andromonoecious, and non-climacteric, producing non-aromatic, high sugar, firm-fleshed
fruits with a long shelf life, and which are preferred in
certain markets. These differences make breeding strategies and objectives quite different for the two groups of
melons.
A melon TILLING platform generated from a monoecious climacteric cantalupensis genotype has recently
become available [30], and has proven to be useful for
improving the shelf life of climacteric melons. A TILLING platform generated in an inodorus background
might represent a useful resource for functional studies
and for breeding non-climacteric melons.
In this paper, we present the development of an EMSmutagenized population from an andromonoecious nonclimacteric inodorus melon type (Cucumis melo L. subspecies melo cv Piel de Sapo). In total, 2,368 M2 families
were obtained. A diversity of interesting mutant phenotypes were observed in the field. In addition, several
mutant families have been identified in several genes
that were selected based on their potential contribution
to fruit quality and disease resistance.
Methods
EMS mutagenesis of melon seeds
The double haploid line, M62-113 (from Semillas Fito
S.A.), belonging to the Piel de Sapo commercial type
(Cucumis melo subsp melo var inodorus), was used as
the starting cultivar. This is the parent of the melon
genetic map and is one of the parents of the DHL line
selected for the sequencing of the melon genome. Different batches of a total of ~12,000 M62-113 seeds
were treated with the alkylant mutagen ethyl methanesulfonate (EMS). Different EMS concentrations were
tested, and 1% was finally selected and tested on a larger batch of seeds. Seeds were first hydrated with tap
water (16h), and were then soaked in tap water containing EMS concentrations (18 h). The treated seeds
were thoroughly rinsed in tap water twice for 4 hours.
After washing, seeds were placed on trays over wet
paper, and were kept in a germination chamber overnight. ~5,000 mutant plants of this first (M1) generation were transplanted at the greenhouse. Plants were
grown in two localities, in Barcelona (by Semillas Fito
S.A. and IRTA) and in Valencia (by COMAV-UPV).
Each M1 plant was selfed (1 fruit per plant) giving rise
to the M2 seed.
TILLING procedure
Twenty seeds were sown per M2 family, and the germination percentage was scored. Finally, a total of 2,368
Page 2 of 11
mutagenized M2 families, which produced at least 10
viable seedlings in at least two plantings, were sampled
for DNA extraction. 1-cm discs of tissue from 10 individual plants per M2 were pooled, and total DNA was
extracted, quantified, diluted and organized into twentyfive 96-well plates. Using equivalent amounts of DNA
from individual M2 families, the samples were pooled
four-fold and organized into a 96-well format. Seeds of
all pooled M2 populations are maintained at the Genebank of the COMAV-UPV, coded as F (S. Fito),
I (IRTA) or C (COMAV-UPV) plus the corresponding
number.
Primer sets were designed from 3 genes that were
selected based on their potential contribution to fruit
quality and disease resistance: Cm-eIF4E (translation
initiation factor 4E) and Cm-eIF(iso)4E (translation initiation factor E, Isoform) which are involved in resistance to
viruses [31], and Cm-PDS (Phytoene Desaturase), which
is involved in carotenoid synthesis [32]. The CODDLe
program (Codons Optimized to Deliver Deleterious
Lesions) [33] was used to select the regions most likely to
harbor deleterious changes induced by EMS (Table 1).
These 3 genes were tilled in the Piel de Sapo population
as well as in a set of 2,483 available lines of the cantalupensis TILLING population reported in [30]. Four additional genes involved in fruit ripening were tilled in the
Piel de Sapo population: Cm-ACO1 (ACC oxidase 1),
involved in the conversion of ACC into ethylene [34];
Cm-NOR (non-ripening), a transcription factor related to
ethylene-sensitive/insensitive phenotypes [35]; Cm-DET1
(de-tiolated-1), a negative regulator of light-mediated
responses that affects carotenoid and flavonoid pathways
in tomato and other crops [36,37]; and Cm-DHS (Deoxyhypusine Synthase), mutations of which delay fruit softening in tomato [38]. Primer sets used for these
additional genes are the same as those used in [30].
The amplicons analyzed for each gene are indicated in
Figures 1 and 2.
Nested PCR was used to improve the specificity of
amplification. The PCR reactions were performed in a
25 μl volume consisting of dH 2O, 1× PCR buffer (Promega Corp, Madison, WI), 2.5 mM MgCl 2 , 0.2 mM
dNTPs, 1 U Taq polymerase, 0.4 mM forward and
reverse primers, unlabeled in the first reaction, and
700 nm and 800 nm 5’ labeled (MWG Biotech AG,
Germany) in the second reaction, and 10 ng DNA. The
thermocycling conditions were 95°C for two minutes for
initial denaturing, followed by 35 cycles of 95°C for
20 seconds, 63-69°C (specific for each primer pair) for
one minute, 72°C from 30 seconds to one minute and
one cycle of 72°C for five minutes. PCR products
(~0.2 μl) were separated in 1% agarose gels. The PCR
products were heated and cooled in a thermocycler (a
gradient starting at 94°C and decreasing 0.1°C per second
T
HGIm
O
(2)m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
Page 3 of 11
Table 1 Primer pairs used to amplify the 3 target genes tilled in both EMS-mutant populations
Gene
Forward (*LabelledIRD700)
Reverse (*LabelledIRD800)
Cm-eIF4E
1st amplicon
GACTCAAACGCCTAACAGAAAATC
CTATCTCACCAAGTTTCCTAAATT
*GAGGGCGGTGCCATTCTTCTTCGG
*TCCCTAAATCGAACCAAGAAACGCC
Cm-eIF4E
2nd amplicon
TTGTTTCGTGTTGACATGTCCATC
AGCATGCATTTCACCTCATTGGCT
*TGCTTGGCTGTTAATTTATCTCTGC
*GTCAAGTACAGAACAAGAATCTGAG
Cm-eIF(iso)4E
Cm-PDS
GATCGATAATTTCCCTTTTC
TGACAGGCTTAAGCACTATG
*ACCCCAATTCATTCTAGGG
*TGTGCAAGCGCAACAAGGTAC
CAATGGGATGCTTAGATCAAC
TCCTTCCAAAATTACCCTAG
*GGATTTCTCGGAGTGACTCGG
*GCTCTCGAGCAACTAGCACT
Nested PCR was used in all cases with labeled primers in the second PCR.
to 4°C) to form the heteroduplexes. Once the heteroduplexes were formed, the products were treated with Endo
I. Digestions were performed in 30μl of final volume with
dH2O, 3μl digestion buffer, 150 ng of PCR product and
3μl of EndoI (diluted 1/5000). The products were incubated at 45°C for 20 minutes to digest mismatches in the
heteroduplex. Digestion was stopped by adding 5μl of
0.15 M EDTA and placing the reaction on ice. After the
digestion was completed, the products were filtered
through a Millipore MultiScreen filter plate that was
packed with hydrated Sephadex G-50 medium beads
(Amersham Biosciences). Samples were concentrated in
the Speed Vac at 65°C for 45 minutes. Loading dye was
added and the PCR products were denatured and loaded
onto a polyacrylamide gel attached to a LI-COR 4300
DNA Analyzer (LICOR, Lincoln, NE) for separation.
A total of 903 pools were assayed (592 four-fold pools of
the Piel the Sapo population and 311 eight-fold pools of
the cantalupensis population). Once a mutation was
revealed in a pool, the corresponding families were
1169
500
1719
Cm-eIF4E
154
299
154
166
1
2
5 6
3
8 9
7
78
126
66
51
4
12
10 11
13
14
15
16
17 18
Cm-eIF(iso)4E
19
1188
Cm-PDS
1125
169
290
46
1476
45
754
188
116
161
20
22
58
21
23 24 25
Figure 1 Gene structure of the target genes screened in both populations,inodorus and cantalupensis, (eIF4E, eIF(iso)4E and PDS).
Boxes represent exons and lines introns. White and black boxes indicate 3’UTR and 5’UTR regions. Amplicons analyzed by TILLING are indicated.
Black and white triangles indicate mutations found in the inodorus Piel de Sapo and cantalupensis Charentais populations, respectively. Numbers
correspond to the mutations described in table 3.
HGJm
(2)O
m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
Page 4 of 11
933
933
107
103
150
Cm-ACO1
125
334
227
291
1272
89
Cm-NOR
184
26
182
314
564
1185
Cm-DET1
72
83
431
776
102
288
933
27
166
123
69
1232
182
28 29
198
136
57
81
30
578
Cm- DHS
624
84
166
93
96
31
754
168
803
106
219
32
87
33
Figure 2 Gene structure of the target genes screened in the inodorus population (ACO-1, NOR, DET-1 and DHS). Boxes represent exons
and lines introns. White and black boxes indicate 3’UTR and 5’UTR regions. Amplicons analyzed by TILLING are indicated. Black triangles indicate
mutations found in the Piel de Sapo population. Numbers correspond to the mutations described in table 3.
analyzed individually to select the mutant M2 family.
Mutation was confirmed by sequencing in the pooled
DNA, in the individual M2 families and in several individual plants used in the segregation studies. The effect of
the mutations was analyzed with SIFT (Sorting Intolerant
from Tolerant, http://blocks.fhcrc.org/sift/SIFT.html)
[39], which predicts whether an amino acid substitution
affects protein function. Scores below 0.05 are predicted
to affect protein function.
Phenotyping of M2 and M3 individuals
Seedling phenotypes were systematically evaluated in the
M2 generation. Additionally, in order to increase the
number of seeds in our mutagenized population, we
obtained the M3 generation. M2 populations that are
being reproduced are also being inspected for phenotypes
that are distinct from the wild type in plant, flowering
and fruit traits. To date, about 800 M2 plants have been
reproduced by S. Fito. M3 plants are also being analyzed
for ethylene-response mutants by growing seedlings in
darkness with and without an air flux containing 10
microL L-1 ethylene, and analyzing the “triple response”.
Results
Generation of an EMS mutant population in the Piel de
Sapo background
We selected 0.5%, 1 and 1.5% for generating a pilot population of ~600 lines, and studied the effect of EMS dosage
on M2 seed viability and seedling vigor. Increased EMS
concentration led to an increased percentage of M2 fruits
in which seeds were non-viable or in which the number
of viable seeds was too low to perform TILLING analysis
(13.24%, 27.75% and 31.38%, respectively). Only a slight
reduction in the average germination rate was observed
in M2 families obtained from 0.5% M1 (93.51%), whereas
higher reductions were obtained in 1% and 1.5% (82.73
and 75.94%, respectively). In addition, seedling growth
was more restricted at the 1.5% concentrations. In contrast, the progeny from the 1%-treated M1 were robust.
Based on these results, 1% EMS was used in the end to
complete the mutagenized melon population. This EMS
dose produces an acceptable level of M1 seed survival,
fertility in M1 plants to set viable M2 seeds, and vigor in
M2 seedlings. From a total of 3,140 M1 fruits finally
HGKm
O
(2)m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
Page 5 of 11
obtained, 2,368 M2 families were sampled for DNA and
used for TILLING purposes.
Phenotyping of the mutagenized population
Ten plants per M2 line were scored for seedling phenotypes. A large number of the mutations affected
cotyledons, with 5% of the M2 families showing variation in their number position and shape. About 4.3%
of the M2 families segregated for “dwarf” or “semidwarf” plants. Albinism and chlorophyll deficiency
occurred in 2.1% of the M2 families. Over 2% of the
families showed alterations in leaves and shoot morphology. Gravitropic mutants and necrosis also
appeared, but were less frequent. Figure 3 shows several seedling phenotypes. M3 families were characterized for plant flowering and fruit traits. Fruit traits of
interest for melon breeding were identified: longer and
rounder fruits than the standard Piel de Sapo fruits,
with a lighter rind color, without the typical rind
spots, and with different degrees of netting. Some lines
have a subtle aroma and showed a color change and
the formation of an abscission layer near maturity,
a
traits that are more similar to climacteric cantalupensis
types than to the standard non-climacteric inodorus
Piel de Sapo. Differences in the dark-grown seedlings’
response to ethylene were also observed. Wild type
M62-113 showed the typical triple response: exaggeration of the apical hook, together with the inhibition of
hypocotyl and root growth as well as the radial swelling of the shoot [40], whereas some M3 families
showed responses of varying intensities (Figure 3).
Identification of mutations in target genes by TILLING
The TILLING approach allowed us to identify nucleotide changes in two of the three gene targets first
screened in the Piel de Sapo population (Table 2). EMSinduced changes were mostly G:C to A:T transitions
(67%) (Table 3), as was expected due to the frequent
alkylation of guanine residues by EMS, thus forcing mispairing with T [41]. Four mutations were exonic, most
of which were determined to be missense mutations,
only one being silent. A graph of the target genes marking the location of the induced polymorphisms is shown
in Figures 1 and 2.
b
Wild type
Wild type
c
+Ethylene
+Air
+Ethylene
+Air
+Ethylene
+Air
+Ethylene
+Air
Wild type
Figure 3 Images of selected mutant phenotypes observed in the melon TILLING population. a) Mutant seedlings found in M2. From left
to right and from top to bottom, variations in cotyledons number and position, alterations in leaf and shoot morphology, albinism and
chlorosis, including virus-like symptoms, dwarfing and altered gravitropism compared with seedling wild type. b) First row: Mutations found in
adult M3 plants, chlorosis, dwarfing and leaf and flower deformation. Second row: alterations in fruits in comparison with wild type, deformation,
chlorosis, rough surface, and pear-shape. Third row: fruit phenotypes of interest for melon breeding, variation in shape, longer and rounder
melons, and melons with a subtle aroma and formation of abscission layer with or without color change. c) Variation in triple response: M2
families showing a more intense response to ethylene than wild type and others segregating for the response.
HGLm
(2)O
m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
Page 6 of 11
Table 2 Comparison of the mutations found by TILLING
in the gene targets screened in the two EMSmutagenized melon populations, Piel de Sapo (PS) and
Charentais (CH)
PS Population
Gene
CH population
Exon/Intron-UTR
Cm-eIF4E
3/1
CmeIF(iso)4E
0/0
5/9
1/0
Cm-PDS
1/1
1/3
TOTAL
4/2
7/12
Four new alleles were reported for Cm-eIF4E, defined
by 4 mutations. Two (in exons 1 and 2) were predicted
to affect protein function according to SIFT (p<0.05).
The segregation of the missense mutations was analyzed
in each mutant M2 family (Table 3) and homozygous
individuals for each mutation are being produced
for phenotypic analysis. Two new alleles were reported
for Cm-PDS. Mutations were detected in the first intron
and in the second exon of the amplicon (Figure 1). The
exonic mutation was predicted to affect protein
function.
The genotype/phenotype segregation was verified for
the Cm-PDS mutations in 15 plants of each M2. The
intronic mutation found in M2 family F1151 (mutation
20 in Figure 1) presented a Mendelian segregation of 4
wild type/7 heterozygous/4 homozygous mutant (fitting
to a 1:2:1 ratio) (Table 3). No associated phenotype was
observed, which agrees with the position of the mutation in an intronic region. The M2 family C384 carried
a missense mutation in exon 2 (mutation 21 in Figure
1). The segregation of M2 was 9 heterozygous/6 wild
type. In this family, we observed that some plants
showed delayed growth and even death at the cotyledon
stage, and therefore could not be sampled. M3 seeds
from a heterozygous M2 plant were used to repeat segregation analysis. Fourteen plants germinated out of the
20 seeds sown. Again, plants with delayed growth were
observed, but we also found a seedling with albino cotyledons and hypocotyl (Figure 4). This seedling was carefully grown until tissue could be sampled for DNA
extraction. The albino was the only plant homozygous
for the mutant allele; the remaining M3 plants were
wild type or heterozygous. This mutant line showed a
similar albino phenotype comparable to the previously
reported phenotypes for PDS disruption [32].
To further evaluate the mutation rate of the Piel de
Sapo population, we extended the TILLING screen to 4
additional genes involved in fruit quality. Mutations were
found for all genes, except for Cm-NOR, with Cm-DET1
being the most mutated gene. Six mutations were exonic,
most determined to be missense, only one silent, but all
were predicted to be tolerated (Figure 2, Table 3).
Mutation efficiency
We used these results to calculate the mutation rate of
the population. A total of 14 point mutations were
detected out of the total 11,534 bp analyzed. Previous
studies reported the difficulty in tracking mutations
on the ends of the fragments (~100 bp) [42]. Therefore,
200 bp was subtracted off each amplicon, and we considered the length screened in this study to be 9,134 bp. The
overall mutation density was then calculated by dividing
the total base pairs screened, which includes the sum of
the total length of the 12 amplicon sizes × the total number of individuals screened, by the total number of mutations revealed by TILLING ((9,134 × 2,368)/14 =
1,544,950). It was calculated to be ~1/1.5 Mb. The mutation frequency as determined by TILLING is coherent
with the moderate level of phenotypic mutants found in
our population.
In order to compare the inodorus and the cantalupensis
populations, the same genes were screened in a set of
2,483 lines of the Charentais population [30]. The number of mutations was higher, and new alleles were found
for the three genes (Table 2 Figure 1). The spectrum of
observed changes was similar to that found in the Piel de
Sapo population (Table 3). Only one mutation was found
for Cm-eiF(iso)4E. Cm-eIF4E was also the most mutated
gene with 14 mutations: seven intronic, two in the 3’UTR, and five exonic. Of the exonic mutations, two,
located in exons 1 and 2, were not tolerated (p<0.05).
One intronic mutation was located in the first nucleotide
of intron 3, and may affect a splicing site. TILLING analysis also revealed four mutations in Cm-PDS, one of
which was not tolerated. The 19 point mutations discovered in the 3,871 bp analyzed in the Charentais population make a mutation rate of ~1/401 Kb ((3,071 × 2,483)/
19 = 401,331). This is about 3 times higher than that
found in the Piel de Sapo population. This high mutation
rate in the cantalupensis population has also been
observed by screening additional genes in the final version of the population, which consists of 4,023 lines [30].
Discussion
The TILLING platform reported here is the first ever
developed in an andromonoecious non-climacteric inodorus melon genetic background. The mutation rate in
the Piel de Sapo population was moderate. It is less
mutated than the cantalupensis population that was
developed using a different genotype and a different protocol for treating the seeds, which resulted in increased
seed viability at higher EMS doses (1 to 3%). Our attempt
was the first with Piel de Sapo melons, and results suggest that our EMS treatment causes a high level of seed
lethality. Regarding the protocol, Charentais seeds were
not hydrated before being treated. Seed hydration has
been reported to activate cellular division and induce the
HGMm
O
(2)m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
Page 7 of 11
Table 3 Details of the mutations found by TILLING in the gene targets screened in the two EMS-mutagenized melon
populations, Piel de Sapo (PS) and Charentais (CH)
Gene/
population
Amplicon size
(bp)
Identified
mutants
N° Exon/
1
Intron
Position
AA
change
M2
Family
M2 segregation (WT/
H/M)2
Cm-eIF4E/PS
500
1
1
A 74 G
R 25 K
F2036
9/7/8
0.00
1169
3
5/3/0
0.00
Cm-eIF4E/CH
500
1169
3
11
Exon 1
SIFT
score
2
Exon 2
G 385 A
E 129 K
F57
3
Exon 2
G 465 A
T 155 T
F1880
4
Intron 2
G/C
5
Exon 1
G 289 A
E 97 K
53
6
Exon 1
C 294 T
F 98 F
2532
7
Intron 1
G/A
8
Exon 2
C 401 T
A 134 V
1229
0.10
9
Exon 2
G 407 A
G 136 E
444
0.00
F959
0.02
1420
10 Intron 2
C/A
11 Intron 2
C/T
1287
12 Intron 3
G/A first nt of
intron
13 Intron 3
C/T
14 Intron 3
C/T
15 Exon 4
G 616 A
16 Intron 4
C/T
2035
17 3’UTR
T/C
1368
18 3’UTR
G/A
2163
1115
splicing
1667
2646
1233
E 206 K
2060
0.09
222 TGA
CmeIF(iso)4E/
PS
1014
0
-
CmeIF(iso)4E/
CH
1014
1
19 Exon 1
Cm-PDS/PS
1188
2
Cm-PDS/CH
1188
Cm-ACO1/PS
933
Cm-NOR/PS
Cm-DET1/PS
1185
776
Cm-DHS/PS
4
G 128 A
R 43 K
M1587
1.00
20 Intron 1
C/T
F1151
4/7/4
21 Exon 2
C 560 T
S 227 F
C384
6/9/0
22 Exon 1
C 395 T
S 172 F
1182
23 Intron 1
C/T
2495
24 Intron 1
T/C
1460
25 Intron 1
C/T
1226
0
-
933
1
26 Exon 4
C 728 T
1272
0
1
27 Exon 2
T 402 A
L 135 L
I76
2
28 Exon 4
G 760 A
V 255 I
29 Exon 4
C 848 T
S 284 L
30 Intron 6
G 2981 A
T 243 I
C142
0.00
0.01
10/6/3
1.00
F201
2/11/2
0.37
F1876
2/4/3
0.25
1232
2
F1248
31 Intron 7
C 3211 T
578
1
32 Exon 1
G 184 A
V 62 I
F 1728
754
1
33 Exon 3
G 610 A
V 204 I
C348
C487
13/20/0
0.58
2/6/0
0.27
1
Number of mutations corresponds to those represented in Figures 1 and 2
For some mutations, it was difficult to obtain homozygous MM plants as seedlings showed altered development.
2
repair of DNA damage [43]. Differences in seed structure
between genotypes, as well as an increase in the efficiency
and accuracy of the repair of the induced lesions may
explain the differences between populations. Perhaps,
HGNm
using a higher EMS dosage and/or an extended length of
mutagen application, combined with a more intense
wash length, and a more accurate seedling nursery would
have increased mutation efficiency. Other authors that
(2)O
m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
Page 8 of 11
M/M
WT/WT
WT/M
Figure 4 Verification of genotype/phenotype relationship in the Cm-PDS gene. Chromatograms of wild type (WT), homozygous mutants
(M) and heterozygous are shown for the C 560 T transition altering codon 227 S (serine) to F (phenylalanine) and predicted to affect the protein
function. Segregation of the mutation was confirmed in M3 seeds from a heterozygous M2 plant. The albino was the only plant homozygous for
the mutant allele; the remaining M3 plants were wild type or heterozygous.
also found variable results in mutation frequency when
using EMS within a crop suggest the use of alternative
mutagens as a way for getting a higher density of mutations with less toxicity to the treated seeds [20].
Despite this higher mutation rate in the Charentais
population, the efficiency of both populations in producing mutations predicted to be damaging to the proteins
in Cm-eIF4E and Cm-PDS was similar. The new mutations found in Cm-eIF4E could be of interest, more so
when this factor is highly conserved in eukaryotes. The
natural diversity of Cm-eIF4E has been studied in
melon and related species by EcoTILLING [31] and no
variation has been reported within Cucumis melo,
except for the nsv mutation (a point mutation of
Leu228-His in exon 5), which controls resistance to
Melon necrotic spot virus [44]. Variants in exons 1 to 3
were identified using EcoTILLING in another Cucumis
species, Cucumis zeyheri, a wild relative of melon, isolated from the cultivated species by strong crossability
barriers. Therefore, our new Cm-eIF4E alleles represent
non-transgenic variants absent in nature. However it
remains to be demonstrated if these mutations will lead
to a gain of function, thereby producing new functional
phenotypes, or to a loss of function. In other crops,
recessive resistance to virus results from defective forms
of eIF4E, for example in pepper (pvr2), pea (sbm1),
tomato (pot-1), lettuce (mo1) and barley (rym) [45-47].
In most cases, resistance results from a few amino acid
changes clustered in two neighboring regions of the
eIF4E structure, located near the structural pocket
involved in CAP-binding [48]. Some of the coding
changes identified in our TILLING assay (in exons 1
and 2) are located near these two regions, close to the
conserved tryptophan residues required for CAP-binding activity (Figure 5). The other coding mutations are
found in different regions of the gene, but most affect
highly conserved aminoacids. EcoTILLING has also
shown that most natural variation in Cm-ACO1 occurs
in exons 1, 2 and 3 [49]. No variants are found in large
germplasm collections in exon 4. The effect of the identified mutation in exon 4 on ethylene production needs
to be demonstrated.
The mutants found in Cm-DET1 and Cm-DHS are the
first reported for these genes in inodorus melons. It has
been demonstrated that phenotypes of the tomato
mutants high pigment-2dg (hp-2dg) and hp-2j are
caused by lesions in the DET1 gene. Point missense
mutations and intron mutations directing alternative
splicing have been reported in both the N-terminus and
the C-terminus of the protein in Arabidopsis and
tomato, suggesting that both ends of the protein are
important for this function [50,51]. It has been reported
that suppression of DHS delays loss of tissue integrity in
senescing tomato fruit, leading to an extended shelf life.
Further analysis will indicate if melon mutants CmDET1 and Cm-DHS have alterations in fruit senescence.
Segregation studies are now being performed along with
phenotype analysis of the mutant lines.
HGOm
O
(2)m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
Page 9 of 11
Figure 5 Localization of eIF4E amino acids differing between virus-susceptible and virus-resistant genotypes of pepper (Capsicum spp.,
pvr2), tomato (Lycopersicon spp., pot1), lettuce (Lactuca spp., mo1), pea (Pisum sativum, sbm1), melon (Cucumis melo, nsv) and barley
(Hordeum vulgare, rym) in the predicted amino acid sequences (protein alignment of the susceptible genotypes). Black boxes indicate amino
acids differing between susceptible and resistant genotypes. The two protein regions with clustering of amino acid substitutions are highlighted in
pink and blue. In red are the mutations detected by TILLING in our PS population, in green, the mutations detected in the Charentais population. The
conserved tryptophan residues reported to be required for cap-binding activity are underlined in the Pisum sequence [48].
Conclusions
The TILLING approach worked in inodorus melons as a
way of identifying new heritable variants in candidate
genes that are different from those present in natural
populations. The cosegregation of a mutation predicted
to alter the functionality of PDS with the albino phenotype expected for PDS disruption suggests that this is a
promising approach for advancement in reverse melon
genetics. It is also useful to analyze TILLING populations
phenotypically, in order to use the phenotypes of interest
readily in crop improvement. The novel fruit phenotypes
could be of interest to diversify the market supply of Piel
HHFm
de Sapo melons. The information presented here will
also be useful for creating new inodorus melon populations with a higher mutation rate. Completion of the
melon genome sequence will provide many potential target genes of interest that may be functionally studied,
facilitating future genomic and breeding studies.
