101 A dvances in Environmental Biology, 3(1): 101-106, 2009 ISSN 1995-0756

101
A dvances in Environmental Biology, 3(1): 101-106, 2009
ISSN 1995-0756
© 2009, A merican-Euras ian Network for Scientific Information
ORIGINAL ARTICLE
T his is a refereed journal and all articles are professionally screened and reviewed
Genetic Relatedness among Romanian Olive Trees and Varieties Using Amplified
Fragment Length Polymorphism (AFLP) Markers
1
Ibrahim Mohammad Rawashdeh, 1Na s a b Q. Rawashdeh,
Amri, Saleh Shdaifat and 4Abdel Rahman Al-Tawaha.
2
Abdel Nabi Fardous ³Ahmed
1
Medicinal plants and biodiversity program, National C enter for Agricultural Research and Technology
Transfer (NCARTT), P. O. Box 639. Baq’a, Jordan.
2
water management in dry lands and environmental biophysics, (NCARTT)
³Internati o n a l C enter for Agriculture Research in the Dry Areas (ICARDA), West Asia office, Amman.
4
Olive program, NCART, P. O. Box 639. Baq’a, Jordan.
4
Department of Biological Sciences, Al Hussein Bin Talal University, Maan, P.O. Box 20, Jordan.
Ibrahim M ohammad Rawas hdeh,; Genetic Related n e s s among Romanian Olive Trees and Varieties
Us ing A mplified Fragment Length Polymorphis m (A F LP) M arkers ,Am.-Eurasian J. Sustain. Agric.,
3(1): 101-106, 2009
ABS TRACT
A mplified fragment length polymorphis m (A FLP) a nalys is was us ed to s tudy the genetic variations among
and within different individuals o f Ro ma n ian olive trees from two regions Tafilah and A jlun along with s ix
varieties . Four primer combinations were us ed which pro d u ced about 538 amplified fragment. Dendrogram and
matrix of s imilarity were obtained by the Unweighted Pair-Group M ethod analys is (U P GM A ). A jlun Romanian
o liv e trees formed a s eparated group and mos t of Tafilah trees were formed with a s eparated grou p . H ig h
s imilarity (39%) among all tes ted individuals was obs erved between two s amples of A jlun. A mong varieties
Ras ee and Improved Nabali were s howed high s imilarit y (25%). A FLP confirmed that their is a genetic
variations within Romania n o liv e tres s which lead to enlarge the s tudying thos e trees morphologically and
biochemically to entering p ro mis e o n es within breeding programs in the future. Finally, A FLP has a powerful
tool for detecting ge n e t ic v a riability among cultivars and s pecies help in known the pedigree of relatives and
ances tors .
Key words: A FLP, Olive, Jordan, Romanian trees and Polymorphis m.
Introduction
O live (Olea europaea L.) is an evergreen t re e
grown at temperate regions with altitude ranged from
(400m to 1500m) above s ea level (a . s . l.). The
produ c tion is bas ed on the climatic conditions , tree
cu ltivation as well the phys iological s tate of trees ,
thes e lead to phenomenon known "alternate bearing".
In Jordan, olive culture is one o f the mos t important
agriculture activities , the olive plantations accounted
more than 10 million trees , where the Nabali Ba la d i
is the mos t wides pread and the dominant cultivar.
Oil production was exceeded the needs that allow
for government to export the s urplus into neig h b o r
countries and Europe markets . M oreover, Jordan
oliv e oil cons idered the bes t among other countries
bas ed on the p e rcentage of PH. However, mos t
framers s aid that the old trees (Roma n ia n ) are bes t
than t h e in t ro d u c e d v a rie t ie s t a kin g into
cons iderations the fruit quality and oil perc e n tage.
This s aying oriented the direction to s t u d y t he
genetic relatednes s among thos e and introduced
varieties us ing the molecular markers .
Corres ponding Author
Ibrahim M ohammad Rawashdeh, M edicinal plants and biodiversity program, National Center
for Agricultural Research and Technology Transfer (NCARTT), P. O. Box 639. Baq’a,
Jordan.
