107 A dvances in Environmental Biology, 3(1): 107-112, 2009 ISSN 1995-0756

107
A dvances in Environmental Biology, 3(1): 107-112, 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 Variation among Sumac (Rhus Coriaria L.) Sample s Collected from Three
Locations in Jordan as Revealed by Aflp Markers
1
Ibrahim Mohammad Rawashde h , 2 Abudel Latif Ghzawi,
Khairallh, 4Abdel Rahman Al-Tawaha and 3Bannur Salama.
1
Nasab Q. Rawashdeh,
3
Kamal
1
Medicinal plants and biodiversity program, National Center for Agricultural Research and extention (NCARE),
P. O. Box 639. Baq’a, Jordan.
2
Biotechnologists, Hashimiah University.
3
Manager GEF/WB project conservation of medicinal and herbal plants of Jordan.
³Bannur Salama, Biotechnology research centers, Tripoli, Libya. Banor2004@ yahoo.com
4 Department of Biological Sciences, Al Hussein Bin Talal University Ma’an, P.O. Box 20, Jordan.,
Ibrahim M ohammad Rawas hdeh, A budel Latif Ghzawi, Na s a b Q . Rawas hdeh, Kamal Khairallh, A bdel
Rahman A l-Tawaha and Bannur Salama; Genetic Variation among S u ma c (Rhus Coriaria L.) Samples
Collected from Three Locations in Jordan as Revealed by A flp M arke rs : A m .-Eurasian J. Sustain.
Agric., 3(1): 107-112, 2009
ABS TRACT
Sumac (Rhus coriaria) is a medicinal plant grown in upper elevations and dis tributed between A jlun,
Jeras h and Irbid areas of Jordan. A mplified Fragment Length Polymorphis m (A FLP) analys is was us ed to s tudy
the genetic relations hip among thirty s amples of s umac (Rhus coriaria L.) trees collected from the Northern
regions in Jordan. Three o f s e v e n primer pair combinations , produced 42 polymorphic bands . The UPGM A
method was employed to cons truct a dendrogram bas ed on the Jaccard's s imilarity coefficient. A high s imilarity
index (1.00) was s howed betwe e n two s amples of Jeras h, while the lowes t s imilarity (0.12) was regis tered
between the Irbid s amples . A dendrogram s how e d fo u r g ro ups , with the Jeras h s amples more clos ely related
compared to the res t of the s amples . The A jloun and Irbid s a mp le s fo rme d one group. A FLP is cons idered a
powerful tool for as s es s ing the genetic relatednes s among either known or unknown s amples , s p e c ie s and
individuals .
Key words: A FLP, s umac, Rhus, Jordan and polymorphis m.
Introduction
Rhus coriaria commonly called s umac or
s umach, is a p e re n nial plant that belongs to the
anacardiaceae family, one of approximately 250
s pecies of the Rhus genus . This s pecies is widely
dis tributed in uplands with elevation (1200m a.s .l)
mainly in A jloun, Irb id and Jeras h areas in Jordan.
It is characterized by deciduous s pirally arranged
leaves , pinnately compound, dens ely panicle or s pike
flowers with red color and dens e clus ters of re d d is h
drupes fruits . It is propagated by new s prouts from
rhizomes or s eeds (Sumac. http:// e n .wikipedia.org/
wiki/ s umac).
R h u s coriaria s pecies was planted at t h e
roads ides , ornamental and fore s t protected areas to
cons erve the s oil fro m eros ion. M oreover, it was
us ed in folk medicine to alleviate s t o ma ch problems ,
diarrhea, bleeding and s kin problems [12].
In Turkey and other A rabic countries , the s umac
berry was us ed as a cooking s p ice (http://
unitproj.library.ucla.edu/bioned/s pice/index.cfm); in
Corres ponding Author
Ibrahim M ohammad Rawashdeh, M edicinal plants and biodiversity program, National Center for
Agricultural Research and extention (NCARE), P. O. Box 639. Baq'a, Jordan.
E-mail: [email protected].
Adv. Environ. Biol., 3(1): 107-112, 2009
addition it is cons idered an imp ortant ingredient of
t h y me (Zatter) and s es ame mix with olive oil which
is des ired by many people w ith breakfas t dis hes .
Özcan and Hacis eferogullari[12] reported that s umac
is a very popular condiment us ed as a majo r s ouring
agent , mixed with fres hly cut onions and cons idered
as appetizer. Jordanian people collected the fruits and
flowers and p roces s ed them to become edible and
then marketed to improve their income.
However, the water extract of s umac Rhus
coriaria has a pos itive effect on the pos itive gram
bacteria[9,10], als o its extract was s hown to inhibit
the formation of hydroperoxide in natural peanut oil
s tored at 65ºC for 35 days [2].
