Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(4): 85-92, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-405 Citation: S.T. Nabavi, F. Farahan, M. Sheidai, K. Poursakhi, M.R. Naeini (2019) Population genetic study of Ziziphus jujuba Mill.: Insight in to wild and cultivated plants genetic structure. Caryologia 72(4): 85-92. doi: 10.13128/ caryologia-405 Published: December 23, 2019 Copyright: © 2019 S.T. Nabavi, F. Farahan, M. Sheidai, K. Poursakhi, M.R. Naeini. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Population genetic study of Ziziphus jujuba Mill.: Insight in to wild and cultivated plants genetic structure Seyyedeh Tahereh Nabavi1, Farah Farahan2, Masoud Sheidai3,*, Katay- oun Poursakhi1, Mohammad Reza Naeini4 1 Department of Horticulture Science, Isfahan (Khorasgan) Branch, Islamic Azad Univer- sity, Isfahan, Iran 2 Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran 3 Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran 4 Department of Horticulture Crops Research, Qom Agricultural and Natural Resources Research and Education Center,AREEO, Qom, Iran *Corresponding author: msheidai@yahoo.com Abstract. Ziziphus jujuba (jujube) of buckthorn family (Rhamnaceae) is an important medicinal crop plant cultivated in different provinces of Iran. It has also wild popula- tions in some geographical areas. We carried out population genetic study on 8 popu- lations of cultivated versus wild jujuba by using ISSR molecular markers to produce data on population genetic structure, gene flow, and genetic variability in the studied populations. We also aimed to investigate genetic differentiation between wild and cul- tivated plants and identify the potential gene pools of this medicinal plant species. The studied populations had a moderate genetic variability and were grouped in two major groups by PCoA plot. AMOVA revealed significant genetic difference among these cultivars. Mantel test showed significant correlation between genetic distance and geo- graphical distance in the studied populations. PCoA analysis showed genetic differen- tiation between wild and cultivated plants within each province. STRUCTURE analysis identified two potential gene pools for jujube cultivars. Data obtained may be used in genetic conservation and future breeding programs of this medicinal plant species in the country. Keyword. Ziziphus jujube, ISSR, STRUCTURE. INTRODUCTION The genus Ziziphus Mill. of the buckthorn family (Rhamnaceae), con- tains about 40 species that are deciduous evergreen trees or shrubs and are distributed in the tropical and subtropical regions of the world (Sing et al. 2007). South and Southeast Asia are considered to be the center of both evo- lution and distribution of Ziziphus species (Sing et al. 2007). These plant spe- cies are of medicinal value and are known to be self-incompatible and pro- duce inter-specific hybrids (Asatryan and Tel-Zur 2013, 2014). 86 Seyyedeh Tahereh Nabavi et al. Z. jujuba (jujube) is one of the well known species of the genus with great medicinal value. It is mainly distributed in southwestern Asia. Traditional use of the species dates back to 2,500 years ago, as revealed in the original Chinese materia medica records. The fruit, seed, and bark are used to alleviate stress and insomnia and as appetite stimulants, digestive aids, antiarrhythmics, and contraceptives (Vahedi et al. 2008). The fruit is eaten fresh or dried and made into can- dy; tea, or syrup (Gupta et al. 2004; Jiang et al. 2007). Moreover, some specific saponins, as well as ethyl acetate and water extracts of the fruit and bark, have explored the potential cytotoxicity of jujube. These extracts bring about apoptosis and differential cell cycle arrest, moreo- ver, activity against certain human cancer cell lines has been demonstrated in vitro (Lee et al. 2004; Huang et al. 2007;Vahedi et al. 2008). Ziziphus jujube is an important plant species to the mankind, due to which its cultivation and conservation gained high importance within recent years. Moreover, as jujube has wide geographical distribution and forms many local populations, it is important to be studied from population genetic point of view. The species with extensive geographical distribution can be adapted to adverse environmental conditions and harbor different gene content that may be used in future breeding programs and