E-mail: [email protected].
Adv. Environ. Biol., 3(1): 101-106, 2009
Changes in lifes tyles and cooking habits towards
healthy eating, enviro n me ntal concerns , exotic
cuis ine, and convenience food have opened n e w
market opportunities for olive oil products [11]. Olive
oil is the principal s ource of fat in the M editerranean
die t , which has been as s ociated with a lower
incidence of coronary heart dis eas e and cert ain
cancers [15]. A dulteration is , the adding cheaper
materials from other oil products , the bigges t
problem faced the cons umers and the directions now
toward to have organic farming to ge t h igh quality
and guarantee production.
Traditionally, morphological and phys iological
traits were us ed to identify olive cultivars , more
recently, molecular markers have been fo u nd to be a
us eful tool for cultiv ar identification and determining
patterns of relatednes s amo ng cultivars . Recently,
DNA –bas ed techniqu e s have been developed to
detect genetic variations wit hin and among varieties ,
p o p u la t ions and s pecies . Ran d o m a mp lifie d
polymorphic DNA (RA PD) us ed for detecting genetic
variation in mutants of Nabali olive cultivar [13]. In
a d d it io n , RA P D t e c h n iq u e w a s u s e d t o
c h a ra cterization olive varie t ie s [16], g e n e t ic
relations hips [6,2], cultivars analys is [10,3], genetic
divers ity and A lbania n o live germplas m [4,5] and
identification cultivars [8].
A mplified Fragment Length Polymo rp h is m
(A FLP) markers have been developed by Vos et al.,
[14] and widely s pread due to its reliability and
robus tnes s . The us e of A FLP has s ome advantages in
terms of us e in the identification of d ia g nos tic or
s pecific markers , it has s everal advantages , it does n’t
require previous knowledge of the DNA s equence,
gen erate reproducible fingerprinting profiles , allows
the amplification of a h ig h number of DNA
fragments per reaction and enabling the detectio n of
s pecific amplified fragments [14,9].
A FLP markers w ere us ed to s tudy the relation
between olive cultiv a rs from W es tern M editerranean
[1] and dis criminated among olive varieties [5].
The purpos e of this s tudy was to detect the
genetic relatednes s among the olive Romanian trees
a n d olive cultivars were grown in Jordan by us in g
A FLP markers .
Materials and methods
-Plant material
Leaves s elected ra n d o mly from around the trees
o f e ighteen olive Romanian trees and s ix varietie s
were collected from private farms North (A jlu n - A lHas himiah) and s outh (Tafilah) regions of Jordan,
while leaf s amples of olive varieties were collected
from A l-Hus s ein nurs ery -M inis try of A griculture
during A ugus t 2006. Olive s amples from Tafilah
region were s tored at refrigerator until proces s ing for
DNA is olation, but samples of varieties and thos e
from North were us ed fres hly.
102
DNA isolation
DNA was obtained after ground the s tored leaves
in liquid n it rogen (-196ºC), the quality and quantity
of genomic DNA were detected on agaros e gel. Total
cellular DNA was extracte d us ing anew-modified
CTA B is olation protocol. A pproximately (20 to 30
mg) fres h and s tored leaves were ground and mixed
with 750 µl of fres hly and preheated of (2x CT A B
s olution with 0.8g PVPP) in 2ml tubes then pla c ed
a t 65º C for 30 min. the mixture was mixed with 750
µl of chlorop h o rm/ is oamyl alcohol (24:1) vortex a
fe w s econds , thos e centrifuged at 14000g for 20 min.
Upper ph a s e (s upernatant) was placed in 2ml tubes
with 600ml is opropanol, tubes were s haked until the
thread o f DNA appeared, then centrifuged for 20 min
at 14000g. The s olution poured and tubes left to dry,
the n 600 µl of cooled 70% ethanol added and placed
at refrigerator (–20ºC) overnight. Next day ethanol
w a s p o ured, tubes dried and 100µl of TE added an d
p la c ed at 65ºC for 30min. Four micolitter of RN A s e
(10mg/ml) were added per tube and left for 45min at
37ºC.
