Genetic Diversity among Some Walnut (Juglans regia L.) Genotypes by SSR Markers

The food needs for increasing population, climatic changes, urbanization and industrialization, along with the destruction of forests, are the main challenges of modern life. Therefore, it is very important to evaluate plant genetic resources in order to cope with these problems. Therefore, in this study, a set of ninety-one walnut (Juglans regia L.) accessions from Central Anatolia region, composed of seventy-four accessions and eight commercial cultivars from Turkey, and nine international reference cultivars, was analyzed using 45 SSR (Simple Sequence Repeats) markers to reveal the genetic diversity. SSR analysis identified 390 alleles for 91 accessions. The number of alleles per locus ranged from 3 to 19 alleles with a mean value of 9 alleles per locus. Genetic dissimilarity coefficients ranged from 0.03 to 0.68. The highest number of alleles was obtained from CUJRA212 locus (Na = 19). The values of polymorphism information content (PIC) ranged from 0.42 (JRHR222528) to 0.86 (CUJRA212) with a mean PIC value of 0.68. Genetic distances were estimated according to the UPGMA (Unweighted Pair Group Method with Arithmetic Average), Principal Coordinates (PCoA), and the Structure-based clustering. The UPGMA and Structure clustering of the accessions depicted five major clusters supporting the PCoA results. The dendrogram revealed the similarities and dissimilarities among the accessions by identifying five major clusters. Based on this study, SSR analyses indicate that Yozgat province has an important genetic diversity pool and rich genetic variance of walnuts.


Introduction
The walnut plant, botanically known as Juglans regia L., is categorized in genus Juglans and belongs to the Juglandaceae family. The Juglans genus has more than 20 species [1]. J. regia L., known as the common walnut (English or Persian walnut), is a long-lived, deciduous, monoecious, open-pollinated, and generally dichogamous plant. J. regia is a diploid plant with a haploid chromosome number of n = 16 [2].
Numerous studies have been conducted on walnut fruits concerning biochemical, phytochemical, and antioxidant characteristics, and their contribution to human health and nutrition. Walnut kernel has a high amount of protein, fat, vitamins, minerals, and polyphenols, therefore, placing walnut in the human diet is inevitable [3,4]. Walnut has

DNA Extraction
Total genomic DNA was isolated from fresh leaves by the CTAB method described by Doyle and Doyle [32] with some modifications [11]. The Qubit Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) was used to quantify isolated DNA. Followed by diluting the extracted DNA to 10 ng/µL for SSR-PCR reactions, the samples stored at −20 • C.

SSR-PCR Reactions
Eight walnut cultivars were used for testing amplification success and degree of polymorphism of forty-eight previously published SSR primer pairs (Table 2). Finally, 45 SSR primer pairs were selected for the characterization of 91 walnut accessions. All SSR-PCR reactions were done based on a three-primer strategy according to Scheulke [33] with minor modifications. A total volume of 12. When the PCRs were completed, the reactions were subjected to denaturation for capillary electrophoresis in an ABI 3130xl genetic analyzer (Applied Biosystems Inc., Foster City, CA, USA (ABI)) using a 36-cm capillary array with POP7 as the matrix (ABI). Samples were denatured by mixing 0.5 µL (in 6-FAM and VIC labeled primers) or 1.0 µL (in NED and PET labeled primers) of the amplified product, 0.3 µL of the size standard and 9.7 µL of Hi-Di formamide. The ABI data collection software 3.0 was used for resolving the fragments, and then SSR fragment analysis was done using the GeneMapper 4.0 (Applied Biosystems Inc. Bedford, MA, USA).

Genetic Diversity
The effective number of alleles (Ne), expected heterozygosity (He), the number of alleles per locus (Na), and observed heterozygosity (Ho) were calculated using the GenAlEx version 6.5 program [34]. PIC for the loci was calculated using PowerMarker software version 3.25 [35].