Acknowledgements and Funding
The authors thank Torben Jahrmann and Eulàlia Fitó (Semillas Fitó S.A.) for
selfing the M1 plants and obtaining M3 seed from the M2 families, Fatima
Dahmani for sequencing Cm-eIF4E/PS mutants, Montserrat Saladié for
providing the sequences of Cm-ACO1 and Cm-NOR from the M62-113 melon
line and Diego Bergareche for obtaining data on the M2 segregation. This
(2)O
m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
work was supported by projects GEN2003-20237-C06-02/03, funded by the
Ministerio de Educación y Ciencia (Spain), and GEN2006-27773-C2-1/2,
funded by the ERA-PG programme.
Author details
1
IRTA, Centre de Recerca en Agrigenòmica CSIC-IRTA-UAB, Carretera de
Cabrils Km 2, 08348 Cabrils (Barcelona), Spain. 2Institute for the Conservation
and Breeding of Agricultural Biodiversity (COMAV-UPV), Universitat
Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain. 3Instituto
de Biología Molecular y Celular de Plantas (IBMCP), Universitat Politècnica de
València (UPV)-Consejo Superior de Investigaciones Científicas (CSIC), Ciudad
Politécnica de la Innovación (CPI), Ed. 8E, C/Ingeniero Fausto Elio s/n, 46022
Valencia, Spain. 4Unité de Recherche en Génomique Végétale (INRA-URGV), 2
rue Gaston Crémieux CP 5708, 91057 Evry Cedex, France. 5Department of
Plant Science, School of Agriculture and Biology, Shanghai Jiaotong
University, 200030 Shanghai, People’s Republic of China.
Authors’ contributions
JG-M, MG and AJM performed the EMS mutagenesis. FN and BP contributed
to the development and conservation of the M2 seed. BP, CR and CE
extracted the DNA and performed the phenotypic analysis. DNA pooling,
TILLING screens and analysis, and PDS mutant phenotypic analysis were
done by MG, CT and MX. MX and MP participated in the sequencing of the
eIF4E mutants in the M2 population. JG-M, AB, BP, and FN coordinated the
study. BP, MG, CE, and JG-M were primarily responsible for drafting and
revising the manuscript with contributions from co-authors. All authors read
and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 15 March 2011 Accepted: 11 August 2011
Published: 11 August 2011
References
1. The International Cucurbit Genomics Initiative (ICuGI). [http://www.icugi.
org].
2. González-Ibeas D, Blanca J, Roig C, González-To M, Picó B, Truniger V,
Gómez P, Deleu W, Caño-Delgado A, Arús P, Nuez F, García-Mas J,
Puigdomènech P, Aranda MA: MELOGEN: an EST database for melon
functional genomics. BMC Genomics 2007, 8:306.
3. Fita A, Picó B, Monforte A, Nuez F: Genetics of Root System Architecture
Using Near-isogenic Lines of Melon. J Am Soc Hortic Sci 2008,
133:448-458.
4. Fernandez-Silva I, Eduardo I, Blanca J, Esteras C, Picó B, Nuez F, Arús P,
Garcia-Mas J, Monforte AJ: Bin mapping of genomic and EST-derived
SSRs in melon (Cucumis melo L.). Theor Appl Genet 2008, 118:139-150.
5. Deleu W, Esteras C, Roig C, González-To M, Fernández-Silva I, Blanca J,
Aranda MA, Arús P, Nuez F, Monforte AJ, Picó MB, Garcia-Mas J: A set of
EST-SNPs for map saturation and cultivar identification in melon. BMC
Plant Biol 2009, 9:90.
6. Mascarell-Creus A, Cañizares J, Vilarrasa J, Mora-García S, Blanca J, GonzálezIbeas D, Saladié M, Roig C, Deleu W, Picó B, López-Bigas N, Aranda MA,
Garcia-Mas J, Nuez F, Puigdomènech P, Caño-Delgado A: An oligo-based
microarray offers novel transcriptomic approaches for the analysis of
pathogen resistance and fruit quality traits in melon (Cucumis melo L.).
BMC Genomics 2009, 10:467.
7. Blanca JM, Cañizares J, Ziarsolo P, Esteras C, Mir G, Nuez F, Garcia-Mas J,
Pico B: Melon transcriptome characterization. SSRs and SNPs discovery
for high throughput genotyping across the species. Plant Genome 2011,
4(2):118-131.
8. González VM, Benjak A, Hénaff EM, Mir G, Casacuberta JM, Garcia-Mas J,
Puigdomènech P: Sequencing of 6.7 Mb of the melon genome using a
BAC pooling strategy. BMC Plant Biology 2010, 10:246.
9. Moreno E, Obando JM, Dos-Santos N, Fernández-Trujillo JP, Monforte AJ,
Garcia-Mas J: Candidate genes and QTLs for fruit ripening and softening
in melon. Theor Appl Genet 2007, 116:589-602.
10. Essafi A, Díaz-Pendón JA, Moriones E, Monforte AJ, Garcia-Mas J, MartínHernández AM: Dissection of the oligogenic resistance to Cucumber
mosaic virus in the melon accession PI 161375. Theor Appl Genet 2009,
118:275-284.
Page 10 of 11
11. Comai L, Henikoff S: TILLING: practical single-nucleotide mutation
discovery. Plant J 2006, 45:684-94.
12. Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, Jamai A, El-Mellouki T,
Liu S, Ritchie R, Nielsen N, et al: TILLING to detect induced mutations in
soybean. BMC Plant Biol 2008, 8(1):9.
13. Dalmais M, Schmidt J, Le Signor C, Moussy F, Burstin J, Savois V, Aubert G,
de Oliveira Y, Guichard C, Thompson R, Bendahmane A: UTILLdb, a Pisum
sativum in silico forward and reverse genetics tool. Genome Biol 2008, 9:
R43.
14. Dierking EC, Bilyeu KD: New sources of soybean meal and oil
composition traits identified through TILLING. BMC Plant Biol 2009, 9:89.
15. Perry J, Brachmann A, Welham T, Binder A, Charpentier M, Groth M,
Haage K, Markmann K, Wang TL, Parniske M: TILLING in Lotus japonicus
identified large allelic series for symbiosis genes and revealed a bias in
functionally defective ethyl methanesulfonate alleles toward glycine
replacements. Plant Physiol 2009, 151(3):1281-1291.
16. Caldwell DG, McCallum N, Shaw P, Muehlbauer GJ, Marshall DF, Waugh R:
A structured mutant population for forward and reverse genetics in
Barley (Hordeum vulgare L.). Plant J 2004, 40(1):143-150.
17. Henikoff S, Bradley JT, Comai L: TILLING. Traditional mutagenesis meets
functional genomics. Plant Physiol 2004, 135:630-636.
18. Wu JL, Wu C, Lei C, Baraoidan M, Bordeos A, Madamba MR, RamosPamplona M, Mauleon R, Portugal A, Ulat VJ, et al: Chemical- and
irradiation-induced mutants of indica rice IR64 for forward and reverse
genetics. Plant Mol Biol 2005, 59(1):85-97.
19. Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D: A reverse
genetic, nontransgenic approach to wheat crop improvement by
TILLING. Nat Biotechnol 2005, 23:75-81.
20. Till BJ, Cooper J, Tai TH, Colowit P, Greene EA, Henikoff S, Comai L:
Discovery of chemically induced mutations in rice by TILLING. BMC Plant
Biol 2007, 7:19.
21. Xin Z, Wang ML, Barkley NA, Burow G, Franks C, Pederson G, Burke J:
Applying genotyping (TILLING) and phenotyping analyses to elucidate
gene function in a chemically induced sorghum mutant population.
BMC Plant Biol 2008, 8:103.
22. Dong C, Dalton-Morgan J, Vincent K, Sharp P: A modified TILLING method
for wheat breeding. Plant Genome 2009, 2:39-47.
23. Sestili F, Botticella E, Bedo Z, Phillips A, Lafiandra D: Production of novel
allelic variation for genes involved in starch biosynthesis through
mutagenesis. Mol Breeding 2010, 25:145-154.
24. Watanabe S, Mizoguchi T, Aoki K, Kubo Y, Mori H, Imanishi S, Yamazaki Y,
Shibata D, Ezura H: Ethylmethanesulfonate (EMS) mutagenesis of
Solanum lycopersicum cv. Micro-Tom for large-scale mutant screens.
Plant Biotech 2007, 24:33-38.
25. Elias R, Till BJ, Mba Ch, Al-Safadi B: Optimizing TILLING and Ecotilling
techniques for potato (Solanum tuberosum L). BMC Res Notes 2009, 2:141.
26. Piron F, Nicolaı M, Minoıa S, Piednoir E, Moretti A, Salgues A, Zamir D,
Caranta C, Bendahmane A: An induced mutation in tomato eIF4E leads to
immunity to two Potyviruses. PLoS ONE 2010, 5(6):e11313.
27. Himelblau E, Gilchrist EJ, Buono K, Bizell C, Mentzer L, Vogelzang R,
Osborn T, Amasino RM, Parkin IAP, Haughn : Forward and reverse genetics
of papid cycling Brassica oleracea. Theor Appl Genet 2009, 118:953-961.
28. Stephenson P, Baker D, Girin T, Perez A, Amoah S, King GJ, Østergaard L: A
rich TILLING resource for studying gene function in Brassica rapa. BMC
Plant Biol 2010, 10:62.
29. Pitrat M: Melon (Cucumis melo L.). In Handbook of Crop Breeding Vol I.
Vegetables. Edited by: Prohens J, Nuez F. New York:Springer; 2008:283-315.
30. Dahmani-Mardas F, Troadec Ch, Boualem A, Leveque S, Alsadon AA,
Aldoss AA, Dogimont C, Bendahman A: Engineering Melon Plants with
Improved Fruit Shelf Life Using the TILLING Approach. PLoS ONE 2010, 5:
e15776.
31. Nieto C, Piron F, Dalmais M, Marco CF, Moriones E, Gómez-Guillamón ML,
Truniger V, Gómez P, Garcia-Mas J, Aranda MA, Bendahmane A: EcoTILLING
for the identification of allelic variants of melon eIF4E, a factor that
controls virus susceptibility. BMC Plant Biol 2007, 7:34.
32. Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu LJ: Disruption of
phytoene desaturase gene results in albino and dwarf phenotypes in
Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin
biosynthesis. Cell Res 2007, 17:471-482.
33. Codons Optimized to Deliver Deleterious Lesions (CODDLe). [http://www.
proweb.org/input].
HHGm
O
(2)m
González et al. BMC Research Notes 2011, 4:289
http://www.biomedcentral.com/1756-0500/4/289
Page 11 of 11
34. Lasserre E, Bouquin T, Hernández JA, Bull J, Pech JC, Balague C: Structure
and expression of three genes encoding ACC oxidase homologs from
melon (Cucumis melo L.). Mol Gen Genet 1996, 251(1):81-90.
35. Giovannoni JJ: Fruit ripening mutants yield insights into ripening control.
Curr Opin Plant Biol 2007, 10:1-7.
36. Davuluri GR, van Tuinen A, Mustilli AC, Manfredonia A, Newman R,
Burgess D, Brummell DA, King SR, Palys J, Uhlig J, Pennings HMJ, Bowler C:
Manipulation of DET1 expression in tomato results in
photomorphogenic phenotypes caused by post-transcriptional gene
silencing. Plant J 2004, 40:344-354.
37. Wei S, Li X, Gruber MI, Li R, Zhou R, Zebarjadi A, Hannoufa A: RNAimediated suppression of DET1 alters the levels of carotenoids and
sinapate esters in seeds of Brassica napus. J Agric Food Chem 2009,
57(12):5326-5333.
38. Wang TW, Zhang CG, Wu W, Nowack LM, Madey E, Thompson JE:
Antisense suppression of deoxyhypusine synthase in tomato delays fruit
softening and alters growth and development DHS mediates the first of
two sequential enzymatic reactions that activate eukaryotic translation
initiation factor-5A. Plant Physiol 2005, 138:1372-1382.
39. Ng PC, Henikoff S: SIFT: predicting amino acid changes that affect
protein function. Nucleic Acids Res 2003, 31(13):3812-3814.
40. Guzman P, Ecker JR: Exploiting the triple response of Arabidopsis to
identify ethylene-related mutants. The Plant Cell 1990, 2:513-523.
41. Henikoff S, Comai L: Single-nucleotide mutations for plant functional
genomics. Ann Rev Plant Biol 2003, 54:375-401.
42. Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH,
Enns LC, Burtner C, Johnson JE, Odden AR, et al: Spectrum of chemically
induced mutations from a large-scale reverse genetic screen in
Arabidopsis. Genetics 2003, 164(2):731-740.
43. Britt AB: DNA damage and repair in plants. Annu Rev Plant Physiol Plant
Mol Biol 1996, 47:75-100.
44. Truniger V, Nieto C, González-Ibeas D, Aranda M: Mechanism of plant
eIF4E-mediated resistance against a Carmovirus (Tombusviridae): capindependent translation of a viral RNA controlled in cis by an (a)
virulence determinant. Plant J 2008, 56(5):716-727.
45. Gao Z, Johansen E, Eyers S, Thomas CL, Ellis THN, Maule AJ: The potyvirus
recessive resistance gene, sbm1, identifies a novel role for translation
initiation factor eIF4E in cell-to-cell trafficking. Plant J 2004, 40(3):376-385.
46. Kang BC, Yeam I, Frantz JD, Murphy JF, Jahn MM: The pvr1 locus in
Capsicum encodes a translation initiation factor eIF4E that interacts with
Tobacco etch virus VPg. Plant J 2005, 42(3):392-405.
47. Ruffel S, Gallois J, Lesage M, Caranta C: The recessive potyvirus resistance
gene pot-1 is the tomato orthologue of the pepper pvr2-eiF4 genes. Mol
Genet Genom 2005, 274(4):346-353.
48. Nicaise V, German-Retana S, Sanjuán R, Dubrana MP, Mazier M,
Maisonneuve B, Candresse T, Caranta C, LeGall O: The Eukaryotic
Translation Initiation Factor 4E Controls Lettuce Susceptibility to the
Potyvirus Lettuce mosaic virus1. Plant Physiol 2003, 132:1272-1282.
49. Esteras C, Pascual L, Saladie M, Dogimont C, Garcia-Mas J, Nuez F, Picó B:
Use of Ecotilling to identify natural allelic variants of melon candidate
genes involved in fruit ripening. Proceedings Plant GEM8 Lisbon 2009.
50. Levin I, Frankel P, Gilboa N, Tanny S, Lalazar A: The tomato dark green
mutation is a novel allele of the tomato homolog of the DEETIOLATED1
gene. Theor Appl Genet 2003, 106:454-460.
51. Kolotilin I, Koltai H, Tadmor Y, Bar-Or C, Reuveni M, Meir A, Nahon S,
Shlomo S, Chen L, I Levin: Transcriptional profiling of high pigment-2dg
tomato mutant links early fruit plastid biogenesis with its
overproduction of phytonutrients. Plant Physiol 2007, 145:389-401.
doi:10.1186/1756-0500-4-289
Cite this article as: González et al.: Towards a TILLING platform for
functional genomics in Piel de Sapo melons. BMC Research Notes 2011
4:289.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
T
HHHm
T
(2)O
m
T
BMC Genomics
BioMed Central
Open Access
Research article
MELOGEN: an EST database for melon functional genomics
Daniel Gonzalez-Ibeas1, José Blanca2, Cristina Roig2, Mireia González-To3,
Belén Picó2, Verónica Truniger1, Pedro Gómez1, Wim Deleu3,
Ana Caño-Delgado4, Pere Arús3, Fernando Nuez2, Jordi Garcia-Mas3,
Pere Puigdomènech4 and Miguel A Aranda*1
Address: 1Departamento de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS)- CSIC, Apdo.
correos 164, 30100 Espinardo (Murcia), Spain, 2Departamento de Biotecnología, Instituto de Conservación y Mejora de la Agrodiversidad
Valenciana (COMAV-UPV), Camino de Vera s/n, 46022 Valencia, Spain, 3Departament de Genètica Vegetal, Centre de Recerca en Agrigenòmica
CSIC-IRTA, Carretera de Cabrils Km2, 08348 Cabrils (Barcelona), Spain and 4Departament de Genètica Molecular, Centre de Recerca en
Agrigenòmica CSIC-IRTA, Jordi Girona 18-26, 08034 Barcelona, Spain
Email: Daniel Gonzalez-Ibeas - [email protected]; José Blanca - [email protected]; Cristina Roig - [email protected]; Mireia GonzálezTo - [email protected]; Belén Picó - [email protected]; Verónica Truniger - [email protected]; Pedro Gómez - [email protected];
Wim Deleu - [email protected]; Ana Caño-Delgado - [email protected]; Pere Arús - [email protected]; Fernando Nuez - [email protected];
Jordi Garcia-Mas - [email protected]; Pere Puigdomènech - [email protected]; Miguel A Aranda* - [email protected]
* Corresponding author
Published: 3 September 2007
BMC Genomics 2007, 8:306
doi:10.1186/1471-2164-8-306
Received: 8 May 2007
Accepted: 3 September 2007
This article is available from: http://www.biomedcentral.com/1471-2164/8/306
© 2007 Gonzalez-Ibeas et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Melon (Cucumis melo L.) is one of the most important fleshy fruits for fresh consumption. Despite
this, few genomic resources exist for this species. To facilitate the discovery of genes involved in essential traits,
such as fruit development, fruit maturation and disease resistance, and to speed up the process of breeding new
and better adapted melon varieties, we have produced a large collection of expressed sequence tags (ESTs) from
eight normalized cDNA libraries from different tissues in different physiological conditions.
Results: We determined over 30,000 ESTs that were clustered into 16,637 non-redundant sequences or
unigenes, comprising 6,023 tentative consensus sequences (contigs) and 10,614 unclustered sequences
(singletons). Many potential molecular markers were identified in the melon dataset: 1,052 potential simple
sequence repeats (SSRs) and 356 single nucleotide polymorphisms (SNPs) were found. Sixty-nine percent of the
melon unigenes showed a significant similarity with proteins in databases. Functional classification of the unigenes
was carried out following the Gene Ontology scheme. In total, 9,402 unigenes were mapped to one or more
ontology. Remarkably, the distributions of melon and Arabidopsis unigenes followed similar tendencies, suggesting
that the melon dataset is representative of the whole melon transcriptome. Bioinformatic analyses primarily
focused on potential precursors of melon micro RNAs (miRNAs) in the melon dataset, but many other genes
potentially controlling disease resistance and fruit quality traits were also identified. Patterns of transcript
accumulation were characterised by Real-Time-qPCR for 20 of these genes.
Conclusion: The collection of ESTs characterised here represents a substantial increase on the genetic
information available for melon. A database (MELOGEN) which contains all EST sequences, contig images and
several tools for analysis and data mining has been created. This set of sequences constitutes also the basis for an
oligo-based microarray for melon that is being used in experiments to further analyse the melon transcriptome.
HHIm
O
(2)m
Background
Melon (Cucumis melo L.) is an important horticultural
crop grown in temperate, subtropical and tropical regions
worldwide. Melon is among the most important fleshy
fruits for fresh consumption, its total production in 2004
exceeding 874 million metric tons, of which 72.5% are
produced in Asia, 11.7% in Europe, 8.4% in America and
6.1% in Africa, being a significant component of fresh
fruit traded internationally [1]. Melon belongs to the
Cucurbitaceae family, which comprises up to 750 different
species distributed in 90 genera. Species in this family
include watermelon, cucumber, squash and marrow, all
of them cultivated essentially because of their fruits, but
this family also includes species of interest for other reasons, as, for example, their contents in potentially therapeutic compounds (e.g. Momordica charantia) [2]. Melon
is a diploid species, with a basic number of chromosomes
x = 12 (2x = 2n = 24) and an estimated genome size of 450
to 500 Mb [3], similar in size to the rice genome (419 Mb)
[4,5] and about three times the size of the Arabidopsis
genome (125 Mb) [6]. Melon has been classified into two
subspecies, C. melo ssp. agrestis and C. melo ssp. melo with
India and Africa being their centres of origin, respectively
[7,8].
Melon has a great potential for becoming a model for
understanding important traits in fruiting crops. Melon
fruits have wide morphological, physiological and biochemical diversity [7,9] which can be exploited to dissect
biological processes of great technological importance,
among them flavour development and textural changes
that occur during fruit ripening. The contemporary melon
cultivars can be divided into two groups, climacteric and
nonclimacteric, according to their ripening patterns [10].
Climacteric fruits are characterized by rapid and profound
changes during ripening associated to increased levels of
respiration and release of ethylene, whereas the nonclimacteric varieties do not produce ethylene and have long
shelf-life. Analyses of climacteric and nonclimacteric melons have illustrated the process of aroma formation [1114] and the temporal sequence of cell wall disassembly
[15-17]. Melon can be also a very useful experimental system to analyse other aspects of fundamental plant biology. For example, melon and other cucurbits have been
used to analyse the development of the plant vasculature
and the transportation of macromolecules through it [1820], and different interactions between melon and pests
and pathogens have been characterised with varying
depths [21-27].
Important genetic tools have been described for melon, as
for example linkage genetic maps [28,29] and the development of a genomic library of near isogenic lines (NILs)
from an exotic accession [30]; also, biotechnology is feasible in melon [31-33]. However, the great majority of
genes involved in the aforementioned traits are yet to be
identified in melon. Partial sequencing of cDNA inserts of
expressed sequence tags (ESTs) have been used as an effective method for gene discovery. By sequencing clones
derived from RNA from different sources, and/or by normalizing cDNA libraries, the total set of genes sampled
can be maximized. Bioinformatic analysis, annotation
and clustering of sequences could yield databases which
mining can be used to select candidate genes implicated
in traits of interest. EST collections can also serve to construct microarrays useful for identifying sets of plant genes
expressed during different developmental stages and/or
responding to environmental stimuli [34,35]. In addition,
EST collections are good sources of simple sequence
repeats (SSRs) and single-nucleotide polymorphisms
(SNPs) that can be used for creating saturated genetic
maps [36,37]. Thus, EST collections have been generated
for many plant species, being the most comprehensive
those of Arabidopsis[6] and rice [38]. Fruit crops have
been less extensively surveyed, but important collections
are publicly available for several species, including tomato
[39], apple [40], grape [41] and citrus [42].
Despite the importance of the family Cucurbitaceae, relatively little EST information is currently available: only
16,039 nucleotide sequences have been annotated from
the whole Cucurbitaceae family in the publicly accessible
GenBank database as of November 2006; out of these,
12,180 correspond to the Cucumis genus and 6,061 to
melon. These numbers are in sharp contrast with the data
available for families composed of other important food
crops like Solanaceae (1,020,102 sequences), Fabaceae
(1,466,518 sequences), Brassicaceae (1,010,148 sequences
excluding Arabidopsis), Vitaceae (449,478) and Rosaceae
(390,066 sequences). Here we describe a public EST
sequencing project in melon. We report the determination and analysis of 30,675 high-quality melon ESTs,
sequenced from eight normalized cDNA libraries corresponding to different tissues in different physiological
conditions. We have classified the sequences into functional categories and described SSRs and SNPs of potential use in genetic maps and marker-assisted breeding
programs. A database which contains all EST sequences,
contig images and several tools for analysis and data mining has been created. In addition, we have analyzed the
EST melon dataset to identify candidate genes potentially
coding microRNAs or involved in fruit maturation processes and pathogen defence. The pattern of transcript
accumulation in different physiological conditions has
been characterised by Real-Time-qPCR for 20 of these candidate genes.
HHJm
T
(2)O
m
Results
EST Sequencing and Clustering
Eight cDNA libraries were constructed using material
from "Piel de Sapo" Spanish cultivars, the C-35 cantaloupe line (both belonging to Cucumis melo L. ssp. melo)
and the accession pat81 of C. melo L. ssp. agrestis (Naud.)
Pangalo. The sources of RNA to construct each library
were fruits of 15 and 46 days after pollination (dap),
leaves, photosynthetic cotyledons inoculated with Cucumber mosaic virus (CMV), healthy roots and Monosporascus
cannonballus Pollack et Uecker (the causal agent of melon
vine decline) infected roots (Table 1). Approximately
3,700 sequences were determined from each library by
single-pass 5' sequencing, except for the library prepared
from CMV infected cotyledons for which approximately
6,600 sequences were determined, yielding a total of
33,292 raw sequences. Processing to eliminate vector
sequences, low quality chromatograms and sequences of
less than 100 base pairs (bp) gave rise to 29,604 good
quality expressed sequence tags (ESTs) (Table 2) implying
a cloning success of approximately 89%. The average
edited length was 674 bp, and only a 6.4% of the
sequences had less than 350 bp.
Clustering of the sequences using default parameters of
the EST analysis pipeline EST2uni [43] yielded 6,023 tentative consensus sequences (also called contigs) and
10,614 unclustered sequences (also called singletons),
with a total of 16,637 non-redundant sequences or unigenes (Table 2). All good quality ESTs were used for clustering, independently of the melon genotype of origin,
because single nucleotide polymorphisms (SNPs) were
expected among genotypes. The number of ESTs per unigene was between 1 and 44 (1 case), with an average of 1.8
ESTs per contig, as a high proportion of contigs (4,886 out
of 6,023) contained less than 5 ESTs and contigs with
more than 8 ESTs were scarce (Fig. 1A). Therefore, redundancy values were notably low (around 16%). The unigene length varied between 101 bp and 2,664 bp,
averaging 751 bp (Fig. 1B). Library specific unigenes were
about one third of the total for each library (Table 2). A
second round of clustering yielded 14,480 unigene clusters, referred to as superunigenes. A web integrated data-
base that contains all EST sequences, contig images and
several tools for analysis and data mining has been created
and named MELOGEN [44]. Codon usage was estimated
using this EST collection. As expected, the codon usage of
melon was very similar to that of Arabidopsis and other
dicots. The preferred stop codon was UGA occurring in
the 48% of the sequences. Suppression of the CG dinucleotide in the last two codon positions is very frequent in
dicots, possibly as a consequence of methylation of C in
the CG dinucleotide, resulting in an increased mutation
rate [45]; in agreement with these data, the ratio XCG/
XCC for melon was 0.52, very similar to the corresponding figure for tomato (0.58), pea (0.51), potato (0.48) and
other dicots [45].
Libraries obtained from tissues inoculated with M. cannonballus were expected to contain sequences from the
fungus. To estimate the proportion of sequences of fungal
origin in these libraries, BLAST analyses against a database
with plant and fungal sequences were carried out [46].
Only 56 sequences from these libraries were found to
have a more significant similarity with fungal sequences
than with plant sequences (Table 3). Consequently, these
sequences were considered of fungal origin [46].
SSRs and SNPs
We have analysed the nature and frequency of microsatellites or simple sequence repeats (SSRs) in the melon
sequence dataset. A search for repeats of two, three or four
nucleotides in the dataset yielded 1,052 potential SSRs.
