2018 ASHS Annual Conference

The decreasing costs of genome sequencing and the availability of well-assembled genome sequences for many crops has facilitated the identification and development of simple sequence repeats (SSR) markers. The genome sequence of inbred spinach line Sp75 (168X coverage) is publicly available. Genome sequences of six Sp75 chromosomes (463.4 Mb) were used to search for SSRs using the MISA program. The search criteria were set for the minimum number of repeats of 6, 5, 4, 4, 4 for di-, tri-, tetra-, penta-, and hexa- repeats respectively, and the maximal number of nucleotides interrupting two SSRs were set to 1. In total, 42,155 SSRs were identified from Sp75 chromosome scaffolds in 40,552 loci. Stepwise removal of compound loci (1450), di-repeats loci (15, 942), and repeat motifs containing only A and T (10, 529) reduced the number of unique SSR loci to 12, 631. The remaining tri-, tetra-, penta-, hexa- repeats SSRs were sorted according to the chromosomes. Subsequently, SSR motifs along with 250 bp from either side of the motif were extracted, which serves as the reference sequence. A whole-genome resequencing (30x) of 30 additional spinach genotypes was completed. Paired-end Illumina reads from the resequenced accessions will be aligned against the SSRs containing reference sequence using BWAmem. Visualization of aligned sequence in the Tablet software allows in silico identification of polymorphic loci. A subset of polymorphic loci identified following a visual inspection using the Tablet program will be amplified using fluorescent primers and the allele sizes will be determined using capillary electrophoresis. We plan to use the newly developed polymorphic SSRs to fingerprint spinach genotypes and to conduct genetic diversity assessment of USDA spinach germplasm.