Population Relationship
The dendrogram, based on shared allele genetic distance was constructed using the UPGMA (Unweighted Pair Group Method with Arithmetic Average) method implemented in Molecular Evolutionary Genetics Analysis (MEGA) Program v. 10.2.2 [36]. Principal Coordinates (PCoA) based clustering was also done using the GenAlEx version 6.5 program. STRUCTURE 2.3.4, the model-based software, was used for population structure and identification of admixed individuals [37]. In this model, a number of populations (K) are considered to be available, which each of them is characterized by a set of allele frequencies at each locus. Individuals in the sample are given to populations (clusters), or jointly to more populations if their cultivars indicate that they are admixed. Ln P (D) values (logarithm probability for each K) were applied to determine the Delta K indicating the probable population number. The term of Delta K is calculated by the change ratio of logarithm probability (∆K = 2 to ∆K = 10). In the diagram, the highest K of Delta K confers the information about the probable population number.

Polymorphism Levels of SSR Loci
Of the screened 48 SSR primer pairs, three failed in the amplification, and the 45 remaining SSR markers generated polymorphic alleles for the eight walnut accessions tested, and they were consequently used for their genetic characterization ( Table 2).
The genetic diversity analysis of walnut accessions included the average number of alleles (Na), number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and the polymorphism information content (PIC) ( Table 3). Following the statistical analysis of the 45 polymorphic loci, in total, 390 alleles were detected for all studied accessions, and the number of alleles varied between 3 to 19 alleles per locus with a mean value of 9. The highest number of allele was obtained from the CUJRA212 locus (Na = 19). The number of effective alleles (Ne) ranged from 1.89 (JRHR224485) to 7.92 (CUJRA212) with a mean of 4.06. The observed heterozygosity (Ho) changed from 0.34 (JRHR225388) to 0.96 (JRHR227254) with a mean of 0.65. Observed heterozygosity (He) was the highest in the CUJRA212 loci. The average value of expected heterozygosity (He) was 0.73 and the highest value (0.87) was seen in CUJRA212 locus. Polymorphism information content (PIC) of the loci ranged from 0.42 (JRHR222528 and JRHR224485) to 0.86 (CUJRA212) with an average of 0.68 (Table 3).
Principal Coordinate Analysis (PCoA) was done to envision the relationship between the cultivars in more detail. The variations expressed on axes 1, 2, and 3 were 4.60, 4.25, and 3.01%, respectively. The first principal component accounts for 11.86% of the total variance. Observation of the resultant matrix of PCoA (Figure 2b), showed that these walnut accessions were sorted into five main clusters. PCoA Cluster-I included eight commercial cultivars (USA, France, and Unknown), PCoA Cluster-II included nine commercial cultivars (USA and Turkey) and 13 accessions (Turkey), and PCoA Cluster-III, IV, and V included a totally 61 accessions (Turkey). Foreign commercial cultivars sorted into Cluster-I, and all Turkish commercial cultivars and one foreign cultivar (Midland) were sorted into Cluster-II. The remaining sixty-one accessions sorted into Cluster-III, IV, and V. The results of the PCoA showed that all accessions are separated from each other. Overall patterns of genetic differentiation denoted using the UPGMA (Figure 2a and b) and PCoA were in accordance with each other.
Structural genetic analysis was performed in 91 walnut accessions using 45 amplified loci by STRUCTURE and STRUCTURE HARVESTER programs. The highest value of Delta K (ΔK) was obtained at ΔK = 5 (Figure 3). ΔK = 5 corresponded to the most possible number of populations in the study. Thus, all cultivars are categorized into five major clusters similar to the UPGMA and the PCoA based clustering results (Figure 2a and b).  (Table S1).
Principal Coordinate Analysis (PCoA) was done to envision the relationship between the cultivars in more detail. The variations expressed on axes 1, 2, and 3 were 4.60, 4.25, and 3.01%, respectively. The first principal component accounts for 11.86% of the total variance. Observation of the resultant matrix of PCoA (Figure 2b), showed that these walnut accessions were sorted into five main clusters. PCoA Cluster-I included eight commercial cultivars (USA, France, and Unknown), PCoA Cluster-II included nine commercial cultivars (USA and Turkey) and 13 accessions (Turkey), and PCoA Cluster-III, IV, and V included a totally 61 accessions (Turkey). Foreign commercial cultivars sorted into Cluster-I, and all Turkish commercial cultivars and one foreign cultivar (Midland) were sorted into Cluster-II. The remaining sixty-one accessions sorted into Cluster-III, IV, and V. The results of the PCoA showed that all accessions are separated from each other. Overall patterns of genetic differentiation denoted using the UPGMA (Figure 2a,b) and PCoA were in accordance with each other.
Structural genetic analysis was performed in 91 walnut accessions using 45 amplified loci by STRUCTURE and STRUCTURE HARVESTER programs. The highest value of Delta K (∆K) was obtained at ∆K = 5 (Figure 3). ∆K = 5 corresponded to the most possible number of populations in the study. Thus, all cultivars are categorized into five major clusters similar to the UPGMA and the PCoA based clustering results (Figure 2a,b). The dendrogram of the relationships of accessions was very similar to structural genetic analysis.