Approximately, 6% of the unigenes contained at least one
of the considered SSRs motifs, with repeats of three
nucleotides being prevalent (Table 4). The maximum and
minimum lengths of the repeats were 68 and 17 nucleotides, respectively, and the average length was 26 nucleotides. The most common repeat among dinucleotides was,
by far, the AG repeat, constituting the 83% (Table 4).
Repeats of AT and AC dinucleotides followed, with
approximately 9% and 7%, respectively. Among the trinucleotide repeats, the most frequent was AAG (66%, Table
4), and the least frequent was ACT (0.6%, Table 4).
Among tetranucleotide repeats, the most frequent was
AAAG (51%, Table 4). A high proportion of SSRs (29.5%)
Table 1: Description of cDNA libraries
Name
Subspecies/cultivar/accession
Tissue/physiological condition
15d
46d
A
AI
CI
HS
PS
PSI
Ssp. melo cv. "Piel de Sapo" T-111
Ssp. melo cv. "Piel de Sapo" T-111
Ssp. agrestis accession pat81
Ssp. agrestis accession pat81
Ssp. melo var. cantaloupe accession C-35
Ssp. melo var. cantaloupe accession C-35
Ssp. melo cv. "Piel de Sapo" Piñonet torpedo
Ssp. melo cv. "Piel de Sapo" Piñonet torpedo
Fruit 15 days after pollination
Fruit 46 days after pollination
Roots
Roots infected with M. cannonballus
Photosynthetic cotyledons infected with CMV
Leaves
Roots
Roots infected with M. cannonballus
HHKm
O
(2)m
Table 2: EST statistics
Library
Raw
sequences
Good-Quality
ESTs
EST length
Singletons
Contigs
Unigenes
Redundancy (%)
Library-specific
unigenes
Novelty (%)
15d
46d
A
AI
CI
HS
PS
PSI
3,936
3,840
3,936
3,647
6,605
3,648
3,840
3,840
33,292
3,582
3,493
3,666
3,255
5,664
3,012
3,377
3,555
29,604
608.1 ± 175.2
583.0 ± 161.1
700.0 ± 185.4
756.3 ± 137.1
651.4 ± 205.7
669.3 ± 171.1
679.9 ± 198.7
749.3 ± 156.2
1,009
1,000
1,289
928
2,089
939
1,179
1,279
10,614
1,930
1,854
1,900
1,688
2,590
1,609
1,766
1,826
6,023
2,939
2,854
3,189
2,616
4,679
2,548
2,945
3,105
16,637
18
18
13
20
17
15
13
13
1,100
1,063
1,365
1,005
2,264
998
1,258
1,363
37
37
43
38
48
39
43
44
were found in open reading frames (ORFs), though an
analysis of the localization of di-, tri- and tetranucleotides
separately showed that di- and tetranucleotides localised
preferentially in untranslated regions (UTRs), whereas trinucleotides localised in both, UTRs and ORFs (Table 5).
Single nucleotide polymorphisms (SNPs) are the most
abundant variations in genomes and, therefore, constitute
a powerful tool for mapping and marker-assisted breeding. We initially identified in the melon sequence dataset
14,074 single nucleotide sequence variations and therefore potential SNPs (pSCH; Table 6) distributed in 4,663
contigs; however, these variations would include highquality SNPs (pSNP) but also sequencing errors and
mutations introduced during the cDNA synthesis step.
Using more stringent criteria, these figures were substantially reduced: Putative SNPs were annotated only when
the least represented allele was present in at least two EST
sequences from the same genotype in a given contig and
showing the same base change. Two accessions of the
same cultivar (cv. "Piel de sapo") represented 47.3% of
the sequences, but more than one half of the sequences
were from two other more distant genotypes, the C-35
cantaloupe accession (29.3%) and the pat81 agrestis
accession (23.4%). Thus, a total of 356 high-quality SNPs
were found in 292 contigs, averaging 1.2 SNPs per contig.
Table 3: ESTs showing significant similarity with fungal
sequences
Library
Number of ESTs
15d
46d
A
AI
CI
HS
PS
PSI
Total
0
0
2
26
0
1
1
30
60
Transitions were much more common than transversions.
There were 117 AG and 112 CT transitions compared with
28 AC, 37 AT and 33 GT transversions (Table 6). CG transversions were not detected. The MELOGEN database [44]
includes a tool for designing oligonucleotide primers to
amplify the region containing the polymorphism to generate the corresponding molecular marker.
Functional annotation
In order to identify melon unigenes potentially encoding
proteins with known function, we carried out a BLASTX
analysis [47] of the sequence dataset against the databases
listed in Table 7. Out of the 13,019 unigenes with a hit
with proteins in databases, 11,431 (68.7%) unigenes
showing an E value of 1e-10 were annotated. On the
other hand, 31.3% of the unigenes did not show significant similarity to any protein in the databases and, therefore, were not annotated.
Additionally, we performed a functional classification of
the unigenes following the Gene Ontology scheme. Gene
Ontology provides a structured and controlled vocabulary
to describe gene products according to three ontologies:
molecular function, biological process and cellular component [48]. To do that, we added GO terms based on the
automated annotation of each unigene using the Arabidopsis database [6]. A summary of the results with the
percentage of unigenes annotated in representative categories corresponding to the GO slim terms [48] is shown,
as well as a comparison of the distribution of melon and
Arabidopsis unigenes (Fig. 2). The distributions of melon
and Arabidopsis unigenes follow similar tendencies, suggesting that the melon dataset is representative of the
whole melon transcriptome. In total, 9,402 unigenes
could be mapped to one or more ontologies, with multiple assignments possible for a given protein within a single ontology. A high percentage of unigenes in both
species was classified as "unknown function". Out of the
9,791 assignments made to the cellular component category, 25.8% corresponded to membrane proteins and
17.8% to plastidial proteins (Fig. 2A). Under the molecu-
HHLm
(2)O
m
!!! Table 4: Simple sequence repeats (SSRs) statistics*
Number of di-pSSR
%
AG
AT
AC
Total
205
23
18
246
83.3
9.4
7.3
100
Dinucleotide repeat
Figure 1 statistics
Unigenes
Unigenes statistics. (A) Distribution of melon ESTs among
unigenes (contigs and singletons). (B) Size distribution of
melon unigenes.
lar function category, assignments were mainly to catalytic
activity (23.0%) and to hydrolase activity (14.7%) (Fig.
2B). The distribution of unigenes under the biological
process category was more uniform, with 19.9% of assignments to cellular process and 12.7% to biosynthesis (Fig.
2C).
We have also identified 6,673 (40.1%) melon unigenes
with an ortholog in the Arabidopsis database, and a
HMMER motif has been assigned to 4,655 (28.0%) unigenes by comparisons with the Pfam database [49] (Table
7). All these results are compiled in the MELOGEN database, which also contains direct links to the databases
used to carry out analyses.
Genes potentially encoding microRNAs
Central to RNA silencing are small RNA molecules
(sRNAs) that can arise from endogenous or exogenous
sources from precursors with double-stranded RNA
(dsRNA) pairing. One class of such sRNAs are microRNAs
(miRNAs), which originate from endogenous long selfcomplementary precursors that mature in a multi-step
process involving many enzymes [50,51]. Recently, a
Trinucleotide repeat
Number of tri-pSSR
%
AAC
AAG
AAT
ACC
ACG
ACT
AGC
AGG
ATC
CCG
Total
41
471
22
18
14
4
25
54
48
17
714
5.7
66.0
3.1
2.5
2.0
0.6
3.5
7.6
6.6
2.4
100
Tetranucleotide repeat
Number of tetra-pSSR
%
AAAC
AAGG
AATC
AATG
AATT
ATCG
ACTC
AAGC
ACAT
AAAG
AAAT
Total
8
7
4
3
2
2
1
1
1
47
16
92
8.7
7.6
4.3
3.3
2.2
2.2
1.1
1.1
1.1
51.1
17.3
100
*The number of di-, tri- and tetranucleotide repeats identified in the
melon database is shown for the complete set of putative SSRs
(pSSRs).
comprehensive strategy to identify new miRNA homologs
in EST databases has been developed [52,53]. We have followed this strategy to identify potential melon miRNAs. A
total of 20 ESTs that contained homologs to miRNAs in
the microRNA Registry database [54] were identified and
grouped into 12 contigs and, after manual inspection of
secondary foldback hairpin structure, 5 unigenes were
selected (Table 8). Contig sequences varied between 536
and 840 nucleotides long, and had negative folding free
energies of -206.8 to -160.8 kcal mol-1 (Table 8) according
to MFOLD [55], which are in the range of the computational values of Arabidopsis miRNA precursors [52]. Their
predicted secondary structures showed that there were at
least 16 nucleotides paired between the sequence of the
potential mature miRNA and its opposite arm (miRNA*)
in the corresponding hairpin structure (Fig. 3). The location of the potential miRNAs varied among ESTs, 4 were
found in the sense orientation of the EST, 1 was found in
HHMm
O
(2)m
Table 5: Localization of simple sequence repeats (SSRs) with respect to putative initiation and termination codons in the melon
sequence dataset*
5'-UTR
ORF
3'-UTR
Other†
Total
Dinucleotide repeats
Trinucleotide repeats
Tetranucleotide repeats
All SSRs analyzed
No.
%
No.
%
No.
%
No.
%
65
0
13
1
79
82.3%
0.0%
16.5%
1.3%
100.0%
121
99
13
1
234
51.7%
42.3%
5.6%
0.4%
100.0%
18
2
9
0
29
62.1%
6.9%
31.0%
0.0%
100.0%
204
101
35
2
342
59.6%
29.5%
10.2%
0.6%
100%
*Only full length melon unigenes were used for this analysis. Full length unigenes were automatically selected by checking the presence of the start
codon by comparison of the 5'-terminal region of the unigene with the corresponding Arabidopsis gene used for annotation. †Imprecise localization
of the SSR with respect to putative initiation or termination codons.
the antisense orientation. We have also searched for
potential targets of the potential miRNAs in the melon
EST dataset, identifying 3 of them (Table 8). However,
minimal folding free energy indexes (MFEIs) [53] were
below the -0.85 cut-off value proposed by Zhang et al.
[53] only for m12 (Table 8). Potential melon miRNA m12
has a precursor of 536 nt in length and codes for a melon
ortholog of the Arabidopsis miR319. miR319 targets a
transcription factor of the TCP family [56,57]; in the
melon dataset, an ortholog of this Arabidopsis gene has
been found in a unigene annotated as a TCP transcription
factor. In this case, the melon miRNA and its potential target have a pattern of paired/non-paired bases between the
target and the miRNA identical to the corresponding target-miRNA pattern in Arabidopsis (data not shown).
Genes potentially encoding pathogen resistance and fruit
quality traits
Pathogens affect severely the productivity of melon crops.
Three of the cDNA libraries sequenced here correspond to
pathogen-infected tissues and, thus, should contain transcripts from genes whose expression is induced in
response infection. We have carried out a bioinformatics
search for homologs of genes involved in pathogen resistance response (see [58] for a review) and virus susceptibility [59-61], finding among them at least one melon
ortholog to the Arabidopsis FLS2 receptor [62], several
unigenes potentially encoding disease resistance proteins
as well as mitogen-activated protein kinases, homologs to
translation initiation factors constituting potential virus
susceptibility factors, etc. [see Additional file 1].
Fruit development and ripening are the most important
processes determining the fruit quality traits of fleshy
fruits like melon. At present most of the molecular and
genetic data available about fruit development and ripening come from tomato [63,64] and Arabidopsis [65,66].
In recent years, several genes and quantitative trait loci
controlling fruit quality traits have been described in
melon [67,68]. As for developmental processes,
homologs to genes involved in melon fruit development,
ripening and quality have been found in the melon dataset. These include several MADS-box genes, homologs to
the fw2.2 and ovate QTLs [69,70], several homologs to
Table 6: Single nucleotide polymorphisms (SNPs) statistics*
Variation
Number
%
pSNP transversions:
pSNP transitions:
pSCH transversions:
pSCH transitions:
127
229
4,273
9,801
35.7
64.3
30.4
69.6
Mutation
Number of pSNP
Mutation
Number of pSCH
A<->C
A<->G
A<->T
C<->G
C<->T
G<->T
28
117
37
29
112
33
A<->C
A<->G
A<->T
C<->G
C<->T
G<->T
859
5,860
1,897
431
3,941
1,086
*Type and number of transition and transversions are shown for putative single nucleotide variations in sequence (pSCH) and for putative highquality single nucleotide polymorphism (pSNP) identified in the melon database.
HHNm
(2)O
m
Table 7: Functional annotation statistics
A. Number of unigenes with BLAST hits
Database*
Number of unigenes
%
Arabidopsis
Cucurbitaceae
Uniref90
Any database
11,724
5,340
11,893
13,019
70.5
32.1
71.5
78.3
Database*
Number of unigenes
%
Pfam
4,655
28.0
Database*
Number of unigenes
%
Arabidopsis
6,673
40.1
B. Number of unigenes with HMMER hits
C. Number of unigenes with orthologue
* Databases searched were: Arabidopsis [6,109]; Uniref90 [110,115]; Cucurbitaceae: all cucurbitaceae sequences available in the National Center for
Biotechnology Information (NCBI); any database: results using Arabidopsis, cucurbitaceae and Uniref90 databases all together; Pfam [49,112].
members of the SBP-box gene family to which the major
tomato ripening gene COLORLESS NON-RIPENING
belongs [71], several ACC synthase and ACC oxidase
genes, unigenes from several cell wall-metabolism
enzymes, etc [see Additional file 1].
Expression analysis of selected ESTs by Real-Time-qPCR
The accumulation of transcripts for 20 selected genes was
analyzed by reverse transcription Real-Time-qPCR. ESTs
for this analysis were preferentially chosen among those
showing significant similarity with genes related to
response to infection and fruit quality characteristics in
melon and other species, and included CTL1, EIF4A-2,
EIF4E, EIN4, GA2OX1, HSP101, HSP70, IAA9, LSM1,
LUT2, NCBP, SVP, HIR, TCH4, TIP4, TOM1, TOM2A,
TOM3, UGE5 and WRKY70 (Table 9). Preliminary experiments were carried out to choose between GAPDH and
CYCLOPHILIN (CYP7) RNAs as endogenous controls;
results showed that the CYP7 RNA levels varied the least
among treatments (data not shown) and, therefore, transcript accumulation levels were expressed relative to CYP7
RNA levels.
Figure 4A illustrates the alteration of the RNA accumulation levels of selected genes that occurred in photosynthetic cotyledons after CMV infection. A significant
increase in the level of transcripts from HSP101, HSP70,
HIR, TOM2A, WRKY70 and EIN4 was observed; for
HSP101, HSP70, WRKY70 and EIN4, transcript accumulation levels in inoculated cotyledons were up to five times
greater than in uninoculated controls (Fig. 4A). All of
these genes, except TOM2A, have been shown to be
responsive to virus infection in other hosts [72-74]. Notably, the expression of EIF4E, known to be required for
MNSV multiplication [27], remained unaltered. A shutoff
of host gene expression also occurs in association with
virus infection [75]; for the set of genes analysed here,
only GA2OX1 and NCBP responded to CMV infection
with a reduction in the accumulation of their transcripts.
The response of selected genes in roots inoculated with M.
cannonballus was analysed in melon genotypes known to
be susceptible (cultivar "Piel de sapo"; Fig. 4B) and partially resistant (accession pat81 of C. melo L. ssp. agrestis;
Fig. 4C) to the infection by this fungus. The patterns of
transcript accumulation resulted clearly different for both
genotypes. For pat81 (resistant), transcription factors
WRKY70 and SVP increased their expression between 2
and 3 times after inoculation; other stress-inducible genes
(HSP101, HSP70) showed only a moderate increase (Fig.
4C). For "Piel de sapo" (susceptible), accumulation of
WRKY70 and SVP transcripts only increased about 1.5
times after inoculation whereas the expression of HSP101
showed a marked increase (Fig. 4B). It is also worth noting the differential response of the GA2OX1 gene in the
two genotypes. Expression of GA2OX1 increased about
1.5 times in pat81 roots after the M. cannonballus attack,
whereas it decreased in "Piel de sapo" roots after fungal
infection (compare Figs. 4B and 4C).
Comparison of patterns of transcript accumulation at two
stages of fruit development showed increased levels of
HHOm
(
HIFm
%%
*
#$!
%
%
*
* %
%
#$!
% % % &$!
%
%
O
(2)m
!
"
!
"
)
'
!
"
products2 of melon and Arabidopsis unigenes according to the Gene Ontology scheme for functional classification of gene
Distribution
Figure
Distribution of melon and Arabidopsis unigenes according to the Gene Ontology scheme for functional classification of gene
products.
(2)O
m
!!! Table 8: Potential melon miRNAs
Name
Potential mature miRNA
sequence (5'->3')*
Melogen unigene†
Precursor folding free
energy (kcal mol-1)§
MFEI
(kcal mol-1)
miRNA
family
Potential target in melon
m2
m4
m7
m8
m12
ugaagcugccagcaugaucu
ugauugagccgugccaauauc
ucggaccaggcuucauucccc
uugacagaagauagagagcac
uuggacugaagggagcucccu
bCL2353Contig1
bPSI_40-F10-M13R_c
bA_31-D02-M13R_c
bCI_04-H02-M13R_c
b15d_24-H05-M13R_c
-206.8
-195.0
-160.8
-188.2
-163.6
-0.67
-0.66
-0.70
-0.74
-0.86
miR167
miR171
miR166
miR157
miR319
-bHS_39-C12-M13R_c
-bCI_30-A09-M13R_c
bCL2243Contig1
*Nucleotide sequences correspond to potential mature miRNA sequences as deduced from BLAST searches using known plant miRNA sequences
[54] and analysis of secondary structure predictions [52]. †Accession numbers (MELOGEN database) of potential precursors of melon miRNAs are
given. §Computational values of folding free energies have been calculated using MFOLD 3.1 [55]. Minimal folding free energy indexes (MFEIs) have
been calculated as described by Zhang et al. [53].
gene expression for 9 of the analysed genes. This was particularly evident for HSP70, TOM2A, TOM3, EIN4 and
IAA9. In contrast, decreased levels of transcript accumulation were observed for the other 11 genes.
genes for experiments aimed at understanding important
processes involved in fruit development and resistance to
viral and fungal pathogens. Also, data presented here provide an important tool for generating markers to saturate
melon genetic maps.
Discussion
In this paper we provide an initial platform for functional
genomics of melon by the identification of more than
16,000 unigenes assembled from almost 30,000 ESTs
sequenced from 8 melon cDNA libraries. It is probably
premature to estimate the proportion of melon genes represented in this dataset, but based on available data for
other plant species (i.e. Arabidopsis and rice), it is likely
that the melon unigene set characterised here represents
approximately between half and one-third of the number
of expressed, protein coding genes of melon. Libraries
were constructed from various tissue types, but with a bias
towards fruit development and pathogen-infected tissues.
Data from these libraries will become a useful resource of
Potential3precursors of melon microRNAs
Figure
Potential precursors of melon microRNAs. (A) Stem
loop sequence of putative precursor miRNA corresponding
to unigene bCI_04-H02-M13R_c. (B) Stem loop sequence of
putative precursor miRNA corresponding to unigene
b15d_24-H05-M13R_c. The mature miRNA sequences are
shown in bold.
In contrast to typical EST gene-sampling strategies
reported previously, we have found a low degree of redundancy in the sequences determined. The process of clustering reduced the number of sequences to 56%, from
29,604 good quality ESTs to 6,023 contigs and 10,614 singletons. Contigs with more than 8 ESTs were scarce, the
majority of them being formed by 3 or 2 ESTs. Redundancy of the sequences derived from each library ranged
from 13% to 20%, with singletons constituting approximately one third of the unigenes determined per library.
This low redundancy is probably due to the success of the
normalization process, responsible for the suppression of
superabundant transcripts specific for a given tissue or
condition. Normalization precludes in silico analysis of
gene expression, but greatly increases the number of unigenes that can be determined by reducing redundancy
[76]. Here we have used a recently described normalization protocol which is based on the cleavage of DNA or
DNA-RNA duplexes by a specific DNase [77]; this process,
in our hands, has proven simple, reproducible and efficient. Another factor that has contributed to the low
redundancy values obtained has been the sequencing of
libraries from very distinct tissues. Thus, the number of
library specific unigenes was about one half of the total
number of unigenes contributed by each library, suggesting that further sequencing of the libraries still has the
potential to provide a good number of new, non-redundant sequences.
cDNA sequences are a useful source of SSRs, which are
excellent molecular markers due to their high degree of
polymorphism. A common feature of cDNA sequences
obtained from plants is the high frequency of SSRs that
they contain [36]. We have identified more than 1,000
potential SSRs in the melon dataset, with approximately
HIGm
O
(2)m
Table 9: Transcripts selected from the database for gene expression analysis by Real Time qPCR
Gene
Melogen unigene
Sequence length (bp) Arabidopsis locus Aminoacid
similarity (%)
Annotation (HMMR domain)
CTL1
CYP 7
EIF4A-2
EIF4E
EIN4
GA2OX1
HSP101
HSP70
IAA9
bCL1465Contig1
1,387
AT1G05850
66.8
bCL3337Contig1
bCL2906Contig1
bCL4710Contig1
bCL1742Contig1
bCL1313Contig1
bCI_38-F11-M13R_c
bPSI_41-D06-M13R_c
bCL1341Contig1
801
1,146
815
829
1,454
726
819
1,672
AT5G58710
AT1G54270
AT4G18040
AT3G04580
AT1G78440
AT1G74310
AT5G09590
AT5G65670
78.4
92.6
77.5
55.7
59.9
73.4
86.8
51.2
LSM1
bHS_37-F11-M13R_c
766
AT3G14080
72.4
LUT2
NCBP
SVP
bCL3563Contig1
bCL183Contig1
bCL2852Contig1
1,082
1,081
826
AT5G57030
AT5G18110
AT2G22540
75.5
85.3
54.9
Chitinase-like protein 1, similar to class I chitinase
(Glyco_hydro_19)
Peptidyl-prolyl cis-trans isomerase, cyclophilin
Eukaryotic translation initiation factor 4A-2 (DEAD)
Eukaryotic translation initiation factor 4E 1 (IF4E)
Ethylene receptor (Response_reg)
Gibberellin 2-oxidase (2OG-FeII_Oxy)
Heat shock protein 101 (AAA_2)
Heat shock protein 70/HSC70-5 (HSP70)
Auxin-responsive protein/indoleacetic acid- induced
protein 9 (AUX_IAA)
Small nuclear ribonucleoprotein/snRNP, putative/Sm
protein, putative, similar to U6 snRNA-associated
Sm-like protein
Lycopene epsilon cyclase (Lycopene_cycl)
Novel cap-binding protein (IF4E)
Short vegetative phase protein, MADS box
transcription factor related cluster (SRF-TF)
Band 7 family protein, strong similarity to
hypersensitive-induced response protein (Band_7)
Xyloglucan:xyloglucosyl transferase/xyloglucan
endotransglycosylase/endo- xyloglucan transferase/
TCH4
Tonoplast intrinsic protein (MIP)
Transmembrane protein-related/TOM1 (DUF1084)
Senescence-associated family protein
Tobamovirus multiplication protein 3/THH1
(DUF1084)
UDP-glucose 4-epimerase/UDP-galactose 4epimerase/Galactowaldenase (Epimerase)
WRKY family transcription factor, DNA-binding
protein (WRKY)
HIR
bCL144Contig1
1,266
AT1G69840
89.7
TCH4
bCL1212Contig1
1,228
AT5G57560
65.9
TIP4
TOM1
TOM2A
TOM3
bA_23-B12-M13R_c
bCL4416Contig1
bCL3115Contig1
bPSI_36-E03-M13R_c
751
780
1,113
857
AT2G25810
AT4G21790
AT1G32400
AT1G14530
50.5
75.4
60.1
UGE5
bCL1153Contig1
1,313
AT4G10960
70.9
WRKY70
bA_25-B01-M13R_c
889
AT3G56400
42.1
6% of the melon unigenes containing di-, tri- or tetranucleotide repeats. A clear bias toward AG and AAG repeats
existed, that account for 67% of the SSRs. In contrast, the
GC repeat was not found in the melon dataset. A similar
bias toward AG and against CG repeats has been identified in Arabidopsis and other plant species [40,78]. As
proposed at least in one other instance [40], this may be
due to the tendency of CpG sequences to be methylated
[79], which potentially might inhibit transcription.
Another interesting feature of melon SSRs relates to their
pattern of localization with respect to putative initiation
and termination codons. It is known that the UTRs of
transcribed sequences are richer in SSRs than coding
regions, particularly at the 5'-UTRs [36,40]. However, in
the melon dataset, a high proportion of SSRs (29.5%)
were found in ORFs. An analysis of the localization of di, tri- and tetranucleotide repeats separately showed that
di- and tetranucleotides were preferentially located in
UTRs, whereas trinucleotides localised in both, UTRs and
ORFs, consistently with maintenance of the ORFs coding
capacity. Thus, the prevalence of trinucleotide repeats in
the melon dataset (71%) explains this result.
We identified in the melon sequence dataset 356 highquality SNPs. Since non-redundant sequences analysed
here encompassed 4.5 Mb, one SNP was found every
12,000 pb of sequence. This small figure is probably due
to the limited number of melon genotypes used and the
low redundancy found among libraries. In fact, when the
frequency of SNPs is computed in relation to the length
and number of contigs containing SNPs, the corresponding value (one SNP in every 616 bp of sequence) is of the
same order of magnitude as values previously calculated
for melon (441 bp; [80]) and other plant species [40].
With the advent of high-throughput detection systems,
the SSRs and SNPs identified here will constitute an
important resource for mapping and marker-assisted
breeding in melon and closely related crops.
As an approach to the function of melon unigenes, we carried out a bioinformatics analysis based on BLASTX and
matches with the Pfam database [49]. The proportion of
melon unigenes with no similar sequences in databases
was quite high, suggesting that the melon dataset may
encompass an important number of melon-specific
HIHm
(2)O
m
!
!
!
!
Figure 4 analyzed by Real Time qPCR
Transcripts
Transcripts analyzed by Real Time qPCR. (A) Pattern of transcripts accumulation in CMV-infected melon cotyledons (CI)
relative to that of healthy cotyledons (CS). (B) Pattern of transcripts accumulation in M. cannonballus infected roots of C. melo
L. cv. "Piel de sapo" (PSI) relative to that of healthy roots (PS). (C) Pattern of transcripts accumulation in M. cannonballus
infected roots of C. melo L. ssp. agrestis (AI) relative to that of healthy roots (A). (D) Pattern of transcripts accumulation in
fruits of 15 days after pollination of C. melo L. cv. "Piel de sapo" (15d) relative to that of fruits of 46 days after pollination (46d).
cy: cyclophilin endogenous control; see Table 9 for the rest of genes.
