Use of ISSR markers to assess the genetic diversity in wild medicinal Ziziphus spina-christi (L.) Willd. collected from different regions of Saudi Arabia

ABSTRACT

Ziziphus spina-christi (sidr) is a shrub, sometimes a tree, native to a vast area of Africa stretching from Mauritania to West Africa. In the Kingdom of Saudi Arabia, it is an exotic medicinal plant for many diseases. The aim of this study was to assess the genetic diversity within and among 34 accessions of Z. spina-christi collected from different regions of Saudi Arabia. The amplification of genomic DNA with 11 inter-simple sequence repeat (ISSR) primers yielded 105 scorable loci, of which 93.4% were found to be polymorphic. The observed number of alleles (na), effective number 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, H_t (0.266 ± 0.0289) was close to the average intrapopulation genetic diversity, H_s (0.2199 ± 0.0216). A high level of gene flow (N_m = 2.37) between populations, reflecting high genetic differentiation (G_st = 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.

KEYWORDS:

• ISSR markers
• genetic diversity
• AMOVA
• Ziziphus spina-christi

Introduction

The genus Ziziphus belongs to the family Rhamnaceae with about 85 species. Ziziphus spina-christi (L.) Willd. (sidr) is one of the species of Zizyphus found in Saudi Arabia. It is a wild and cultivated plant distributed in the Middle East, Pakistan and in the North and East of Africa. Z. spina-christi can grow either as a tree or as a shrub. The leaves are short, the flowers are pedunculated and the yellow or red fruits are edible.[1] Z. spina-christi is a medicinal plant and its leaf extract (peptide and cyclopeptide alkaloids) has neuroprotective and therapeutic roles against pentylenetetrazol convulsant effect.[2] In Saudi Arabia, it is used for the treatment of many diseases like wounds, ulcers, etc. Mizrahi et al. [3] reported that Ziziphus species are cultivated in hot and arid regions. Some pharmacological screening studies indicated that Z. spina-christi leaves appears to be a safe alternative to lower the blood glucose level.[4] Regarding the reproductive biology of the Ziziphus genus, many studies have shown self-incompatibility and synchronous protandrous dichogamy.[5,6] Asatryan and Tel-Zur [7] observed that three studied Ziziphus species, Z. jujuba, Z. mauritiana and Z. spina-christi, are xenogamous and self-incompatible. Therefore, an understanding of the genetic diversity of the Ziziphus genus is an important objective, since genetic diversity is a primordial component of biodiversity and it is related to the geographic distribution of the species.

In the assessment of genetic diversity, molecular markers based on DNA have many advantages. Among the polymerase chain reaction (PCR)-based marker techniques, inter-simple sequence repeats (ISSR) are one of the simplest and widely used markers.[8] ISSR markers are considered as a dominant marker; two phenotypes can be distinguished at each locus following the Mendelian theory.[9] Ratnaparkhe et al. [10] suggested that ISSR markers could provide important information for genome analysis of plant species as a marker associated with disease resistance gene clusters. This marker involves amplification of genomic DNA by PCR using a primer typically attached at the 5’ or 3’ end with 1–4 arbitrary, often degenerate bases extended into the flanking sequences.[11] ISSR markers offer great potential for differentiating closely related cultivars and the generated bands are very repeatable on duplicate samples.[12] ISSR markers have been used for characterisation of germplasm,[13] for assessment of genetic diversity and phylogenetic studies of different species,[14,15] for identification of DNA markers linked to agronomic traits [10] and for plant breeding.[16]

In this paper, we report the use of ISSR markers to assess the genetic diversity among and within wild populations of Z. spina-christi collected from different regions of Saudi Arabia.

Materials and methods

Sample collection

The leaf samples of 34 accessions of Z. spina-christi were collected from different regions of Saudi Arabia (Table 1). The young leaves were collected in silica gel for good quality and quantity of genomic DNA.

Table 1. Details of selected Z. spina-christi populations from Saudi Arabia.