establishing genetic-rich germ plasm collections (Sheidai et al. 2013, 2014, 2016). Different molecular markers were used to investi- gate the genetic diversity in Z. jujuba cultivars or wild individuals. For instance, random amplified polymor- phic DNA (RAPD), amplified fragment length poly- morphisms (AFLP), sequence-related amplified poly- morphisms (SR AP), simple sequence repeats (SSR), inter-simple sequence repeats (ISSR), and chloroplast microsatellite (Cp-SSR) markers were used to study cul- tivar relationships and genetic variability (see for exam- ple, Zhao and Liu 2003; Peng et al. 2000; Liu et al.2005; Wang et al. 2007; Singh et al. 2007; Wang et al. 2014; Zhang et al. 2014; Huang et al. 2015). Population genetic study is an important step for genetic evaluation of medicinally important species as it gives insight on the genetic structure, genetic diver- sity and gene flow versus genetic fragmentation of these plant species. It also produces data on the number of potential gene pools for conservation and breeding strat- egies (Sheidai et al. 2013, 2014, 2016). Therefore, the aim of present study was to produce data on genetic diversity, population genetic structure and to compare the culti- vars and wild populations of Ziziphus jujuba of Iran. We investigated 150 plants of both cultivated as well as wild jujube growing in 23 localities within 8 provinces. For genetic study we used ISSR molecular markers, as these markers are very useful tool to detect genetic polymorphism, are inexpensive and readily adaptable technique for routine germplasm fingerprinting. They can be used to illustrate genetic relationship between accessions or genotypes and construction of genetic linkage maps (Sheidai et al. 2013, 2014, 2016). The suit- ability of ISSRs was reported by Alansi et al. (2016), who studied genetic diversity in populations of Ziziphus spi- na-christi (L.) Willd. MATERIAL AND METHODS Plant materials In total 80 plants were studied in 8 provinces (Fig. 1). Ten plants were randomly selected in each population and used for molecular studied. ISSR assay For molecular studies, the fresh leaves were ran- domly collected from 53 randomly selected plants in the studied area and were dried in silica gel powder. The genomic DNA was extracted using CTAB-activated charcoal protocol (Križ man et al., 2006). The extraction procedure was based on activated charcoal and polyvi- Figure 1. Distribution map of Zizphus jujube populations studied. 87Population genetic study of Ziziphus jujuba Mill.: Insight in to wild and cultivated plants genetic structure nylpyrrolidone (PVP) for binding of polyphenolics dur- ing extraction and under mild extraction and precipita- tion conditions. This promoted high-molecularweight DNA isolation without interfering contaminants. Qual- ity of extracted DNA was examined by running on 0.8% agarose gel. Ten ISSR primers, UBC 807, UBC 810, UBC 811, UBC 834,CAG(GA)7, (CA)7AC, (CA)7AT, (CA)7GT (GA)9A, and (GA)9T, commercialized by the University of British Columbia, were used. PCR reactions were per- formed in a 25-μL volume containing 10 mMTris-HCl buff er at pH 8, 50 mM KCl, 1.5 mM MgCl2 , 0.2 mM of each dNTP (Bioron, Germany), 0.2 μM of a single primer, 20 ng of genomic DNA, and 3 U of Taq DNA polymerase (Bioron). Amplification reactions were per- formed in a Techne thermocycler (Germany) with the following program: 5 min for initial denaturation step at 94 °C, 30 s at 94 °C, 1 min at 55 °C, and 1 min at 72 °C. Th e reaction was completed by a fi nal extension step of 7 min at 72 °C. The amplification products were visual- ized by running on 2% agarose gel, followed by ethidium bromide staining. The fragment sizes were estimated using a 100-bp molecular size ladder (Fermentas, Ger- many). The experiment was replicated 3 times and con- stant ISSR bands were used for further analyses. Data analyses The ISSR bands obtained were treated as binary characters and coded accordingly (presence = 1, absence = 0). The numbers of private versus common alleles were determined. The shared loci among populations were determined by POPGENE ver. 1.3 (2000). Genetic diver- sity parameters like, New gene diversity (He), Shannon information index (I), the number of effective alleles, and percentage of polymorphism (Weising 2005), were determined by using GenAlex 6.4 (Peakall and Smouse, 2006). For