HIIm
O
(2)m
sequences. However, the proportion specific sequences
might be overestimated because blasting has been made
with unigene sequences, which in many cases do not
cover the complete length of the transcript. We performed
a functional classification of the unigenes following the
Gene Ontology scheme, which is one of the more versatile
and complete systems for functional classification [48]. A
comparison of the distributions of melon and Arabidopsis unigenes in GO categories showed that both followed
similar tendencies, suggesting that the melon dataset is
representative of the whole melon transcriptome. This is
remarkable, as the number of different libraries
sequenced has been relatively small; again, this is probably due to the success of the normalization process. We
have also carried out specific searches for genes involved
in pathways of particular relevance in melon, such are
resistance response and fruit development, identifying a
remarkable number of melon candidates. For example, an
ortholog of the flagellin receptor FLS2 from Arabidopsis
[62] has been identified, together with 163 candidate
RLKs that may have critical roles in pathogen recognition
or diverse signalling processes. Similarly, up to 8 MADSbox gene homologs with potential roles in development
have been found in the melon dataset. Moreover, a bioinformatics approach [52,53] allowed the identification of
potential precursors of melon miRNAs together with several potential targets in the melon dataset. This finding
opens the door to biotechnology approaches based on the
use of artificial miRNAs to specifically silence melon
genes [81,82].
The transcript accumulation analysis for the 20 selected
genes revealed important changes in gene expression associated with pathogen infection and fruit development. For
virus infection, the accumulation of transcripts remained
unaltered for 12 genes, but showed a significant increase
for 6 genes and a decrease for 2. Among the set of genes
analysed, TOM2A and EIF4E were known to code for virus
susceptibility factors [27,83]; the expression of TOM2A
was increased after CMV infection, consistently with its
requirement by the virus, but this was not the case for
EIF4E. Different hypotheses can explain this result: since
EIF4E is an abundant, housekeeping protein, increased
expression may not be essential for virus multiplication;
alternatively, CMV may not use EIF4E in melon or may
use a factor coded by a different member of the 4E family;
it may also be that timing of the sampling for this experiment was not appropriate to detect such an effect, as
requirement of EIF4E might occur very early during virus
multiplication. In the cases of infection of susceptible
("Piel de sapo") and resistant (pat81) melons by M. cannonballus, more extensive alterations in gene expression
seemed to occur in the susceptible than in the resistant
accession. Significantly, for the susceptible accession,
stress responsive genes (e.g. HSP101) appeared to be max-
imally induced, whereas for the resistant accession, a gene
encoding a WRKY70 transcription factor, potentially
involved in resistance response, was induced to high levels. Significantly, expression of GA2OX1 increased about
1.5 times in pat81 after the M. cannonballus attack,
whereas it decreased in "Piel de sapo". GA2ox is a major
gibberellin (GA) catabolic enzyme, with an important
role in controlling GA levels in plants. Hormones control
many plant developmental processes, and strong evidence
indicates that hormone signalling is involved in the regulation of root growth and architecture [84,85]. The differential response of the GA2OX1 gene in the two melon
genotypes is consistent with an enhanced root growth in
pat81 after infection [86]. Notably, other genes involved
in hormone-mediated signalling pathways, such as the
IAA9 gene, did not show such differential response to M.
cannonballus infection in both genotypes. In the case of
fruit development, differences in the expression of
selected genes between immature and ripening fruits
appeared to be even sharper than in the cases of healthy
and pathogen-infected tissues. Specific roles during fruit
development for HSP70, TOM2A and TOM3 have not
been identified, though an increased expression has been
shown at least in the case of TOM2A in tomato [87]. The
ethylene receptor gene EIN4 showed a two-fold increase
in expression. EIN4 is the ortholog of Arabidopsis EIN4
and tomato LeETR4 [88,89]. In tomato, LeETR4 is also
highly expressed in ripening fruit, suggesting that it
responds by modulating ethylene signalling during ripening [63]. The MADS-box gene (SVP) showed about a fourfold decrease in expression. This gene is the ortholog of
tomato JOINTLESS, which specifies the abscission zone in
tomato. In tomato fruit microarray hybridizations, the
expression of JOINTLESS also decreased from 7 to 57 DAP
[87], in agreement with our data for melon. The lycopene
epsilon cyclase (LUT2) and xyloglucan endotransglycosylase (TCH4) genes showed an approximately four-fold
decrease in expression during melon fruit development.
These findings fit with the patterns of expression of these
genes in tomato, where their transcript levels decrease to a
non-detectable level in the ripe fruits [90,91].
Conclusion
In summary, this collection of ESTs represents a substantial increase on the information available for melon. The
dataset contains SSR and SNP markers that can be used for
breeding, as well as a significant number of candidate
genes that can be experimentally tested for their roles in
various important processes. This set of genes constitutes
also the basis for a microarray for melon that is being used
in experiments to further analyse fruit development and
maturation and responses to pathogen infections.
HIJm
(2)O
m
Methods
Plant material
The cDNA libraries were prepared using material from
four different melon genotypes: the line T-111 (Semillas
Fitó, Barcelona, Spain), which corresponds to a Piel de
Sapo breeding line, the Piel de Sapo cultivar "Piñonet torpedo" (Semillas Batlle, Barcelona, Spain), the accession C35 of the germplasm collection of La Mayora-CSIC
(EELM-CSIC, Málaga, Spain), which corresponds to a cantaloupe-type of melon, and the accession pat81 of C. melo
L. ssp. agrestis (Naud.) Pangalo maintained at the germplasm bank of COMAV (COMAV-UPV, Valencia, Spain)
(Table 1). Seeds of line T-111 were germinated at 30°C for
two days and plants were grown in a greenhouse in peat
bags, drip irrigated, with 0.25-m spacing between plants.
Fruits of 15 and 46 days after pollination were collected
and mesocarp tissues were recovered and used for RNA
extractions. Root samples were from Piel de sapo and
pat81 plants, both healthy and inoculated with M. cannonballus. Piel de sapo is fully susceptible to the infection
by this fungus whereas pat81 has been shown to be partially resistant [92,93]. Seeds were pre-germinated in Petri
dishes. After 4 days, seedlings were transplanted to 0.5-l
pots filled with sterile soil substrate and grown in a greenhouse (20–35°C, 60–85% relative humidity). Inoculations were carried out by adding 50 colony-forming units
(CFU) of M. cannonballus per gram of sterile soil as
described by Iglesias et al. [94]. Fourteen days after inoculation, healthy and inoculated roots were collected for
RNA extraction. The presence of the fungus and the infection levels were assessed by real-time quantitative PCR as
described by Picó et al. [95]. CMV infected cotyledons
were collected from plants of the C-35 accession. In this
case, seeds were pregerminated in Petri dishes for 24 h at
28°C in the dark, planted in 0.5-l pots and maintained in
an insect-proof green house (20–28°C, 45 to 85% relative
humidity) for 6 to 7 days, until the first true leaf started
emerging. At this stage, cotyledons were mechanically
inoculated with CMV following standard procedures [96].
Inoculated cotyledons were harvested 4 days after inoculation and used for RNA extractions. Dot-blot hybridisation [97] was used to check infection by CMV. Plants of
the C-35 accession were also used for collecting healthy
leaves. Plants were maintained in the greenhouse for 21
days, and second and third leaves above cotyledons were
harvested for RNA extractions.
Construction of cDNA libraries and EST sequencing
Total RNA was prepared as described by Aranda et al. [98].
Poly(A+) RNA from total RNA was purified using MicroPoly(A+) Purist (Ambion, Austin, TX, USA), a celluloseoligo(dT)-based method. Integrity and quality of both
total and poly(A+) RNA were tested by gel electrophoresis.
cDNA libraries were constructed with the SMART cDNA
Library Construction kit (Clontech, Mountain View, CA,
USA), using a modified primer to include a Sfi I enzyme
restriction site. A normalization step was carried out with
TRIMMER kit (Evrogen, Moscow, Russia). After normalization, a cDNA fractionation step was performed with SizeSep 400 Spun Columns (Amersham Biosciences,
Buckinghamshire, England). cDNA was digested with Sfi
I, generating Sfi IA-Sfi IB cohesive ends for directional
cloning into a modified version of BlueScript SK plasmid
vector (Stratagene, La Jolla, CA, USA). Ligation products
were transformed into E. coli electrocompetent cells
DH10B (Invitrogen, Carlsbad, CA, USA) by electroporation. The titer of the libraries was evaluated by plating an
aliquot on LB agar plates with ampicillin at 100 g ml-1.
Only libraries of 105 cfu ml-1 or more were considered as
acceptable. Prior to large scale sequencing, the average
insert size was estimated by restriction analyses of 24 plasmid DNA minipreps per library from randomly picked
colonies.
Sequencing was carried out from the 5'-end of the inserts
without library amplification using the universal M13
reverse primer. An external custom service was contracted
for this task (Macrogen Inc., Seoul, Korea). Approximately
6,000 clones were sequenced from the CI library, and
3,500 clones were sequenced from each of the other
libraries (Table 2). Sequences obtained in this work can be
found in GenBank [accession numbers AM713476 to
AM743079] and MELOGEN [44].
Bioinformatics
EST sequences were automatically trimmed, clustered and
annotated using the EST2uni analysis pipeline [43].
EST2uni compromises the analysis pipeline written in
PERL [99], a database (MySQL) [100] and a web site to
browse the results coded in PHP [101]. Thus, for the EST
pre-processing step, base calling was performed with
Phred [102], low quality regions and vector sequences
were trimmed with Lucy [103], and repeats and low complexity regions were masked with RepeatMasker [104] and
Seqclean [105]. Further vector contamination was also
eliminated with Seqclean using NCBI's UniVec [106].
High-quality EST sequences were then assembled to
obtain the unigene set using Tgicl [105].
Detection of SSRs was performed using Sputnik [107].
Putative SNPs were annotated when the least represented
allele was present in two EST sequences or more. ORFs
were predicted in the ESTs with the aid of the ESTScan
software [108].
For functional annotation, comparisons against the Arabidopsis (TAIR) [109] and Uniref [110] databases were carried out using BLASTN or BLASTX for nucleotide or
protein sequences, respectively. Functional domains were
searched with HMMPFAM [111] using the Pfam database
HIKm
O
(2)m
[112]. The Gene Ontology (GO) classification [48] was
derived from the BLASTN results against the Arabidopsis
proteome. Also, a bi-directional BLASTN comparison was
performed in order to obtain a set of putative orthologs
with Arabidopsis. Finally, a set of superunigenes was
obtained grouping different unigenes with the same
expected mRNA target, as judged by extensive sequence
overlapping.
To assess codon usage, we generated a set of melon
sequences predicted to contain full-length coding regions.
These sequences were subjected to BLASTX and, after
manual inspection, sequences showing a high similarity
to Arabidopsis proteins were selected to ensure that no
sequences containing frame-shift errors were included in
the analysis. From this smaller dataset, which included
588 sequences, ORFs were defined and a codon usage
table was created. Codon usage was calculated from
sequences using the GCUA program [113]. All codons
were found in the dataset, with the least frequent codon
represented 134 times.
To identify potential melon miRNAs, the 33,292 melon
ESTs were subjected to a BLAST search against mature
sequences of known miRNAs from the miRNA Registry
Database (released January 2007) [54] using BLASTN
[47]. ESTs with only 0–1 mismatched nucleotides with
known miRNAs were considered. Selected ESTs were subjected to a BLAST search against protein databases in order
to remove potential protein-coding sequences. ESTs pertaining to the same melon unigene of the MELOGEN
database were grouped. The secondary structures of the
unigenes encoding potential miRNA precursors were predicted with the web-based tool MFOLD [55], using
default parameters. In each case, only the lower energy
structure was selected for visual inspection, as previously
described [52,114]. In order to select unigenes with perfect or near-perfect secondary foldback hairpin structures,
only sequences with a maximum size of 3 nucleotides for
a bulge in the miRNA sequence and with at least 16 paired
nucleotides between the mature sequence and the opposite arm were considered as potential miRNA candidates.
In addition, the minimal folding free energy index (MFEI)
for each sequence was calculated following Zhang et al.
[53].
Gene expression analyses
Real time quantitative PCR was performed with an AB
7500 System (Applied Biosystems, Foster City, CA, U.S.A)
to quantify mRNA corresponding to some transcripts of
interest, in the tissues and physiological conditions used
for library construction. Twenty ESTs representing these
transcripts were chosen from the database and used to
generate gene-specific primers (Table 10) with Primer
Express Software (Applied Biosystems). The chemistry
used for PCR product detection was the Power SYBR green
dye (Applied Biosystems) and ROX as passive reference.
CYCLOPHILIN served as endogenous control (sequence
extracted from the database), Ct was the method of calculation to perform relative quantification, and three
technical replicates were carried out and considered for
statistical analysis. Melting curves analyses at the end of
Table 10: Primer sequences for Real Time-qPCR analysis of transcript accumulation
Gene
Primer sequence (5'->3') Forward
Reverse
CTL1
CYP 7
EIF4A-2
EIF4E
EIN4
GA2OX1
HSP101
HSP70
IAA9
LSM1
LUT2
NCBP
SVP
HIR
TCH4
TIP4
TOM1
TOM2A
TOM3
UGE5
WRKY70
tgggccatgttggctctaag
cgatgtggaaattgacggaa
ttcccgaggtttcaaagatca
ttcggttccttcccttccat
tgcaacgtgactgctgtttct
tagggcaaatcggttagcga
aacgtatggtgcggattgaca
gctgaggcgtaccttggaaa
gacggaaagccaggttcaag
ctacttcgagatgggcggaa
gctggcgtggaacactcttt
cgtcggtctgcttaatttgca
cgaggcaggtcacgttctct
tgacgggctcagagacagtg
ggagggtagccttgagggaat
tccttgctggtgtcggatc
gggagaaggaagaaacttcatgag
ctcctcagcagccgaagaaa
aatggagttcgggctgttgt
gcgaaagtgtccaaaagcca
ggattgctcctggcctgac
ctcccgtgacaactccatca
cggtgcataatgctcggaa
ccaatgcttctggtggcact
ccgccgatgtagctttcatc
tctggcatgtgaagatccaaga
ccaaatgcaaaccgattgaa
tccaccttcatggtatccaacat
atcctgccagcatcctttgt
cccctccatactcactttcacaa
tcaccaacgatcaccctttca
cgaatgccttcaatgtccagt
cgcctacgaactccattgaca
agagaagacgaggagcgcaa
gttccagggacgttttcagc
ctggacattgctcgacaacaa
cgtttgccaataacgcattg
gcgtcaaaagcggataaagc
tggacccgctaaaacaccac
aaggcaaggcttggcatgt
acacaagctttttgcatccgt
tcggctgcttttcttcgatc
HILm
(2)O
m
the process and No Template Controls (NTC) were carried
out to ensure product-specific amplification and no
primer-dimer quantification. A control reaction as for
reverse transcription but without the enzyme was performed to evaluate genomic DNA contamination.
!!! 4.
5.
6.
Authors' contributions
Daniel Gonzalez-Ibeas prepared RNAs for two libraries,
constructed the eight libraries, carried out the gene expression analysis by Real-Time-qPCR and participated in the
bioinformatics analyses and in the drafting of the manuscript. José Blanca carried out the bioinformatics analyses,
EST database and web page, and participated in the drafting of the manuscript. Cristina Roig and Belén Picó prepared RNAs for the root libraries and participated in the
drafting of the manuscript. Mireia González-To, Wim
Deleu and Jordi Garcia-Mas prepared RNAs from melon
fruits and participated in the drafting of the manuscript.
Pere Puigdomènech is the main coordinator of The MELOGEN Project and participated in the conception of the
study together with Pere Arús, Fernando Nuez, Jordi Garcia-Mas and Miguel A. Aranda. Miguel A. Aranda is the
principal investigator or this work, supervised it and wrote
the manuscript. All authors read and approved the final
manuscript.
Additional material
7.
8.
9.
10.
11.
12.
13.
14.
Additional file 1
Genes potentially encoding pathogen resistance and fruit quality traits.
Genes were identified in the melon data set by comparison with the Arabidopsis database [6,109]. A brief description, the corresponding Arabidopsis locus and the HMMR domain identified are given for each
unigene.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-8-306-S1.pdf]
15.
16.
17.
18.
19.
Acknowledgements
This work was supported by grants from Ministerio de Educación y Ciencia
(Spain) (GEN2003-20237-C06) and Consejería de Educación y Cultura
(Región de Murcia, Spain) (BIO2005/04-6436). Wim Deleu, Cristina Roig
and Daniel Gonzalez-Ibeas are recipient of a postdoctoral fellowship from
the Centre de Recerca en Agrigenòmica CSIC-IRTA (Spain), a Juan de la
Cierva grant from Ministerio de Educación y Ciencia (Spain) and a predoctoral fellowship from Ministerio de Educación y Ciencia (Spain), respectively.
20.
21.
22.
References
1.
2.
3.
FAOSTAT Agriculture data [http://faostat.fao.org/default.aspx]
Jayasooriya AP, Sakono M, Yukizaki C, Kawano M, Yamamoto K,
Fukuda N: Effects of Momordica charantia powder on serum
glucose levels and various lipid parameters in rats fed with
cholesterol-free and cholesterol-enriched diets. J Ethnopharmacol 2000, 72:331-336.
Arumuganathan K, Earle ED: Nuclear DNA content of some
important plant species. Plant Mol Biol Rep 1991, 9:208-218.
23.
24.
Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook
J, Sessions A, Oeller P, Varma H, others: A Draft Sequence of the
Rice Genome (Oryza sativa L. ssp. japonica). Science 2002,
296:92-100.
Yu J, Hu S, Wang J, Wong GK-S, Li S, Liu B, Deng Y, Dai L, Zhou Y,
Zhang X, others: A Draft Sequence of the Rice Genome
(Oryza sativa L. ssp. indica). Science 2002, 296:79-92.
Arabidopsis Genome Initiative: The Arabidopsis Information
Resource (TAIR): a comprehensive database and web-based
information retrieval, analysis, and visualization system for a
model plant. Nucleic Acids Res 2001, 29:102-105.
Kirkbride JH: Biosystematic monograph of the genus Cucumis (Cucurbitaceae) Boone, North Carolina: Parkway Publishers; 1993.
Garcia-Mas J, Monforte AJ, Arus P: Phylogenetic relationships
among Cucumis species based on the ribosomal internal
transcribed spacer sequence and microsatellite markers.
Plant Syst Evol 2004, 248:191-203.
Liu L, Kakihara F, Kato M: Characterization of six varieties of
Cucumis melo L. based on morphological and physiological
characters, including shelf-life of fruit.
Euphytica 2004,
135:305-313.
Miccolis V, Saltveit ME: Morphological and physiological
changes during fruit growth and maturation of seven melon
cultivars. J Am Soc Hort Sci 1991, 116:1025-1029.
Shalit M, Katzir N, Tadmor Y, Larkov O, Burger Y, Shalekhet F, Lastochkin E, Ravid U, Amar O, Edelstein M, others: Acetyl-CoA:
Alcohol acetyltransferase activity and aroma formation in
ripening melon fruits. J Agric Food Chem 2001, 49:794-799.
Bauchot AD, Mottram DS, Dodson AT, John P: Effect of Aminocyclopropane-1-carboxylic Acid Oxidase Antisense Gene on
the Formation of Volatile Esters in Cantaloupe Charentais
Melon (Cv. Védrandais). J Agric Food Chem 1998, 46:4787-4792.
Flores F, El Yahyaoui F, de Billerbeck G, Romojaro F, Latche A, Bouzayen M, Pech JC, Ambid C: Role of ethylene in the biosynthetic
pathway of aliphatic ester aroma volatiles in Charentais
Cantaloupe melons. J Exp Bot 2002, 53:201-206.
Yahyaoui FEL, Wongs-Aree C, Latche A, Hackett R, Grierson D, Pech
JC: Molecular and biochemical characteristics of a gene
encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening. FEBS J
2002, 269:2359-2366.
Hadfield KA, Bennett AB: Polygalacturonases: Many Genes in
Search of a Function. Plant Physiol 1998, 117:337-343.
Rose JKC, Hadfield KA, Labavitch JM, Bennett AB: Temporal
sequence of cell wall disassembly in rapidly ripening melon
fruit. Plant Physiol 1998, 117:345-361.
Bennett AB: Biochemical and genetic determinants of cell wall
disassembly in ripening fruit: A general model. Hortscience
2002, 37:447-450.
Haritatos E, Keller F, Turgeon R: Raffinose oligosaccharide concentrations measured in individual cell and tissue types in
Cucumis melo L leaves: Implications for phloem loading.
Planta 1996, 198:614-622.
Volk GM, Turgeon R, Beebe DU: Secondary plasmodesmata formation in the minor-vein phloem of Cucumis melo L and
Cucurbita pepo L. Planta 1996, 199:425-432.
Gomez G, Torres H, Pallas V: Identification of translocatable
RNA-binding phloem proteins from melon, potential components of the long-distance RNA transport system. Plant J
2005, 41:107-116.
Chen JQ, Rahbé Y, Delobel B, Sauvion N, Guillaud J, Febvay G: resistance to the aphid Aphis gossypii: behavioural analysis and
chemical correlations with nitrogenous compounds. Entomol
Exp Appl 1997, V85:33-44.
Luo MZ, Wang YH, Frisch D, Joobeur T, Wing RA, Dean RA: Melon
bacterial artificial chromosome (BAC) library construction
using improved methods and identification of clones linked
to the locus conferring resistance to melon Fusarium wilt
(Fom-2). Genome 2001, 44:154-162.
Klingler J, Powell G, Thompson GA, Isaacs R: Phloem specific
aphid resistance in Cucumis melo line AR 5: effects on feeding behaviour and performance of Aphis gossypii. Entomol Exp
Appl 1998, 86:79-88.
Marco CF, Aguilar JM, Abad J, Gomez-Guillamon ML, Aranda MA:
Melon resistance to Cucurbit yellow stunting disorder virus
HIMm
O
(2)m
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
is characterized by reduced virus accumulation. Phytopathology 2003, 93:844-852.
Diaz JA, Nieto C, Moriones E, Truniger V, Aranda MA: Molecular
characterization of a Melon necrotic spot virus strain that
overcomes the resistance in melon and nonhost plants. Mol
Plant-Microbe Interact 2004, 17:668-675.
Klingler J, Creasy R, Gao LL, Nair RM, Calix AS, Jacob HS, Edwards
OR, Singh KB: Aphid resistance in Medicago truncatula
involves antixenosis and phloem-specific, inducible antibiosis, and maps to a single locus flanked by NBS-LRR resistance
gene analogs. Plant Physiol 2005, 137:1445-1455.
Nieto C, Morales M, Orjeda G, Clepet C, Monfort A, Sturbois B, Puigdomenech P, Pitrat M, Caboche M, Dogimont C, others: An eIF4E
allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon. Plant J 2006, 48:452-462.
Perin C, Hagen L, Conto VD, Katzir N, Danin-Poleg Y, Portnoy V,
Baudracco-Arnas S, Chadoeuf J, Dogimont C, Pitrat M: A reference
map of Cucumis melo based on two recombinant inbred line
populations. Theor Appl Genet 2002, 104:1017-1034.
Gonzalo MJ, Oliver M, Garcia-Mas J, Monfort A, Dolcet-Sanjuan R,
Katzir N, Arus P, Monforte A: Simple-sequence repeat markers
used in merging linkage maps of melon (Cucumis melo L.).
Theor Appl Genet 2005, 110:802-811.
Eduardo I, Arus P, Monforte AJ: Development of a genomic
library of near isogenic lines (NILs) in melon (Cucumis melo
L.) from the exotic accession PI161375. Theor Appl Genet 2005,
112:139-148.
Ayub R, Guis M, BenAmor M, Gillot L, Roustan JP, Latche A, Bouzayen M, Pech JC: Expression of ACC oxidase antisense gene
inhibits ripening of cantaloupe melon fruits. Nat Biotechnol
1996, 14:862-866.
Guis M, Roustan JP, Dogimont C, Pitrat M, Pech JC: Melon biotechnology. Biotechnol Genet Engng Rev 1998, 15:289-311.
Gaba V, Zelcer A, Gal-On A: Cucurbit biotechnology – The
importance of virus resistance. In Vitro Cell Dev Biol Plant 2004,
40:346-358.
Rudd S: Expressed sequence tags: alternative or complement
to whole genome sequences? Trends Plant Sci 2003, 8:321-329.
Alba R, Fei Z, Payton P, Liu Y, Moore SL, Debbie P, Cohn J, D'Ascenzo
M, Gordon JS, Rose JKC, others: ESTs, cDNA microarrays, and
gene expression profiling: tools for dissecting plant physiology and development. Plant J 2004, 39:697-714.
Morgante M, Hanafey M, Powell W: Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes.
Nat Genet 2002, 30:194-200.
Rafalski JA: Novel genetic mapping tools in plants: SNPs and
LD-based approaches. Plant Sci 2002, 162:329-333.
Ouyang S, Zhu W, Hamilton J, Lin H, Campbell M, Childs K, ThibaudNissen F, Malek RL, Lee Y, Zheng L, others: The TIGR Rice
Genome Annotation Resource: improvements and new features. Nucleic Acids Res 2007, 35:D883-D887.
Fei ZJ, Tang XM, Alba R, Giovannoni J: Tomato Expression Database (TED): a suite of data presentation and analysis tools.
Nucleic Acids Res 2006, 34:D766-D770.
Newcomb RD, Crowhurst RN, Gleave AP, Rikkerink EHA, Allan AC,
Beuning LL, Bowen JH, Gera E, Jamieson KR, Janssen BJ, others: Analyses of expressed sequence tags from apple. Plant Physiol 2006,
141:147-166.
Goes da Silva F, Iandolino A, Al Kayal F, Bohlmann MC, Cushman MA,
Lim H, Ergul A, Figueroa R, Kabuloglu EK, Osborne C, others: Characterizing the Grape Transcriptome. Analysis of Expressed
Sequence Tags from Multiple Vitis Species and Development of a Compendium of Gene Expression during Berry
Development. Plant Physiol 2005, 139:574-597.
Forment J, Gadea J, Huerta L, Abizanda L, Agusti J, Alamar S, Alos E,
Andres F, Arribas R, Beltran JP, others: Development of a citrus
genome-wide EST collection and cDNA microarray as
resources for genomic studies. Plant Mol Biol 2005, 57:375-391.
