Assessment of Genetic Diversity using EST Derived SSR Markers in Pomegranate (Punica granatum L.) Germplasms

Pomegranate (Punica granatum L.) is an economically important perennial crop, with high nutritional, medicinal and ornamental importance. Total 2417 dbEST sequences were downloaded from NCBI and after pre-processing, 1152 sequences were assembled by CAP3. On evaluating by MISA tool, 175 sequences were found to be possessing SSR motifs and further subjected to BlastX hit, 125 sequences were annotated with gene ontology numbers. The distribution of SSR motifs highlighted di-nucleotide repeats as the highest (59.8%) followed by tri-nucleotide (37.7%). Total 124 successful primer pairs were designed and on the basis of unigene function, randomly 32 primer pairs were selected for validation on 46 germplasm. Eight EST-SSR primers were found to be polymorphic which generated 433 scorable bands with average of 54 bands per primer. The PIC and heterozygosity ranged from 0.178 to 0.555 and 0.198 to 0.625 respectively. Thirteen out of 20 gSSR primers were found to be polymorphic which generated 627 scorable bands with average of 48 bands per primer. The PIC and heterozygosity ranged from 0.175 to 0.374 and 0.194 to 0.498 respectively. Clustering pattern of pooled data of EST-SSR and gSSR showed major two clusters A and B having similarity coefficient of 0.37 to 1.00. The results of cluster analysis revealed grouping patterns based on geological distributions and derivation relationships. Thus, the developed EST derived SSR markers based on functional annotation of sequences could be very useful for various research areas in pomegranate, such as identification of the economically important pomegranate cultivars, study evolutionary origin analysis, genetic linkage map construction and marker-assisted selection for breeding.