Biotechnology & Biotechnological Equipment

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Use of ISSR markers to assess the genetic diversity

in wild medicinal Ziziphus spina-christi (L.) Willd.
collected from different regions of Saudi Arabia

Saleh Alansi, Mohamed Tarroum, Fahad Al-Qurainy, Salim Khan &

Mohammad Nadeem

To cite this article: Saleh Alansi, Mohamed Tarroum, Fahad Al-Qurainy, Salim Khan &
Mohammad Nadeem (2016) Use of ISSR markers to assess the genetic diversity in wild medicinal
Ziziphus�spina-christi (L.) Willd. collected from different regions of Saudi Arabia, Biotechnology &
Biotechnological Equipment, 30:5, 942-947, DOI: 10.1080/13102818.2016.1199287

To link to this article: https://doi.org/10.1080/13102818.2016.1199287

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BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT, 2016
VOL. 30, NO. 5, 942
947
http://dx.doi.org/10.1080/13102818.2016.1199287

ARTICLE; BIODIVERSITY AND ECOSYSTEMS

Use of ISSR markers to assess the genetic diversity in wild medicinal Ziziphus
spina-christi (L.) Willd. collected from different regions of Saudi Arabia
Saleh Alansi, Mohamed Tarroum, Fahad Al-Qurainy, Salim Khan and Mohammad Nadeem
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia

ABSTRACT ARTICLE HISTORY

Ziziphus spina-christi (sidr) is a shrub, sometimes a tree, native to a vast area of Africa stretching Received 29 October 2015
from Mauritania to West Africa. In the Kingdom of Saudi Arabia, it is an exotic medicinal plant for Accepted 6 June 2016
many diseases. The aim of this study was to assess the genetic diversity within and among 34 KEYWORDS
accessions of Z. spina-christi collected from different regions of Saudi Arabia. The amplification of ISSR markers; genetic
genomic DNA with 11 inter-simple sequence repeat (ISSR) primers yielded 105 scorable loci, of diversity; AMOVA; Ziziphus
which 93.4% were found to be polymorphic. The observed number of alleles (na), effective number spina-christi
of alleles (ne), Nei’s gene diversity (h) and genetic diversity estimated by Shannon’s information
index (I) were 1.93, 1.44, 0.26 and 0.41, respectively. The total genetic diversity, Ht (0.266 § 0.0289)
was close to the average intrapopulation genetic diversity, Hs (0.2199 § 0.0216). A high level of
gene flow (Nm D 2.37) between populations, reflecting high genetic differentiation (Gst D 0.1739).
The analysis of molecular variance showed that the maximum value of genetic variation was found
within populations (90%), whereas a low value of genetic variance was observed among
populations. The analysis using the unweighted pair-group method with arithmetic averages
clustered the population from Farasan Island as an out-group due to its geographical origin. The
obtained results demonstrate that the ISSR markers may be used for evaluation of the genetic
diversity due to their efficiency in revealing polymorphism even in closely related germplasm and
may help in Ziziphus genome analysis.

Introduction Z. jujuba, Z. mauritiana and Z. spina-christi, are xenoga-

mous and self-incompatible. Therefore, an understand-
The genus Ziziphus belongs to the family Rhamnaceae
ing of the genetic diversity of the Ziziphus genus is an
with about 85 species. Ziziphus spina-christi (L.) Willd.
important objective, since genetic diversity is a primor-
(sidr) is one of the species of Zizyphus found in Saudi Ara-
dial component of biodiversity and it is related to the
bia. It is a wild and cultivated plant distributed in the
geographic distribution of the species.
Middle East, Pakistan and in the North and East of Africa.
In the assessment of genetic diversity, molecular
Z. spina-christi can grow either as a tree or as a shrub.
markers based on DNA have many advantages. Among
The leaves are short, the flowers are pedunculated and
the polymerase chain reaction (PCR)-based marker tech-
the yellow or red fruits are edible.[1] Z. spina-christi is a
niques, inter-simple sequence repeats (ISSR) are one of
medicinal plant and its leaf extract (peptide and cyclo-
the simplest and widely used markers.[8] ISSR markers
peptide alkaloids) has neuroprotective and therapeutic
are considered as a dominant marker; two phenotypes
roles against pentylenetetrazol convulsant effect.[2] In
can be distinguished at each locus following the Mende-
Saudi Arabia, it is used for the treatment of many dis-
lian theory.[9] Ratnaparkhe et al. [10] suggested that
eases like wounds, ulcers, etc. Mizrahi et al. [3] reported
ISSR markers could provide important information for
that Ziziphus species are cultivated in hot and arid
genome analysis of plant species as a marker associated
regions. Some pharmacological screening studies indi-
with disease resistance gene clusters. This marker
cated that Z. spina-christi leaves appears to be a safe
involves amplification of genomic DNA by PCR using a
alternative to lower the blood glucose level.[4] Regard-
primer typically attached at the 5’ or 3’ end with 1
4
ing the reproductive biology of the Ziziphus genus,
arbitrary, often degenerate bases extended into the
many studies have shown self-incompatibility and syn-
flanking sequences.[11] ISSR markers offer great poten-
chronous protandrous dichogamy.[5,6] Asatryan and
tial for differentiating closely related cultivars and the
Tel-Zur [7] observed that three studied Ziziphus species,