EST2uni
[http://www.melogen.upv.es/genomica/web_estpipe/
index.php]
MELOGEN database [http://www.melogen.upv.es]
Sterky F, Bhalerao RR, Unneberg P, Segerman B, Nilsson P, Brunner
AM, Charbonnel-Campaa L, Lindvall JJ, Tandre K, Strauss SH, others:
A Populus EST resource for plant functional genomics. Proc
Natl Acad Sci USA 2004, 101:13951-13956.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
Hsiang T, Goodwin PH: Distinguishing plant and fungal
sequences in ESTs from infected plant tissues. J Microbiol Methods 2003, 54:339-351.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs. Nucleic Acids Res 1997,
25:3389-3402.
The Gene Ontology Consortium: Gene Ontology: tool for the
unification of biology. Nat Genet 2000, 25:25-29.
Finn RD, Mistry J, Schuster-Bockler B, Griffiths-Jones S, Hollich V,
Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R, others:
Pfam: clans, web tools and services. Nucleic Acids Res 2006,
34:D247-D251.
Carrington JC, Ambros V: Role of microRNAs in plant and animal development. Science 2003, 301:336-338.
Bartel DP: MicroRNAs: Genomics, biogenesis, mechanism,
and function. Cell 2004, 116:281-297.
Zhang BH, Pan XP, Cannon CH, Cobb GP, Anderson TA: Conservation and divergence of plant microRNA genes. Plant J 2006,
46:243-259.
Zhang BH, Pan XP, Cox SB, Cobb GP, Anderson TA: Evidence that
miRNAs are different from other RNAs. Cell Mol Life Sci 2006,
63:246-254.
Griffiths-Jones S: The microRNA Registry. Nucleic Acids Res 2004,
32:D109-D111.
Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003, 31:3406-3415.
Palatnik JF, Allen E, Wu XL, Schommer C, Schwab R, Carrington JC,
Weigel D: Control of leaf morphogenesis by microRNAs.
Nature 2003, 425:257-263.
Gustafson AM, Allen E, Givan S, Smith D, Carrington JC, Kasschau
KD: ASRP: the Arabidopsis Small RNA Project Database.
Nucleic Acids Res 2005, 33:D637-D640.
Chisholm ST, Coaker G, Day B, Staskawicz BJ: Host-microbe interactions: Shaping the evolution of the plant immune
response. Cell 2006, 124:803-814.
Kushner DB, Lindenbach BD, Grdzelishvili VZ, Noueiry AO, Paul SM,
Ahlquist P: Systematic, genome-wide identification of host
genes affecting replication of a positive-strand RNA virus.
Proc Natl Acad Sci USA 2003, 100:15764-15769.
Diaz-Pendon JA, Truniger V, Nieto C, Garcia-Mas J, Bendahmane A,
Aranda MA: Advances in understanding recessive resistance
to plant viruses. Mol Plant Pathol 2004, 5:223-233.
Robaglia C, Caranta C: Translation initiation factors: a weak
link in plant RNA virus infection. Trends Plant Sci 2006, 11:40-45.
Gomez-Gomez L, Boller T: FLS2: An LRR receptor-like kinase
involved in the perception of the bacterial elicitor flagellin in
Arabidopsis. Mol Cell 2000, 5:1003-1011.
Giovannoni JJ: Genetic regulation of fruit development and ripening. Plant Cell 2004, 16:S170-S180.
Tanksley SD: The genetic, developmental, and molecular
bases of fruit size and shape variation in tomato. Plant Cell
2004, 16:S181-S189.
Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E,
Yanofsky MF: Assessing the redundancy of MADS-box genes
during carpel and ovule development. Nature 2003, 424:85-88.
Dinneny JR, Weigel D, Yanofsky MF: A genetic framework for
fruit patterning in Arabidopsis thaliana. Development 2005,
132:4687-4696.
Pitrat M: 2002 gene list for melon. Cucurbit Genet Coop Rep 2002,
25:76-93.
Monforte AJ, Oliver M, Gonzalo MJ, Alvarez JM, Dolcet-Sanjuan R,
Arus P: Identification of quantitative trait loci involved in fruit
quality traits in melon (Cucumis melo L.). Theor Appl Genet
2004, 108:750-758.
Frary A, Nesbitt TC, Frary A, Grandillo S, van der Knaap E, Cong B,
Liu JP, Meller J, Elber R, Alpert KB, others: fw2.2: A quantitative
trait locus key to the evolution of tomato fruit size. Science
2000, 289:85-88.
Liu JP, Van Eck J, Cong B, Tanksley SD: A new class of regulatory
genes underlying the cause of pear-shaped tomato fruit. Proc
Natl Acad Sci USA 2002, 99:13302-13306.
Manning K, Tor M, Poole M, Hong Y, Thompson AJ, King GJ, Giovannoni JJ, Seymour GB: A naturally occurring epigenetic mutation
in a gene encoding an SBP-box transcription factor inhibits
tomato fruit ripening. Nat Genet 2006, 38:948-952.
HINm
(2)O
m
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
Aranda MA, Escaler M, Wang D, Maule AJ: Induction of HSP70 and
polyubiquitin expression associated with plant virus replication. Proc Natl Acad Sci USA 1996, 93:15289-15293.
Escaler M, Aranda MA, Roberts IM, Thomas CL, Maule AJ: A comparison between virus replication and abiotic stress (heat) as
modifiers of host gene expression in pea. Mol Plant Pathol 2000,
1:159-167.
Whitham SA, Yang CL, Goodin MM: Global impact: Elucidating
plant responses to viral infection. Mol Plant-Microbe Interact
2006, 19:1207-1215.
Aranda M, Maule A: Virus-induced host gene shutoff in animals
and plants. Virology 1998, 243:261-267.
Bonaldo MDF, Lennon G, Soares MB: Normalization and subtraction: Two approaches to facilitate gene discovery. Genome Res
1996, 6:791-806.
Zhulidov PA, Bogdanova EA, Shcheglov AS, Vagner LL, Khaspekov
GL, Kozhemyako VB, Matz MV, Meleshkevitch E, Moroz LL, Lukyanov
SA, others: Simple cDNA normalization using kamchatka
crab duplex-specific nuclease. Nucleic Acids Res 2004, 32:e37.
Zhang LD, Yuan DJ, Yu SW, Li ZG, Cao YF, Miao ZQ, Qian HM, Tang
KX: Preference of simple sequence repeats in coding and
non-coding regions of Arabidopsis thaliana. Bioinformatics
2004, 20:1081-1086.
Finnegan EJ, Genger RK, Peacock WJ, Dennis ES: DNA methylation
in plants. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:223-247.
Morales M, Roig E, Monforte AJ, Arus P, Garcia-Mas J: Singlenucleotide polymorphisms detected in expressed sequence
tags of melon (Cucumis melo L.). Genome 2004, 47:352-360.
Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH:
Expression of artificial microRNAs in transgenic Arabidopsis
thaliana confers virus resistance.
Nat Biotechnol 2006,
24:1420-1428.
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D: Highly
specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 2006, 18:1121-1133.
Yamanaka T, Imai T, Satoh R, Kawashima A, Takahashi M, Tomita K,
Kubota K, Meshi T, Naito S, Ishikawa M: Complete inhibition of
tobamovirus multiplication by simultaneous mutations in
two homologous host genes. J Virol 2002, 76:2491-2497.
Malamy JE: Intrinsic and environmental response pathways
that regulate root system architecture. Plant Cell Environ 2005,
28:67-77.
Radi A, Dina P, Guy A: Expression of sarcotoxin IA gene via a
root-specific tob promoter enhanced host resistance against
parasitic weeds in tomato plants. Plant Cell Rep 2006,
25:297-303.
Dias RDS, Pico B, Espinos A, Nuez F: Resistance to melon vine
decline derived from Cucumis melo ssp agrestis: genetic
analysis of root structure and root response. Plant Breeding
2004, 123:66-72.
Tomato expression database [http://ted.bti.cornell.edu]
Sakai H, Hua J, Chen QHG, Chang CR, Medrano LJ, Bleecker AB,
Meyerowitz EM: ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis. Proc Natl Acad Sci USA 1998,
95:5812-5817.
Tieman DM, Klee HJ: Differential expression of two novel
members of the tomato ethylene-receptor family. Plant Physiol 1999, 120:165-172.
Ronen G, Cohen M, Zamir D, Hirschberg J: Regulation of carotenoid biosynthesis during tomato fruit development: Expression of the gene for lycopene epsilon-cyclase is downregulated during ripening and is elevated in the mutant
Delta. Plant J 1999, 17:341-351.
Catala C, Rose JKC, Bennett AB: Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early
tomato fruit growth. Plant Physiol 2000, 122:527-534.
Esteva J, Nuez F: Field resistance to melon dieback in Cucumis
melo L. Cucurbit Genet Coop 1994, 17:76-77.
Iglesias A, Pico B, Nuez F: A temporal genetic analysis of disease
resistance genes: resistance to melon vine decline derived
from Cucumis melo var. agrestis.
Plant Breeding 2000,
119:329-334.
Iglesias A, Picó B, Nuez F: Artificial inoculation methods and
selection criteria for breeding melons against vine decline.
Acta Hortic 2000, 510:155-162.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
Picó B, Roig C, Fita A, Nuez F: Detección de Monosporascus cannonballus en raíces de melón mediante PCR cuantitativa en
tiempo real. Acta Port Hortic 2005, 7:169-176.
Hull S: Matthews's plant Virology San Diego: Academic Press; 2002.
Sambrook J, Rusell DW: Molecular cloning A laboratory manual. New
York: CSHL PRESS; 2001.
Aranda MA, Escaler M, Thomas CL, Maule AJ: A heat shock transcription factor in pea is differentially controlled by heat and
virus replication. Plant J 1999, 20:153-161.
PERL [http://www.perl.org]
MySQL [http://www.mysql.com]
PHP [http://www.php.net]
Ewing B, Hillier L, Wendl MC, Green P: Base-calling of automated
sequencer traces using Phred. I. Accuracy assessment.
Genome Res 1998, 8:175-185.
Chou HH, Holmes MH: DNA sequence quality trimming and
vector removal. Bioinformatics 2001, 17:1093-1104.
RepeatMasker [http://www.repeatmasker.org]
DFCI Gene Indices Software Tools [http://compbio.dfci.har
vard.edu/tgi/software/]
NCBI's UniVec
[http://www.ncbi.nlm.nih.gov/VecScreen/Uni
Vec.html]
Sputnik
[http://capb.dbi.udel.edu/main/ssr-proj/ssr-file/descrip
tion.html]
ESTScan software
[http://www.isrec.isb-sib.ch/ftp-server/ESTS
can/]
TAIR: the Arabidopsis information resource [http://www.ara
bidopsis.org/]
Uniref [http://www.ebi.ac.uk/uniref/]
Eddy SR: Profile hidden Markov models. Bioinformatics 1998,
14:755-763.
Pfam database [http://www.sanger.ac.uk/Software/Pfam/]
McInerney JO: GCUA (General Codon Usage Analysis). Bioinformatics 1998, 14:372-373.
Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP: MicroRNAs in plants. Genes Dev 2002, 16:1616-1626.
Apweiler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, Ferro S,
Gasteiger E, Huang H, Lopez R, Magrane M, others: UniProt: the
Universal Protein knowledgebase. Nucleic Acids Res 2004,
32:D115-D119.
Publish with Bio Med Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical researc h in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
BioMedcentral
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
HIOm
O
(2)m
BMC Plant Biology
BioMed Central
Open Access
Research article
A set of EST-SNPs for map saturation and cultivar identification in
melon
Wim Deleu†1, Cristina Esteras†2, Cristina Roig2, Mireia González-To1,
Iria Fernández-Silva1, Daniel Gonzalez-Ibeas3, José Blanca2,
Miguel A Aranda3, Pere Arús1, Fernando Nuez2, Antonio J Monforte1,4,
Maria Belén Picó2 and Jordi Garcia-Mas*1
Address: 1IRTA, Centre de Recerca en Agrigenòmica CSIC-IRTA-UAB, Carretera de Cabrils Km 2, 08348 Cabrils (Barcelona), Spain, 2COMAV-UPV,
Institute for the Conservation and Breeding of Agricultural Biodiversity, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia,
Spain, 3Departamento de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS)- CSIC, Apdo.
correos 164, 30100 Espinardo (Murcia), Spain and 4Instituto de Biología Molecular y Celular de Plantas (IBMCP) UPV-CSIC, Ciudad Politécnica
de la Innovación Edificio 8E, Ingeniero Fausto Elio s/n, 46022 Valencia, Spain
Email: Wim Deleu - [email protected]; Cristina Esteras - [email protected]; Cristina Roig - [email protected]; Mireia GonzálezTo - [email protected]; Iria Fernández-Silva - [email protected]; Daniel Gonzalez-Ibeas - [email protected];
José Blanca - [email protected]; Miguel A Aranda - [email protected]; Pere Arús - [email protected]; Fernando Nuez - [email protected];
Antonio J Monforte - [email protected]; Maria Belén Picó - [email protected]; Jordi Garcia-Mas* - [email protected]
* Corresponding author †Equal contributors
Published: 15 July 2009
BMC Plant Biology 2009, 9:90
doi:10.1186/1471-2229-9-90
Received: 31 March 2009
Accepted: 15 July 2009
This article is available from: http://www.biomedcentral.com/1471-2229/9/90
© 2009 Deleu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: There are few genomic tools available in melon (Cucumis melo L.), a member of the Cucurbitaceae, despite
its importance as a crop. Among these tools, genetic maps have been constructed mainly using marker types such as
simple sequence repeats (SSR), restriction fragment length polymorphisms (RFLP) and amplified fragment length
polymorphisms (AFLP) in different mapping populations. There is a growing need for saturating the genetic map with
single nucleotide polymorphisms (SNP), more amenable for high throughput analysis, especially if these markers are
located in gene coding regions, to provide functional markers. Expressed sequence tags (ESTs) from melon are available
in public databases, and resequencing ESTs or validating SNPs detected in silico are excellent ways to discover SNPs.
Results: EST-based SNPs were discovered after resequencing ESTs between the parental lines of the PI 161375 (SC) ×
'Piel de sapo' (PS) genetic map or using in silico SNP information from EST databases. In total 200 EST-based SNPs were
mapped in the melon genetic map using a bin-mapping strategy, increasing the map density to 2.35 cM/marker. A subset
of 45 SNPs was used to study variation in a panel of 48 melon accessions covering a wide range of the genetic diversity
of the species. SNP analysis correctly reflected the genetic relationships compared with other marker systems, being able
to distinguish all the accessions and cultivars.
Conclusion: This is the first example of a genetic map in a cucurbit species that includes a major set of SNP markers
discovered using ESTs. The PI 161375 × 'Piel de sapo' melon genetic map has around 700 markers, of which more than
500 are gene-based markers (SNP, RFLP and SSR). This genetic map will be a central tool for the construction of the
melon physical map, the step prior to sequencing the complete genome. Using the set of SNP markers, it was possible
to define the genetic relationships within a collection of forty-eight melon accessions as efficiently as with SSR markers,
and these markers may also be useful for cultivar identification in Occidental melon varieties.
Page 1 of 9
(page number not for citation purposes)
HJFm
(2)O
m
BMC Plant Biology 2009, 9:90
Background
Single-nucleotide polymorphisms (SNPs) are the most
frequent type of variation found in DNA [1] and are valuable markers for high-throughput genetic mapping,
genetic variation studies and association mapping in crop
plants. Several methods have been described for SNP discovery [2]: SNP mining from expressed sequence tag (EST)
databases [3]; based on array hybridization [4] or amplicon resequencing [5]; from the complete sequence of a
genome [6] and more recently, using high-throughput
sequencing technologies [7]. The discovery of SNP markers based on transcribed regions has become a common
application in plants because of the large number of ESTs
available in databases, and EST-SNPs have been successfully mined from EST databases in non-model species
such as Atlantic salmon [8], catfish [9], tomato [10] and
white spruce [11].
Melon (Cucumis melo L.) is an important crop worldwide.
It belongs to the Cucurbitaceae family, which also includes
cucumber, watermelon, pumpkin and squash. The melon
genome has an estimated size of 450 Mb [12] and is a diploid with a basic chromosome number of x = 12. In recent
years research has been carried out to increase the genetic
and genomic resources for this species, such as the
sequencing of ESTs [13], the construction of a BAC library
[14], the development of an oligo-based microarray [15]
and the development of a collection of near isogenic lines
(NILs) [16]. Genetic maps have also been reported for
melon, but they have been constructed with different
types of molecular markers and genetic backgrounds [1721], making it difficult to transfer markers from one map
to another. The aim of the International Cucurbit Genomics Initiative (ICuGI) [22], currently in progress, is to
obtain a consensus genetic map by merging genetic maps
available using a common set of SSRs as anchor markers.
A double haploid line (DHL) population from the cross
between the Korean accession PI 161375 (SC) and the inodorus type 'Piel de sapo' T111 (PS) was the basis for the
construction of a genetic map with 221 co-dominant,
transferable RFLP and SSR markers [21]. New EST-derived
SSR markers, added to this map using a bin-mapping
strategy with only 14 mapping individuals, gave a new
map with 296 markers distributed in 122 bins and a density of 4.2 cM/marker [21]. There is a need for saturating
the SC × PS genetic map with more markers that are amenable for large-scale genotyping, as are SNPs. In a preliminary experiment with melon, amplicon resequencing of
34 ESTs in SC and PS was used for SNP discovery, obtaining a frequency of one SNP every 441 bp and one indel
every 1,666 bp [23]. The availability of more than 34,000
melon ESTs from normalized cDNA libraries from different melon genotypes and tissues [13] is a valuable
resource for the identification of SNPs to be added to the
current genetic map.
http://www.biomedcentral.com/1471-2229/9/90
Genetic markers can also be used for variability analysis
studies. In melon, there have been several attempts to elucidate intraspecific relationships among melon germplasm, using isozyme [24], RFLP [25], RAPD [26], AFLP
[27] and SSR [28] markers, with SSRs the preferred marker
for fingerprinting and genetic variability analysis in melon
[28]. Due to the absence of a known set of SNPs in the
species, this marker has not been compared with other
types for variability analysis. It would be of special interest
to have a set of these markers for a high-throughput system to identify the germplasm used in breeding programs,
mainly from inodorus and the cantalupensis melon types.
The objectives of this work were to increase the marker
resolution in the melon genetic map, discovering ESTSNPs in a melon EST database, and to study the performance of a subset of EST-SNPs for variability analysis in a
collection of melon accessions.
Results and discussion
SNP discovery
Two strategies were used to discover SNPs in melon. The
first was based on producing amplicons from randomly
selected melon ESTs and resequencing the parental lines
of the melon genetic map PI 161375 (SC) × 'Piel de sapo'
T111 (PS). Primers were designed from 223 melon ESTs
(Table 1). After discarding primers that did not amplify a
PCR product, amplicons that did not produce high quality
sequences and monomorphic amplicons, 93 ESTs
(56.3%) showed at least one polymorphism between SC
and PS.
The second strategy was the validation of in silico SNPs
from the ICuGI database [22]. Three hundred and sixty-six
in silico SNPs found in the database were selected, belonging to two types of SNPs: pSNP and pSCH (Table 1; see
methods). Primers were designed from 269 ESTs containing pSNP and 97 containing pSCHs. Putative in silico SNPs
were validated in 51.8% and 21.3% of the amplicons for
pSNPs and pSCHs, respectively. In some instances additional SNPs were detected in the sequenced regions, giving a slightly higher percentage of polymorphic
amplicons (69.7% and 31.3% for pSNP and pSCH amplicons, respectively). From the ESTs reported by GonzalezIbeas et al. [13], 47.3% were obtained from two accessions of the 'Piel de sapo' cultivar type (Pinyonet and PS),
and the remainder from two genotypes, the C-35 cantaloupe accession (29.3%) and the pat81 agrestis accession
(23.4%). The pSNPs and pSCHs were deduced from this
set of EST sequences, with a high proportion found
between pat81 and 'Piel de sapo', and SNPs experimentally validated after resequencing amplicons from PS and
SC. SC belongs to the agrestis melon type as the accession
pat81 but has a different origin, so, as expected not all the
SNPs were conserved between SC and PS, giving a pSNP
validation of 51.8%. On the other hand, only 21.3% of
Page 2 of 9
(page number not for citation purposes)
T
HJGm
O
(2)m
BMC Plant Biology 2009, 9:90
http://www.biomedcentral.com/1471-2229/9/90
Table 1: Amplicons designed from ESTs for SNP discovery
Amplicons
Failed
Monomorphic
Polymorphic
Polymorphic amplicons*
In silico SNP validation
Random ESTs
223
58
72
93
56.3%
in silico pSNPs
269
41
69
159
69.7%
51.8%
in silico pSCHs
97
14
57
26
31.3%
21.3%
TOTAL
589
113
198
278
58.4%
ESTs were selected at random or chosen because they contained pSNPs or pSCHs in the MELOGEN database. Columns show the number of
amplicons that failed to amplify or gave bad quality sequences, and monomorphic and polymorphic amplicons between SC and PS. The percentages
of polymorphic amplicons and in silico SNPs that were validated are shown in the last two columns. (*) Polymorphic amplicons rate was calculated
without considering failed amplicons.
the pSCHs were validated, indicating that many may represent sequencing errors or mutations introduced during
the cDNA synthesis procedure. The SNPs in a subset of
amplicons containing in silico SNPs between 'Piel de Sapo'
and pat81 were validated using different genotyping
methods (see below) rather than resequencing in PS and
SC.
A total of 368 amplicons (random and containing in silico
SNPs) were resequenced in PS and SC and produced
177.5 kb of melon DNA, with 431 SNPs and 59 short
indels, at an average of one SNP every 412 bp and one
indel every 3.0 kb, (Table 2). This is in agreement with the
values obtained in a previous small-scale experiment
using the same two melon accessions, which gave one
SNP every 441 bp and one indel every 1.6 kb [23]. SC and
PS belong to the agrestis (C. melo ssp. agrestis) and inodorus
(C. melo ssp. melo) melon groups, respectively, which are
two of the more distant groups in the species [28]. This
may explain the relatively high frequency of SNPs
between the cultivars.
SNP detection
Various detection methods were used for genotyping the
SNPs in each EST. A restriction site around the SNP position, different in the parental sequences, was used to
develop a CAPS marker for 103 EST-SNPs. When more
than one SNP was discovered in one amplicon, we
selected the most suitable SNP for detection using CAPS.
When no restriction enzyme was available to produce a
CAPS marker, we used the SNaPshot SNP detection system. Seventy-seven EST-SNPs were genotyped with SNaP-
shot. For 14 ESTs, PS and SC gave a different amplicon
size, so they could be genotyped as SCAR markers. Four
EST-SNPs were genotyped using DNA sequencing and two
were converted into dCAPS. The SNP detection method
used for each mapped EST-SNP is shown in Additional file
1.
SNP variability
Forty-five SNPs (see Additional file 2) were randomly chosen to study their variability in a set of melon accessions
of worldwide cultivar and botanical types (see Additional
File 3). The inodorus cultivars were overrepresented in
order to assess whether SNPs between distant melon
accessions (SC and PS) were also variable among more
closely related genotypes.
All SNPs were polymorphic and the mean major allele frequency was 0.69 (Table 3). Only one SNP (AI_24-H05)
had a rare allele (frequency = 0.08), whereas the frequencies of the two alleles were similar in 28 SNPs (major
allele frequency < 0.65). Average gene diversity (He) was
0.4 (ranging from 0.14 to 0.5). Forty-three SNPs yielded
He > 0.20, demonstrating that most of the chosen SNPs
were highly informative, as found for SNPs in rye [29] but
contrasting with crops such as soybean [30] and wheat
[31] where SNPs yielding rare alleles are more frequent.
The mean gene diversity index for SNPs was considerably
lower than the values reported for SSRs in melon (e.g. PIC
= 0.58 [21], He = 0.66 [28]). To ensure the difference was
not due to sampling, gene diversity indexes were estimated using a subset of genotypes that had been included
Table 2: Frequency of SNPs and indels found after resequencing EST-derived amplicons
Amplicons sequenced in SC and PS
Length sequenced (bp)
SNPs
bp per SNP
indels
bp per indel
Reference
368
177,518
431
411.9
59
3,008.8
this report
34
15,000
34
441.2
9
1,666.6
[23]
Data from a previous report using the same two melon parental lines is shown as a comparison.
Page 3 of 9
(page number not for citation purposes)
HJHm
(2)O
m
BMC Plant Biology 2009, 9:90
http://www.biomedcentral.com/1471-2229/9/90
Table 3: Gene diversity indexes for SNP and SSR alleles using all, inodorus or genotypes described in a previous study [28]
Genotypes
Marker type
Major allele frequency
Ho
He
He range
all
SNP
0.69
0.10
0.40
0.14–0.50
inodorus
SNP
0.85*
0.07
0.15
0–0.50
group used in [28]
SNP
0.63
0.09
0.47
0.16–0.50
group used in [28]
SSR
0.47
0.14
0.64
0.51–0.83
Ho, observed heterozygosity; He expected heterozygosity. * Major allele frequency was only calculated for polymorphic SNPs.
in a previous study with SSRs [28] (see Additional file 3).
The differences in gene diversity were confirmed, demonstrating that they were intrinsic to the different marker
type. SNPs are biallelic, implying that the He value can
not exceed 0.5, whereas SSRs are multiallelic and so it can
be higher. Haplotypes may yield higher gene diversity values than individual SNPs and provide more efficient
application of SNP markers [29].
All inodorus genotypes could be distinguished with the set
of SNPs, although polymorphism was notably reduced
(Table 3). Fourteen SNPs were monomorphic and 18 were
informative (minor allele frequency > 0.1). As most of the
SNPs were discovered between the agrestis and inodorus
cultivar and not within inodorus, we expected the SNP polymorphism within inodorus to be lower. Nevertheless,
these results demonstrate that SNPs discovered using a
germplasm sample can be successfully transferred to different germplasm samples in melon.
The genetic relationships among accessions based on SNP
polymorphism were investigated by cluster analysis. The
NJ dendrogram (Figure 1) fits very well with previous classifications using different markers [26,28,32]. Comparing
the common genotype set in [28], the average pair-wise
distances based on SNPs and SSR were 0.47 and 0.64,
respectively. The correlation between the two distance
matrices was 0.73 (P < 0.00001) according to Mantel's
test, confirming that the current SNP set is as effective as
SSRs in establishing genetic relationships among melon
accessions, as shown in species such as rye [29] and soybean [30].
The population structure was estimated using the STRUCTURE software [33]. The a posteriori probability of the data
increased rapidly from K = 1 to 4 and begun to reach a plateau for K = 5, inferring that our collection can be divided
in five populations. Genetic variability among melon
germplasm seems to be highly structured. The subdivision
of the accessions in 5 populations agrees with the botanical classification and the cluster analysis (Figure 1): group
1 included all the inodorus cultivars from Spain; group 2, a
diverse group of traditional inodorus landraces and similar
ones from the Near-East region such as elongated (chate
and flexuosus) and Asiatic ananas and chandalak types;
group 3, modern cantalupensis cultivars; group 4, mainly
traditional varieties and wild melons from India and
Africa and group 5 included conomon accessions from the
Far East. The population structure should be taken into
account when establishing a collection of genotypes for
association mapping studies in melon and models including population structure should be used [34]. Alternatively, melon collections without structure, as we found
with the inodorus melon accessions included in our studies, could be used.
