Development and identification of SSR molecular markers based on whole genomic sequences of Punica granatum

Objective The number of polymorphic SSR markers developed through cDNA library is limited. In order to promote studies on genetic diversity, genetic mapping and variety identification, it is necessary to develop molecular marker loci.

Method In this study, SSRs with 1−6 nucleotide repeats were searched from the published whole genome sequences of Punica granatum using MISA and the characterization of SSR sequences and their polymorphisms were analyzed.

Result (1) A total of 146 445 SSR loci were detected in whole genome sequences of Punica granatum. Then content of mono-nucleotide repeat SSRs was the highest, accounting for 51.95%; in contrast, the content of hexa-nucleotide repeat SSRs was the lowest, only 0.38%. In all SSR sequences, most of bases were A/T, showing a bias in SSR sequences. (2) The range of SSR length ranged from 10 to 252 bp, with an average of 15.48 bp. The SSR length among different types of SSRs varied a lot, and the abundance of SSR sequences tended to decrease with the increase of repeat number. (3) In designed 140 pairs of primers including different types of SSRs, valid fragments could be produced with 119 pairs in 12 germplasms of Punica granatum and 41 pairs could produce polymorphic fragments with 0.007−0.566 polymorphic information content (PIC). We further screened 15 pairs of primers with polymorphic and stable fragments, which detected 44 alleles in these germplasms of Punica granatum, with average of 2.933 3 alleles per SSR locus.

Conclusion SSR markers can be developed in large-scale based on whole genome sequences of Punica granatum and a large number of SSR primers also can be identified for genetic diversity analysis, genetic mapping and variety identification. This study provides lots of SSR information and marker resources for researching genetics and breeding of Punica granatum.