Introduction
The genus Ziziphus Mill. (Family Rhamnaceae) is composed of 40 species that are widely distributed in the tropical and subtropical regions of the world, with South and Southeast Asia as their centre of evolution and distribution (Singh et al., Reference Singh, Sharma and Singh2007). Ziziphus species are deciduous evergreen trees or shrubs and are well known as a source of medicine plant (Asatryan and Tel-Zur, Reference Asatryan and Tel-Zur2013). The fruit is eaten fresh or dried and made into candy, tea or syrup (Gupta et al., Reference Gupta, Mazumder, Vamsi, Sivakumar and Kandar2004; Jiang et al., Reference Jiang, Huang, Chen and Lin2007). 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, moreover, activity against certain human cancer cell lines has been demonstrated in vitro (Lee et al., Reference Lee, Park and Lee2004; Huang et al., Reference Huang, Kojima-Yuasa, Norikura, Kennedy, Hasuma and Matsui-Yuasa2007; Vahedi et al., Reference Vahedi, Fathi Najafi and Bozari2008).
Ziziphus jujuba (jujube) is one of the well-known medicinal species of the genus (Asatryan and Tel-Zur, Reference Asatryan and Tel-Zur2013). It is mainly distributed in southwestern Asia. It has been traditionally used as medicine since 2500 years ago, in China. The fruit, seed and bark of this plant species are used to alleviate stress and insomnia and as appetite stimulants, digestive aids, antiarrhythmics and contraceptives (Vahedi et al., Reference Vahedi, Fathi Najafi and Bozari2008). Ziziphus jujuba 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.
Population genetic study is an important step for genetic evaluation of medicinally important species as it gives insight on the genetic structure, genetic diversity and gene flow vs genetic fragmentation of these plant species. It also produces data on the number of potential gene pools for conservation and breeding strategies (Sheidai et al., Reference Sheidai, Taban, Talebi and Noormohammadi2016). Various molecular markers have been used to perform the above tasks such as, amplified fragment length polymorphism (AFLP), simple sequence repeats (SSRs), inter-simple sequence repeats (ISSRs), start codon targeted (SCoTs), retrotransposon-microsatellite amplified polymorphism (REMAPs) etc. (e.g. Saboori et al., Reference Saboori, Noormohammadi, Sheidai and Marashi2019).
Different molecular markers have been used for population genetic investigation and phylogenetic studies in Z. jujuba. For example, Farahani et al. (Reference Farahani, Sedighzadegan, Sheidai and Koohdar2019) and Nabavi et al. (Reference Nabavi, Farahani, Sheidai, Poursakhi and Naeini2019) revealed good level of genetic diversity among wild/ uncultivated populations of jujube by using ISSR, SRAP and IRAP molecular markers which can be used in conservation and breeding of this important horticultural crop plant within Iran. As well as Reche et al. (Reference Reche, García-Martínez, Carbonell, Almansa, Hernández, Legua and Amorós2019) revealed ISSR markers was able to differentiate the cultivars jujube, so they are useful for genetic studies of Z. jujuba specie. Similarly, the genetic relationships between different Z. jujuba cultivars and/ or wild jujuba individuals was studied by using random amplified polymorphic DNA (RAPD), AFLP, sequence-related amplified polymorphisms (SRAP), SSR, ISSR and chloroplast microsatellite (Cp-SSR) markers (see for example, Peng et al., Reference Peng, Shu, Sun and Peng2000; Liu et al., Reference Liu, Peng, Peng, Zhou and Dai2005; Singh et al., Reference Singh, Sharma and Singh2007; Wang et al., Reference Wang, Tian, Wang, Sui, Li and Huang2007; Wang et al., Reference Wang, Liu, Ma, Liu, Tang and Wu2014; Zhang et al., Reference Zhang, Huang, Yin, Lian and Li2014; Huang et al., Reference Huang, Yang, Zhang, Yin, Liu and Li2015).