genetic grouping of the studied cultivated and wild plants, Nei genetic distance was determined (Weis- ing, 2005), and used in clustering as well as ordination methods (Podani 2000). Genetic differentiation of the studied populations was determined by AMOVA after 1000 permutations as performed in GenAlex 6.4 (Peakall and Smouse, 2006). The Mantel test (Podani, 2000) after 5000 permutation was performed to study the associa- tion between genetic distance and geographical distance of the studied populations. Genetic structure of the populations was studied by model-based clustering as performed by STRUCTURE software ver. 2.3 (Pritchard et al., 2000). We used the admixture ancestry model under the correlated allele frequency model. A Markov chain Monte Carlo simula- tion was run 20 times for each value of K (1-8) after a burn-in period of 10 5. Data were scored as dominant markers and analysis followed the method suggested by Falush et al. (2007). For the optimal value of K in the popula- tion studied, we used The STRUCTURE Harvester web- site (Earl and von Holdt, 2012) was used to perform the Evanno method to identify the proper value of K (Evan- no et al., 2005). To study genetic differentiation between wild and cultivated plants, we performed PCoA (Princi- pal coordinate analysis) analysis within each province. RESULTS We obtained 31 ISSR bands (Loci) in total (Table 1). The highest number of bands (17 bands) occurred in population 1 (Soth Khorasan), and 2 (Fars) (16 bands), respectively. Some of the populations had private bands with population 4 (Sistan-o-Baloochestan) having the highest number (4 private bands). Few common bands occurred in the studied populations too. These are shared alleles among these populations. Genetic diversity parameters determined in Z. juju- ba populations are presented in Table 3. The percentage of genetic polymorphism obtained ranged from 3.25 in population 7 (Golestan) to 51.61 in population 2 Fars). A moderate level of genetic polymorphism (>30%) also occurred in populations 3, and 4 (DNorth-Khorasan, andSistan-o-Baloochestan, respectively). The highest mean value of New gene diversity (He) occurred in pop- ulations 1 to 4 (0.10-0.16, Table 2). Table 1. Details of ISSR bands in Z. Jujube populations. Population Pop1 Pop2 Pop3 Pop4 Pop5 Pop6 Pop7 Pop8 No. Bands 16 17 13 15 12 10 8 13 No. Bands Freq. >= 5% 16 17 13 15 12 10 8 13 No. Private Bands 1 2 0 4 0 1 0 1 No. LComm Bands (<=50%) 6 7 6 5 4 3 3 5 88 Seyyedeh Tahereh Nabavi et al. Detailed analysis of ISSR loci revealed that 16 ISSR loci (50% of all ISSR loci), have high Gst value I.e. >0.50 (equivalent of Fst). This indicates that, these loci are dif- ferent in the studied populations and lead to population genetic differentiation. This ISSR locus had a low value of Nm and therefore, they are not shared by all the pop- ulations. On the contrary, 14 ISSR loci had Nm value >1, and low Get value. They are the common alleles shared by the studied populations. The mean Nm value of the studied populations was 0.38, which is very low and indicates lack of extensive gene flow among the studied populations. The Nei’s genetic identity and genetic distance of the studied populations are provided in Table 3. Genet- ic similarities between 0.70 to 0.96% were observed in the studied populations. The highest genetic identity occurred between populations 1 and 2 (0.96%). Genetic differential of Z. Jujube populations Based on Nei genetic distance, PCoA plot was con- structed for the studied cultivars and wild populations, separately (Fig. 2). The plot constructed for the cultivars, placed Z. Jujube populations in two main groups. Pop- ulations 2, 3 and 4 formed the first main group, while populations 1, 6, 7, and 8, comprised the second major group. Some trees in population1 and 5 were intermixed in both groups. This is due to within population genetic variability and the common shared alleys in these two populations. Similarly, PCoA analysis of the wild populations revealed that these populations differ genetically from each other as they are placed in separate groups (Fig. 3). Therefore, both cultivated and wild plants of the studied provinces are genetically differentiated from each other. Moreover, AMOVA produced significant genetic difference among Z. Jujube populations (PhiPT = 0.57, P = 0.001). AMOVA revealed that 57% of total