DNA quantitation was meas ured us ing machine
of S2100 UV/VIS DIODE-A rray-Spect rophotometer,
Vers ion 1.7.
AFLP procedure
Th e A FLP procedure was performed as
previous ly des cribed (Vos e e t al., 1995). Olive DNA
(500ng) was double diges ted with EcoRI and Tru91
(an is ochizomer o f MseI) and double s tranded
adaptors were ligated to the ends of DNA fragments ,
generat in g template DNA for s ubs equent PCR
a mp lification (p re -a mp lific a t io n fo llo w e d b y
s elective). Res t riction and ligation reactions were
carried ou t s imultaneous ly in a s ingle reaction tube
[14].
To c a rry out the reaction, an enzyme mas ter mix
for 16 reactions was prepared containing 2µl Ec o R I
buffer, 2µl Tru91 buffer, 0.25µl EcoRI enzyme
(12u/µl), 0.25µl Tru91 enzyme (12u/µl), 0.625µl
1mg/ml BSA , 9.875µl d d H 2O and 5µl genomic DNA
(500ng ), t he mix was centrifuged for a few s econds
then s tored at 37ºC for 3 hr. The res triction-ligation
reactions cons is t e d of 0.5 µl 10x T4 buffer, 0.2µl T4
DNA ligas e (3u/µl), 0.5µl Tru 9 1 a daptor forward,
0.5µl Tru91 adaptor backward, 0.5µl EcoRI adaptor
forward, 0.5µl EcoRI adaptor backward and 2.3µl
ddH2O. Thos e dis tributed 5µl for the res triction
tubes , then s tored at 37ºC for 3hr.T he res trictionligatio n reactions were diluted 1: 4, then us ed in preamplification s tep . The Tru91complementary primer
had a 3'-C and the EcoRI compleme n t a ry primer had
a 3'-A .
The pre-amplification (pres elective) mix was
prepared by adding 2µl of 5-fo ld diluted DNA from
the res triction-lig a t ion reaction, 2.5µl 10xbuffer
(PCR), 2.5µl dNTPs (5mM ), 1µl A FLP pres elective
Adv. Environ. Biol., 3(1): 101-106, 2009
primer (EcoRI+ A -3'), 1µl A FLP pres e lective primer
(Tru91+ C-3'), 0.2µl Taq polymeras e (5u/µl) and
15.8µl ddH2O. The pre-amplification (pres elective)
amplification was carried out in a th e rmal cycler
prog rammed at 72ºC for 2min followed by 20 cycles
of 94ºC for 30 s , 56ºC for 40s and 72ºC for 50s and
finally incubated at 4º C. The pre-amplification DNA
w a s diluted 5-fold with ddH2O and s elective
amplifica t io ns were carried out by us ing different
EcoRI and Tru91 primer combination (A lpha DNA ).
Primers s elected fo r the s election amplification were
from available A FLP s elective primers that purchas ed
before and s tore d at –20ºC at NCA RTT lab. The
EcoRI primers contained thre e s elective nucleotides
with s equence (Table 1) while the Tru91 primers had
the s elective nucleotides s tarting with G (Table 1).
For the s elective amplification, the reactions were
s et up as follows : 2µl of 5-fold diluted pre-s elective
amplification reaction product, 1.0µl Tru91 p rimers ,
1.0µl EcoRI primers , 2µl of 10 x buffer, 2µl dNTPs
(0.25mM ), 0.2µlT a q polymeras e (5u/µl), 0.25µl of
M gCl2 (15mM ) and 11.55ddH2O.
