Assessing Genetic Variability and Population Structure of Ziziphus Nummularia as Characterized by Phenotypic Traits and SSR Markers with Climatic Distribution


 Genus of Ziziphus (family Rhamnaceae), approximately 170 species of prodigious economic and ecological importance. Studies on the genetic diversity within Pakistani Ziziphus species are limited, and to date, no single report on the application of DNA barcoding attempt for identification of Ziziphus species is available in the literature. Therefore, the current study was designed to biogeographic distribution, to assess diversity based on phenotypic traits, SSRs markers, and relationships among Ziziphus nummularia (Burm.f.) Wight & Arn. genotypes. For the first time we use ArcGIS modeling, and their cartographic function for the identification of diversity in climatic variables such as temperature, mean diurnal, humidity, precipitation, and precipitation seasonality and the using of these techniques to determine the important variable which is responsible for the recent distribution of the Ziziphus species. A total of 11 phenotypic traits were noted and have significant phenotypic variation among the traits. The study has used 40 simple sequence repeats (SSR) markers for gaining insights into the genetic diversity within 180 genotypes of Z. nummularia. Successful amplification was achieved with 27 SSRs and was applied for understanding the population structure and relationships among the genotypes. A total of 120 alleles were amplified from Z. nummularia genotypes collected from three districts of the Malakand division, alleles per locus ranged from 2 to 6, averaging 4.4286. whereas polymorphism information content (PIC) from 0.332 to 0.794 in locus JSSR-490 and JSSR-97, within mean value was 0.671 per locus, expected heterozygosity (He), was 0.575, observed heterozygosity (Ho), 0.6618 and average gene diversity 0.494. Flow estimates (6.415) indicated frequent gene flow within Z. nummularia genotypes. Analysis of molecular variance (AMOVA) revealed high genetic variability (80%). The genetic relationship inferred from the neighbor-joining (NJ) phylogeny separate genotypes into three clusters and Bayesian model-based STRUCTURE analyses and PCoA analysis resolved all genotypes and indicted that the KP, populations, Swat and Buner, represent excesses of two different migration routes, with one designated from Swat and Dir (L). The overall results indicated the prevalence of genetic variability and relationships among Z. nummularia across geographical boundaries has retained unique alleles and this may facilitate the development of agronomically desirable and genetically improved Ziziphus cultivars and collections that can help achieve efficient conservation.


Introduction
Ziziphus species family (Rhamnaceae), which has the most important and as well have been the dual function to avail tolerance to drought and drought through various physiological mechanisms including the stimulation of the oxidative metabolic pathway, regulation of osmotic pressure, and sensitive stomata closure to minimize dehydration during adverse conditions (Arndt et al. 2001), and other hand important values as used against for the control of the various hypoglycemic agent, antihypertensive agent, anti-in ammatory agent, antimicrobial agent, antioxidant agent, antitumor agent, liver protective agent, and its pharmacological properties as immune system stimulant (Hammer et al. 2001). Z. nummularia (Burm.f.) Wight & Arn., a perennial herb commonly called small ber and wild ber which is native to southeastern Pakistan and India (Singh et al. 2015), most important and highly notably species of the Ziziphus by their known valuable values of nutritional, medically and traditionally of different parts such as fruits, leaves, root and tubers for the used various aliments i.e. ulcers, pulmonary, fevers, wounds of healing, liver troubles asthma, roots are cooling and used to treat biliousness and headache. The bark is used to treat boils and is effective against diarrhea, blood puri cation, antioxidants, and dysentery, etc (Hammer et al. 2001). Different online works of literature studies rewarded for more consideration on genetic diversity such as morphological and molecular diversity as well as of their identi cation, e.g., resistance to different diseases, stress, and high yield production, the limited studies were recorded on the genetic diversity (Ren et al. 2015).