Population code	Location	Number of plant samples	Geographical coordinates
1	At-taif	9	21° 16′ 13″ N, 40° 24′ 57″ E
2	Riyadh	12	24° 38′ 26″ N , 46° 46′ 22″ E
3	Farasan Island	5	16°48′N 41°54′E
4	Jizan	8	16° 53′ 21N, 42° 33′ 4E

DNA extraction

DNA was extracted from 0.2 g of leaf tissue using the modified CTAB (cetyl trimethylammonium) bromide method of Khan et al.[17] The material was ground to a fine powder, using liquid nitrogen with a pestle and mortar. The fine powder of each sample was transferred in a 2-mL Eppendorf tube and suspended in 700 μL of extraction buffer with 3% CTAB (w/v), 100 mmol/L Tris-HCl, pH 8, 2 mol/L NaCl, 25 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 8, 3% β-mercaptoethanol (v/v) and 3% polyvinylpyrrolidone (w/v). The suspension was mixed for 5 min and incubated in a water bath at 65 ºC for 30 min. The mixture was then removed from the water bath, cooled and an equal volume of chloroform:isoamyl alcohol (24:1) was added. The suspension was mixed for 10 min, followed by centrifugation at 10,000 rpm for 10 min at room temperature. The supernatant was collected, transferred to another 1.5 mL tube and precipitated with 2/3 volume ice-cold isopropanol for 1 h at −20 °C. The DNA was pelleted by centrifugation at 10,000 rpm for 10 min at 4 °C. The pellet was washed with 70% alcohol, dried at room temperature and then suspended in Tris-EDTA (TE) buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8). The isolated DNA was assayed by Nanodrop 8000 spectrophotometer (Thermo Scientific) and then used for PCR amplification.

PCR amplification

Amplification of genomic DNA was performed with ISSR primers (Table 2). The PCR bead (GE Healthcare, UK) was used for the amplification of DNA. Each reaction was performed in 25 μL reaction volume containing 20 μL of deionized sterile water, 4 μL of DNA (50 ng) and 1 μL of 10 pmol/L of ISSR primer. After mixing the PCR components, the reaction was carried out in an Applied Biosystems Thermal Cycler (Singapore) using the following conditions: initial denaturation at 94 ºC for 4 min, followed by 40 cycles of denaturation for 1 min, annealing step at 45 ºC for 1 min, extension at 72 ºC for 1 min and final extension at 72 ºC for 7 min. The amplified fragments were electrophoresed in 1.5% agarose gel (Hoefer, Richmond CA, USA). The gel was documented using a Syngene BIO IMAGING system (Ingenius L, UK).

Table 2. ISSR primers used for PCR amplification.

Label	Primers	Sequence	Annealing temperature, °C
1*	(CA)6GG	5′-CACACACACACAGG-3′	43
4	(GA)8A	5′-GAGAGAGAGAGAGAGAA-3′	48
6*	(CA)6AG	5′-CACACACACACAAG-3′	40
7*	(CA)6GT	5′-CACACACACACAGT-3′	40
8	(AC)8T	5′-ACACACACACACACACT-3′	51
9	(AG)8YC	5′-AGAGAGAGAGAGAGAGYC-3′	55
10	(AG)8YA	5′-AGAGAGAGAGAGAGAGYA-3′	53
14	(TG)8RT	5′-TGTGTGTGTGTGTGTGRT-3'	53
20*	(CA)8GT	5′-CACACACACACACACAGT-3′	53
21	(GA)8YG	5′-GAGAGAGAGAGAGAGAYG-3′	53
23	(CT)8RC	5′-CTCTCTCTCTCTCTCTRC-3′	55
27	(GT)6RC	5′-GTGTGTGTGTGTRC-3′	48
40	(AG)8GT	5′-AGAGAGAGAGAGAGAGGT-3′	52
43	(AC)8AG	5′-ACACACACACACACACAG-3′	54
46	(AC)8TG	5′-ACACACACACACACACTG-3′	52

Note: R = A, G; Y = C, T

*These primers gave poor amplification.