Due to medicinal importance of Z. jujuba, the present study was performed with the following aims:1- To produce data on genetic diversity in Z. jujube cultivars, 2- To investigate discriminating power of the SCoT and REMAP markers by Gst and NM analysis for fingerprinting of Z. jujube.
Materials and methods
Plant materials
We randomly selected 100 trees of 10 geographical populations (10 trees per population) and used for population genetic investigation (Table 1 and online Supplementary Fig. S1).
Table 1. Ziziphus jujuba population in REMAP and SCoT studies
Scot and REMAP assay
Fresh leaves were put to dry in silica gel powder. Cetyltrimethyl-ammonium bromide -activated charcoal protocol (CTAB) was applied to extract the genomic DNA (Križman et al., Reference Križman, Jakše, Baričevič, Javornik and Prošek2006). The extraction was done by activating charcoal and poly vinyl pyrrolidone for binding of polyphenolics during extraction; for mild extraction and precipitation conditions, the high-molecular weight DNA isolation was boosted without the interference of impurities. The extracted DNA was examined in terms of quality and quantity by running on 0.8% agarose.
3 REMAP primer combinations, derived from one single IRAP primer (NIKITA) with 3 ISSR primers ((CA)7GT, (GA)9 T, (GA)9C) were tested on plants samples. Using a 25 μl volume containing 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany); 50 mM KCl; 10 mM Tris-HCl buffer at pH;8 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of each primer, polymerase chain reaction (PCR) was implemented.
The following programme was used for amplification of nuclear region in a PCR reaction: 5 min initial denaturation step 94°C, followed by 40 cycles of 1 min at 94°C; 1 min at 53.5°C and 2 min at 72°C. The reaction was completed by a final extension step of 7 min at 72°C. The amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. The fragment size was estimated by using a 100 bp molecular size ladder (Fermentas, Germany).
Four primers (SCOT1, SCOT2, SCOT36 and SCOT41) based on Collard and Mackill (Reference Collard and Mackill2009) for monocotyledons plants were selected. These primer sequences are: SCoT1: CAACAATGGCTACCACCA, SCoT2: CAACAATGGCTACCACCC, SCoT36: GCAACAATGGCTACCACC and SCoT41: CAATGGCTACCACTGACA.
PCR reaction mixture with total volume of 25 μl contained 10 mM Tris-HCl buffer (pH = 8), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of dNTP (Bioron, Germany), 0.2 μM of primer, 20 ng genomic DNA and 1U of Taq DNA polymerase (Bioron, Germany).
The amplification reactions were performed in Techne thermocycler (Germany) with the following programme: 5 min at 94°C, 40 cycles of 1 min at 94°C, 1 min at 49–58°C (SCOT1 50°C, SCOT2 49°C, SCOT36 50°C, SCOT41 58°C) and 1 min at 72°C and a final cycle of 7 min at 72°C. The amplification products were visualised by running on 2% agarose gel, stained with sybergreen (Powerload, Kosar Co. Iran). The fragment size was estimated by using a 100 bp molecular size ladder (Fermentas, Germany).
Data analyses
The SCoT and REMAP bands obtained were treated as binary characters and coded accordingly (presence = 1, absence = 0). Detrended correspondence analysis (DCA) was used to evaluate suitability of SCoT bands obtained for population genetic analysis (Hill MO, Gauch, Reference Hill and Gauch1980). The number of private bands vs common bands and genetic diversity parameters like: The percentage of allelic polymorphism, allele diversity (Weising et al., Reference Weising, Nybom, Wolff and Kahl2005), Nei’ gene diversity (He) and Shannon information index (I) (Weising et al., Reference Weising, Nybom, Wolff and Kahl2005), were determined. We used GenAlex 6.4 for these analyses (Peakall and Smouse, Reference Peakall and Smouse2006). Discriminating power of REMAP and SCoT markers investigated by Gst and NM analysis as implemented in POPGENE32. The Neis’ genetic distance was also being determined among these populations. Mantel test was also determined to illustrate if genetic distance is associated with geographical distance in the studied populations (Mantel, Reference Mantel1967).