Se le c t ive amplification was carried out in a
thermal cycler programmed at 94ºC for 2min,
follo w ed by 13 cycles of 94ºC for 20s , 68ºC (0.7ºC
/cycle) fo r 30s and 72ºC for 1min, and 23 cycles of
94ºC for 30s , 59º C for 30s and 72ºC for 1min and
fin al incubation at 4ºC.The s amples were loaded on
6% polyacrylamide gel o n A pelex gel s equencing
apparatus (vertical). The s elective amplifica tion
reaction product (6µl) w a s mixed with 4ul of loading
buffer (98% deionized fo ramide, 10mM EDTA (pH:
8), 0.05% b romofenol, 0.05% xylene cyanol), from
which 6µl was finally loaded on the gel.
Four primer pair combinations (EcoRI- / Tru91-)
were us ed in analys is (Table 2), s ix olive varie ties
and eighteen s amples o f Romanian trees from each
A jlun and Tafilah regions were s ubjected to the
s e le c t iv e a mp lific a t io n w it h t h e s e p rime r
combinations , and treated under the s ame conditions
by the s ame res earc h e r. To determine the s ize of the
A FLP fragments , we us ed an A F LP DNA marker
(50bp s tep ladder) (Promega), ranging in length fro m
50 - 800bp.
Data analysis
A FLP polymorphic bands were s cored as pres ent
(1) o r abs ent (0) an es timates of s imilarity among all
varieties w e e calculated according to Nei and Li,
[12] definition o f s imilarity Sij= 2a/ 2a+b+c, where
S ij is the s imilarity between t w o v a rie t ie s
(in d ividuals ) i and j, a is the number of bands
pres ent in both individuals , b is the number of bands
pres ent in j and abs ent in i. The matrix of s imilarity
was analyzed by the Unweighted Pair-Group M ethod
(UPGM A ) and the d e n d rogram was obtained us ing
s ps s . Vers ion 10. Program.
103
Res ults and Dis cus s ion
Only one primer combinatio n s o ut of five, A GCCA T, w a s failed to produce at leas t one amplified
fragment. T h e A FLP fingerprinting of the s ix olive
v a rieties and eighteen s amples of Romanian trees
were collected from each A jlun and Tafia lh regions
(Table 1), tes ted us ing four random primer pair
combinations (Table 2 and 3), revealed a total
number of 1119 amplified D N A fragments , 538 out
of t h em turned to be polymorphic and were
dis tributed acros s the entire lanes . The av erage
percentage of polymorphis m ranged from 25% for
EA A C-M CCT (Tafialh Romania n t rees ) to 71% for
EA GC-M CA C for (Tafialh Romanian trees ) (Table 3)
for olive v a rieties high percentage of polymorphis m
s howed by EA CT-M CA A and t he lowes t percentage
of polymorphis m is 45% for EA A C-M CCT (Table
3). S o me bands wee s pecific to one or more of A jlun
and Tafialh als o the s ame to olive varieties .
V a r iations among olive varieties, Ajlun and Tafila h
Romanian olive trees
Dendrogram cons tructed by UPGM A derived
from s imilarity matrix des cribed above, s howed five
main dis tinct clus ters (Figure 1). The firs t clus ter was
formed twelve olive s amples of A jlun. The s e c ond
clus ter formed two individuals from A jlu n . The third
group formed four s a mples of A jlun, four s amples of
Tafilah and two s amples of Tafilah with one A jlun
s ample . The four clus ter formed two s amples of
Tafilah Romanian trees with one s ample of A jlun.
The fifth clus ter can be divided into tw o groups , the
firs t included two s ub–s ub clus ters ; one formed olive
variety K18 with one individual tree from Tafila, the
s econd formed two indiv id uals of Tafilah and Nabali
Baladi s howed s eparated grou p (Figure 1). The
s econd s ub-s ub clus ter is included two groups ; the
first has two Tafilah individuals w h ich are more
clos ely and one s ample has s eparated group.
The s ec ond group of the fifth clus ters were
formed three groups the firs t formed Nas ohee variety
an d o ne individual Tafilah s ample als o it was wholly
s eparated fro m this group (Figure 1). The s econd
group s howed that Ras ee and imp roved Nabali more
c los ely but s howed s haring with one A jlun s a mp le
and all of them collected in one gro u p . W ithin this
group one s ample of Tafilah individuals was s howed
s eparated from other. O n e s ample of Tafilah was
s howed a s eparated one among all s a mp les within
this group.