These results demonstrate that SNPs discovered using a
small germplasm sample can be transferred to different
cultivar groups, being useful for depicting genetic relationships as well as for cultivar identification.
SNP mapping using a bin-mapping strategy
Two hundred and seventy-eight SNP-containing ESTs
(Table 1) plus twelve additional SNP-containing ESTs previously discovered between the two parental lines [22]
were used for mapping in the SC × PS genetic map using
14 DHLs of the melon bin-mapping population [21]. In
total, 199 EST-derived SNPs were mapped, yielding 200
new markers (Figures 2 and 3). F112 produced two SCAR
markers (F112a and F112b) that mapped to groups I and
V, respectively. Our previous melon bin-map contained
296 markers distributed in 122 bins, with a density of 4.2
cM/marker and 2.4 markers per bin [21]. With the addition of 35 candidate genes previously reported for resistance to virus and fruit ripening [23,35,36] and the SNPs
now described, the new bin-map contains 528 markers,
distributed in 145 bins, with an increased density of 2.35
cM/marker and 3.64 markers per bin. The SNP-based
markers defined 23 new bins with an average bin length
of 8.55 cM. Some of the new bins were located in regions
with poor marker density in the previous SC × PS melon
map [21], such as HS_30-B08 in group XI, AI_12-B08 in
group VII, A_38-F04 in group VI or P06.05 in group III.
Essentially the new version of the melon bin-map is a
gene-based map, with 412 markers (78%) obtained from
Page 4 of 9
(page number not for citation purposes)
HJIm
O
(2)m
BMC Plant Biology 2009, 9:90
http://www.biomedcentral.com/1471-2229/9/90
K=5
SC
HER
GIN
PAT
FREE
CHT
TRI
KAK
SEN5
MOM
ZA1
ANN
INB
YC
CAR
DUL
C35
VED
VER
DOU
JPN
FLEX
ALF
KIZ
CHAN
EIN
AYN
KRK
CHA
ERI
ACD
VLV
VVG
AMD
BBL
VHC
TNI
AMC
ACA
BBE
HND
RMO
VCU
T111
AMA
PSPO
PPS
VVT
0.05
Figure
Dendogram
of
45 SNPs
1 inand
48population
melon accessions
structure based on the variability
Dendogram and population structure based on the
variability of 45 SNPs in 48 melon accessions. The
neighbor-joining (NJ) tree based on Nei genetic distances
[44] for the selected melon accessions is shown on the right.
The subdivision based on STRUCTURE is shown on the left;
each accession on the NJ is colored according to its group
assignation defined from STRUCTURE analysis.
gene sequences. Additionally, 114 RFLPs derived from
ESTs were previously mapped in an F2 population from
the cross SC × PS [37], and their approximate position can
also be plotted in the corresponding bin-map. As a large
proportion of the markers are codominant and based on
gene sequences, this makes this map a very useful tool for
melon breeding and comparative analysis in cucurbit species.
With the advent of next generation sequencing technologies, SNP discovery has become more feasible in nonmodel crop species, allowing the discovery of thousands
of SNPs in a single experiment [7]. In Eucalyptus grandis
more than 23,000 SNPs were discovered using 454
sequencing technology, with a validation rate of 83%
[38]. In melon, a preliminary analysis of 100,000 reads
obtained after 454 sequencing of leaf cDNAs from SC and
PS produced more than 1,000 SNPs (Garcia-Mas, unpublished). This indicates that the use of next generation
sequencing technologies is the next step towards saturation of the melon genetic map.
Conclusion
The set of 200 SNP markers discovered and mapped have
increased the marker resolution of the melon genetic map
by defining new bins. The genetic map contains more
than 500 gene-based codominant markers (SNPs, RFLPs
and SSRs), which can be used as anchor points with other
genetic maps in this species. This genetic map is also a useful resource for comparative mapping in the Cucurbitaceae, for the construction of the melon physical map and
for sequencing the melon genome. Additionally, the set of
SNPs has proven to be as useful as microsatellites for studying genetic relationships in melon and for varietal identification.
Methods
Plant material and DNA extraction
The parent lines of the melon double haploid line (DHL)
mapping population, PI 161375 'Songwan Charmi' (SC)
and 'Piel de sapo' line T111 (PS), were used for SNP discovery [20]. Fourteen DHLs from the SC × PS segregating
population were used to bin-map the SNP set [21]. The 48
melon genotypes selected for analysis with a subset of
SNPs (see Additional file 3) were obtained from the germplasm collection maintained at COMAV (Valencia, Spain)
and from a previous study of germplasm variability using
SSRs [28]. DNA from all genotypes was extracted using a
modified CTAB method [27]. DNA of the forty-eight
melon accessions was extracted from leaves of five individuals per accession to take into account the genetic variability within heterogeneous accessions.
SNP discovery and detection
SNPs were discovered using two different strategies.
Firstly, random ESTs were selected from the International
Cucurbit Genomics Initiative (ICuGI) webpage [22].
Primer pairs were designed from each EST using the
Primer3 software [39] with an average length of 20 nucleotides, a melting temperature around 60°C and an
expected PCR product of 500–700 bp. Genomic DNA
from the parental lines of the melon mapping population
was amplified with each primer pair as previously
described [23]. Amplified fragments were purified with
Sepharose columns and sequenced using the ABI Prism
BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) in an ABI Prism 3130
sequencer (Applied Biosystems, Foster City, CA, USA).
Sequences were aligned and screened for polymorphism
with the Bioedit software [40]. Putative SNP positions
were visually verified on the sequence chromatogram, and
Page 5 of 9
(page number not for citation purposes)
HJJm
(2)O
m
BMC Plant Biology 2009, 9:90
I
0.0
II
MC216 CmPABP
0.0
ECM61
MC51 CmEIL1
FR13B20
32.0
78.0
MG1 CMCTN86
CMGAN92 CM101A
47.0
CMCT505 CMCCA145
MC309 ECM233
ECM58 ECM139 CmPG4 52.0
AI_34-A07 PSI_04-D07
AI_09-G08 AI_17-E07
PSI_07-A04 F112a P01.16
ECM199
65.0
ECM173 PSI_35-C01
CMCTN53 ECM60b
ECM60c TJ26
FR10P24 PSI_12-D08
AI_09-D03
CSWCT11
FR12I13
TJ27
ECM110 CmERS1
F116
AI_05-G01
20.0
24.0
PS_10-C09 AI_14-H05
P12.74 P05.79 P01.41
CmeIF(iso)4G-1 CmEIL3
CM24 CMAGN68 CMGA108
PSI_03-B09 GCM331
PS_02-H06 A_25-G05
CmXTH2
MC273 ECM223
AI_04-E05
GCM548
57.0
MC332
MC248
AI_14-E02
70.0
75.0
78.0
80.0
IV
AI_24-G04 HS_10-A02
AI_14-B01 P12.96
GCM190 MC127
0.0
CSWCT10
AI_18-E05
CmEIL4 CmEXP2
TJ12b CMTAN66a CMBR83
TJ30 TJ31 MC244 GCM106
14.0
CM11 CMGA128 CMBR95
AI_08-F10 A_21-C11
CSWCT16T CMBR105 MC54
CMTA170A CMBR100
25.0
AI_33-H11
P06.15 46d_37-H067
33.0
0.0
MG34A
P12.50
57.0
CMGAN94 GCM186
MC69
ECM181
MC233
20.0
25.0
MC220
ECM60a AI_06-G01
AI_14-F04
ECM205 MC365
ECM125 MC215 F028
TJ10 PS_14-A11
AI_33-E02 PS_08-G08
AI_37-A07
70.0
72.0
PSI_27-C02 CmeIF4A-3
56.0
58.0
60.0
64.0
72.0
MC211 CM131 CmeIF4A-2
81.0
90.0
91.0
95.0
CM47
CMBR89
MC344
46d_11-A08
TJ12a GCM246
AI_03-F03 AI_03-E11
98.0
108.0
MC284 CMBR35 CMAGN79
ECM53 CMAGN73 ECM185
ECM106 CM122 ECM198 86.0
ECM122 GCM336
CI_35-H04 AI_10-B10
P12.94 CmnCBP P01.11 93.0
MC219
97.0
CMTCN6
ECM134
A_23-C03
108.0
CMTC168
MC60
FR12J11
123.0
127.0
CT02B CMCTN50
PSI_28-E12
AI_37-E06
ECM137
AI_09-E07
AI_17-B12
MC294
MC212
GCM168
ECM191
ECM138
AI_11-E06
P05.27
A_03-H09
137.0
VI
PSI_10-B04
CSCCT571
ECM108
ECM97 CmXTH4
PS_34-C02 A_31-E10
PS_25-E09
40.0
MC296
ECM208
CMCTN5
ECM51 CmACS5
MC252
V
12.0
CMTCN66b GCM448
CMTCN18 MC226
AI_02-C08 CmETR2
ECM184
GCM262
26.0
CMAGN61
CMAGN52
34.0
ECM197 ECM124
PSI_20-A04
GCM101
MC376
86.0
104.0
0.0
9.0
10.0
MC134
ECM85
63.0
MC340 PS_09-H05
F216 SSH1A13
MC279
AI_09-F07
18.0
41.0
III
CM149
ECM230
12.0
http://www.biomedcentral.com/1471-2229/9/90
116.0
41.0
A_18-A08
15d_17-G01
CMATN101
TJ37 CMTCN9
50.0
CMCTN35
CMAT35
HS_11-A09 15d_14-B01
AI_08-G09 P02.22
CI_19-H12 SSH9G15
ECM129 ECM92 MC256
CmeIF(iso)4E
ECM142 CMGAN3 MC4 70.0
ECM203 GCM622 CmETR1
ECM109 GCM295 CmeIF4G
PS_15-B02 PS_03-B08
MC8 ECM52
FR11A2
MC21 GCM303 GCM255
ECM81 GCM302 ECM135
CMTCN41 CMCT123
CI_56-B01 FR14P22
AI_19-F11 PS_19-B07
AI_03-B03 15d_29-E06
MC224 AI_05-H08
HS_20-C04 HS_05-B07
A_38-F04
CMTCN2
MRGH63
F112b
CSWTA02 CSWCTT02T
AI_13-H12
CMBR15 CMBR107
CMTAA166
97.0
ECM132 ECM178
GCM112 AI_13-F02
PS_28-B07 P01.45
HS_06-D02 CmERF3
MC42
105.0
CMTAAN100
CMBR123
CMCTN38
115.0
ECM231 HS_33-D11
PSI_19-F05 PS_07-E07 CmEthInd
GCM155
135.0
CMCTN4 PSI_11-D12
141.0
Figure 2 bin map of Cucumis melo obtained by selective genotyping of fourteen DHLs
EST-SNP
EST-SNP bin map of Cucumis melo obtained by selective genotyping of fourteen DHLs. Linkage groups are represented by vertical bars, divided in bins defined by the joint genotype of the selected DHLs. The mapped SNPs in this report are
shown in bold. Underlined markers are candidate genes previously reported [23,35,36]. The other markers have been
described in [21]. Genetic distances are shown on the left, indicating the position of the last marker included in the bin according to the framework map in [21]. Markers defining new bins are shown in italics. The hypothetical position of the last marker
of these bins is indicated by a dashed horizontal line within the linkage group bar, without the genetic distance.
the genomic sequences compared with the original EST
sequence to identify any introns. In the second strategy, in
silico SNPs previously identified [13] using EST2uni [41]
were classified as i) pSNPs, corresponding to SNPs present
in at least two EST sequences from the same genotype in a
given contig and with the same base change and ii)
pSCHs, corresponding to single nucleotide variations in
sequence that did not follow the above criteria for pSNPs.
Selected pSNPs and pSCHs were verified in most cases
after resequencing the parental lines of the melon mapping population. For a small subset, the SNP was verified
with an appropriate SNP detection method.
Bioedit software was used to generate restriction maps
from sequences obtained from SC and PS. SNPs (or
indels) showing differential restriction maps were used to
develop cleaved amplified polymorphic sequence (CAPS)
markers. When no differential restriction maps were available, the ABI Prism SNaPshot ddNTP Primer Extension Kit
(Applied Biosystems) was used for SNP genotyping [23].
Markers F112, 46d_11-A08, FR12J11, 15d_17-G01,
P01.45, PSI_26-B12, F012, PS_18-F05, PS_16-C09, F088,
A_02-H11, AI_13-G03 and FR15D10 produced amplicons of different sizes in the parental lines, which were
not sequenced and were genotyped as sequence characterized amplified region markers (SCARs) after electrophoresis in agarose gels or using a LI-COR IR2 sequencer (Li-Cor
Inc, Lincoln, Nebraska, USA). Markers PSI_12-D08 and
PSI_35-F11 were converted into dCAPS markers [42].
Markers F028, F149, F080 and PSI_25-B05 were genotyped using direct sequencing.
Page 6 of 9
(page number not for citation purposes)
HJKm
O
(2)m
BMC Plant Biology 2009, 9:90
VII
5.0
8.0
VIII
ECM50 F072
GCM181 MC373
ECM79 AI_05-F11
CmeIF(iso)4G-2
A_06-A03
AI_12-B08 AI_27-F07
PSI_26-B12
3.0
6.0
16.0
MC311
MC44
CMAGN75 ECM182
ECM227
AI_25-C11 F271
P06.69
39.0
47.0
48.0
51.0
55.0
60.0
IX
15d_01-B03 A_30-G06
CSGA057 CMBR24 CMBR7
ECM88 P4.35 PS_28-E01
F125 PSI_29-D11
0.0
F080 PS_18-F05
TJ3 MC301
ECM217
ECM128 PSI_23-A11
HS_25-A10 CmAco3
13.0
MC68
TJ2 CmACS3
CI_33-B09
29.0
ECM221 AI_02-A08
MC253 CMTCN30
PSI_33-F04 HS_04-F11
41.0
CSAT425B ECM84
TJ38 ECM77 CMCAN90
CMBR53 CMBR27 P06.02
AI_03-G06 CmACS2
CMAGN21 CMBR84 GCM521
54.0
F149 F012
CSWCT12T CI_08-C08
CM139 AI_08-H11
ECM204
68.0
76.0
http://www.biomedcentral.com/1471-2229/9/90
CMGA15 ECM172 CmERF2
PSI_37-G01 CI_37-H11
77.0
CMTC13 AI_21-G05
AI_21-D08 P01.8
35.0
MC356 MC11 MC78
ECM200
CMAT141
PSI_25-H03
94.0
0.0
10.0
17.0
ECM150 ECM177
MRGH7 CM98
ECM66
PSI_12-C05 AI_17-B03
AI_39-A12 PSI_23-G11
31.0
FR18J20 A_17-A08
F036 AI_04-D08
35.0
54.0
57.0
61.0
PSI_21-D01
TJ33
0.0
CMTCN62
MC146 ECM210 P05.50
CMTC160 ECM63
ECM183 P4.39 A_02-H01
MC17 CSWCT01
CSWCT22A
HS_30-B08
CMTCN67
MC264 CMGAN12
MC375 CMAGN45
CmXTH5
CMGA172 ECM228
MC112 MC67 CM40
CMTA134B CMCTN65
CMGA165 ECM232
GCM153 ECM49
ECM116 ECM220
ECM101 GCM344
AI_38-B09 F088
46d_21-E02 CmEXP3
CM101B
CMTCN8
31.0
34.0
42.0
HS_39-A03
CMATN56 CmEXP1
97.0
TJ29
MC330
HS_23-D06 FR15D10
CSWCT18B CmeIF4A-1 29.0
HS_35-E11 AI_22-A08
A_05-A02 FR12O13 CmACO5
MC231 ECM164
HS_02-E07 PS_24-E03
PSI_41-B07
49.0
CMATN89 MC349
CMGA104 P06.79
PSI_35-H10
66.0
MC325
71.0
CMTCN14
CMAGN33 CMAGN32
CMAGN80
ECM123
ECM218
P02.03 FR14F22
MC93
MC265
ECM147
ECM145
A_08-D10 15d_27-B02
AI_13-G03
93.0
CSAT425A
MC286
CI_57-E03
53.0
62.0
CM91
CMTCN1
CMCTN7
AI_21-E10
CmERF1 CmPME3
MC132 GCM206
PSI_22-B02 PSI_12-D12
5A6U nsv MC320
ECM67 ECM105
CmeIF4E AI_35-A08
AI_09-G07 15d_01-B03
FR12P24
17.0
76.0
102.0
XII
ECM175 MC149 ECM78 0.0
PS_16-C09 PSI_35-F11 2.0
PS_33-E12 AI_36-F12
8.0
CMCTN71 CMCTN19
69.0
ECM55
MC138
99.0
MC92 CMTC47
ECM186 P05.64
A_04-B10 FR13O21
CMACC146 GCM241
MC208 CMAG59 F013
A_32-B01 CI_58-C10
XI
HS_23-E06 ECM86 ECM82
PS_15-H02 PS_40-E11
MRGH21
A_08-H06
ECM56
F129
MC125 PS_19-E06
P05.15 AI_16-D09
X
81.0
P02.75
AI_08-F01 A_20-H12
AI_35-E03 P01.17
MC237
CMATN22
110.0
Figure 3 bin map of Cucumis melo obtained by selective genotyping of fourteen DHLs
EST-SNP
EST-SNP bin map of Cucumis melo obtained by selective genotyping of fourteen DHLs. Linkage groups are represented by vertical bars, divided in bins defined by the joint genotype of the selected DHLs. The mapped SNPs in this report are
shown in bold. Underlined markers are candidate genes previously reported [23,35,36]. The other markers have been
described in [21]. Genetic distances are shown on the left, indicating the position of the last marker included in the bin according to the framework map in [21]. Markers defining new bins are shown in italics. The hypothetical position of the last marker
of these bins is indicated by a dashed horizontal line within the linkage group bar, without the genetic distance.
SNP mapping
SNPs and indels were mapped by selective genotyping
using the bin-mapping strategy [43], adapted for the
melon mapping population [21]. Fourteen out of 72
DHLs from the melon mapping population were selected
to obtain the maximum resolution with a minimum
number of genotypes. SNPs and indels were placed in the
bin map by visual inspection of the genotypes predicted
by the markers and genotypes in the bin set.
Genetic variability analysis
Forty-five SNPs from 44 amplicons (two SNPs were
selected from F241) were chosen for genetic variability
analysis. SNPs were genotyped as CAPS or by pyrosequencing as shown in Additional file 2. Thirty SNPs,
described in Additional file 1, were used. Twelve SNPs
that were not polymorphic between SC and PS were also
included in the variability analysis, and the primers for
each amplicon are provided in Additional file 2. The SNPs
CmERF1, CmPm3 and CmXTH5 have been previously
described [36].
Eight SNPs were genotyped by minisequencing the region
surrounding the polymorphism (two SNPs were detected
for F241 in the same reaction). Pyrosequencing was performed using a PSQ™ HS 96 system (Pyrosequencing AB,
Uppsala, Sweden) following the manufacturers' instructions. Primers were designed with the Pyrosequencing™
Assay Design Software (Biotage AB, Uppsala, Sweden).
One of the amplifying primers was 5' end labeled with
biotin, allowing the immobilization of the fragment onto
M-280 streptavidin coated Sepharose™ dynabeads (Dynal
AS, Oslo, Norway). The genotyping primer was hence
designed to anneal several nucleotides upstream of the
SNP. After denaturation of the streptavidin-captured PCR
fragments, the single stranded DNA fragments were
released into the wells of the PSQ HS 96 plate. Pyrosequencing was performed using the PSQ HS SNP Reagent
kit (Pyrosequencing AB, Uppsala, Sweden), and bioluminometric quantification of pyrophosphate (Ppi) released
as a result of nucleotide incorporation during DNA synthesis was measured with the PSQ™ HS 96 system.
Page 7 of 9
(page number not for citation purposes)
HJLm
(2)O
m
BMC Plant Biology 2009, 9:90
http://www.biomedcentral.com/1471-2229/9/90
Allele frequencies, major allele frequency, gene diversity
(measured as expected heterozygosity, He [44]), genetic
distances and neighbor-joining (NJ) tree were calculated
using Powermarker 3.25 [45]. The NJ tree was plotted
with MEGA 3.0 [46]. Distance matrices were compared by
the Mantel test [47].
Additional file 2
SNP markers used for genotyping the melon accessions. The EST from
where the SNPs were discovered, the genotyping method (CAPS or pyrosequencing), linkage group where the marker maps to, and source of the
marker are given. For unmapped SNP markers, the amplicon primer
sequences are given. For SNP markers genotyped using pyrosequencing,
forward, reverse and internal primers were used for genotyping. 5'bio: Forward or reverse primer was 5' labeled with biotine. ST1: Additional file 1.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712229-9-90-S2.xls]
The number of populations in our collection was deduced
with the STRUCTURE software [33]. This package uses a
Bayesian clustering approach to identify subpopulations
and to assign individuals to these populations on the
basis of their genotypes. Given a sample of individuals, K
populations are assumed (where K may be unknown) and
individuals are assigned to these populations. A posteriori
probability for each K (Pr(K)) can be calculated, which is
very small for K values lower than the appropriate value.
Usually, the researcher fixes a minimum K (for example K
= 1), recording Pr(K) after the analysis, and tests increasing Ks, plotting K against Pr(K). The final K is defined
when Pr(K) reaches a plateau for higher K values. Consequently, in the current report, several number of populations (from K = 1 to 8) were tested with the software and
the total number of populations was set when the probability reached a plateau for higher K.
Additional file 3
Forty-eight melon accessions that were examined in this study. Plant
assignation (or common name), code used in the current study, accession
number from the respective gene banks, cultivar group, origin and seed
bank donor (COMAV, Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Valencia, Spain; USDA/ARS/NCRPIS, North Central Regional Plant Introduction Station, Ames, IA, USA; IPK, Institute
of Plant Genetics and Crop Plant Research, Gatersleben, Germany;
INRA, Institute Nationale de la Recherche Agronomique, Montfavet, Avignon, France; Semillas Fitó SA, Barcelona, Spain; ARO, Agricultural
Research Organization, Ramat Yishay, Israel) are specified for each genotype. Accessions marked with (*) were previously used by Monforte et al.
(2003) for an SSR study.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712229-9-90-S3.xls]
Authors' contributions
WD discovered and mapped the SNPs and performed the
genotyping for the variability analysis. CE and MGT discovered and mapped SNPs. CR discovered SNPs. IFS
mapped SNPs. DGI identified and selected in silico SNPs.
JB carried out the bioinformatics analyses for in silico
SNPs. AJM performed the variability analysis, coordinated
the SNP mapping and participated in the drafting of the
manuscript. MBP prepared DNAs for the melon accessions and participated in the genotyping for the variability
analysis and in the drafting of the manuscript. JGM, PA,
FN, MBP and MAA were involved in the conception of the
study. JGM is the principal researcher of this work, supervised it and wrote the manuscript. All authors read and
approved the final manuscript.
Acknowledgements
This work was supported by a grant from the Ministerio de Educación y
Ciencia (Spain) (GEN2003-20237-C06). WD is recipient of a postdoctoral
fellowship from the Centre de Recerca en Agrigenòmica CSIC-IRTA-UAB
(Spain). CR is recipient of a Juan de la Cierva grant from the Ministerio de
Educación y Ciencia (MEC) (Spain). DGI and CS are recipients of pre-doctoral fellowships from MEC (Spain). IFS is recipient of a pre-doctoral fellowship from INIA (Spain). We are grateful to Armand Sanchez and Anna
Mercader (UAB) for their help with the pyrosequencing analysis.
References
1.
2.
3.
Additional material
Additional file 1
SNPs markers mapped in the SC × PS genetic map. Shown here, for
each SNP marker: the EST and accession number from where it was
obtained; best BlastX hit and E-value for each EST; amplicon primer
sequences; SNP/indel position; SNP detection method; linkage group and
BIN where the marker maps to. asequence available in [22] or http://
www.melogen.upv.es without accession number. bSNPs published by
Morales et al (2004). cSNP position is provided when located in exons
and referred to EST in first column. dthird primer was used for SNaPshot
genotyping.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712229-9-90-S1.xls]
4.
5.
6.
7.
8.
Brookes AJ: The essence of SNPs. Gene 1999, 234(2):177-186.
Ganal MW, Altmann T, Roder MS: SNP identification in crop
plants. Curr Opin Plant Biol 2009, 12(2):211-217.
Batley J, Barker G, O'Sullivan H, Edwards KJ, Edwards D: Mining for
single nucleotide polymorphisms and insertions/deletions in
maize expressed sequence tag data. Plant Physiol 2003,
132(1):84-91.
Borevitz JO, Liang D, Plouffe D, Chang HS, Zhu T, Weigel D, Berry
CC, Winzeler E, Chory J: Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res 2003,
13(3):513-523.
Choi IY, Hyten DL, Matukumalli LK, Song Q, Chaky JM, Quigley CV,
Chase K, Lark KG, Reiter RS, Yoon MS, et al.: A soybean transcript
map: gene distribution, haplotype and single-nucleotide polymorphism analysis. Genetics 2007, 176(1):685-696.
Velasco R, Zharkikh A, Troggio M, Cartwright DA, Cestaro A, Pruss
D, Pindo M, Fitzgerald LM, Vezzulli S, Reid J, et al.: A high quality
draft consensus sequence of the genome of a heterozygous
grapevine variety. PLoS ONE 2007, 2(12):e1326.
Barbazuk WB, Emrich SJ, Chen HD, Li L, Schnable PS: SNP discovery via 454 transcriptome sequencing.
Plant J 2007,
51(5):910-918.
Hayes B, Lærdahl JK, Lien S, Moen T, Berg P, Hindar K, Davidson WS,
Koop BF, Adzhubei A, Høyheim B: An extensive resource of sin-
Page 8 of 9
(page number not for citation purposes)
HJMm
O
(2)m
BMC Plant Biology 2009, 9:90
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
gle nucleotide polymorphism markers associated with
Atlantic salmon (Salmo salar) expressed sequences. Aquaculture 2007, 265:82-90.
Wang S, Sha Z, Sonstegard TS, Liu H, Xu P, Somridhivej B, Peatman
E, Kucuktas H, Liu Z: Quality assessment parameters for ESTderived SNPs from catfish. BMC Genomics 2008, 9:450.