CONTACT Mohamed Tarroum [email protected]

© 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
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 BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 943

generated bands are very repeatable on duplicate sam- assayed by Nanodrop 8000 spectrophotometer (Thermo
ples.[12] ISSR markers have been used for characterisa- Scientific) and then used for PCR amplification.
tion of germplasm,[13] for assessment of genetic
diversity and phylogenetic studies of different species,
[14,15] for identification of DNA markers linked to agro- PCR amplification
nomic traits [10] and for plant breeding.[16] Amplification of genomic DNA was performed with ISSR
In this paper, we report the use of ISSR markers to primers (Table 2). The PCR bead (GE Healthcare, UK) was
assess the genetic diversity among and within wild pop- used for the amplification of DNA. Each reaction was per-
ulations of Z. spina-christi collected from different formed in 25 mL reaction volume containing 20 mL of
regions of Saudi Arabia. deionized sterile water, 4 mL of DNA (50 ng) and 1 mL of
10 pmol/L of ISSR primer. After mixing the PCR compo-
nents, the reaction was carried out in an Applied Biosys-
Materials and methods
tems Thermal Cycler (Singapore) using the following
Sample collection conditions: initial denaturation at 94  C for 4 min, fol-
lowed by 40 cycles of denaturation for 1 min, annealing
The leaf samples of 34 accessions of Z. spina-christi were
step at 45  C for 1 min, extension at 72  C for 1 min and
collected from different regions of Saudi Arabia (Table 1).
final extension at 72  C for 7 min. The amplified
The young leaves were collected in silica gel for good
fragments were electrophoresed in 1.5% agarose gel
quality and quantity of genomic DNA.
(Hoefer, Richmond CA, USA). The gel was documented
using a Syngene BIO IMAGING system (Ingenius L, UK).
DNA extraction
DNA was extracted from 0.2 g of leaf tissue using the Data analysis
modified CTAB (cetyl trimethylammonium) bromide
The data from the ISSR marker analysis was scored for
method of Khan et al.[17] The material was ground to a
presence (1) and absence (0) of bands. Faint and unclear
fine powder, using liquid nitrogen with a pestle and mor-
bands were not counted. The POPGEN 32 software was
tar. The fine powder of each sample was transferred in a
used to measure the following parameters: observed
2-mL Eppendorf tube and suspended in 700 mL of