Genetic divers ity has b een s tudied bas ed on the
morphological, phys iological and chemical analys is .
Goulão et al. [6], reported that the traditio nal
methods for characterization and as s es s ment of
g e n e t ic v a riability bas ed on morp h o lo g ic a l,
phys iological and agronomic traits are o ft en not
adequate.
However, recently molecular analy s is s olved the
problem in a few hours compared to mo nths or
years . M any molecular markers techniques s uch as
A mplified Fragment Leng t h Polymorphis m A FLP,
were us ed to dis tinguis h between individuals , s pecies ,
acces s ions and varieties . A FLP marke rs have been
developed by Vos et al.,[16] and widely s p re a d due
to its reliability and robus tnes s . This technique bas ed
on four s teps res triction/ ligation, pre-s elective,
s elective and gel electrophores is .
The us e of molecular markers is s peeding up
plant breeding and clarifying , c o nfirming or even
reformulat ing the s ys tematic taxonomy of s everal
groups of organis ms [15]. A FLP markers complement
the traditional morpholo g ical and phonological
des criptors us ed for the regis tration of new cultivars .
They als o contribute to the protec t ion of intellectual
property and allow the certification of clonally
prop a g a ted varieties [4]. The A FLP method[16] has
be e n widely employed in res earch of plants , fungi
and bacteria[1]. The A FLP technique have been us ed
t o s t u d y t he genetic rela t io n s h ip s a mo n g
M editerranean pis tacia s pecies [5], apple cultivars [6],
Miscanthus[7], Brassica nigra acces s ions [11] and
European Rubus [14]. In addition, in t ra s p e c ific
divers ity reported in wo ody plants us ing A FLP
markers is very high s uch as in olive[13].
The aim of this s tudy was to examine the
genetic relations hip among s umac s amples that were
colle c t e d from different s ites in Jordan bas ed on
A FLP markers .
Materials and methods
Plant material
Leaves were collected randomly fro m 10 s umac
trees from Jordan namely, Irbid, A jloun and Jeras h
(Table 1).
108
DNA isolation
Total cellular DNA was extracted us ing a
modified CTA B is olation proto col. A pproximately
(30 mg) fre s h leaves was ground in liquid nitrogen
and mixed wit h 750 µl of fres h and preheated 2x
CTA B s olution with 0.8g PVPP in 2ml tube s t h e n
placed at 65ºC for 30 min. The mixture was mixed
with 750 µl of chlo ro phorm/ is oamyl alcohol (24:1)
then vortexe d a few s econds , then centrifuged at
14000g for 20 min. The upp e r p has e (s upernatant)
was placed in 2ml tubes with 600ml is opropanol and
the tubes were then s haked u n t il the thread of DNA
appeared before they were centrifuged for 20 min at
14000g. The s olution wa s poured off and the tubes
left to dry before adding 600 µl of cold 70% e t h a n o l
and then placed overnight at –20ºC. The ethanol was
then poured off and the tubes dried . 100µl of TE
was added and placed at 65ºC for 30min. Four
mic olitter of RNA s e (10mg/ml) were added per tub e
and left for 45min at 37ºC. DNA q u a n t itation was
pe rformed us ing a S2100 UV/VIS DIODE-A rraySpectrophotometer, Vers ion 1.7.
AFLP procedure
The A FLP procedure was perfo rmed as
previous ly des cribed by Vos e et al.[16]. Sumac DNA
(500ng) was double diges ted with EcoRI and Tru91
(an is ochizome r o f MseI) and double s tranded
adaptors were ligated to the ends of DNA fragments ,
gene ra ting 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 trictio n and ligation reactions were
carried out s imultaneou s ly in a s ingle reaction
tube[16].
To carry o ut the reaction, an enzyme mas ter mix
fo r 35 reactions was prepared containing 2µl EcoRI
buffer, 2µl Tru91 buffer, 0.25µl EcoRI enzyme
(12u/µl), 0.25µl Tru91 enzyme (12u/µl), 0.625µl
1mg/ml BSA , 9.875µl dd H 2 O a nd 5µl genomic DNA
(500ng), the mix was centrifuged for a few s econds
then s tored at 37ºC for 3 hr. The res trictio n -ligation
re actions cons is ted of 0.5µl 10x T4 buffer, 0.2µl T 4
DNA ligas e (3u/µl), 0.5µl Tru91 adaptor forward,
0.5µl Tru91 adaptor revers e, 0.5µl EcoRI adaptor
forward, 0.5µl EcoRI adaptor revers e and 2.3µl
ddH 2 O. Th en dis tributed 5µl for the res triction tubes ,
then s tored at 37ºC for 3hr. The res triction-ligation
reactions were dilute d 1:4, then us ed in preamplification s tep. The Tru91 complementary primer
had a 3'-C and the EcoRI complementary p rimer had
a 3'-A .