genetic variability occurred among populations while, 43% of genetic variability was due to within population difference. Paired-sample AMOVA also produced sig- nificant difference among the studied populations. These results indicate that although the studied Z. jujube cul- Table 2. Genetic variability parameters determined in Ziziphus jujube populations based on ISSR markers (populations numbers are according to Fig. 1). Pop N Na Ne I He uHe P% Pop1 10.000 0.968 1.240 0.223 0.146 0.154 45.16% Pop2 10.000 1.065 1.262 0.252 0.164 0.172 51.61% Pop3 10.000 0.742 1.180 0.161 0.107 0.112 32.26% Pop4 10.000 0.871 1.193 0.177 0.115 0.121 38.71% Pop5 10.000 0.613 1.141 0.125 0.084 0.088 22.58% Pop6 10.000 0.484 1.105 0.091 0.061 0.065 16.13% Pop7 10.000 0.290 1.028 0.021 0.015 0.016 3.23% Pop8 10.000 0.710 1.167 0.145 0.097 0.102 29.03% N = No. Of studied plants, Na = No. Of polymorphic alleles, Ne = Effective No. of alleles, He = New gene diversity, uHe = Unbiassed gene diversity, and P% = Percentage of polymorphism. Table 3. Nei genetic identity versus genetic distance in the Z. Jujube populations (populations numbers are according to Fig1. Nei’s genetic identity (above diagonal) and genetic distance (below diago- nal). pop ID 1 2 3 4 5 6 7 8 1 **** 0.9609 0.8920 0.8411 0.8574 0.8735 0.8445 0.9128 2 0.0399 **** 0.9189 0.8675 0.8526 0.8032 0.7677 0.8477 3 0.1143 0.0846 **** 0.9076 0.8505 0.7602 0.7187 0.7955 4 0.1731 0.1422 0.0969 **** 0.8741 0.7263 0.6824 0.7415 5 0.1538 0.1595 0.1619 0.1345 **** 0.8011 0.7621 0.7748 6 0.1353 0.2192 0.2742 0.3198 0.2218 **** 0.9434 0.9539 7 0.1691 0.2644 0.3303 0.3821 0.2717 0.0583 **** 0.9548 8 0.0913 0.1652 0.2288 0.2991 0.2552 0.0472 0.0462 **** Figure 2. PCoA plot of ISSR data in Z. Jujube populations. Figure 3. PCoA plot of Z. Jujube wild populations based on ISSR data. 89Population genetic study of Ziziphus jujuba Mill.: Insight in to wild and cultivated plants genetic structure tivars and wild populations differ genetically from each other, but also some degree of within population of genetic variability do occur in each population. Wild versus cultivated Z. Jujuba plants In the other attempt, we investigated the genetic dif- ferentiation of wild versus cultivated plants within each locality. In three provinces namely, 1- Fare, 2- Golestan, and 3-Kerman, both cultivated and wild plants were present. The comparison of ISSR bands in these plants revealed almost complete genetic differentiation of wild and cultivated plants in Fars province, while in two oth- er provinces, they were genetically differentiated to some degree (Fig. 4). This indicates that these two types of Z. jujube, are not genetically alike and we may have still nov- el genes in wild plants that can be introduced in to culti- vated plants genome. These genetic variability are of high importance in medicinal plant conservation and breeding. Assocition between genetic diversity and geographical fea- tures Correlation analysis performed did not show signifi- cant association between gene diversity with either alti- tude or latitude in the studied populations (Fig. 5). The same hold true for percentage of genetic polymorphism. This may happen due to cultivation practice and selec- tion made by local gardeners which interfere with local natural adaptation. However, Mantel test (Fig. 6) between geographical distance (combined distance of longitude and altitude) and genetic distance produced significant correlation (P<0.01). Therefore, with increase in geographical dis- tance, an increase in genetic difference of the popula- tions occurred. This is called isolation by distance (IBD). This indicates that the combined effect of geographical features as well as genetic background of the studied cultivars bring about significant genetic differentiation among Z. Jujube populations. Genetic structure of Z. Jujube populations The genetic structure of the studied popula- tions and degree of genetic admixture among popula- tions were determined by STRUCTURE analysis. The STRUCTURE plot (Fig. 7) revealed presence of differ- ent allele combinations (differently colored segments) in the Z. Jujube populations. However, some degree of shared common alleles (similarly colored segments) was observed in populations 1, 2 and 3, and also in popula- tions 6, 7, and 8. Populations 4 and 5 contained distinct allele combinations. Figure 4. PCoA plot of wild versus cultivated Z. Jujube plants with- in Fars province. Figure 5. Correlation analysis of genetic diversity and genetic poly- morphism with geographical features in Z. Jujube populations. Figuse 6. Mantel test plot between genetic distance and geographi- cal distance of Z. Jujube populations. 