Information regarding genetic diversity was important for the usage of genetic resources in the breeding of wild Ziziphus species. To expand the phylogenetic genetic or genetic diversity of Ziziphus species for off coming breeding systems which are most and as well as it is necessary to realize genetic diversity and a wider population structure of released varieties and germplasm. However, in comparison with a large amount of morphological and This is the rst attempt to study genotypes distribution modeling to predict the present and future habitat and various environmental variables, Furthermore, we assess the genetic diversity and structure analysis based on SSR molecular markers and morphological parameters of 180 Z. nummularia genotypes collected from various climatic regions of Pakistan. This study will be useful to optimize the management of Ziziphus and in choosing diverse parental genotypes for a future breeding program and conservation. petiole length (PL), internode length (InL), stem diameter (StD), fruit width (FtW), fruit diameter (FtD), fruit length (FtL). In these districts, were collected 180 genotypes of Z. nummularia.

Genomic DNA extraction
Total genomic DNA was extracted from the young leaves of selected genotypes using a modi ed CTAB (Cetyltreimethyl ammonium bromide) method (Liu et al. 2014). Fresh leaves of Ziziphus species were ground to powder and placed into Eppendorf tube where 800 μL of extraction buffer (3% CTAB, 5 M NaCl, 0.5 M EDTA, 1 M Tris-HCl pH 8.0) with 2 μL β-mercaptoethanol were added and incubated at 65°C for 30 min. Incubation was followed by adding equal volumes of chloroform: isoamyl alcohol (24:1) before centrifugation at 12,000 rpm for 10 min. Genomic DNA from the aqueous phase was precipitated by using ice-cold 100% ethanol at −20 °C for 30 min and centrifugation at 12,000 rpm for 10 min. The supernatant was discarded, and the pellet washed with 70% ethanol twice and centrifuged at 12,000 rpm for 5 min. The concentration and quality of the extracted genomic DNA were checked using spectrophotometers (Thermo sher scienti c, USA) and by loading 5 μL of DNA on 1.0% Agarose gel.

PCR reaction for SSR markers
A total of 27 pairs of SSR primers were applied to 180 genotypes of Z. nummularia (Table 1). SSRs were designed using the online program Primer 3 (http://www.frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi), annealing temperature was set from 50-60°C, the optimal length of markers was 20bp and preferably with 50% GC content. Polymerase chain reaction (PCR) was performed in a volume of 12.5 μL containing 0.5 μL (~50 ng) genomic DNA, 6.3 μL of 2×Taq Master Mix (CWBIO, Beijing), and 0.5 μL of 10 μmol/L each of forward and reverse primers. The reaction was set for 94°C of denaturation for 3 min; 27 cycles of 94°C for 30 s, 50-60°C for 30 s, and 72°C for 30 s; and a nal extension at 72°C for 10 min. PCR products were visualized using 8% denaturing polyacrylamide gel electrophoresis (PAGE) in 1×TBE buffer, and running at 200 V for 50 min, followed by silver staining for 10 min (Trans Gen Biotech, China). The data analysis was performing by using different software and layers for the modeling and genetic diversity the world-climatic dataset was used for the data obtaining the bioclimatic layers of the selected studies regions, while the composition of the layers was annual precipitation, mean diurnal range, annual mean temperatures and pre-seasonality for this ArcGIS 10 software was used for thematic map-making and modeling. The morphological data for the selected genotypes traits were further analyzed for various statistical parameters were analyzed for mean, maximum, minimum, by using the statistics software, Excel, 2016.
