*2.5. Calculation of Clustering*

The heterozygosity (He), observer gene number (Na), effective alleles (Ne), observed heterozygosity (Ho), and the Shannon–Weaver index (I) were calculated using Pop-gen software v.1.32 (Canada, University of Alberta). Polymorphic information content (PIC) of SSR markers was computed using EXCEL (China, WPS of JINSHAN). When the PIC of an SSR marker was below 0.25, it was considered as a low polymorphic marker, and a marker was considered highly polymorphic if its PIC was above 0.5.

These amplification bands of each SSR primer were separated using polyacrylamide gel-electrophoresis. The band patterns were visualized with silver staining, and gel images were taken with a digital camera. In the same location, the presence of a band was marked as "1", the absence of a band was marked as "0", and a missing band was marked as "−1". In this study we used Genalex-6 software [31] to conduct the matrix calculation of SSR marker data which had been assigned a value, then transformed it into a triangle matrix, saved it as a mega-file, finally, imported the mega-file into the Mega-6.0 software (USA, Tamura, K team), and selected the unweighted pair group method with arithmetic (UPGMA) algorithm in the "phylogeny" dropdown menu to draw the cluster diagram [32].

The software Structure v.2.3 (USA, UChicago; Britain, Oxon) was used to analyze the population structure [33,34]. An admixture model and correlated allele frequencies were used to estimate the number of the populations. For each of the K-values (ranging from 1 to 5), ten independent runs were performed with a burn-in period of 100,000 followed by 500,000 Markov chain Monte Carlo runs. The optimal K-values depends on the peak of K = mean (|Ln"P(D)|)/(sdLnP(D)). Based on the structure results, the most probable K-value was analyzed using Structure Harvester (http://taylor0.biology.ucla.edu/struct\_ harvest/, 2020).
