Analysis of genetic diversity and population structure of walnut (Juglans regia L.) in Central Asia ecological region based on SSR markers

Abstract:【Objective】In the distribution areas of walnut (Juglans regia L.) in Central Asia, research- ers in different countries have studied the genetic diversity of walnut in their own countries. However, there have been few studies on the germplasm of walnut in several regions, especially the related studies between Xinjiang and many other countries in Central Asia. In this study, genetic diversity and popu- lation genetic structure of 137 walnut germplasm resources collected from four regions of Ili, Kashgar, Hotan and Aksu and six regions of Kyrgyzstan and Tajikistan were analyzed by SSR (Simple Sequence Repeat) molecular markers. The genetic diversity and genetic relationships of walnut germplasm in six regions of Xinjiang and Central Asia were explored at the molecular level, in order to provide material references for further revealing the origin of walnut in the Central Asian ecology-region.【Methods】137 accessions of walnut germplasm resources were collected a in six areas with individual random sam- pling, including 19 of Kashgar region, 15 of Aksu area, Hotan region in 21, 31 of Ili region, 37 of Kyr-gyzstan, 13 of Tajikistan. The fresh walnut leaves collected were wiped clean and immediately put into the self-sealed bag containing silica gel for preservation. The DNA of each material was extracted by Plant Genomic DNA Extraction Kit (Tiangen Company). After testing the quality and concentration of DNA,30 accessions of randomly selected walnut germplasms were used to construct the six group’s genomic DNA mixed gene pool, using published 64 SSR primers .The PCR products were detected by 1% agarose gel electrophoresis, and the bright and clear primers were screened and amplified. The 14 pairs of primers screened were used for PCR amplification of all the test materials. PCR products were sepa- rated with 6% non-denatured polyacrylamide gel electrophoresis. The bands were detected by silver staining. The amplification of each primer in each individual was counted. Genetic diversity indices including the number of alleles (Na), the number of effective alleles (Ne), Shannon's information index (I) and allele frequency were calculated by Popgene32 software, and the genetic structure indices were calculated. The results included genetic differentiation coefficient (Gst), gene flow between populations (Nm), Nei’s standard genetic distance (GD) and genetic consistency (GI). Clustering analysis of 137 walnut germplasm was carried out using NTSYS- PC2.10 program and the map was made. 【Results】A total of 130 alleles were detected by 14 pairs of SSR primers, with the range of variation ranging from 6 to 13. An average of 9.285 7 alleles could be detected by each pair of SSR primers. The percentage of 14 SSR loci ranged from 0.625 7 to 0.865 1, with an average of 0.763 4. The average Shannon's diversity index (I) was 0.459 8. The mean value of total genetic diversity (Ht) was 3.248 1. The mean value of gene diversity (Hs) within the population was 2.108 9; The genetic differentiation index (Gst) between populations ranged from 0.057 8 to 0.406 8, with an average of 0.259 5. The mean value of gene flow (Nm) was 3.017 4; All allele frequencies were not uniformly distributed. UPGMA clustering results showed that 137 samples were divided into three groups when the genetic similarity coefficient was 0.75. The first group consisted of 42 samples, including 16 samples from Aksu, 21 samples from Hotan and 5 samples from Kyrgyzstan. The second group contained 44 accessions, including 12 samples from Ili and 32 samples from Kyrgyzstan. The third group contained 51 samples, including 19 samples from Kashgar, 13 samples from Tajikistan and 19 samples from Ili. To a certain extent, the clustering results indicated the distance of the genetic relationship among the populations, and the overall distribution were related to the geographical distribution, and there was a certain gene exchange among the different regions. The analysis results of genetic distance and genetic consistency were consistent with the tree cluster diagram constructed by UPGMA method based on genetic distance frequency.【Conclusion】14 pairs of SSR primers with clear bands and strong polymorphism in walnut were screened , The genetic diversity of the six regions detected by SSR markers was high, among walnut germplasm resources from different geographical distribution in the Central Asian ecological region, and the level of genetic differentiation among populations was high. The gene exchange among walnut communities was high, which might be caused by the long-distance dispersal of walnut seeds by human or birds The walnut germplasm in Ili region would be likely the origin of walnut in Central Asia.