Genome-wide characterization and development of simple sequence repeat markers for genetic studies in pomegranate (Punica granatum L.)

Abstract

Genome-wide characterization and development of first set of polymorphic Class I SSRs markers in pomegranate (Punica granatum L.) through in silico analysis using the draft genome sequence of pomegranate cv. Dabenzi (296 Mb) as reported by Qin et al. (2017). The availability of the draft genome sequence of pomegranate cv. Dabenzi presents unprecedented opportunities for the development of largescale genomic resources, such as DNA markers for genotyping applications. In this study, we identify a new set of highly polymorphic simple sequence repeat (SSR) markers by targeting the SSR motif lengths of ≥ 24 bp. A total of 1,73,633 SSRs were identified in the 296-Mb pomegranate genome assembly, reflecting an average density of 527.97 SSRs/Mb. Of these, 43,853 SSRs belong to Class I category (> 20 bp). Concerning the abundance of repeat types in the current dataset, dinucleotide (NN) repeats (31.19%) were the dominant class among all SSRs identified in the genome, followed by tetranucleotide (NNNN: 20.5%) and trinucleotide repeats (NNN: 16.8%). The top two SSR motifs in NN category were AT/AT (64.90%) and AG/CT (28.51%), whereas AAT/ATT (34.66%) and AAG/CTT (28.91%) were the most abundant among NNN repeats. Primer pairs were designed for a total of 2856 Class I SSRs and 110 primers were then assayed initially on eight pomegranate genotypes for polymorphism survey. Polymorphic fragments were obtained for 82 SSRs (77.36%), of which a subset of 13 informative SSRs was further employed to investigate genetic diversity among 46 pomegranate genotypes. Approaches, such as population structure, cluster and PCA elucidated genetic relationships among 46 diverse pomegranate genotypes. In summary, here we developed the first set of genome-wide SSRs in pomegranate that will serve as a powerful genomic tool for future genetic studies. These SSRs have widespread applications in QTL mapping and marker-assisted selection for breeding.