The SSR binary data obtained from the PCR ampli cation of the Z. nummularia

Bioclimatic layers
The current studies data were downloaded from the world-climatic database and ArcESRI information was applied to incorporate the data. The annual mean temp, annual precipitation, mean diurnal range, and pre-seasonality layers were applying on the mention areas of the Malakand division KP, Pakistan, while the GPS data were recorded at the point of collection where the collected samples were found the different regions such as healthy and mature plants. The annual mean temperature from low to high was noted by the different color layers while annual precipitation layers were presented in (Fig 1A and B). The mean diurnal range and precipitation seasonality from low to high layers were noted by colors and while shows the overall temperature range and their distribution of the Z. nummularia in the selected three different regions of Malakand division, KP, Pakistan. Data maps were shown in ( Phenotypic diversity and correlation among the traits 11 phenotypic traits were recorded for 180 Z. nummularia genotypes collected from various locations of Malakand division, which was found in variation in fruits, leaves, color etc. (Fig 4), the phenotypic diversity was using different statistical analysis. Descriptive statistics of the morphological parameters are summarized in (Table 2). The %CV was calculated for phenotypic traits in genotypes was 90.55%, for ZNDR (Dir (L) was 54.86, for ZNBU (Buner) was 11, Signi cant variation was found for the branching (BR) among the genotypes of (3) regions, the highest value was observed for genotypes collected from ZNST (Swat) (69.46%) followed by ZNDR (Dir (L) (41.84 %) while, the lowest value was recorded for the genotypes collected from ZNBU (8.766%). For LL in genotypes collected from (Swat) was (57.47%), ZNDR (Dir L) (50.80%), for ZNBU (9.97%). Furthermore, the highest variation was observed for LW> LT> PL> InL> StD> FtW. The number of FtL varied for genotypes collected from ZNST, ZNDR, ZNBU (56.10%, 45.31%, and 7.47%), respectively. Furthermore, the Pearson correlation coe cient revealed a signi cant positive as well as a negative association (p = 0.05 and 0.01) among the studied traits of Z. nummularia (Table 3). Several traits revealed strong interrelationships within phenotype categories, particularly leaf traits with yield contributing traits and a few traits correlating with other categories, such as inherently linked growth and phenology-related traits (Tables 2 and 3).   Genetic diversity and population structure The current research works total of 27 SSRs pair primers were successfully ampli ed size of the PCR bands ranged from 137 bp up to 240 bp (JSSR-181 repeat motif as CTT-17 and JSSR-460 as AG-11). Total 120 alleles were reported from 27 SSRs markers average allele was 4.42 per locus. The allele frequency at each SSRs ranged from 0.510 to 1.190   Note; * Expected homozygosity and heterozygosity were computed using, ** Nei's (1973) expected heterozygosity*BP= Polymorphic bands * ne = Effective number of alleles, * I = Shannon's Information index, * Nm = Gene ow estimated from Fst = 0.25(1 -Fst)/Fst.
The population structure analysis was carried out for the 180 Z. nummularia genotypes collected from three geographical regions of KP, Pakistan, were estimated by the user of Bayesian clustering system probabilistically assigned individuals to respective populations there have three types of clustering were identi es which showed relationship among them of different geographical origin, K values were estimated as the mean of the nal estimations of L″ (K) found the middle value of more than 20 runs isolated by the standard deviation of L(K), Δ K = m(| L″(K) |)/s[L(K)] (Pritchard et al. 2000). For the given ranges, i.e., 1 to 20 and the highest values were recorded in K=3 and K=5 consequently, the analysis of the STRUCTURE software was performed for the K=3 which largely grouped by the genotypes, according to Pritchard et al. (2010) and Bayes' rule estimation of K, values give a comparative estimation of LnP(D) and the smallest value is regarded as almost correct. The estimation of ΔK, values that the peak reached K=5, according to the formula our result shows clear peaks of ΔK at K=3 of Z. nummularia genotypes accordingly (Fig 5). In the absence of clear-cut origins of the accessions, a non-strati ed strategy was adopted for the genetic structure analysis. Our results showed a clear peak for ΔK at K = 3, where all the accessions were roughly divided into three major groups, with some admixture among groups (Fig 6). ΔK value was recorded in K = 3 which were suggested that the 180 Z. nummularia genotypes were approximately divided into three Pops (Pop =1, Pop =2 and POP=3) accordingly, (Fig 1 and 2 (Table 5).

Neighbor-joining (NJ) and Principal coordinate analysis (PCoA)
The neighbor-joining system was used for the composite phylogenetic tree for the 180 Z. nummularia genotypes collected from three geographical regions. Viz, ZNST, ZNDR and ZNBU (Swat, Dir, and Buner) of KP, Pakistan, largely agreed with the results of the STRUCTURE analysis showed that, in general, the genotypes from the different regions were clustered together represented three clusters i.e. Cluster 1, cluster 2 and cluster 3, (Fig 7). Cluster 1 was the largest group which comprised mainly the ZNBU, 16 genotypes from ZNST regions and 27 genotypes from ZND region, Cluster, 2 contained a total of 40 genotypes, 9 from ZNBU, 03 were ZNDR and 28 from ZNST regions, Cluster, 3 comprised most genotypes ZNST (Swat), from ZNBU (Buner). The 54 Z. nummularia genotypes were separated into two sub-clusters within the cluster (Fig 3), sub-cluster 2 comprised only ZNST, Z. nummularia genotypes subcluster 1 included ZNBU and ZNDR respectively, however, and there is very strong support for clustering genotypes with related geographical origins.