The pre-amplification (pres elective) mix was
prepared by adding 2µl of 5-fold d iluted DNA from
the res triction-ligat io n reaction, 2.5µl 10xbuffer
(PCR), 2.5µl dNTPs (5mM ), 1µl A FLP pres elective
p rimer (EcoRI+ A -3'), 1µl A FLP pres elective primer
(Tru91 + C-3'), 0.2µl Taq polymeras e (5u/µl) and
15.8µl ddH2 O. The pre-amp lification (pres elective)
Adv. Environ. Biol., 3(1): 107-112, 2009
109
Table 1: Oligonucleotides adaptors and primer combinations used for AFLP analysis.
Name
Sequence
EcoRI adaptor
5'- CT CGT AGACT GCGT ACC-3'
3'- AAT T GGT ACGCAGT C-5'
Tru91 adaptor
5'-GACGAT AGT CCT GAG-3'
3'-T ACT CAGGACT CAT -5'
Primers used in pre-amplification
EcoRI+1-A
5'-GACT GCGT ACCAAT T CA-3'
Tru91+1-C
5'-GAT GAGT CCT GAGT AAC-3'
Primers used in selective amplification
EcoRI+3-ACA
5'-GACTGCGTACCAATTC+ACA-3'
EcoRI+3-AAG
5'-GACTGCGTACCAATTC+AAG-3'
EcoRI+3-AAC
5'-GACTGCGTACCAATTC+ATA-3'
EcoRI+3-ATA
5'-GACTGCGTACCAATTC+ATA-3'
EcoRI+3-AAT
5'-GACTGCGTACCAATTC+AAT-3'
MseI (an isochizomer of T ru91)
MseI+3-CAT
5'-GAT GAGT CCT GAGT AAC+CAT -3'
MseI+3-CAG
5'-GAT GAGT CCT GAGT AAC+CAG-3'
MseI+3-CAA
5'-GAT GAGT CCT GAGT AAC+CAA-3'
Primer pair combinations (EcoRI/ MseI).
1-ACA/CAT
2-AAG/CAG
3-AAG/CAT
4-AAC/CAT
5-AAC/CAG
6-AT A/CAA
7-AAT /CAT
amplification was carried out in a thermal cycler
programmed at 72ºC for 2min followed by 20 cyc les
of 94ºC for 30 s ec, 56ºC for 40 s ec an d 72ºC for 50
s ec and finally incubated a t 4º C. The preamplification DNA w as diluted 5-fold with ddH2 O
and s elective amplifications were carried out by us ing
different EcoRI and Tru91 primer combinations
(A lpha DNA ).
Primers s electe d for the s election amplification
were from available A FLP s elective primers that
were purchas ed and s tored at –20ºC at NCA RE lab.
T h e E c o RI primers contained three s elective
nucleotides with s equence (Table 1) while the Tru91
primers had the s e le ctive 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 pres elective amplification reaction product, 1.0µl Tru91
primers , 1.0µl EcoRI primers , 2µl of 10 x b u ffer, 2µl
dNTPs (0.25mM ), 0.2µlTaq polymeras e (5u/µl),
0.25µl of M gCl2 (15mM ) and 11.55ddH2 O.
Selective amplification was carried out in a
thermal cycler programmed at 94º C for 2min,
followed by 13 cycles of 94ºC for 20 s ec, 68ºC for
30 s ec and 72ºC for 1min, and 23 cycles of 94ºC for
30 s e c , 59ºC for 30 s ec and 72ºC for 1min a n d a
final incubatio n a t 4ºC. The s elective amplification
reaction product (6µl) was mixe d w ith 4ul of loading
buffer (98% de io nized foramide, 10mM EDTA (pH:
8), 0.05% bromofenol, 0.05% xylene c y a nol), from
w hich 6µl was finally lo a d e d o n t o a 6%
poly acrylamide gel run on a vertical gel s equencing
apparatus (Cleaver, Scientific, Ltd.).
Nin e primer combinations (EcoRI- / Tru91-)
were us ed in analys is (Table 1), thirty s amples from
s umac t re e s were s ubjected to the s elective
amplification with thes e primer combinations , and
treated under the s ame conditions . To det e rmine the
s ize of the A FLP fragments , we us ed a n A FLP DNA
marke r (50bp s tep ladder) (Promega), ranging in
length from 50 to 800bp.
Data analysis
A FLP polymorphic bands were s cored as pres ent
(1) or abs ent (0) and es timates of s imilarity among
all tes ted s amples were calculated according to N e
and Li, (1979). The matrix of s imilarity was analyzed
by the Unweighted Pair-Group M ethod (UP GM A )
and the d e n d rogram was obtained by us ing SPSS
program, vers ion 10.