Grouping of the species was done by different clustering and ordination methods such as NJ and Multidimensional scaling (MDS) (Podani, Reference Podani2000). PAST version 2.17 (Hammer et al., Reference Hammer, Harper and Ryan2012) was used for multivariate analysis.
SCOT data was also analysed by TCS Networking as implemented in Pop ART (Population Analysis with Reticulate Trees) program (http://popart.otago.ac.nz). In order to investigate the province's separation of the studied cultivars, SCOT data were also analysed by the MDS plot in the PAST ver. 32 program.
Results
SCoT results
DCA plot (Not figure), revealed that SCot molecular markers are scattered along the plot and therefore are distributed throughout the genome and are suitable for population genetic studies. The primary analysis of SCoT molecular data in the studied jujube cultivars revealed that these cultivars produced 70 loci or bands. In total, each cultivar studied contained 9–22 SCoT bands (online Supplementary Fig. S2). Six cultivars had 2–5 private/ specific bands due to their peculiar genetic structure. These cultivars are namely, No.1–2 and4, No. 7–8 and No. 10. Such specific bands may be formed due to specific genetic content either through local adaptation or cultivation practice and selection.
Genetic diversity parameters determined in the studied cultivars are provided in Table. The genetic polymorphism varied from 1.92% in cultivar No. 1 and 3 to 25.00% in the cultivar No. 7. The mean value of genetic polymorphism, is indicative of low genetic variability within each cultivar. The other genetic diversity parameters like Shanon information index (I) as well as gene diversity (He), were also very low in the studied jujube cultivars (online Supplementary Table S1).
The AMOVA test showed significant genetic differences among Z. jujube populations (P = 0.001). The results show that the populations of Z. jujube have been genetically distinguished from each other using SCoT marker (online Supplementary Fig. S3).
Nei genetic distance obtained among cultivars ranged from 0.11 to 0.66. We obtained a significant correlation between genetic distance and geographical distance of the studied populations (p. <0.01), by Mantel test.
Different ordination and clustering methods like MDS and NJ produced similar results; therefore, only MDS plot is presented here. MDS and NJ plots (Fig. 1 and online Supplementary Fig. S4) grouped the specimens of each population together, separated from each other. This means that these cultivars are genetically differentiated from each other. This is in agreement with AMOVA results which showed significant genetic difference (P < 0.05) among these cultivars.
Figure 1. MDS plot of SCoT marker in Ziziphus jujube.
The TCS plot (Fig. 2) also confirmed the results of MDS on the separation between cultivars. In this plot, the lines that are placed horizontally on each branch represent the number of the locus that varies between the cultivars. Although the cultivars are well separated from each other, the difference between the loci represents a variation within them.
Figure 2. TCS plot of SCoT marker in Ziziphus jujube.
REMAP results
DCA plot (Not figure), revealed that REMAP molecular markers are scattered along the plot and therefore are distributed throughout the genome and are suitable for population genetic studies. The primary analysis of REMAP molecular data in the studied jujube cultivars revealed that these cultivars produced 60 loci or bands (online Supplementary Fig. S5).
Six cultivars had 1–3 private/ specific bands due to their peculiar genetic structure. These cultivars are namely, No.2, No. 3, No. 6, No. 7, No. 9 and No. 10. Such specific bands may be formed due to specific genetic content either through local adaptation or cultivation practice and selection.
Genetic diversity parameters determined in the studied cultivars are provided in Table. The genetic polymorphism varied from 0.00% in cultivar No. 1 to 9.09% in the cultivar No. 7. The mean value of genetic polymorphism is indicative of low genetic variability within each cultivar. The other genetic diversity parameters like Shanon information index (I) as well as gene diversity (He), were also very low in the studied Ziziphus cultivars (online Supplementary Table S2).