The PCoA (Principal co-ordinate analysis) were divided into three clusters (Fig 8), which contained the assignments composite by ZN clustering method and population structure (Fig 5 and 7) respectively. The majority of Z. nummularia genotypes belonging to cluster I, ZNST genotypes were distributed in the left half of the PCoA plot. While the rest of the Z. nummularia genotypes from district ZNBU and ZNDR regions belonging to clusters II and III are distributed in the right of the PCoA plot. The distribution of cluster I was more widely scattered than clusters II and III, indicating that Z. nummularia genotypes of the Swat region had higher diversity than the genotypes of Buner and Dir regions. The PCoA results corresponded to cluster analysis as the Swat genotypes were close to Dir and Buner genotypes were joined with Buner region of Z. nummularia genotypes.

Analysis of molecular variance (MOVA)
The genetic diversity was recorded within and among the population of different geographical regions and population structure providing arranged data for the hierarchical AMOVA (analysis of molecular variance), the current total genetic variances among the population 8% while differences among individuals within populations contributed 12% to total variance and 72.00% of the total variance occurred within individuals. The determination of FIS value as 0.102, FIT showed the highest level of genetic variation as 0.105 and FST values 0.092 respectively, suggesting that the xation index value of the Z. nummularia genotypes was highly signi cant (P-Value 0.001), (Table 5).

Discussion
Distribution and climate change Z. nummularia and forecast an especially uncertain future for this rare species of Ziziphus species, which is native of Malakand division, KP, Pakistan. It is looking likely that Z. nummularia is going to harshly reduce ranges, as recommended by the up to 99% decreases of the suitable habitats across all the global climatic models during these periods and due to the result, the plant species are more likely and highly risk extinction by the local restriction.
Climatic changes have already affected by several plants species which were distributed in all over the world and mostly tropical and subtropical region of the world has been identi ed as the most sensitive regions in the (remote) of Pakistan, which the projection percentages species loss will be reach to 60% by 2080 (Appleby et al. 2009).
As Hindukush regions range in the northern regions of Pakistan many plant species, one of the Z. nummularia, we are likely going to face a wave of mass extinction of Z. nummularia genus over the coming periods. However, the high level of genetic diversity of the current Z. nummularia genus may eventually favor adaptation and perseverance remains high ambiguous and species growth will depend on factors such as genetic makeup, tness, healthy habitat, and fragmentation while dispersal approaches, While the seed of Z. nummularia by the lacking of obvious adaptations for the long-distance and brochure, is likely the great dispersal mod, it required the mean way like grazing mammals that consume the seeds along with the foliage cannot be ruled out In the current study, the information generated through multivariate analysis and genetic parameters has given an excellent view of genetic diversity for the 11 quantitative traits for the 180 Z. nummularia genotypes collected from different regions (Swat, Dir, and Buner) of KP, Pakistan. The level of variation was enough to discriminate against many genotypes for Z. nummularia from each other in our study. This agreed with many earlier studies involving morphological traits that High estimated obtained for all diversity parameters indicated allelic richness in the analyzed genotypes, which can be utilized in breeding programmed to get the desired plant types for breeding. 120 total polymorphic alleles were recoded while the highest alleles 6 per locus while 2 is lowest alleles are noted and average values as 4.4286 respectively, this information can be helpful for future genetic analysis speci c to linkage groups. The analysis of molecular variance (AMOVA) results showed major and signi cant (80%; P = 0.001) contribution of within-individual difference to a total variation, whereas amongindividual and among-population differences contributed 12 and 8%, respectively.