Res ults and Dis cus s ion
Out of nine p rimer pair combinations that were
tes ted only three pair combinations A TA /CA A ,
A A G/ CA G a nd A A T/CA T s howed amp lifie d
fragments (Table 2). A high number of amplified
fragments 25 an d 17 were detected by the
combinations A TA /CA A and A A G/CA G, res pectively
(Table 3). The primer pair combination A A T/CA T
s howed the lowes t n u mb e r of bands . The s imilarity
ranged from 0.12 % to 1.00 % (Table 2). A high
s imilarity index (1.00) was s howed among the
s amples that we re collected from Jeras h governorate.
Thes e s amples ind icate that thes e s umac trees
probably came from the s ame s ource of s eeds and
nurs ery of Jeras h fores t, which p ropagated them and
then dis tributed to the farmers in the s ame re gion,
but the low e s t s imilarity (0.12 %) regis tered between
s amples of Irbid and Irbid-A jloun (Table 2).
However, the A F LP analys is s how that their is
a genetic variation among the s u ma c trees s tudied.
This judgment bas ed on t h e dendrogram (Fig 1) was
generated among s amples . Four main clus ters were
generated from this dendrogram.
Adv. Environ. Biol., 3(1): 107-112, 2009
Fig. 1:
110
A dendrogram of s umac s amples genera t e d by the UPGM A method us ing the Jaccard's s imilarity
matrix bas ed on A FLP markers obtained with three primer pair combinations .
The firs t group includes three s ubgro ups , the firs t
one formed 4 s amples were more clos ely and the
s econd s ubgroup formed two s amples from Jeras h the
third s howed one individual of Irbid. The s econd
group formed two individuals from Irbid. The third
group has three s ubgroups th e firs t has one s ample
from A jloun a n d one from Jeras h, the s econd group
included t w o s amples from jeras h but the final s ub
group has one A jloun ind iv id u al. The fourth group
included three s ub clus ters the firs t has four s ub- s ub
clus ter, the s econd s ub c lu s ter has three s ub-s ub
clus ters but the res t s ub clus ter has t w o s ub-s ub
clus ter.
High genetic divers ity was found a mo n g the
three p o p u lations collected from three locations
(Figure 1 and Table 2). For Jeras h region, with the
exception of the pair of s a mp le s 25, 24, 28 and 27,
all other entries are genetically dis tinct but they
fo rme d two s eparated groups , the firs t included 7
in dividuals , the s econd included 3 individuals . For
Irbid region, two individuals formed one group, one
individual included with the firs t group, and s even
indiv id uals were grouped with A jloun s amples .
A jloun region relatively s howed varied individuals
through its formed two s amples with Jeras h and eight
s amples with Irbid in dis tinct groups . Hig h s imilarity
was s howed between 17and 18 s amples (Figure 1
and Table 2).
Irbid population has s hown the hig h e s t intra
population divers ity s ince 7 s ub-clu s t e rs can be
vis ualized; five of them included one individual each.
High s imilarity was s howed between one s ample
from A jlo u n (20) and Jeras h (23). The acces s ions 76
and 18 are the clos es t. H ig h g enetic divers ity within
a population was found.
T h e s imilarity matrix
a llo w ed to confirm the high relatednes s between the
s amples in the pairs (24, 25, 26, 27 and 28) of
Jeras h and (17 and 18) of A jloun. T h e variability
that was found may b e re lated to the ecogeographical c o n dition which plays a great role for
creating variation through climatic changes over the
pas t years .
Adv. Environ. Biol., 3(1): 107-112, 2009
111
Table 2: Similarity matrix based on the AFLP markers among thirty trees of sumac collected from three l o cations in Jordan during 2007.
Further s tudy s hould be conducted to s urvey all
of s umac s pecies grown in Jordan and e xt end the
res earch to include the s tudying of the chemicals (for
example, M alic acid, citric acid and tarta ric acid) and
t h e phys ical properties . M ore efficient res ults will be
obtained if all findings c o rre la ted with ecogeographical information and if it is pos s ib le to
compare them with other s p e cies grown within
neighbor cou n tries of Jordan s uch as Syria and
Turkey etc. K n o w le dge of the genetic s tructure of a
la ndrace is fundamental in elaborating s trategies
which involve the local farmers , allowing us to
improve and s afeguard the genetic integrity of
landrace genetic res o u rces [8]. A FLP markers enable
a quick and reliable as s es s ment of intras pecific
genetic variability[3].
In conc lu s ion, A FLP is a us eful tool that can
facilitate the collection and evaluation a c ces s ions or
genetic res ources and s hortening the time needed for
as s es s the genetic variation among and within
individuals and s pecies in the fields .
Ack nowledgements
The a u t hors are grateful to Dr. A bdel Nabi
Fardous the previous of general director of NCA RTT
for his s upport and encouragement of the s cientific
res earch.
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