The AMOVA test showed significant genetic differences among Z. jujube populations (P = 0.001). The results show that the populations of Z. jujube have been genetically distinguished from each other using REMAP marker (online Supplementary Fig. S6).
Nei genetic distance obtained among cultivars ranged from 0.16 to 0.72. We obtained a significant correlation between genetic distance and geographical distance of the studied populations (p. <0.01), by Mantel test.
Different ordination and clustering methods like MDS and NJ produced similar results; therefore. MDS plot (Fig. 3) grouped the specimens of each population together, separated from each other. This means that these cultivars are genetically differentiated from each other. This is in agreement with AMOVA results which showed significant genetic difference (P < 0.05) among these cultivars.
Figure 3. MDS plot of REMAP marker in Ziziphus jujube.
The TCS plot (Fig. 4) also confirmed the results of MDS on the separation between cultivars. In this plot, the lines that are placed horizontally on each branch represent the number of the locus that varies between the cultivars. Although the cultivars are well separated from each other, the difference between the loci represents a variation within them.
Figure 4. TCS plot of REMAP marker in Ziziphus jujube.
Fingerprinting
Discriminating power analysis of molecular markers is important for fingerprinting. For this purpose, Gst and Nm parameters was measured by Popgene analysis. The value of Gst in 23 loci in SCoT and 27 loci in REMA marker in Ziziphus jujube cultivars was 1.00 while the mean Nm value was 0.00, which indicated these markers are suitable molecular tool to investigate genetic structure and genetic finger printing of Ziziphus jujube cultivars.
Discussion
Ziziphus jujuba with good medicinal and food importance (Vahedi et al., Reference Vahedi, Fathi Najafi and Bozari2008) should be studied from genetic and breeding points of view. This tree species grows in different geographical regions of our country and its population genetic study is of immediate importance. Data produced can be utilised in future conservation and breeding programmes.
Population genetic study produces insight on the genetic structure, the stratification vs gene flow, as well as genetic divergence of the populations (Freeland et al., Reference Freeland, Kirk and Petersen2011).
The present study reports genetic variability of jujube cultivars in different localities of Iran. We revealed that multilocus molecular markers like SCoTs and REMAPs are powerful technique for genetic finger printing and discriminating jujuba cultivars and populations. The results obtained by DCA analyses revealed that these molecular markers can be used for genetic fingerprinting in jujube germ plasm.
This is in close agreement with the other reports on these cultivars. For example, Farahani et al. (Reference Farahani, Sedighzadegan, Sheidai and Koohdar2019) and Nabavi et al. (Reference Nabavi, Farahani, Sheidai, Poursakhi and Naeini2019) used ISSRs, SRAPs and IRAP markers to investigate genetic diversity in many cultivars in IRAN. On the other hand, Singh et al. (Reference Singh, Chhajer, Pathak, Bhatt and Kalia2017) investigated genetic variation and relationships among cultivars of Ziziphus mauritiana (Lamk.) native of India by using SCoT, ISSR and ribosomal DNA (rDNA) markers. They reported high level of polymorphism among SCoT (61.6%) and ISSR (61%) markers. 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 populations is great as they are located in different provinces ranging from south to north of the country with no intermediately plant populations among them.
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., Reference Zhang, Huang, Yin, Lian and Li2014).
However, we also noticed good genetic differentiation within each province between wild and cultivated Z. jujuba plants; this is probably due to effects of cultivation practice and artificial selection made by jujube growers in the gardens. Such selection pressure is absent in wild plants.
Conclusion
In the present study, we found the loci with the high value of Gst (1.00) in SCoT and REMAP markers that can be used in fingerprinting of Z. jujuba.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S147926212300031X.
Ethical standards
On behalf of all the co-authors, the author testifies that this article has not been published in whole or in part elsewhere. Data (including images) provided in this paper are not fabricated or manipulated in any way to support our conclusions.
Competing interest
None.