Jaccard´s genetic s imilarity (Table 4) e s timated
w ithin and among all tes ted individuals s howed the
highes t s imila rity values in Ras ee variety and A jlun
s ample (0.182) and the lowes t value (0.000) between
improved Nabali a nd A jlun. High s imilarity was
regis tered between K18 and onTafila h s ample (0.205)
and the lowes t value (0.017).
Adv. Environ. Biol., 3(1): 101-106, 2009
104
Table 1: Olive varieties were collected from Al-Hussein nursery / Ministry of Agriculture in Jordan.
Variety name
Source of collection
1-K18
AL-Hussein nursery
2-Nabali baladi
AL-Hussein nursery
3- Rasee
AL-Hussein nursery
4- Grosadee
AL-Hussein nursery
5- Nasohee
AL-Hussein nursery
6-Improved Nabali
AL-Hussein nursery
Table 2: Oligonucleotides adaptors and primer combinations used for AFLP analysis.
Name
EcoRI adaptor
Tru91 adaptor
Primers used in pre-amplification
EcoRI+1-A
Tru91+1-C
Primers used in selective amplification
EcoRI+3-AGC
EcoRI+3-ACT
EcoRI+3-AAC
MseI (an isochizomer of Tru91)
MseI+3-CAC
MseI+3-CAA
MseI+3-CCT
Primer pair combinations (EcoRI/ MseI).
1-AGC-CAC
2-ACT -CAC
3-AAC-CCT
4-ACT -CAA
/
/
/
/
/
/
Ministry
Ministry
Ministry
Ministry
Ministry
Ministry
of
of
of
of
of
of
Agriculture
Agriculture
Agriculture
Agriculture
Agriculture
Agriculture
Sequence
5'- CT CGT AGACT GCGT ACC-3'
3'- AAT T GGT ACGCAGT C-5'
5'-GACGAT AGT CCT GAG-3'
3'-T ACT CAGGACT CAT -5'
5'-GACTGCGTACCAATTCA-3'
5'-GAT GAGT CCT GAGT AAC-3'
5'-GACT GCGT ACCAAT T C+AGC-3'
5'-GACT GCGT ACCAAT T C+ACT -3'
5'-GACT GCGT ACCAAT T C+AAC-3'
5'-GAT GAGT CCT GAGT AAC+CAC-3'
5'-GAT GAGT CCT GAGT AAC+CAA-3'
5'-GAT GAGT CCT GAGT AAC+CCT -3'
Table 3: Polymorphism rates related t o t h e fo u r A F LP primer combinations among eighteen Romanian olive trees from each T afilah and
Ajlun governorate and six varieties.
Primer combination
Source of genome
T otal number of fragments
Polymorphic fragments
Polymorphism (%)
AGC-CAC
-T afilah-Romanian olive trees
70
50
71
-Ajlun-Romanian olive trees.
86
49
57
-Six olive varieties
67
37
55
ACT -CAA
-T afilah-Romanian olive trees
49
24
49
-Ajlun-Romanian olive trees.
129
44
34
-Six olive varieties
62
45
72
AAC-CCT
-T afilah-Romanian olive trees
154
38
25
-Ajlun-Romanian olive trees.
104
46
44
-Six olive varieties
60
27
45
ACT -CAA
-T afilah-Romanian olive trees
113
59
52
-Ajlun-Romanian olive trees.
172
85
49
-Six olive varieties
53
34
64
T otal: 1119
T otal: 538
Mean: 51
Variations among olive varieties
Dendrogram (Figure 1) was s h owed that the two
following varieties Ras ee and Improved Nabali w ere
more clos ely but formed with A jlun s ample. Nabali
baladi and K18 w e re s hared with Tafilah Romanian
oliv e trees . High s imilarity was s howed between
improved Nabali and Ras ee (0.254) and the lowe s t
was s howed between Nabali Ba ladi and improved
Nabali (0.074).