Yamamoto N, Tsugane T, Watanabe M, Yano K, Maeda F, Kuwata C,
Torki M, Ban Y, Nishimura S, Shibata D: Expressed sequence tags
from the laboratory-grown miniature tomato (Lycopersicon
esculentum) cultivar Micro-Tom and mining for single nucleotide polymorphisms and insertions/deletions in tomato cultivars. Gene 2005, 356:127-134.
Pavy N, Parsons LS, Paule C, MacKay J, Bousquet J: Automated
SNP detection from a large collection of white spruce
expressed sequences: contributing factors and approaches
for the categorization of SNPs. BMC Genomics 2006, 7:174.
Arumuganathan K, Earle ED: Nuclear DNA content of some
important plant species. Plant Mol Biol Rep 1991, 9:208-218.
Gonzalez-Ibeas D, Blanca J, Roig C, Gonzalez-To M, Pico B, Truniger
V, Gomez P, Deleu W, Cano-Delgado A, Arus P, et al.: MELOGEN:
an EST database for melon functional genomics. BMC Genomics 2007, 8:306.
van Leeuwen H, Monfort A, Zhang HB, Puigdomenech P: Identification and characterisation of a melon genomic region containing a resistance gene cluster from a constructed BAC
library. Microcolinearity between Cucumis melo and Arabidopsis thaliana. Plant Mol Biol 2003, 51(5):703-718.
Mascarell-Creus A, Cañizares J, Vilarrasa J, Mora-García S, Blanca J,
Gonzalez-Ibeas D, Saladié M, Roig C, Deleu W, Picó B, et al.: An
oligo-based microarray offers novel transcriptomic
approaches for the analysis of pathogen resistance and fruit
quality traits in melon. BMC Genomics in press.
Eduardo I, Arus P, Monforte AJ: Development of a genomic
library of near isogenic lines (NILs) in melon (Cucumis melo
L.) from the exotic accession PI161375. Theor Appl Genet 2005,
112(1):139-148.
Wang YH, Thomas CE, Dean RA: A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism (AFLP) markers. Theor Appl Genet 1997, 95:791-798.
Danin-Poleg Y, Reis N, Baudracco-Arnas S, Pitrat M, Staub JE, Oliver
M, Arus P, deVicente CM, Katzir N: Simple sequence repeats in
Cucumis mapping and map merging.
Genome 2000,
43(6):963-974.
Perin C, Hagen S, De Conto V, Katzir N, Danin-Poleg Y, Portnoy V,
Baudracco-Arnas S, Chadoeuf J, Dogimont C, Pitrat M: A reference
map of Cucumis melo based on two recombinant inbred line
populations. Theor Appl Genet 2002, 104(6–7):1017-1034.
Gonzalo MJ, Oliver M, Garcia-Mas J, Monfort A, Dolcet-Sanjuan R,
Katzir N, Arus P, Monforte AJ: Simple-sequence repeat markers
used in merging linkage maps of melon (Cucumis melo L.).
Theor Appl Genet 2005, 110(5):802-811.
Fernandez-Silva I, Eduardo I, Blanca J, Esteras C, Pico B, Nuez F, Arus
P, Garcia-Mas J, Monforte AJ: Bin mapping of genomic and ESTderived SSRs in melon (Cucumis melo L.). Theor Appl Genet
2008, 118(1):139-150.
The International Cucurbit Genomics Initiative (ICuGI)
[http://www.icugi.org]
Morales M, Roig E, Monforte AJ, Arus P, Garcia-Mas J: Single-nucleotide polymorphisms detected in expressed sequence tags of
melon (Cucumis melo L.). Genome 2004, 47(2):352-360.
Staub JE, Box J, Meglic V, Horejsi TF, Mccreight JD: Comparison of
isozyme and random amplified polymorphic DNA data for
determining intraspecific variation in Cucumis. Genet Res Crop
Evol 1999, 44:257-269.
Neuhausen SL: Evaluation of restriction fragment length polymorphisms in Cucumis melo. Theor Appl Genet 1992, 83:379-384.
Stepansky A, Kovalski I, Perl-Treves R: Intraspecific classification
of melons (Cucumis melo L.) in view of their phenotypic and
molecular variation. Plant Syst Evol 1999, 217:313-332.
Garcia-Mas J, Oliver M, Gómez H, de Vicente MC: Comparing
AFLP, RAPD and RFLP markers to measure genetic diversity in melon. Theor Appl Genet 2000, 101:860-864.
Monforte AJ, Garcia-Mas J, Arús P: Genetic variability in melon
based on microsatellite variation.
Plant Breeding 2003,
122:153-157.
http://www.biomedcentral.com/1471-2229/9/90
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Varshney RK, Beier U, Khlestkina EK, Kota R, Korzun V, Graner A,
Borner A: Single nucleotide polymorphisms in rye (Secale
cereale L.): discovery, frequency, and applications for
genome mapping and diversity studies. Theor Appl Genet 2007,
114(6):1105-1116.
Yoon MS, Song QJ, Choi IY, Specht JE, Hyten DL, Cregan PB:
BARCSoySNP23: a panel of 23 selected SNPs for soybean
cultivar identification. Theor Appl Genet 2007, 114(5):885-899.
Ravel C, Praud S, Murigneux A, Canaguier A, Sapet F, Samson D, Balfourier F, Dufour P, Chalhoub B, Brunel D, et al.: Single-nucleotide
polymorphism frequency in a set of selected lines of bread
wheat (Triticum aestivum L.). Genome 2006, 49(9):1131-1139.
Monforte AJ, Eduardo I, Abad S, Arus P: Inheritance mode of fruit
traits in melon-heterosis for fruit shape and its correlation
with genetic distance. Euphytica 2005, 144:31-38.
Pritchard JK, Stephens M, Donnelly P: Inference of population
structure using multilocus genotype data. Genetics 2000,
155(2):945-959.
Pritchard JK, Stephens M, Rosenberg NA, Donnelly P: Association
mapping in structured populations. Am J Hum Genet 2000,
67(1):170-181.
Essafi A, Diaz-Pendon JA, Moriones E, Monforte AJ, Garcia-Mas J,
Martin-Hernandez AM: Dissection of the oligogenic resistance
to Cucumber mosaic virus in the melon accession PI 161375.
Theor Appl Genet 2009, 118(2):275-284.
Moreno E, Obando JM, Dos-Santos N, Fernandez-Trujillo JP, Monforte AJ, Garcia-Mas J: Candidate genes and QTLs for fruit ripening and softening in melon.
Theor Appl Genet 2008,
116(4):589-602.
Oliver M, Garcia-Mas J, Cardus M, Pueyo N, Lopez-Sese AL, Arroyo
M, Gomez-Paniagua H, Arus P, de Vicente MC: Construction of a
reference linkage map for melon. Genome 2001, 44(5):836-845.
Novaes E, Drost DR, Farmerie WG, Pappas GJ Jr, Grattapaglia D,
Sederoff RR, Kirst M: High-throughput gene and SNP discovery
in Eucalyptus grandis, an uncharacterized genome. BMC
Genomics 2008, 9:312.
Rozen S, Skaletsky H: Primer3 on the WWW for general users
and for biologist programmers. Methods Mol Biol 2000,
132:365-386.
Hall TA: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.
Nucleic Acids Symp Ser 1999, 41:95-98.
Forment J, Gilabert F, Robles A, Conejero V, Nuez F, Blanca JM:
EST2uni: an open, parallel tool for automated EST analysis
and database creation, with a data mining web interface and
microarray expression data integration. BMC Bioinformatics
2008, 9:5.
Neff MM, Neff JD, Chory J, Pepper AE: dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana
genetics. Plant J 1998, 14(3):387-392.
Howad W, Yamamoto T, Dirlewanger E, Testolin R, Cosson P, Cipriani G, Monforte AJ, Georgi L, Abbott AG, Arus P: Mapping with
a few plants: using selective mapping for microsatellite saturation of the Prunus reference map.
Genetics 2005,
171(3):1305-1309.
Nei M, Tajima F, Tateno Y: Accuracy of estimated phylogenetic
trees from molecular data. II. Gene frequency data. J Mol Evol
1983, 19(2):153-170.
Liu K, Muse SV: PowerMarker: an integrated analysis environment for genetic marker analysis.
Bioinformatics 2005,
21(9):2128-2129.
Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol
Biol Evol 2007, 24(8):1596-1599.
Mantel N: The detection of disease clustering and a generalized regression approach. Cancer Res 1967, 27(2):209-220.
Page 9 of 9
(page number not for citation purposes)
HJNm
(2)O
m
TT
T
M m *m !-,m *-1m !# "-0#1m 32'*'8"-1m .0m *1m +.*'$'!!'-,#1m "#m !"m %#,m .0m B1/3#"m "#m
+32,2#1mYm,m*m!-*3+,m"#m*m'8/3'#0"m#*m,-+ 0#m"#m*-1m!# "-0#1m7mm*m"#0#!&m*1m
1#!3#,!'1m!-00#1.-,"'#,2#1Ymm
-+ 0#m
# "-0m
$ETB$0T ,/+(">-T
m
,20-Gjm
m
!Kdm
m
,20-,G#4jNFFm
m
3**!KdjMFFm
m
$ETC.T ,/+(">-T
m
,20-,Gjm
m
!Idm
m
,20-,G-0jMFFm
m
3**!IdjNFFm
m
$P(1.QET
m
'1-Jm
m
'1-Jm
m
'1-JjMFFm
m
'1-JjNFFm
m
BT
m
GjGm
m
GjGm
m
GjNjMFFm
m
GjNjNFFm
m
T
m
jLm
m
IdjHm
m
JjMFFm
m
jNFFm
m
HJOm
O
(2)m
M!m+.A,m"#mm
%*HmGKm+m
*mKFFm+m
0'1mGFFm+m
#*2',mFYFGPm
T
M"T 09$'!-m "#m *m "'*3!'A,m ,#!#10'm "#m #,8'+m ()m #,m $3,!'A,m "#m *m !-,!#,20!'A,m "#m m
- 2#,'"m#,m*m-.OYm#.#,"'#,"-m"#m*-1m,%m"#m.0-"3!2-m- 2#,'"-1m#,m*mm"# #m3101#m
3,m!-,!#,20!'A,m"#!3"m"#m#,8'+m.0m/3#m*m"'%#12'A,m1#mA.2'+Ym^2-1m.0-.-0!'-,"-1m
.-0m#*m%03.-m"#*mZm0Ym "#*m#,"&+,#_Ym
T
m
m
M#T+.A,m"#m"'*3!'A,m"#m*m#,8'+m()O
m
KFm+m0'1m*m.Nm
GFFm+m*m
GFmxm,*Hm
FYFGPm0'2-,mjGFFm
KFPm*'!#0-*m
HFmx%i+mm!#2'*"-m
T
HKFm
(2)O
m
M$m+.A,m"#m0#!!'A,m.0m*m"'%#12'A,m!-,m()O
O
Gm#.#1m.MZKm
FYGm%Jm
FYFHPm0'2-,m^jGFF_m
FYGm*m
mHm
m
M%m+.A,m"#m#620!!'A,m"#mm-7*#m
m
HPmm
GZJm*m
HF+mm
GFF+m0'1j*m^.mNYFm_m
(3120mmmNYFFZm32-!*40m7m"#(0m#,$0'0Ym',*+#,2#m@"'0mFYHPm"#muj+#0!.2-#2,-*m
m
M&m+.A,m"#m*4"-m-7*#m
m
2,-*mMLPm
!#22-m+A,'!-mGF+m
T
M'm83*m"#m!0%m.0m%#*#1m"#m%0-1m
m
83*m"#m 0-+-$#,-*mFYLPm^.i4_m
*'!#0-*mMFPm^4i4_m
mFYHPm^4i4_m
mFYK6m
mHFm+m
m
M(m+.A,mGFm
m
0'1mGm
mGF+m
!jIHmFYGHm
m
m
M)T *m!-,m*1m1#!3#,!'1m"#m*-1m!# "-0#1m32'*'8"-1m"30,2#m*m1#!3#,!'!'A,m"#m!"m%#,[m
m
O
Y(YGmGO
m
HKGm
O
(2)m
-+ 0#m!# "-0m
#!3#,!'m
m
m
HFMm
m
Hm
m
Im
m
HFNm
m
Im
m
Im
m
Lm
m
Jm
m
Jm
m
m
m
IdjHm
m
m
O
Y(YHmI#-)J>O
T
-+ 0#m!# "-0m
#!3#,!'m
1-Jm
m
1-Jm
m
1-Jm
m
1-Jm
m
T
O
O
Y(YIm;O
T
-+ 0#m!# "-0m
#!3#,!'m
GjGm
m
GjNm
m
GjNm
m
GjHm
m
GjGm
m
GjJm
m
GjKm
m
GjJm
m
GjLm
m
GjKm
m
GjIm
m
GjLm
m
GjIm
m
m
m
T
M*T BBDT 40'#""#1m 31"1m .0m #*m !-m .#02#,#!'#,2#1m *m ,!-m "#m %#0+-.*1+m "#*m
Ym
m
HKHm
(2)O
m
m
>#(&.T#$T
03/.T
.,!0$T+." +T
;1T#$T.0;&$-T
mHKJm
"3"'+m
m
$%,'129,m
mGHKOKGm
+#*-j!&,"*!)m
m
$%,'129,m
JHJm
+#*-j!,2*3.]]m
.(#.#0m
* ,'m
-*jNJm
+#*-j",m
+#*A,m
-*'4'm
KIKm
+#*-j!,2*3.]]m
m
3*%0'm
HFLm
!-,-+-,m
m
&',m
LGNNKJm
!-,-+-,m
-,%2',m
&',m
KGFm
+#*-m
"',(j- '!,m
0-!'m
GJMLm
+#*-m
m
!3"-0m
HNNHIIm
+#*-m
$0m&)'+m
%'.2-m
GONm
!,2*3.#,1'1m
jjGGm
+#*-m
#*-,m,,20'm
#-0%'m
jIGJJHMm
+#*-j!,2*3.]]m
-*(-,'28m
#-0%'m
mGNKGGGm
+#*-j2' '1&]]m
m
&,m
KIm
+#*-j!,2*3.]]m
jjHm
+#*-j!,2*3.]]m
mGIIm
',-"-031m
mIFKm
!,2*3.#,1'1j+#0'm
8C12m,,18m
3,%0>m
mJINm
+-+-0"'!m
&0 3(m
,"'m
HHKm
$*#63-131m
m
,"'m
GHJGGHm
+-+-0"'!m
m
,"'m
mGLJIHFm
!&'2-j%0#12'1m
m
,"'m
mIJJm
$*#63-131m
&'0m20387m
0)m
mIJOm
$*#63-131m
m
0)m
mIKKm
+#*-Zm',-"j1'2'!-m
m
0)m
mGJFLIHm
!,2*3.#,1'1m
m
09,m
mILIm
"8&30mj!&2#m
!0-1#**-m
2*'m
IMHm
+#*-m
!3!3+088-m
2*'m
mHLLOJMm
!2*3.#,1'1m
m
.A,m
mJHFGMLm
!-,-+-,j+)35m
%',1#,j+)35m
.A,m
""$1(>-T
,2*-3.m"#m
#**#%0"m
m^-32&m
*),_m
#*-,m
31!2#**-m
3)-0"',,7#m
1*-4/3'm
0#!'m
32#+*m
3,%0>m
HKIm
O
HKJm
(2)m
jjGFm
+#*-m
+#12,'m
8(129,m
2NGm
%0#12'1m
%0#12'1m.2jNGm
-0#m
HOJm
!,2*3.#,1'1m
&'0m
' 'm
mJOFINNm
!,2*3.m
,),'m
*'m
IINm
!&,"*)m
m
-,%-*'m
$0j!jGm
+#*-j0#2'!3*231m
m
003#!-1m
jHNMm
%0#12'1m
m
'%#0'm
FJNJJm
+#*-m
+#*-m
-023%*m
1!m"#m
jKLGOMm
+#*-m
mGLNm
+#*-m
-0;m
31'm
HKOm
!,2*3.m
.#*1',(m
31'm
mHMLLLFm
!&,"*)m
m
31'm
mKFLJLFm
!,2*3.#,1'1m
)34'1,)m
31'm
mJILKIJm
%0#12'1m
m
#,#%*m
mHNHJJNm
+#*-m
m
30m$0'!m
NHFMm
',-"-031m
@m3*!#m
1.@m
jFJOm
',-"-031m
-,#7j"#5m
1.@m
jjGFOm
',-"-031m
#*A,m!-!m
1.@m
jjGIIm
!,2*3.m
jjMm
',-"-031m
!04*&-m
#*A,m#1!0'2-m
-*-0-1-m
#0"#m%-0"-m
*,!-m
-023%*m
1.@m
1.@m
jjMFm
',-"-031m
jjNm
',-"-031m
jjOFm
',-"-031m
#+.0,'**-m
1.@m
jGONm
',-"-031m
'*-m!00#2#m
1.@m
jGOOm
',-"-031m
*,!-m*'1-m
1.@m
jHJGm
',-"-031m
jHNGGjGjm
1.@m
jKKFm
!,2*3.]]m
#*-,m83!0"-m
1.@m
jLHm
',-"-031m
"30m+0'**m
1.@m
jjGOm
',-"-031m
-**#031m
1.@m
jjHLm
!,2*3.#,1'1m
#*Amm
1.@m
jIIm
',-"-031m
#,"0*m
1.@m
jjIOm
',-"-031m
30#,2#,-m
1.@m
#1!0'230"-m
#,"0*m,#%0-m"#m
',4'#0,-m
1.@m
1.@m
(2)O
m
&mGMm
',-"-031m
*,!-m
&GIm
',-"-031m
&Km
',-"-031m
jjGm
!,2*3.#,1'1m
jGMm
',-"-031m
#*-,m.'@-,!'**-m
1.@m
jHKm
',-"-031m
+0'**-m
1.@m
jjHIm
',-"-031m
*,!-m
1.@m
jjKFm
',-"-031m
-!&3#*-m
1.@m
jjKIm
',-"-031m
mjjHOm
',-"-031m
#*-,m+0'**m
1.@m
jjGm
',-"-031m
*+-,2#m
1.@m
jjIm
',-"-031m
'*-m00#2#m
1.@m
jIOm
',-"-031m
#0"#m.',2-m
1.@m
#*-,m,#%0-m"#m
',4'#0,-m
'.m"#m-0-m
#0"#(-j!1#0-m
07"-m
0%-m"#m
'**!-,#(-1m
+0'**-m
1.@m
1.@m
1.@m
1.@m
1.@m
1.@m
jjJKm
',-"-031m
0'!-m
',-"-031m
0';Am+**-0/3',m
1.@m
0,!#1#2m
m
m
1.@m
YmNHFNm
',-"-031m
,4#0,'8-m
1.@m
YmNHGJm
',-"-031m
-+B,m
1.@m
YmNHGKm
',-"-031m
,4#0,m
1.@m
YmNHGMm
',-"-031m
-*1m
1.@m
YmNHHGm
',-"-031m
-!&3#*-m
1.@m
jjGMm
',-"-031m
+0'**-m-0-m
1.@m
3!jHGm
',-"-031m
0-!&#2m
1.@m
jjIKm
',-"-031m
+0'**-m!,0'-m
1.@m
jjILm
',-"-031m
#,"0*m
1.@m
jjJJm
',-"-031m
#,"0*m20"'-m
1.@m
mjjGILm
',-"-031m
#*A,m+0'**-m
1.@m
jjGIGm
$*#63-131m
*$'!-1m!-+ 0-m
1.@m
jjOm
+#*-m
#*-,m2-)1&m
"1&')'129,m
JGLm
22')m
m
3,'!'m
mGLMFKMm
$*#63-131m
!3)m
30/3>m
mGLOIFKm
',-"-031m
)'0)%!m
30/3>m
+,!&"-m
HKKm
O
m
T
HKLm
(2)m
mGLOIHOm
',-"-031m
5',2#0m27.#m
30/3>m
mGLOIIGm
+#*-Zm',-"-031m
*m2'+ 1m
30/3>m
mGLOILNm
+#*-mZm',-"-031m
11+ #7m
30/3>m
mKFLJKOm
!,2*3.#,1'1m
m
!0,'m
!3+JLNm
+#*-m
2328m3.0#+#m
m
!3+JMJm
+#*-Zm!2*3.#,1'1m
!3+KFFm
!,2*3.#,1'1m
-*"#,m-,#7m
m
,1*IFFIHm
!,2*3.#,1'1m
2-.+0)m
m
1'!jKm
!&,"*!)m
)-2!&m
8 #)'129,m
1'!jLm
+#0'm
)'8'*j303)m
8 #)'129,m
7HNKm
+#*-m
m
3%-1*4'm
mKFKLFHm
%0#12'1m
m
+ 'm
mJNHJHOm
%0#12'1m
m
'+ 3#m
-*"#,m
&+.*',m
m
(2)O
m
T
TT
T
M m-1'!'A,m"#m*1m$+'*'1m+32,2#1m#,m*1mHKm.*!1m/3#m!-,12'237#,m*m.- *!'A,m"#mm
"#m //'#-O '&)O #* -0"m #,m #12#m 20 (-Ym 1m 1'%*1m '"#,2'$'!,m #*m *3%0m "#m !0#!'+'#,2-m "#m *m
$+'*'mHm^[Zm[m#+'**1m'2AmY_Ymg+'*'1m!-,m.-!-mm-m.09!2'!+#,2#m,3*-Ym
m
Gm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
Gm
GFm
HGm
IIm
JHm
KGm
LFm
MIm
NIm
OGm
GFJm
GGMm
m
Hm
GGm
HIm
IJm
JJm
KIm
LJm
MJm
NJm
OHm
GFNm
GGNm
m
Im
GHm
HLm
IKm
JKm
KJm
LKm
MLm
NKm
OJm
GFOm
GGOm
m
Jm
GJm
HMm
ILm
JLm
KKm
LLm
MMm
NLm
OLm
GGGm
GHFm
m
Km
GKm
HOm
IMm
JMm
KLm
LNm
MOm
NMm
OMm
GGIm
GHGm
m
Lm
GLm
IFm
INm
JNm
KMm
MFm
NFm
NNm
OOm
GGJm
GHHm
m
Mm
GMm
IGm
IOm
JOm
KNm
MGm
NGm
NOm
GFFm
GGKm
GHIm
m
Nm
GNm
IHm
JGm
KFm
KOm
MHm
NHm
OFm
GFHm
GGLm
GHKm
Hm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GHLm
GILm
GJOm
GNGm
HFMm
HIGm
HJGm
HKFm
HKOm
HMHm
HOFm
IFJm
m
GHMm
GINm
GKFm
GNIm
HGFm
HIHm
HJHm
HKHm
HLFm
HNHm
HOGm
IFMm
m
GHNm
GIOm
GKMm
GNLm
HHFm
HIJm
HJIm
HKIm
HLGm
HNIm
HOIm
IFNm
m
GIFm
GJHm
GKNm
GOFm
HHGm
HIKm
HJJm
HKJm
HLIm
HNJm
HOLm
IGFm
m
GIGm
GJJm
GKOm
GONm
HHHm
HIMm
HJKm
HKKm
HLJm
HNKm
HONm
IGHm
IGIm
m
GIHm
GJLm
GLIm