Data analysis

The data from the ISSR marker analysis was scored for presence (1) and absence (0) of bands. Faint and unclear bands were not counted. The POPGEN 32 software was used to measure the following parameters: observed number of alleles (na), effective number of alleles (ne), Nei's gene diversity (h) [18], number of polymorphic loci (PL), percentage of polymorphic loci (PPL) and Shannon's information index (I).[19] To analyse the genetic diversity in subdivided populations, we determined the total genetic diversity (H_t), intrapopulation genetic diversity (H_s) and gene differentiation (G_st). The amount of gene flow between populations was calculated using population differentiation [(N_m = 0.5 (1−G_st)/G_st], according to McDermott and McDonald.[20] A dendogram was constructed based on the genetic distance, UPGMA (unweighted pair-group method with arithmetic averages) modified from the NEIGHBOR procedure of PHYLIP software version 3.5. Analysis of molecular variance (AMOVA) was performed on three levels: within populations, among populations and among regions, using the GenAlex cross platform package version 6.1 based on 999 permutations.[21]

Results and discussion

Assessment of genetic diversity is very important for the conservation of plant genetic resources in their natural habitat. Fifteen ISSR primers were used to produce DNA fingerprint profiles. Eleven primers amplified 105 loci across 34 individuals. The remaining primers were considered unsuitable due to poor amplification. Out of these 105 loci, 99 loci were polymorphic, reflecting rich allelic diversity in the sampled populations. The size of the amplified bands ranged between 250 and 3000 bp (Figure 1). The obtained results from the genetic polymorphism analysis in different populations of Z. spina-christi, such as number of observed alleles (na), effective number of alleles (ne), gene diversity (h), Shannon's information index (I), number of polymorphic loci (PL) and percentage of polymorphic loci (PPL), are presented in Table 3. The results showed that within the four studied populations, the number of observed alleles (na) ranged from 1.45 in the population from Farasan Island to 1.773 in the population from the At-taif region. The maximum number of effective alleles (1.525) was found in Population 1 (from At-taif) and the lowest number (1.252) was detected in Population 3 (from Farasan Island). Based on the (h) and (I) values, Population 3 showed a slightly lower genetic diversity, whereas the results for the genetic diversity in Population 2 (from Riyadh) and Population 4 (from Jizan) were convergent. This highly detected polymorphism is in agreement with the findings of Singh et al. [22], who studied the genetic diversity of Z. mauritiana using ISSR markers. A previous study conducted on the screening of the genetic relationships in Chinese Ziziphus, using sequence-related amplified polymorphism (SRAP) markers showed a high polymorphism (98.28%) in the selected individuals.[23] Our analysis of the genetic polymorphism obtained with ISSR markers demonstrated that the highest percentage of polymorphic loci (77.36%), largest number of polymorphic loci (82) and the highest gene diversity (h) and Shannon's information index (0.2967 and 0.4356) were found in Population 1 from At-taif region. The lowest values were observed for Population 3 from Farasan Island. Therefore, the sampled individuals from At-taif region could be considered to possess a higher genetic variation as compared to the other populations, therefore some topographical features of At-taif region (high altitude) might be involved. A large amount of variability was observed in these sampled populations, exhibiting high intraspecific genetic diversity. Our results are in agreement with the findings of Wang et al. [24], through the study conducted on the genetic diversity of castor bean (Ricinus communis L.) based on ISSR markers. This high genetic diversity within each population might also be explained by the predominant dichogamic reproduction of this species. This suggestion is in line with the report of Asatryan and Tel-Zur [7], who revealed that three Ziziphus species, Z. jujuba, Z. mauritiana and Z. spina-christi, were characterised by synchronous protandrous dichogamous flower development.

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).

Table 3. Analysis of genetic polymorphism obtained with ISSR primers in different Z. spina-christi populations.

Population	na	ne	h	I	PL	PPL (%)
1	1.7736	1.5258	0.2967	0.4356	82	77.36 %
2	1.6509	1.3798	0.2197	0.3295	69	65.09 %
3	1.4528	1.2592	0.1539	0.2328	48	45.28 %
4	1.5755	1.3642	0.2092	0.3106	61	57.55 %
Total	1.9340	1.4491	0.2699	0.4167	99	93.40

Note: na, observed number of alleles; ne effective number of alleles; h, Nei's [18] gene diversity; I, Shannon's information index [19] PL, number of polymorphic loci; PPL, percentage of polymorphic loci.