number of alleles (na), effective number of alleles (ne),
extraction buffer with 3% CTAB (w/v), 100 mmol/L Tris-
Nei’s gene diversity (h) [18], number of polymorphic loci
HCl, pH 8, 2 mol/L NaCl, 25 mmol/L ethylenediaminete-
(PL), percentage of polymorphic loci (PPL) and Shan-
traacetic acid (EDTA), pH 8, 3% b-mercaptoethanol (v/v)
non’s information index (I).[19] To analyse the genetic
and 3% polyvinylpyrrolidone (w/v). The suspension was
diversity in subdivided populations, we determined
mixed for 5 min and incubated in a water bath at 65  C
the total genetic diversity (Ht), intrapopulation genetic
for 30 min. The mixture was then removed from the
diversity (Hs) and gene differentiation (Gst). The amount
water bath, cooled and an equal volume of chloroform:
of gene flow between populations was calculated
isoamyl alcohol (24:1) was added. The suspension was
using population differentiation [(Nm D 0.5 (1¡Gst)/
mixed for 10 min, followed by centrifugation at
Gst], according to McDermott and McDonald.[20]
10,000 rpm for 10 min at room temperature. The super-
natant was collected, transferred to another 1.5 mL tube Table 2. ISSR primers used for PCR amplification.
and precipitated with 2/3 volume ice-cold isopropanol Annealing
for 1 h at ¡20  C. The DNA was pelleted by centrifuga- Label Primers Sequence temperature,  C
tion at 10,000 rpm for 10 min at 4  C. The pellet was 1 (CA)6GG 50 -CACACACACACAGG-30 43
4 (GA)8A 50 -GAGAGAGAGAGAGAGAA-30 48
washed with 70% alcohol, dried at room temperature 6 (CA)6AG 50 -CACACACACACAAG-30 40
and then suspended in Tris-EDTA (TE) buffer (10 mmol/L 7 (CA)6GT 50 -CACACACACACAGT-30 40
8 (AC)8T 50 -ACACACACACACACACT-30 51
Tris-HCl, 1 mmol/L EDTA, pH 8). The isolated DNA was 9 (AG)8YC 50 -AGAGAGAGAGAGAGAGYC-30 55
10 (AG)8YA 50 -AGAGAGAGAGAGAGAGYA-30 53
14 (TG)8RT 50 -TGTGTGTGTGTGTGTGRT-3' 53
Table 1. Details of selected Z. spina-christi populations from 20 (CA)8GT 50 -CACACACACACACACAGT-30 53
Saudi Arabia. 21 (GA)8YG 50 -GAGAGAGAGAGAGAGAYG-30 53
23 (CT)8RC 50 -CTCTCTCTCTCTCTCTRC-30 55
Population Number of
27 (GT)6RC 50 -GTGTGTGTGTGTRC-30 48
code Location plant samples Geographical coordinates
40 (AG)8GT 50 -AGAGAGAGAGAGAGAGGT-30 52
1 At-taif 9 21 160 1300 N, 40 240 5700 E 43 (AC)8AG 50 -ACACACACACACACACAG-30 54
2 Riyadh 12 24 380 2600 N , 46 460 2200 E 46 (AC)8TG 50 -ACACACACACACACACTG-30 52
3 Farasan Island 5 16 480 N 41 540 E
Note: R D A, G; Y D C, T
4 Jizan 8 16 530 21N, 42 330 4E 
These primers gave poor amplification.
944 S. ALANSI ET AL.