GOOm
HHIm
HINm
HJLm
HKLm
HLKm
HNMm
HOOm
m
GIJm
GJMm
GLKm
HFGm
HHJm
HIOm
HJMm
HKMm
HLLm
HNNm
IFFm
IGKm
m
GIKm
GJNm
GLNm
HFJm
HHMm
HJFm
HJNm
HKNm
HLMm
HNOm
IFGm
IGMm
Im
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
IGOm
IIHm
IJJm
ILJm
INJm
IOMm
JGGm
JHJm
JIMm
JJMm
JLFm
JMNm
JMOm
m
IHFm
IIIm
IKFm
ILLm
INKm
IONm
JGJm
JHKm
JINm
JJOm
JLHm
m
IHGm
IIJm
IKIm
IMHm
INNm
IOOm
JGKm
JHMm
JIOm
JKFm
JLIm
JNGm
m
IHKm
IIKm
IKJm
IMIm
IOFm
JFIm
JGMm
JHOm
JJFm
JKGm
JLLm
JNHm
m
IHLm
IILm
IKKm
IMMm
IOGm
JFLm
JGNm
JIFm
JJGm
JKIm
JLMm
JNIm
m
IHMm
IINm
IKLm
IMNm
IOHm
JFMm
JGOm
JIIm
JJHm
JKJm
JMIm
JNKm
m
IIFm
IJGm
IKOm
IMOm
IOIm
JFNm
JHFm
JIKm
JJIm
JKNm
JMJm
JNLm
m
IIGm
IJIm
ILFm
INFm
IOKm
JGFm
JHIm
JILm
JJJm
JKOm
JMMm
JNMm
m
HKMm
O
(2)m
Jm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
JNOm
GIm
HGm
HOm
IMm
JMm
KLm
LKm
MJm
NIm
OIm
GFHm
m
Gm
GJm
HHm
IFm
IOm
JNm
KNm
LLm
MKm
NJm
OJm
GFIm
m
Km
GKm
HIm
IGm
JFm
KFm
KOm
LNm
MLm
NKm
OLm
GFJm
m
Lm
GLm
HJm
IHm
JGm
KGm
LFm
LOm
MMm
NLm
OMm
GFKm
m
Nm
GMm
HKm
IIm
JHm
KHm
LGm
MFm
MNm
NMm
ONm
GFLm
m
Om
GNm
HLm
IJm
JIm
KIm
LHm
MGm
MOm
NNm
OOm
GFMm
m
GGm
GOm
HMm
IKm
JJm
KJm
LIm
MHm
NFm
NOm
GFFm
GFNm
m
GHm
HFm
HNm
ILm
JKm
KKm
LJm
MIm
NGm
OFm
GFGm
GFOm
Km
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GGFm
GHFm
GHOm
GIOm
GJOm
GLFm
GLOm
GNFm
GNNm
GOLm
HFKm
HGKm
m
GGGm
GHGm
GIGm
GJFm
GKFm
GLGm
GMFm
GNGm
GNOm
GOMm
HFLm
HGLm
m
GGHm
GHHm
GIIm
GJGm
GKGm
GLHm
GMGm
GNHm
GOFm
GONm
HFMm
HGMm
m
GGJm
GHIm
GIJm
GJHm
GKIm
GLIm
GMIm
GNIm
GOGm
GOOm
HFNm
HGNm
m
GGLm
GHJm
GIKm
GJIm
GKJm
GLJm
GMKm
GNJm
GOHm
HFFm
HGFm
HGOm
m
GGMm
GHKm
GILm
GJJm
GKMm
GLKm
GMLm
GNKm
GOIm
HFGm
HGGm
HHFm
m
GGNm
GHLm
GIMm
GJKm
GKNm
GLMm
GMNm
GNLm
GOJm
HFIm
HGIm
HHGm
m
GGOm
GHMm
GINm
GJNm
GKOm
GLNm
GMOm
GNMm
GOKm
HFJm
HGJm
HHHm
Lm
m
Gm
Hm
m
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
HHIm
HIHm
HJHm
HKGm
HKOm
HLNm
HNFm
HNNm
HOLm
IFMm
IGMm
IHNm
m
HHKm
HIKm
HJIm
HKHm
HLFm
HMGm
HNGm
HNOm
HOMm
IFOm
IGNm
IHOm
m
HHLm
HILm
HJJm
HKIm
HLGm
HMHm
HNHm
HOFm
HONm
IGFm
IGOm
IIFm
m
HHMm
HIMm
HJKm
HKJm
HLHm
HMIm
HNIm
HOGm
IFFm
IGGm
IHFm
IIGm
m
HHNm
HINm
HJMm
HKKm
HLIm
HMJm
HNJm
HOHm
IFHm
IGHm
IHGm
IIHm
m
HHOm
HIOm
HJNm
HKLm
HLKm
HMKm
HNKm
HOIm
IFIm
IGIm
IHHm
IIIm
m
HIFm
HJFm
HJOm
HKMm
HLLm
HMNm
HNLm
HOJm
IFKm
IGKm
IHJm
IIJm
m
HIGm
HJGm
HKFm
HKNm
HLMm
HMOm
HNMm
HOKm
IFLm
IGLm
IHLm
IIKm
Mm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
IILm
IJKm
IKJm
ILHm
IMFm
INGm
IOFm
IOOm
JFNm
JGNm
JHOm
JIMm
m
IIMm
IJLm
IKKm
ILIm
IMIm
INHm
IOGm
JFFm
JFOm
JGOm
JIFm
JINm
m
IINm
IJMm
IKLm
ILJm
IMJm
INIm
IOHm
JFHm
JGFm
JHGm
JIGm
JIOm
m
IIOm
IJNm
IKMm
ILKm
IMKm
INJm
IOJm
JFIm
JGGm
JHHm
JIHm
JJFm
m
IJFm
IJOm
IKNm
ILLm
IMLm
INKm
IOKm
JFJm
JGJm
JHIm
JIIm
JJGm
m
IJGm
IKFm
IKOm
ILMm
IMMm
INLm
IOLm
JFKm
JGKm
JHJm
JIJm
JJHm
m
IJIm
IKHm
ILFm
ILNm
IMNm
INMm
IOMm
JFLm
JGLm
JHKm
JIKm
JJIm
m
IJJm
IKIm
ILGm
ILOm
INFm
INOm
IONm
JFMm
JGMm
JHMm
JILm
JJJm
m
HKNm
(2)O
m
m
Nm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
JJKm
JKJm
JLKm
JMLm
JNKm
JOLm
KFLm
KGMm
KHLm
KIJm
KJHm
KKKm
m
JJLm
JKLm
JLNm
JMMm
JNLm
JOMm
KFMm
KGNm
KHMm
KIKm
KJIm
KKLm
m
JJMm
JKNm
JLOm
JMNm
JNNm
JONm
KFNm
KGOm
KHNm
KILm
KJJm
KKNm
m
JJNm
JKOm
JMFm
JMOm
JNOm
JOOm
KGFm
KHFm
KHOm
KIMm
KJKm
KKOm
m
JJOm
JLFm
JMHm
JNGm
JOFm
KFFm
KGHm
KHGm
KIFm
KINm
KJMm
KLFm
m
JKFm
JLGm
JMIm
JNHm
JOGm
KFGm
KGIm
KHHm
KIGm
KIOm
KKFm
KLIm
m
JKGm
JLHm
JMJm
JNIm
JOJm
KFHm
KGJm
KHIm
KIHm
KJFm
KKGm
KLJm
m
JKIm
JLIm
JMKm
JNJm
JOKm
KFJm
KGLm
KHJm
KIIm
KJGm
KKIm
KLKm
Om
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
KLLm
KMKm
KNJm
KOJm
LFHm
LGFm
LHFm
LIFm
LIOm
LJMm
LKLm
LLJm
m
KLMm
KMLm
KNKm
KOKm
LFIm
LGGm
LHIm
LIGm
LJFm
LJNm
LKMm
LLKm
m
KLNm
KMNm
KNLm
KOLm
LFJm
LGHm
LHJm
LIIm
LJGm
LJOm
LKNm
LLLm
m
KLOm
KMOm
KNMm
KOMm
LFKm
LGIm
LHKm
LIJm
LJHm
LKFm
LKOm
LLMm
m
KMFm
KNFm
KNNm
KONm
LFLm
LGJm
LHLm
LIKm
LJIm
LKGm
LLFm
LLNm
m
KMHm
KNGm
KOFm
KOOm
LFMm
LGLm
LHMm
LILm
LJJm
LKHm
LLGm
LLOm
m
KMIm
KNHm
KOGm
LFFm
LFNm
LGMm
LHNm
LIMm
LJKm
LKIm
LLHm
LMHm
m
KMJm
KNIm
KOIm
LFGm
LFOm
LGNm
LHOm
LINm
LJLm
LKJm
LLIm
LMJm
m
m
m
m
m
m
m
m
m
m
m
m
m
m
GFm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
LMKm
LNIm
LOGm
MFIm
MGHm
MHHm
MIHm
MJIm
Im
GGm
GOm
HOm
m
LMLm
LNJm
LOHm
MFJm
MGJm
MHIm
MIJm
MJJm
Jm
GHm
HFm
IFm
m
LMMm
LNKm
LOIm
MFKm
MGKm
MHJm
MIKm
MJKm
Km
GIm
HGm
IGm
m
LMNm
LNLm
LOKm
MFLm
MGLm
MHMm
MILm
MJLm
Lm
GJm
HHm
IHm
m
LMOm
LNMm
LOMm
MFMm
MGNm
MHNm
MIMm
MJNm
Mm
GKm
HJm
IIm
m
LNFm
LNNm
LOOm
MFNm
MGOm
MHOm
MINm
MJOm
Nm
GLm
HKm
IJm
m
LNGm
LNOm
MFFm
MGFm
MHFm
MIFm
MJGm
Gm
Om
GMm
HLm
IKm
m
LNHm
LOFm
MFHm
MGGm
MHGm
MIGm
MJHm
Hm
GFm
GNm
HNm
ILm
GGm
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
m
IMm
JKm
KJm
LIm
MHm
MMMm
MNKm
MOIm
NFIm
NGGm
NHGm
NIKm
m
INm
JLm
KKm
LJm
MKm
MMNm
MNLm
MOJm
NFJm
NGHm
NHHm
NILm
m
IOm
JMm
KLm
LLm
MLm
MMOm
MNMm
MOKm
NFKm
NGIm
NHKm
NIMm
m
JFm
JNm
KMm
LMm
MMm
MNFm
MNNm
MOLm
NFLm
NGJm
NHLm
NINm
m
JGm
KFm
KNm
LNm
MNm
MNGm
MNOm
MOMm
NFMm
NGMm
NHMm
NIOm
m
JHm
KGm
KOm
LOm
IHJm
MNHm
MOFm
MONm
NFNm
NGNm
NHOm
NJFm
m
JIm
KHm
LFm
MFm
MNIm
MOGm
NFGm
NFOm
NGOm
NIHm
NJGm
m
JJm
KIm
LHm
MGm
MNJm
MOHm
NFHm
NGFm
NHFm
NIJm
NJIm
HKOm
O
(2)m
GHm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
NJJm
NKIm
NLLm
NNIm
OFGm
OGHm
OHKm
OIKm
OJKm
OKOm
OMJm
ONMm
m
NJKm
NKKm
NLOm
NNKm
OFIm
OGIm
OHMm
OILm
OJMm
OLGgm
OMKgm
ONNm
m
NJLm
NLFm
NMGm
NNLm
OFJm
OGKm
OHOm
OIMm
OJOm
OLIm
OMLgm
OOFm
m
NJNm
NLGm
NMJm
NNMm
OFKm
OGMm
OIFm
OINm
OKFm
OLKgm
OMMm
OOHm
m
NJOm
NLHm
NMMm
NOGm
OFLm
OGNm
OIGm
OIOm
OKHm
OLLm
OMOm
OOIm
m
NKFm
NLIm
NMOm
NOHm
OFMm
OHFm
OIHm
OJGm
OKIm
OLNm
ONGm
OOJm
m
NKGm
NLJm
NNFm
NOIm
OFNm
OHGm
OIIm
OJIm
OKMm
OMGm
ONKm
OOKm
m
NKHm
NLKm
NNGm
NONm
OGFm
OHIm
OIJm
OJJm
OKNm
OMHm
ONLm
OOLm
GIm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GFFFm
GFFOm
GFHHm
GFIIm
GFJGm
GFKGm
GFLFm
GFMFm
GFNIm
GFOKm
GGFKm
GGHIm
m
GFFGm
GFGFm
GFHIm
GFIJm
GFJHm
GFKHm
GFLGm
GFMGm
GFNJm
GFOLm
GGGGm
GGHKm
m
GFFHgm
GFGGm
GFHJm
GFIKm
GFJJm
GFKJm
GFLHm
GFMHm
GFNKm
GFOMm
GGGHm
GGHLm
m
GFFIm
GFGHm
GFHLm
GFILm
GFJKm
GFKKm
GFLIm
GFMIm
GFNLm
GFONm
GGGIm
GGHMm
m
GFFJm
GFGJm
GFHMm
GFIMm
GFJLm
GFKLm
GFLKm
GFMJm
GFNMm
GFOOm
GGGJm
GGHNm
m
GFFKm
GFGMm
GFHNm
GFINm
GFJMm
GFKMm
GFLMm
GFMKm
GFNNm
GGFFm
GGGKm
GGIFm
m
GFFLm
GFGNm
GFHOm
GFIOm
GFJOm
GFKNm
GFLNm
GFMMm
GFNOm
GGFGm
GGGLm
GGIGm
m
GFFMm
GFHGm
GFIFm
GFJFm
GFKFm
GFKOm
GFLOm
GFMOm
GFOIm
GGFIm
GGHGm
GGIHm
GJm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GGIIm
GGJLm
GGKNm
GGMHm
GGNJm
GGOMm
GHFNm
GHHGm
GHIHm
GHJOm
GHLOm
GHNNm
m
GGIJm
GGJNm
GGKOm
GGMIm
GGNKm
GHFGm
GHFOm
GHHJm
GHIJm
GHKFm
GHMFm
GHNOm
m
GGIKm
GGJOm
GGLFm
GGMJm
GGNMm
GHFHm
GHGHm
GHHLm
GHILm
GHKGm
GHMGm
GHOIm
m
GGIMm
GGKFm
GGLGm
GGMKm
GGOFm
GHFIm
GHGIm
GHHMm
GHIOm
GHKJm
GHMHm
GHOLm
m
GGINm
GGKGm
GGLIm
GGMMm
GGOGm
GHFJm
GHGJm
GHHNm
GHJHm
GHKLm
GHMKm
GIFFm
m
GGJGm
GGKHm
GGLMm
GGMOm
GGOIm
GHFKm
GHGKm
GHHOm
GHJJm
GHKOm
GHNGm
GIFGm
m
GGJIm
GGKJm
GGLNm
GGNGm
GGOJm
GHFLm
GHGLm
GHIFm
GHJMm
GHLGm
GHNHm
GIFKm
m
GGJKm
GGKKm
GGMFm
GGNIm
GGOKm
GHFMm
GHHFm
GHIGm
GHJNm
GHLIm
GHNMm
GIFOm
GKm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GIGHm
GIHMm
GIJJm
GILIm
GIMOm
GIOGm
GJFMm
GJHFm
GJIFm
GJJMm
GJKLm
GJNFm
GJNIm
m
GIGIm
GIIGm
GIJLm
GILKm
GINHm
GIOHm
GJFOm
GJHHm
GJIGm
GJJNm
GJLFm
m
GIGJm
GIIHm
GIJMm
GILNm
GINIm
GIOLm
GJGFm
GJHIm
GJINm
GJJOm
GJMHm
GJNKm
m
GIGLm
GIIJm
GIKHm
GILOm
GINJm
GJFHm
GJGGm
GJHJm
GJIOm
GJKFm
GJMIm
GJNLm
m
GIGOm
GIIKm
GIKJm
GIMKm
GINLm
GJFIm
GJGHm
GJHKm
GJJFm
GJKGm
GJMJm
GJNMm
m
GIHIm
GIINm
GIKKm
GIMLm
GINMm
GJFJm
GJGJm
GJHLm
GJJGm
GJKHm
GJMMm
GJNNm
m
GIHJm
GIJGm
GIKMm
GIMMm
GINNm
GJFKm
GJGLm
GJHNm
GJJHm
GJKIm
GJMNm
GJOGm
m
GIHKm
GIJHm
GIKNm
GIMNm
GIOFm
GJFLm
GJGOm
GJHOm
GJJLm
GJKKm
GJMOm
GJOHm
HLFm
(2)O
m
GLm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GJOIm
GKFKm
GKGKm
GKHMm
GKJFm
GKKHm
GKLHm
GKNGm
GKOIm
GLFLm
GLHGm
GLIIm
GLIJm
m
GJOKm
GKFLm
GKGNm
GKHNm
GKJGm
GKKIm
GKLIm
GKNHm
GKOJm
GLFOm
GLHJm
m
GJOLm
GKFNm
GKHGm
GKIFm
GKJKm
GKKKm
GKLJm
GKNIm
GKOKm
GLGFm
GLHKm
GLIKm
m
GJOMm
GKFOm
GKHHm
GKIHm
GKJLm
GKKLm
GKLKm
GKNLm
GKOOm
GLGGm
GLHMm
GLILm
m
GJONm
GKGFm
GKHIm
GKIIm
GKJMm
GKKNm
GKLLm
GKNNm
GLFFm
GLGHm
GLHOm
GLIMm
m
GKFFm
GKGGm
GKHJm
GKIJm
GKJNm
GKKOm
GKLMm
GKOFm
GLFIm
GLGIm
GLIFm
GLIOm
m
GKFGm
GKGHm
GKHKm
GKIKm
GKKFm
GKLFm
GKLOm
GKOGm
GLFJm
GLGKm
GLIGm
GLJFm
m
GKFIm
GKGJm
GKHLm
GKINm
GKKGm
GKLGm
GKMJm
GKOHm
GLFKm
GLGNm
GLIHm
GLJGm
GMm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GLJHm
GLKHm
GLLLm
GLNFm
GLNOm
GMFGm
GMGHm
GMHMm
GMIMm
GMJOm
GMKNm
GMMFm
GMMGm
m
GLJJm
GLKIm
GLLOm
GLNGm
GLOFm
GMFHm
GMGIm
GMHNm
GMINm
GMKFm
GMLFm
m
GLJKm
GLKJm
GLMGm
GLNIm
GLOGm
GMFIm
GMGKm
GMHOm
GMIOm
GMKHm
GMLIm
GMMHm
m
GLJLm
GLKKm
GLMHm
GLNJm
GLOKm
GMFJm
GMGNm
GMIGm
GMJGm
GMKIm
GMLJm
GMMIm
m
GLJMm
GLLFm
GLMIm
GLNKm
GLOLm
GMFMm
GMGOm
GMIHm
GMJIm
GMKJm
GMLKm
GMMKm
m
GLJNm
GLLGm
GLMJm
GLNLm
GLOMm
GMFNm
GMHIm
GMIIm
GMJKm
GMKKm
GMLMm
GMMLm
m
GLKFm
GLLHm
GLMLm
GLNMm
GLONm
GMFOm
GMHKm
GMIJm
GMJLm
GMKLm
GMLNm
GMMOm
m
GLKGm
GLLJm
GLMMm
GLNNm
GLOOm
GMGGm
GMHLm
GMIKm
GMJNm
GMKMm
GMLOm
GMNFm
GNm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GMNGm
GMOHm
GNFJm
GNGMm
GNIJm
GNJOm
GNMFm
GNNFm
GNOHm
GOFGm
GOGHm
GOHJm
m
GMNIm
GMOIm
GNFLm
GNGOm
GNIMm
GNKJm
GNMGm
GNNGm
GNOIm
GOFHm
GOGIm
GOHKm
m
GMNJm
GMOLm
GNFNm
GNHFm
GNIOm
GNKMm
GNMHm
GNNHm
GNOJm
GOFIm
GOGJm
GOHLm
m
GMNMm
GMOMm
GNGFm
GNHHm
GNJFm
GNLGm
GNMIm
GNNIm
GNOLm
GOFKm
GOGLm
GOHMm
m
GMNNm
GMONm
GNGHm
GNHIm
GNJIm
GNLJm
GNMJm
GNNJm
GNOMm
GOFLm
GOGOm
GOHNm
m
GMNOm
GNFFm
GNGIm
GNHKm
GNJKm
GNLLm
GNMKm
GNNLm
GNONm
GOFNm
GOHFm
GOIFm
m
GMOFm
GNFGm
GNGJm
GNHMm
GNJLm
GNLMm
GNMLm
GNNMm
GNOOm
GOFOm
GOHGm
GOIGm
m
GMOGm
GNFHm
GNGLm
GNHOm
GNJNm
GNLOm
GNMNm
GNOFm
GOFFm
GOGGm
GOHIm
GOIHm
GOm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
GOIJm
GOJJm
GOKKm
GOMFm
GONHm
GOOKm
HFFOm
HFGNm
HFIHm
HFJGm
HFKHm
HFMGm
HFMHm
m
GOILm
GOJLm
GOKLm
GOMIm
GONIm
GOOLm
HFGFm
HFHHm
HFIIm
HFJHm
HFKLm
m
GOIMm
GOJMm
GOKMm
GOMKm
GONJm
GOOMm
HFGGm
HFHJm
HFIJm
HFJIm
HFKMm
HFMIm
m
GOINm
GOJOm
GOKOm
GOMLm
GONKm
HFFHm
HFGIm
HFHLm
HFILm
HFJJm
HFKNm
HFMKm
m
GOIOm
GOKFm
GOLIm
GOMMm
GONMm
HFFJm
HFGJm
HFHMm
HFIMm
HFJKm
HFLGm
HFMNm
m
GOJFm
GOKGm
GOLJm
GOMOm
GONNm
HFFKm
HFGKm
HFHNm
HFINm
HFJNm
HFLIm
HFMOm
m
GOJGm
GOKHm
GOLMm
GONFm
GOOGm
HFFLm
HFGLm
HFHOm
HFIOm
HFJOm
HFLLm
HFNFm
m
GOJIm
GOKJm
GOLOm
GONGm
GOOJm
HFFNm
HFGMm
HFIGm
HFJFm
HFKGm
HFLMm
HFNHm
m
HLGm
O
(2)m
HFm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
HFNIm
HGFFm
HGHHm
HGIJm
HGJOm
HGKOm
HGMMm
HGNOm
HHFHm
HHGIm
HHHJm
HHIMm
HHINm
m
HFNJm
HGFIm
HGHIm
HGILm
HGKFm
HGLFm
HGMNm
HGOGm
HHFIm
HHGJm
HHHKm
m
HFNMm
HGFOm
HGHJm
HGIMm
HGKGm
HGLGm
HGMOm
HGOIm
HHFJm
HHGKm
HHHMm
HHJFm
m
HFOFm
HGGFm
HGHKm
HGIOm
HGKHm
HGLIm
HGNFm
HGOKm
HHFKm
HHGLm
HHHNm
HHJJm
m
HFOIm
HGGHm
HGHLm
HGJIm
HGKJm
HGLJm
HGNGm
HGOLm
HHFMm
HHGOm
HHHOm
HHJKm
m
HFOKm
HGGJm
HGIFm
HGJJm
HGKLm
HGLKm
HGNJm
HGONm
HHFOm
HHHFm
HHIGm
HHJMm
m
HFOLm
HGGLm
HGIGm
HGJLm
HGKMm
HGLLm
HGNLm
HGOOm
HHGFm
HHHGm
HHIIm
HHJNm
m
HFOOm
HGGNm
HGIIm
HGJNm
HGKNm
HGLMm
HGNMm
HHFFm
HHGHm
HHHHm
HHIKm
HHJOm
HGm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
HHKFm
HHLJm
HHMMm
HHNNm
HIFHm
HIGKm
HIHLm
KHm
GFLm
GOIm
HJLm
IFIm
m
HHKIm
HHLKm
HHMOm
HHNOm
HIFIm
HIGMm
HIHNm
LIm
GGHm
HFGm
HJIm
IFKm
m
HHKJm
HHLNm
HHNFm
HHOGm
HIFKm
HIGOm
HIIHm
LMm
GKKm
HFIm
HJNm
IFLm
m
HHKMm
HHMGm
HHNGm
HHOIm
HIFLm
HIHGm
HIIIm
MKm
GLFm
HFLm
HJOgm
IFOm
m
HHKNm
HHMIm
HHNHm
HHOJm
HIFNm
HIHHm
HIIKm
OIm
GLMm
HGJm
HKGm
IGGm
m
HHLFm
HHMJm
HHNJm
HHOLm
HIGFm
HIHIm
Om
OKm
GMKm
HGKm
HLOm
IGJm
m
HHLGm
HHMKm
HHNLm
HHONm
HIGHm
HIHJm
HFm
GFIm
GMMm
HHLm
HNGm
IHFm
m
HHLIm
HHMLm
HHNMm
HIFGm
HIGIm
HIHKm
HNm
GFKm
GNLm
HILm
HOKm
IHIm
HHm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
IIOm
INOm
KMm
GMJm
INFm
JJGm
JKOm
JOGm
KFGm
MFGm
NLOm
NOJm
m
IJHm
JHHm
LMm
IFJm
INHm
JKFm
JLFm
JOHm
KJLm
MFOm
NMFm
NOKm
m
IJMm
JHNm
NNm
ILNm
IOIm
JKGm
JLJm
JOKm
KMMm
MGHm
NMIm
OGGm
m
IJNm
JKHm
GGHm
ILOm
JGHm
JKHm
JLMm
JOLm
KOFm
MJMm
NMKm
OGJm
m
IKHm
JLOm
GHGm
IMFm
JGIm
JKIm
JMOm
JOMm
KOGm
MMKm
NMLm
OGLm
m
ILHm
JMGm
GHNgm
IMGm
JGOm
JKJm
JNHm
JONm
LGOm
NKJm
NMNm
OHJm
m
IMFm
GFm
GJLm
IMMm
JINm
JKKm
JNIm
JOOgm
LMFm
NLMm
NNHm
OHNm
m
INNm
JOgm
GKLm
IMNm
JJFm
JKLm
JNOm
KFFm
LMMm
NLNm
NOFm
OJFm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
OJHm
ONIm
GGNOm
GHKKm
GHNKm
GIIMm
GIMFm
GIKMm
GKMFm
HGGKm
JMHm
KFMm
HIm
m
m
OJLm
GFGKm
GGOHm
GHKMm
GHNLm
GIJKm
GIMIm
GIKOm
GLKOm
HGJGm
JNJm
KFNm
m
OJNm
GFJNm
GGOOm
GHKNm
GHOJm
GIJNm
GIOOm
GJLKm
GLOHm
GIm
JOIm
KGMm
m
OKGm
GFLJm
GHGFm
GHLFm
GHONm
GIKGm
GJFGm
GJLLm
GMIKm
HKm
JOJm
KGOm
m
OLFm
GFOGm
GHIKm
GHLJm
GIFIm
GIKIm
GJHMm
GJLOm
GMKGm
LGm
JONm
KHFm
m
OLMm
GGFMm
GHIMm
GHLLm
GIGNm
GIKLm
GJIIm
GKFHm
GOKNm
LHm
JOOm
KHIm
m
OLOm
GGGMm
GHJGm
GHMGm
GIHHm
GILHm
GJIJm
GKHFm
HFJMm
ONm
KFIm
KHJm
m
ONHm
GGMGm
GHKHm
GHMHm
GIHLm
GILMm
GJIMm
GKLNm
HFLOm
JIOm
KFLm
KHKm
m
HLHm
(2)O
m
HJm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
GHm
KHLm
KINm
KKJm
KMIm
KNIm
KOLm
HIIMm
HIJKm
HIKJm
HILHm
HIMFm
HIMNm
m
KHNm
KIOm
KKNm
KMJm
KNJm
KONm
HIINm
HIJLm
HIKKm
HILIm
HIMGm
HIMOm
m
KHOm
KJKm
KKOm
KMKm
KNKm
KOOm
HIIOm
HIJMm
HIKLm
HILJm
HIMHm
HINFm
m
KIHm
KJMm
KLHm
KMLm
KNNm
LFGm
HIJFm
HIJNm
HIKMm
HILKm
HIMIm
HINGm
m
KIIm
KJOm
KLKm
KMNm
KNOm
LFIm
HIJGm
HIKFm
HIKNm
HILLm
HIMJm
HINHm
m
KIJm
KKGm
KLOm
KNFm
KOFm
LFJm
HIJHm
HIKGm
HIKOm
HILMm
HIMKm
HINIm
m
KIKm
KKHm
KMFm
KNGm
KOGm
MIm
HIJIm
HIKHm
HILFm
HILNm
HIMLm
HINJm
m
KILm
KKIm
KMHm
KNHm
KOKm
HIILm
HIJJm
HIKIm
HILGm
HILOm
HIMMm
HINKm
GHm
HKm
m
Gm
Hm
Im
Jm
Km
Lm
Mm
Nm
Om
GFm
GGm
HINLm
HIOLm
HJFJm
HJGHm
HJHGm
HJIGm
HJJIm
HJKLm
HJLMm
HJNIm
HJOHm
HJOIm
m
HINMm
HIOMm
HJFKm
HJGIm
HJHIm
HJIHm
HJJJm
HJKNm
HJLOm
HJNJm
m
HINNm
HIONm
HJFLm
HJGJm
HJHJm
HJIIm
HJJLm
HJLGm
HJMGm
HJNKm
m
HIOFm
HIOOm
HJFMm
HJGKm
HJHKm
HJIJm
HJJOm
HJLHm
HJMKm
HJNLm
m
HIOGm
HJFFm
HJFNm
HJGLm
HJHMm
HJIMm
HJKFm
HJLIm
HJMMm
HJNMm
m
HIOHm
HJFGm
HJFOm
HJGMm
HJHNm
HJIOm
HJKIm
HJLJm
HJMOm
HJNNm
m
HIOJm
HJFHm
HJGFm
HJGNm
HJHOm
HJJFm
HJKJm
HJLKm
HJNFm
HJOFm
HIOKm
HJFIm
HJGGm
HJGOm
HJIFm
HJJGm
HJKKm
HJLLm
HJNHm
HJOGm
m
m
m
m
m
m
HLIm
O
m
HLJm
(2)m