90 Seyyedeh Tahereh Nabavi et al. Evanno test produced optimal number of genetic group k = 2. Therefore, 13 studied Ziziphus jujube pop- ulations studied could be grouped in 2 genetic groups. STRUCTURE plot based on k = 2 (Fig. 8), revealed that populations 2-4 comprise the first genetic group, while populations 6-8 comprise the second genetic group. Moreover, populations 1 and 5 stands somewhere in between these two groups. This is in complete agreement with PCoA plot results presented before. DISCUSSION In spite of medicinal importance (Vahedi et al. 2008) and wide geographical distribution of Ziziphus jububa in our country, we had no detailed information on its genetic variability and structure.The present study revealed the presence of a moderate genetic variability in the cultivated populations. It also showed genetic dif- ferentiation between wild versus cultivated plants within each province. Therefore, we can use these plants in a core germ plasm collection of Z. Jujube for conservation and breeding purpose (Sheidai et al. 2013, 2014, 2016). Alansi et al. (2016), studied genetic diversity in pop- ulations of Ziziphus spina-christi (L.) Willd. By using ISSR markers and reported the genetic diversity value of 0.26, total genetic diversity Ht = 0.266, and intra-popu- lation genetic diversity, Hs = 0.2199. In present study, AMOVA revealed significant genet- ic difference among Z. jujube cultivars, and also identi- fied a good level of genetic variability within studied population. Moreover, Gst and Nm results revealed that about 50% of ISSR loci was either private on not shared by all populations, and 50% were exchange in popula- tions via gene flow. This may be to some degree related to out-crossing nature of Z. jujube. Zhang et al. (2015) studied genetic variability and differentiation in cultivated jujube and wild jujube by using SSR molecular markers. They reported high levels of genetic diversity (HE=0.659 and HS=0.674) within populations, and moderate differentiation among stud- ied populations (FST=0.091, RST= 0.068, GST=0.271). They a lso reported a high degree of gene f low (Nm=6.572) and weak correlation between genetic and geographical distances (r 2 =0.026, P>0.05), and suggest- ed that gene flow occurred frequently among popula- tions. AMOVA showed that most of the existing genetic diversity was distributed within populations (88 %), and only 12 % occurred among populations, therefore, the studied populations were not differentiated. On the other hand, Singh et al. (2017) investigated genetic variation and relationships among cultivars of Ziziphus mauritiana (Lamk.) native of India by using start codon targeted (SCoT), ISSR, and ribosomal DNA (rDNA) markers. They reported high level of polymor- phism among SCoT (61.6%) and ISSR (61%) markers. SCoT and ISSR dendrograms delineated all the cultivars of Z. mauritiana into well-supported distinct clusters. These populations were genetically differentiated as also was indicated with high Get values. Difference in the results of these studies is probably due to difference in geographical isolation of the studied populations. In present study, the distance between pop- ulations is great as they are located in different provinc- es ranging from south to north of the country with no intermediately plant populations among them (Fig. 1). Genetic differentiation of the studied populations may be attributed to a combination of adaptation to different environmental conditions and limited capacity for long- distance dispersal (Zhang et al. 2015). However, we also noticed good genetic differentiation within each prov- ince between wild and cultivated Z. Jujube plants; this is probably due to effects of cultivation practice and arti- ficial selection made by jujube growers in the gardens. Such selection pressure is absent in wild plants. In conclusion, we have presented data on genetic variability and genetic structure of both Z. Jujube cul- tivars and wild plants in the country. 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