A dendogram was constructed based on the genetic dis- Table 3. Analysis of genetic polymorphism obtained with ISSR
tance, UPGMA (unweighted pair-group method with primers in different Z. spina-christi populations.
Population na ne h I PL PPL (%)
arithmetic averages) modified from the NEIGHBOR pro-
1 1.7736 1.5258 0.2967 0.4356 82 77.36 %
cedure of PHYLIP software version 3.5. Analysis of molec- 2 1.6509 1.3798 0.2197 0.3295 69 65.09 %
ular variance (AMOVA) was performed on three levels: 3 1.4528 1.2592 0.1539 0.2328 48 45.28 %
4 1.5755 1.3642 0.2092 0.3106 61 57.55 %
within populations, among populations and among Total 1.9340 1.4491 0.2699 0.4167 99 93.40
regions, using the GenAlex cross platform package ver- Note: na, observed number of alleles; ne effective number of alleles; h,
sion 6.1 based on 999 permutations.[21] Nei’s [18] gene diversity; I, Shannon’s information index [19] PL, num-
ber of polymorphic loci; PPL, percentage of polymorphic loci.

Results and discussion

Assessment of genetic diversity is very important for the agreement with the findings of Singh et al. [22], who
conservation of plant genetic resources in their natural studied the genetic diversity of Z. mauritiana using ISSR
habitat. Fifteen ISSR primers were used to produce DNA markers. A previous study conducted on the screening
fingerprint profiles. Eleven primers amplified 105 loci of the genetic relationships in Chinese Ziziphus, using
across 34 individuals. The remaining primers were con- sequence-related amplified polymorphism (SRAP)
sidered unsuitable due to poor amplification. Out of markers showed a high polymorphism (98.28%) in the
these 105 loci, 99 loci were polymorphic, reflecting rich selected individuals.[23] Our analysis of the genetic poly-
allelic diversity in the sampled populations. The size of morphism obtained with ISSR markers demonstrated
the amplified bands ranged between 250 and 3000 bp that the highest percentage of polymorphic loci
(Figure 1). The obtained results from the genetic poly- (77.36%), largest number of polymorphic loci (82) and
morphism analysis in different populations of Z. spina- the highest gene diversity (h) and Shannon’s information
christi, such as number of observed alleles (na), effective index (0.2967 and 0.4356) were found in Population 1
number of alleles (ne), gene diversity (h), Shannon’s from At-taif region. The lowest values were observed for
information index (I), number of polymorphic loci (PL) Population 3 from Farasan Island. Therefore, the sampled
and percentage of polymorphic loci (PPL), are presented individuals from At-taif region could be considered to
in Table 3. The results showed that within the four stud- possess a higher genetic variation as compared to the
ied populations, the number of observed alleles (na) other populations, therefore some topographical fea-
ranged from 1.45 in the population from Farasan Island tures of At-taif region (high altitude) might be involved.
to 1.773 in the population from the At-taif region. The A large amount of variability was observed in these sam-
maximum number of effective alleles (1.525) was found pled populations, exhibiting high intraspecific genetic
in Population 1 (from At-taif) and the lowest number diversity. Our results are in agreement with the findings
(1.252) was detected in Population 3 (from Farasan of Wang et al. [24], through the study conducted on the
Island). Based on the (h) and (I) values, Population 3 genetic diversity of castor bean (Ricinus communis L.)
showed a slightly lower genetic diversity, whereas based on ISSR markers. This high genetic diversity within
the results for the genetic diversity in Population 2 each population might also be explained by the pre-
(from Riyadh) and Population 4 (from Jizan) were dominant dichogamic reproduction of this species.
convergent. This highly detected polymorphism is in This suggestion is in line with the report of Asatryan and

Figure 1. ISSR marker profile generated by primer 4. M, Molecular marker (100 bp DNA ladder, Solis Biodyne, Estonia); Lanes T1
T9
(At-taif); Lanes R1
R12 (Riyadh); Lanes F1
F5 (Farasan Island); Lanes G1
G8 (Jizan).
 BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 945

Table 4. Nei’s analysis of gene diversity in subdivided Table 6. AMOVA of different selected populations of Z. spina-
populations. christi based on ISSR marker analysis.
Ht Hs Gst Nm Source of variation Degrees Sum Estimate Variation
Mean 0.2662 0.2199 0.1739 2.3756 of freedom of squares of variation (%)
St. Dev 0.0289 0.0216 Among region 2 49.607 0.079 1
Among population 1 19.942 1.262 9
Within population 30 365.33 12.170 90
Total 33 434.88 13.518 100
Table 5. Pairwise PhiPT values (above diagonal) and Nei’s
genetic distance (below diagonal) between selected groups.
Population 1 2 3 4

1 0.105 0.138 0.085
2 0.0545 
0.110 0.072 differentiation between the 34 individuals from the differ-