Variations within Ajlun Romanian olive trees
Out of 18 individuals of Romanian oliv e trees ,
twelve individuals were formed a s eparate gro up and
is olated from the whole dendrogram. W ithin this
group, the dend rogram can be divided and formed
two s ub-groups , the firs t includes s ix s amples , but
the s econd formed five s amples , further the firs t
group have two s ub groups , while the s econd formed
two s ub groups , als o four individual trees were
s hare d with four s amples from Tafilah. High
s imilarity (0.39) was s howed between individuals
n umbers (5 and 6) while the lowes t (0.000) w a s
recorded for individuals numbers (10 and 14).
Variations within Tafilah Romanian olive trees
Out of 18 individuals of Roma nian olive trees ,
s ix trees were s howed s eparated gro u p , while two
individuals formed alone, two individuals formed
alone and s h ared with K18. Four individuals formed
alone but s hared with s ame s amples of A jlun. Olive
variety "Nabali Bala di" was formed with the group
of Tafila h , in addition Nas hoe variety s hared with
one Tafilah s ample. Not only Tafilah but als o one
Adv. Environ. Biol., 3(1): 101-106, 2009
105
Fig. 1: Dendrogram generated among Romanian olive Trees and s ix varietie s b y us ing four primer pair
combinations bas ed on A FLP markers . A j.: A jlun s ouree for Romanian trees , T a .: T a fa ilah s ource for
Romanian tree. Impro. Nabal: improved nabali.
s ample fro m A jlun s hared with the s ame group of
Ras ee, Gros adee and improved Nabali. Tafilah. High
s imilarity was s howed between (0.20) betwe e n
number of individuals (22 and 25) but the lowes t
value is (0.000) between individual numbers (25 and
35).
Discussion
The minimum us ing of primer combinations and
s amples are better to have highly polymorphic
amplified fragments and eas ily to dis criminate among
t es ted genotypes . A mong tes ted primers , A CT-CA A
and A GC-CA A were very important primers th a t can
b e u s ed for either s creening wild, landraces
acces s ions and remaining olive varietie s were
unknown or untes ted.
A FLP analys is demons trated that Romanian olive
trees either fromTafilah or A jlu n had high genetic
varia b ility within and among tes ted individuals this
was s howed through dendrogram. M oreover, there is
s ome individuals from A jlun or Tafilah als o s howed
broad genetic variability when th e y are formed a
s eparated group. This variability is related to the
g enetic changes over all pas t years were expos ed t o
through s pontaneous mutations of naturally s election
or s ometimes human capacities .
Lo c a l farmers and s ome res earchers s aid that
their is a relatio n s h ip between Ras ee and improved
nabali, this s tudy was confirmed this s a y ing through
a dendrogram that s howed both were grouped
together. M ore over, Nas ohee v a rie t y which
c ommonly known its origin from Tafilah s o, in our
res ults this va rie t y was grouped with s ome
individuals o f this region. M oreover, in addition to
genetic make up there is als o geographical features ,
is olation an d c limatic conditions als o play a great
role for creating variability within o r a mong
individuals .
A FLP has a p o werful for detecting genetic
variability among cultivars and s pecies als o help in
known the pedigree relatives and ances tors .
References
1.
A ngiolillo, A ., M .O. M e n c u c cini and L. Baldoni,
1999. Olive genetic divers ity as s es s ed us ing
amplified frag me nt length polymorphis ms . Theor
A ppl Genet., 98: 411-421.
Adv. Environ. Biol., 3(1): 101-106, 2009
2.
3.
4.
5.
6.
7.
8.
A ngjelina, B., Šatoviæ Zlatko, Rallo Luis and
Trujillo Is abel, 2004. Optimal us e of RA PD
ma rke rs for identifying varieties in olive (Olea
europaea L.) germplas m collec tions . Journal of
the A merican Society for Horticultural Science,
129(2): 266-270.
