High-throughput SSR marker development and its application in a centipedegrass (Eremochloa ophiuroides (Munro) Hack.) genetic diversity analysis

Centipedegrass (Eremochloa ophiuroides (Munro) Hack.) is a perennial, warm-season C4 grass species that shows great potential for use as a low-maintenance turfgrass species in tropical and subtropical regions. However, limited genetic and genomic information is available for this species, which has impeded systematic studies on the enhancement of its turf quality and resistance against biotic and abiotic stress. In this study, Illumina HiSeq high-throughput sequencing technology was performed to generate centipedegrass transcriptome sequences. A total of 352,513 assembled sequences were used to search for simple sequence repeat (SSR) loci, and 64,470 SSR loci were detected in 47,638 SSR containing sequences. The tri-nucleotides were the most frequent repeat motif, followed by di-nucleotides, tetra-nucleotides hexnucleotides, and pentanucleotides. A total of 48,061 primer pairs were successfully designed in the flanking sequences of the SSRs, and 100 sets of primers were randomly selected for the initial validation in four centipedegrass accessions. In total, 56 (56.0%) of the 100 primer pairs tested successfully amplified alleles from all four centipedegrass accessions, while 50 were identified as polymorphic markers and were then used to assess the level of genetic diversity among 43 centipedegrass core collections. The genetic diversity analysis exhibited that the number of alleles (Na) per locus ranged from 3 to 13, and the observed heterozygosity (Ho) ranged from 0.17 to 0.83. The polymorphism information content (PIC) value of the markers ranged from 0.15 to 0.78, and the genetic distances (coefficient Nei72) between the accessions varied from 0.07 to 0.48. The UPGMA-based dendrogram clustered all 43 core collections into two main groups and six subgroups, which further validated the effectiveness of these newly developed SSR markers. Hence, these newly developed SSR markers will be valuable and potentially useful for future genetic and genomic studies of E. ophiuroides.