The analysis of the inter- and intra-population genetic structure (Table 4) showed that the overall genetic diversity (H_t = 0.266 ± 0.0289) was similar to the intrapopulation genetic diversity (H_s = 0.2199 ± 0.0216). The genetic differentiation among the studied Z. spina-christi populations (G_st) was 0.1739 with effective gene flow observed between populations (N_m = 2.37). According to Nei [25], G_st is classified as low when G_st < 0.05, medium when 0.05 < G_st < 0.15 and high when G_st > 0.15. Thus, the G_st coefficient of Z. spina-christi (G_st = 0.173) would be considered high. The Z. spina-christi species also had a gene flow N_m value greater than 1 (N_m = 2.37), indicating that our sampled populations were not subject to genetic drift.[26] So the high genetic differentiation within populations may be caused by the outcrossing pollination phenomenon. Slatkin and Barton [27] noted that, if the gene flow value is greater than 1, then it is reasonable to prevent substantial differentiation due to genetic drift. Compared to our results, low N_m and G_st have been detected in other Rhamnaceae species,[28] in Liriodendron chinense (Magnoliaceae) and in Lilium cernuum (Liliaceae).[29,30]

Table 4. Nei's analysis of gene diversity in subdivided populations.

	Ht	Hs	Gst	Nm
Mean	0.2662	0.2199	0.1739	2.3756
St. Dev	0.0289	0.0216

The matrix of the pairwise population PhiPT and Nei's genetic distance (Table 5) also reveal the genetic differentiation between the 34 individuals from the different regions analysed in our study. This matrix showed 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 values (0.072, 0.0405) were observed between Population 2 (Riyadh) and Population 4 (Jizan). This was also confirmed by the UPGMA results (Figure 2), which showed separation of Population 3 individuals as an out-group consistent with their source (Island). Interestingly, Population 2 (Riyadh) and Population 4 (Jizan) were much closer despite the vast distance between them (127 km). These values of PhiPT and Nei's genetic distance were low when compared to other plants of different families, such as Rauvolfia serpentina, in which a comparatively larger genetic distance was reported. On the other hand, the individuals of this species were grouped into two clusters and each of them included populations close in geographical origin.[31] When compared to another outcrossing species like Juniperus [32], the pairwise PhiPT genetic distances of Z. spina-christi were the higher.

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
3	0.0908	0.0566	****	0.083
4	0.0568	0.0405	0.0738	****

Figure 2. UPGMA (based on Nei's genetic distance) dendogram showing the relationship between the four studied populations.

The AMOVA revealed that most of the genetic diversity occurred within populations (90%), while the genetic diversity among populations and among regions was 9% and 1%, respectively (Table 6). This indicates that Z. spina-christi is a relatively outcrossing species. The high value of within-population genetic variation in Z. spina-christi is in accordance with other studies in different plant species.[33,34] Overall, the obtained results demonstrate that ISSR markers are suitable for use in genome analysis and genetic diversity studies of Z. spina-christi, as they efficiently generated polymorphism even in closely-related germplasm.

Table 6. AMOVA of different selected populations of Z. spina-christi based on ISSR marker analysis.

Source of variation	Degrees of freedom	Sum of squares	Estimate of variation	Variation (%)
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

Conclusions

The obtained results indicated that the Ziziphus genotypes investigated in this study have wide genetic diversity. High genetic differentiation was found mainly within populations, which may be caused by the outcrossing pollination phenomenon. Cluster analysis using the UPGMA method grouped the population from Farasan Island as an out-group, most probably due to its geographical origin. For better understanding of the genetic diversity of Z. spina-christi, future studies should focus on a larger number of populations and accessions collected from more geographical regions.

Acknowledgements

This study was supported by the Deanship of Scientific Research at King Saud University [project number RGP-014].

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Deanship of Scientific Research [grant number RGP-014].