Discussion
Molecular characteristic was used to elucidate the genetic diversity of 91 accessions.

SSR Polymorphism
In previous studies, Eser et al. [30], Dangl [48], and Orhan et al. [49] used SSRs to determine high genetic diversity and relationships of cultivated walnut accessions. We also present here high polymorphic SSR loci among walnuts (Tables 2 and 3).
Several studies revealed that SSR loci can be beneficial to evaluate molecular fingerprinting of Juglandaceae family species as Juglans regia. For example, in a study by Orhan et al. [49], a total of 135 polymorphic alleles with an average of 6.43 alleles per locus were obtained from 21 SSR primers among the 32 local divers walnut genotypes. Balapanov et al. [48] tested 11 SSR primers for 62 walnut genotypes and a total of 104 alleles were obtained in walnut tested genotypes with a mean value of 9.4. In another study, seven SSR loci were polymorphic in 15 diverse walnut cultivars from Ukraine. A total of 69 alleles were produced with an average of eight alleles per locus [47]. Bernard et al. [46] tested 13 SSR primers for 217 worldwide accessions and 116 alleles were detected with a mean value of 8.9. In an experiment by Vahdati et al. [45], 17 SSR primers were tested for six walnut populations and 147 alleles were detected with a mean value of 5.16. Pop et al. [44] tested seven polymorphic SSR primers for 20 walnut cultivars and reported 6.7 alleles per locus. Mahmoodi et al. [43] tested 16 walnut accessions and five cultivars using nine SSR markers, and they reported 34 alleles with a mean of 4.25. Kim et al. [42] tested eight Korean and 12 foreign walnut cultivars using 12 SSR primer pairs and they obtained an average of 9.6 alleles per locus. In a study, a total of 97 alleles were generated by 32 SSR loci with an average of five alleles per locus [41]. Pollegioni et al. [40] examined 29 Italian walnut genotypes using 12 SSR primers and they obtained a total of 62 alleles and an average of 6.2 alleles per locus. Karimi et al. [39] detected 63 alleles with a mean value of 5.73 from 11 SSR primers in the genetic structure of 105 walnut individuals. Moreover, Dangl et al. [38] tested 14 SSRs for 44 walnut genotypes and the number of alleles per locus ranged from three to eight with an average of 5.2. In the present study, 390 alleles with an average of nine alleles per locus were detected in the genetic characterization of ninety-one accessions. However, Kim et al. [42] and Balapanov et al. [48] reported the highest number of averages as 9.6 and 9.4, respectively. Although there are minor differences in polymorphic alleles in different studies due to the selection of accessions or cultivars, the results obtained in this study are in agreement with previous studies on genetic diversity in walnut accessions achieved by SSRs. Genetic and phylogenetic distance within the genotypes may also affect the polymorphism level detected by the SSR markers due to their out crossing nature.
Our results showed that the level of detected diversity was higher than many studies in the literature, with an average of expected and observed heterozygosities per locus. The reasons for this could be explained by specific characteristics of our populations because they obtained from seeds and showed great diversity due to open pollination.
In the current study, the average He and Ho were found as 0.73 and 0.65, respectively, while Orhan et al. [49] reported them as 0.62 and 0.60, Balapanov et al. [48] found them as 0.75 and 0.67, Khokhlov et al. [47] calculated them as 0.80 and 0.73, Bernard et al. [46] reported them as 0.56 and 0.47, Vahdati et al. [45] found 0.79 and 0.23, Pop et al. [44] calculated them as 0.72 and 0.65, Mahmoodi et al. [43] reported them as 0.62 and 0.63, Ruiz-Garcia et al. [41] found them as 0.57 and 0.51, Pollegioni et al. [40] reported them as 0.64 and 0.60, and Karimi et al. [39] calculated them as 0.66 and 0.68. As a result, in this study, the average He and Ho were found higher compared to many studies in the literature. This might be due to using accessions that were wildly grown by seeds in a region that contains rich genetic resources that exhibited high heterozygosis. Although the genetic diversity of perennial species has resulted in fundamental changes in the mode of reproduction, cultivated walnuts are probably obtained from the selection of seedlings belong to different geographical regions of natural populations over years [50].
Polymorphism information content (PIC) refers to the value of a marker for detecting polymorphism within a population, depending on the number of detectable alleles and the distribution of their frequency [11]. The number of expressed alleles and frequency of alleles per locus which are equivalent to genetic diversity was determined by PIC of which is considered. In the current study, the PIC values were between 0.42 and 0.86 with an average of 0.68, while Orhan et al. [49] [39] reported 0.49 and 0.85 with an average of 0.68. As a result, in this study, the average PIC was found lower compared to the study conducted by Vahdati et al. [45]. The differences among studies may derive from using different SSR markers, or the locations of samples that may collect from much more diverse areas, even different continents [51]. However, higher genetic diversity values were obtained in this study compared to other studies in the literature. Pollegioni et al. [52] also reported a clear longitudinal trend of walnut genetic diversity in Eurasia, with loss of allelic richness and heterozygosity in Europe and reducing effective population size. However, the researchers reported a high degree of genetic diversity in walnuts in the Eurasia region including Turkey. These results supported the high genetic diversity and allelic richness exhibited in this study. Because, firstly, Turkey is located in the eastern Mediterranean basin survived after the Last Glacial Maximum, which sheltered rich genetic resources [53], secondly accessions collected from the Yozgat province have not been studied before and are unique wild accessions. However, the evolution of walnuts during decades also accounts for genetic diversity among accessions and cultivars. Although humans played a role in shaping the modern genetic structure, biogeographic events such as climate changes and socio-economic pressures should not be neglected in the evolution of walnut [53]. Productivity of adult walnut trees for at least 40 years, and surviving the nuts up to two years under simple storage conditions makes it easier to be transported short-and long-distance by humans causing the wide distribution of walnut worldwide [50].
On the other hand, the introduction of walnut to its new habitats may be dispersed easier by similarities in human language over large geographic areas. This may lead to the genetic homogenization of different populations known as the human role in walnut evolution by merging the plant biology and germplasm dispersal processes data with human cultural and linguistic diversity [54].