A ngjelina, B., Šatoviæ Zlatko, Trujillo Is abel
and Rallo Luis , 2004. Genetic re lations hips of
Spanis h olive cultivars us ing RA PD ma rkers .
HortScience, 39(5): 948-951
A ngjelina, B., Šatoviæ Zlatko, Rallo Luis and
Trujillo
Is abel, 2002. Genetic divers ity and
relations hips in olive
(O lea europaea L.)
ge rmp la s m c o lle c t ions as determined by
ra n d o mly a mp lified p o ly mo rp h ic D N A .
Theoretical and A pplied Gene tics , 105(4): 638644.
A ngjelina, B., Šatoviæ Zlatko, Cipria n i Guido,
Baldon i
Luciana, Tes tolin, Raffaele, Rallo
Luis
and Trujillo Is a bel, 2003. Comparative
s tudy of the dis criminatin g capacity of RA PD,
A F LP a n d S SR markers a n d o f t h e ir
effectivenes s in es tablis hing genetic relations hips
in olive. Theoretical and A pplied Genetics , 107
(4): 736-744.
Bes nard, G., P. Bara dat and A . Berville, 2001.
Genetic relations hips in the olive (Olea europaea
L.) reflects mu ltilocal s election of cultivars .
Theor A ppl Genet., 102: 251-258.
A ngjelina, B., Šatoviæ Zlatko, Is maili Hajri,
Panajoti
Dhimitër, Rallo Luis and Trujillo
Is a b el. 2003. RA PD genetic dive rs it y o f
A lbanian olive germplas m and its relations hips
with other M editerranean co u ntries . Euphytica,
130(3): 387-395.
Bogani, P., D. Cavalier, R. Petruccelli, L.
Pols inelli and G. Ros elli, 1994. Identification of
o live tree cultivars by us ing random amplifie d
polymorphic DNA . A cta Horticulturae, 356: 98101.
106
9.
10.
11.
12.
13.
14.
15.
16.
Diaz, S., Carmen Pire, Juan Ferre r a n d M ari
Jos e Bonete, 2003. Identification of Phoenix
d a c tylifera L. varieties bas ed an amplified
fragment length polymorphis m (A FLP) makers .
Cellular & M olecular Biology Letters , 8(4): 891899.
Fabbri, A ., J.I. Hortmaza, and V.S. Polito, 1995.
Random amplified po ly morphic DNA analys is of
olive (O l e a europaea L.) cultivars . J. A mer. Soc.
Hort. Sci., 120(3): 538-542
M artínez, M .G., Zulema A rgonés and Nigel
Poole, 2002. A repos itioning s trate g y for olive
oil in the UK market. A gribus ines s , 18(2): 163180.
Nei, M . and W .H. Li, 1979. M athematical mo d el
for s tudying genetic variation in terns of
res triction endonucleas es . Proc. Natl. A cad. Soc.
USA ., 76: 5269-5273.
Rawas hds eh, I., 2003. Detecting genetic variation
in mutants of oliv e c ultivar Nabali us ing RA PDPCR technique in Jordan . Diras t, A gricultural
Sciences , 30(2): 133-136.
Vos , P., R. Hogers , M . Bleeker, M . Reijans . T.
Van de lee, M . Hornes , A . F ijt ers , J. Pot, J.
Pele man, M . Kuiper and M . Zabeau, 1995.
A FLP: Anew technique for DNA fingerprinting.
Nucleic acid Res ., 23: 4404- 4414.
Vis ioli, F., A ndrea Poli and Clandio Galli, 2002.
A ntioxidant and other biological a c t ivities of
phe n o ls from olives and olive oil. M edicinal
Res earch Reviews , 22(1): 65- 75.
W ies man, Z., 1998. M olecular characterization of
common olive varieties in Is rael and the W es t
bank us ing randomly amplified polymorphic
DNA (RA PD) ma rkers . J. A mer. Soc. Hort. Sci.,
123 (5): 837-841.