For the population, structure performs a Bayesian genetic and admixture by modal STRUCTURE software was assigned all Z. nummularia genotypes of all three different geographical origins into three structure genetic clusters. In addition to assigning individuals to different clusters based on allele frequencies, it also detected the extent of admixture within genotypes of the three genetic clusters obtained with STRUCTURE, clusters Pop 2 broadly contained Z. nummularia genotypes collected from Dir region, (ZNDR), respectively, while Pop 1 and Pop 3 were different types which were a mixture of ZNBU and ZNST regions genotypes. This clustering of pea accessions agreed with the study carried out by Jing et al. (2012) who also showed robust clustering at K= 3, although the germplasm analyzed by them contained different species and subspecies. The mixing seems dominant over the factors such as the self-pollination behavior of the crop and the wide geographical range/barriers. One of the striking observations of the STRUCTURE analysis was that the genotype of ZNDR represented no admixture with the ancestries, with the genotypes of ZNST and ZNBU begin differently from that of other pure ancestries. This indicates that these were probably the regions from which a large amount of dispersal of genotypes has taken place in previous times.
The molecular phylogenetic and PCoA analysis was applied for the identi cation of Z. nummularia genotypes and they were found in ideal molecular markers for the making of molecular phylogenetic relationships in Z. nummularia genotypes of the different geographical origin of KP, Pakistan. The N-J Phylogenetic tree distributed all Z. nummularia genotypes into three clusters, which was in correspondence with our previous studies and while reported studies (Jing et al. 2012;Kumari et al. 2013), who also reported the same number of groups from different plants genotypes from different geographical regions. Furthermore, assignment of genotypes into groups appeared independent of geographic origins but in cluster 1 and cluster 3 has been most of the genotypes were collected from ZNST and ZNBU regions of KP, while cluster 2 were having the ZNDR region of Z. nummularia genotypes from distant geographical locations which obstructed their speci city to geographic origins. Similar results were obtained by Ahmad et al. (2010) in cluster analysis where they found intermixing of Z. nummularia genotypes in different groups. When analyzed concerning gene pools detected by STRUCTURE, the groupings of the N-J tree appeared stronger with a few exceptions where cluster I, 17 genotypes from cluster I from Dir region, while 30 genotypes from cluster III from Buner region, while in cluster II having 3 genotypes from (ZNST 1, ZNST 9 and ZNST 43), respectively, and 9 from cluster I (ZNBU 1, ZNBU 29, ZNBU 33, ZNBU 3, ZNBU 9, ZNBU 43, ZNBU 7, ZNBU 19 and ZNBU 32) respectively, cluster III having a mixture of all three regions genotypes 17 genotypes from cluster I and 15 from cluster II ZNDR, From this, it can be inferred that the clustering of Z. nummularia genotypes was based on genetic stocks regardless of geographic proximities. This could be conveniently ascribed to the movement of germplasm from one location to another from ancient times, the records for which are missing, and presently formal germplasm exchange agreements are in place between different regions as described by Kuleung et al. (2006) and Ahmad et al. (2010). Thus, the present study further suggests that, despite its self-pollinating nature, there is sample intermixing of Z. nummularia genotypes collected from KP, Pakistan, which has resulted in maintaining diversity to a greater extent than can be surveyed and utilized by breeders to develop new improved varieties in the Z. nummularia, the N-J result were complemented with PCoA analysis.
The implication for potential breeding and conservation The global application of molecular markers (Molecular genetic diversity), technologies are attracting more and more common practices to the estimation and study of many aspects of plant genetic resources management (Inouye, 2008). The current study SSRs markers analysis was used to validate taxonomic origin, to determine the distribution of genetic diversity across the collection area of the wild Ziziphus species.
The eld assessment of the collection revealed some interesting traits useful for Ziziphus species genetic improvement (Ferris et al. 2017). Achieving genetic gain through conventional breeding takes a very long time for Z. nummularia because approximately four to ve years is required to complete one cycle of eld data collection and evaluation for the species. Therefore, appropriate planning to preserve individual palms that possess traits of interest should be in place to ensure long-term accessibility. The values of the current potential collection where determined genetic diversity is preserved in a reduced land size can be applied. The establishment of such a collection is an ideal option for oil palm as it increases the e ciency of conservation and allows for more effective access to genetic materials. The results presented above facilitate the identi cation of unique populations or rich allelic individual palms as well as populations that exhibit high genetic variation (Veltman et al. 2019).
