Development of genomic SSR markers for characterization of genetic diversity in wild pomegranate germplasm

Wild pomegranate is a high-value crop for pharmaceutical industries. The lack of genetic and genomic resources in this medicinally important plant requires the development of simple sequence repeat (SSR) markers necessary for breeding programmes. In the present study, genomic sequences obtained using the Illumina platform revealed the presence of 8,000 sequences having genomic simple sequence repeats (gSSRs), with dinucleotide repeats being the most abundant (50.96%). A total of 103 marker pairs were randomly designed for the validation of 45 wild pomegranate accessions for diversity analysis. The amplicon size of developed gSSRs ranged between 141 and 297 base pairs. From a total of 57 polymorphic primers, 21 SSR pairs revealed 100% polymorphism. The existence of high genetic diversity (average Na-0.506, He-0.395, Ho-0.246) indicated the presence of heterozygotes in the wild pomegranate population. The polymorphism information content value ranged from 0.01 to 0.53, with the Shannon index values being between 0.09 and 1.60. Further, the genetic dissimilarity value ranged from 0.05 to 0.45, which grouped the wild pomegranate accessions into three clusters with two out-groups through a neighbour-joining analysis. The analysis of molecular variance indicated 25 and 75% of the total variation between and within the accessions, and population genetic structure research grouped the wild pomegranate accessions into four populations, which were partly bolstered by a principal component analysis and a neighbour-joining tree. Cross-species amplification demonstrated that out of 57 SSRs, 26 SSR loci had the transferability rate in cultivated pomegranate cultivars Ganesh and Dholka and 18 in Kandhari and Bedana, whereas the transferability rate was between 13.46 and 26.92% for plants from other families. Unique alleles were observed for both cultivated pomegranates and three cross-species tested in the present investigation. This is the first report where developed genomic SSRs would contribute to the genomic resources that will provide the basis for breeding-aided programmes for wild pomegranates.