3 0.0908 0.0566 0.083 ent regions analysed in our study. This matrix showed

4 0.0568 0.0405 0.0738
that the highest PhiPT value and the genetic distance
(0.138, 0.0908) were observed among Population 1 (At-
taif) and Population 3 (Farasan Island), whereas the lowest
Tel-Zur [7], who revealed that three Ziziphus species, values (0.072, 0.0405) were observed between Population
Z. jujuba, Z. mauritiana and Z. spina-christi, were charac- 2 (Riyadh) and Population 4 (Jizan). This was also con-
terised by synchronous protandrous dichogamous firmed by the UPGMA results (Figure 2), which showed
flower development. separation of Population 3 individuals as an out-group
The analysis of the inter- and intra-population genetic consistent with their source (Island). Interestingly, Popula-
structure (Table 4) showed that the overall genetic diver- tion 2 (Riyadh) and Population 4 (Jizan) were much closer
sity (Ht D 0.266 § 0.0289) was similar to the intrapopula- despite the vast distance between them (127 km). These
tion genetic diversity (Hs D 0.2199 § 0.0216). The values of PhiPT and Nei’s genetic distance were low
genetic differentiation among the studied Z. spina-christi when compared to other plants of different families, such
populations (Gst) was 0.1739 with effective gene flow as Rauvolfia serpentina, in which a comparatively larger
observed between populations (Nm D 2.37). According genetic distance was reported. On the other hand, the
to Nei [25], Gst is classified as low when Gst < 0.05, individuals of this species were grouped into two clusters
medium when 0.05 < Gst < 0.15 and high when and each of them included populations close in geo-
Gst > 0.15. Thus, the Gst coefficient of Z. spina-christi graphical origin.[31] When compared to another outcross-
(Gst D 0.173) would be considered high. The Z. spina- ing species like Juniperus [32], the pairwise PhiPT genetic
christi species also had a gene flow Nm value greater distances of Z. spina-christi were the higher.
than 1 (Nm D 2.37), indicating that our sampled popula- The AMOVA revealed that most of the genetic diver-
tions were not subject to genetic drift.[26] So the high sity occurred within populations (90%), while the genetic
genetic differentiation within populations may be diversity among populations and among regions was 9%
caused by the outcrossing pollination phenomenon. and 1%, respectively (Table 6). This indicates that Z.
Slatkin and Barton [27] noted that, if the gene flow value spina-christi is a relatively outcrossing species. The high
is greater than 1, then it is reasonable to prevent sub- value of within-population genetic variation in Z. spina-
stantial differentiation due to genetic drift. Compared to christi is in accordance with other studies in different
our results, low Nm and Gst have been detected in other plant species.[33,34] Overall, the obtained results dem-
Rhamnaceae species,[28] in Liriodendron chinense onstrate that ISSR markers are suitable for use in genome
(Magnoliaceae) and in Lilium cernuum (Liliaceae).[29,30] analysis and genetic diversity studies of Z. spina-christi,
The matrix of the pairwise population PhiPT and as they efficiently generated polymorphism even in
Nei’s genetic distance (Table 5) also reveal the genetic closely-related germplasm.

Figure 2. UPGMA (based on Nei’s genetic distance) dendogram showing the relationship between the four studied populations.
946 S. ALANSI ET AL.

Conclusions fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica).

Theor Appl Genet. 1996;92:40
45.
The obtained results indicated that the Ziziphus geno- [10] Ratnaparkhe M, Tekeoglu M, Muehlbauer F. Inter-simple-
types investigated in this study have wide genetic diver- sequence-repeat (ISSR) polymorphisms are useful for find-
sity. High genetic differentiation was found mainly ing markers associated with disease resistance gene clus-
within populations, which may be caused by the out- ters. Theor Appl Genet. 1998;97:515
519.
[11] Zietkiewicz E, Rafalski A, Labuda D. Genome fingerprinting
crossing pollination phenomenon. Cluster analysis using
by simple sequence repeat (SSR)-anchored polymerase
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183.
san Island as an out-group, most probably due to its geo- [12] Fang D, Roose M. Identification of closely related citrus
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diversity of Z. spina-christi, future studies should focus on Theor Appl Genet. 1997;95:408
417.
a larger number of populations and accessions collected [13] Charters Y, Wilkinson M. The use of self-pollinated proge-
nies as ‘in-groups’ for the genetic characterization of
from more geographical regions. cocoa germplasm. Theor Appl Genet. 2000;100:160
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[14] Ajibade S, Weeden N, Chite S. Inter simple sequence
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Acknowledgements Vigna. Euphytica. 2000;111:47
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[15] Joshi S, Gupta V, Aggarwal R, et al. Genetic diversity and
This study was supported by the Deanship of Scientific
phylogenetic relationship as revealed by inter simple
Research at King Saud University [project number RGP-014].
sequence repeat (ISSR) polymorphism in the genus Oryza.
Theor Appl Genet. 2000;100:1311
1320.
[16] Reddy MP, Sarla N, Siddiq E. Inter simple sequence repeat
Disclosure statement (ISSR) polymorphism and its application in plant breeding.
No potential conflict of interest was reported by the authors. Euphytica. 2002;128:9
17.
[17] Khan S, Qureshi MI, Kamaluddin TA, et al. Protocol for iso-
lation of genomic DNA from dry and fresh roots of medici-
Funding nal plants suitable for RAPD and restriction digestion. Afr J
Biotechnol. 2007;6:175
178.
Deanship of Scientific Research [grant number RGP-014]. [18] Nei M. Molecular evolutionary genetics. New York (NY):
Columbia University Press; 1987.
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