Genome-wide simple sequence repeats (SSR) markers discovered from whole-genome sequence comparisons of multiple spinach accessions

The availability of well-assembled genome sequences and reduced sequencing costs have enabled the resequencing of many additional accessions in several crops, thus facilitating the rapid discovery and development of simple sequence repeat (SSR) markers. Although the genome sequence of inbred spinach line Sp75 is available, previous efforts have resulted in a limited number of useful SSR markers. Identification of additional polymorphic SSR markers will support genetics and breeding research in spinach. This study aimed to use the available genomic resources to mine and catalog a large number of polymorphic SSR markers. A search for SSR loci on six chromosome sequences of spinach line Sp75 using GMATA identified a total of 42,155 loci with repeat motifs of two to six nucleotides in the Sp75 reference genome. Whole-genome sequences (30x) of additional 21 accessions were aligned against the chromosome sequences of the reference genome and in silico genotyped using the HipSTR program by comparing and counting repeat numbers variation across the SSR loci among the accessions. The HipSTR program generated SSR genotype data were filtered for monomorphic and high missing loci, and a final set of the 5986 polymorphic SSR loci were identified. The polymorphic SSR loci were present at a density of 12.9 SSRs/Mb and were physically mapped. Out of 36 randomly selected SSR loci for validation, two failed to amplify, while the remaining were all polymorphic in a set of 48 spinach accessions from 34 countries. Genetic diversity analysis performed using the SSRs allele score data on the 48 spinach accessions showed three main population groups. This strategy to mine and develop polymorphic SSR markers by a comparative analysis of the genome sequences of multiple accessions and computational genotyping of the candidate SSR loci eliminates the need for laborious experimental screening. Our approach increased the efficiency of discovering a large set of novel polymorphic SSR markers, as demonstrated in this report.