The present ndings are strongly suggested that there is wide genetic diversity are present in the wild within genotypes of Z. nummularia collected from the area of KP, Pakistan, conservation approach addressed to preserve single outstanding individuals is necessary, namely for the importance of heritage trees which often become one of the few objects of great natural value in areas under the impact of land degradation and the falling into the decay of rural areas (Veltman et al. 2019). Knowledge of genetic variation within and among populations provides information essential in the formulation of appropriate management strategies for conservation (Sharma et al. 2016). The natural areas where the species of interest occur may provide relevant information to develop sampling strategies that will maximize the probability of collecting genetically distinct samples. Based on the results of the present study, the probability of sampling identical or very similar genotypes of Z. nummularia increases when sites are sampled less than 20 km apart even though even very distinct samples, albeit in a low frequency, can be obtained from the same site, a distance of 20 km between collection sites could be used as a general guideline in future collection missions to maximize the diversity captured for Z. nummularia and while in the current result represented that the Pakistani Z. nummularia genotypes are exceptionally diversity/variable that this can be endorsed to diver's neighborhood genetic basics, speci c reproducing weights and the restricted trade of hereditary material (Uddin et al. 2021). The exceptional nature of the Pakistani Ziziphus species, uncovered by our results, backs the case for the execution of more extraordinary characterization and preservation procedures and potential rearing for Ziziphus species (Z. nummularia).

Conclusion
The effect and the incensement of global climate changes in recent years, while the reason of the climate changes heavy rainfall has caused oods (disaster) in various areas and weather station over the world measured the increasing concentration of atmospheric CO 2 up to 400 pp, the highest in millions of years mostly the climate changes exceedingly highly effects on plants and wildlife, respectively. Hear, Z. nummularia from different geographical locations of Pakistan and China were investigated for their genetic variability using 27 SSR markers and reported extensive diversity within the wild collection with signi cant positive correlation and descriptive analysis showed a high coe cient of variation between the study's morphological traits was noted. We rst time establish SSRs ngerprints 180 genotypes and provide a comprehensive data of genetic diversity and their population structure and their relationships addressed with their geographical/climatic distribution was addressed. Better understanding of plants genetic diversity is essential to all conservation and management effort. Remarkably, wise utilization of conserved plant germplasm in a gene bank collection needs knowledge of the genetic diversity that is present as well. Ensuring sustainable food security is a multi-faceted challenge, involving much more than just increasing food production; it is also about protecting the useful diversity of wild. The results shown here may provide valuable resources for future Ziziphus breeding programs. Further, the application of the recently evolved high-throughput platforms and phenomics tools could enable plant breeders to identify and exploit the available plant genetic resources in environmentally sustainable ways. In brief, the current result will be highly bene cial in designing studies relating to Ziziphus species (Z. nummularia) genotypes phylogeny as well as to nd the biological origin and formulating future breeding programs.

Declarations
Authors Contribution: All authors have contributed as follows: NU performed the molecular biology experiments; NU, MN and NA wrote the paper, NM, MUK and MR helped in reviewing. All authors read, contributed to, and approved the nal manuscript. Figure 1 Showed that the ArcGIS mapping of a bioclimatic variable on the selected regions, showing variation in (A), annual mean temperature and (B), annual precipitating. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Showed that the ArcGIS mapping of a bioclimatic variable on the selected regions, showing variation in (A), Prec-Seasonality and (B), Mean diurnal range, respectively. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 3
Applied ArcGIS mapping of a bioclimatic variable on the selected regions, (A), distribution in the selected remote regions of Z. nummularia genotypes and (B and C), showing accrued temperature ranges. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 4
Photographs collected Z. nummularia fruits and leaves from Malakand Division, KP, Pakistan A to F designated diversity in fruits shape/size and colour and G to I designated for Z. nummularia leaves diversity Showed that the Delta K values for the analysis STRUCTURE of Z. nummularia genotypes, was calculated according to Evanno et al., (2000), is plotted against the number of modeled gene pools (K)