Genetic Relationships among Walnut Accessions
The results of SSR-based structural genetic analysis, the UPGMA, and the PCoA based clustering were similar to the genetic relationships of walnut accessions (Figure 2a-c). Seventeen commercial cultivars were collected from different origins (USA, French, Unknown, and Turkey). As reported by Orhan et al. [49], Khokhlov et al. [47], Pop et al. [44], Mahmoodi et al. [43], Kim et al. [42], Ruiz-Garcia et al. [41] some of these commercial cultivars were clustered in same groups which agreed with the reports of the current study ( Figure 2). This result in this study provides that SSRs used in this study were enough to discriminate against the cultivars. The powerful dendrogram was obtained in this study that matches relationships reported among commercial walnut cultivars in previous studies [41][42][43]49]. The results are also imply that geographic barriers have shaped the distribution of walnut genetic resources.

Conclusions
In this study, walnut accessions analyzed by three different genetic analyses revealed that all ninety-one cultivars, which were fingerprinted using 45 SSR markers, were grouped at clusters with the same geographic origins. The assessment of the molecular diversity of walnut accessions is considerable for the optimal development of programs aiming the conservation of cultivated and wild genotypes in their ecosystems. SSR markers were distinctively capable of identifying all the studied walnut accessions in this study and proved that SSR markers are a potential tool for utilization in walnut breeding programs, genetic diversity, and germplasm characterization. These results could provide essential information for further understanding the genetic differentiation and utilization strategies for walnut germplasm. These markers as trustworthy tools can be applied for the evaluation of genetic diversity and relationships of walnut accessions in future molecular studies.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/su13126830/s1, Table S1: Walnut Table. Author Contributions: M.G., S.K. and M.Z. were responsible for in silico SSRs and SSR data analysis. M.G., S.E., T.N. and G.B. coordinated and organized all research activities. M.Z., H.K. and M.A.G. performed DNA extractions and PCR reactions. All authors contributed to writing and editing the manuscript. All authors read and approved the published version of the manuscript.

Conflicts of Interest:
The authors declare that they have no conflict of interest.