Abstract
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.
Highlights
Simple sequence repeats (SSRs) are a valuable source of genetic markers because of their abundance, high rate of polymorphism, ubiquitous distribution throughout the genome, codominant inheritance, high extent of allelic diversity, and ease of assay by polymerase chain reactions (PCR) [1,2]
According to the classification basis for the marker loci informativeness level proposed by Botstein et al (1980) [58], all the E. ophiuroides simple sequence repeat (SSR) validated in the present study indicated a moderate level (0.50 > polymorphism information content (PIC) > 0.25) to a high level of informativeness (PIC > 0.5), with an average PIC value of 0.58, with the exception of TJIB.Eo_016 (PIC = 0.15), which was considered a poor marker in these respects
The present work represents a substantial advance in the identification of a large number of informative SSR loci in E. ophiuroides by high-throughput RNA sequencing technology based on the Illumina HiSeq 2000 platform
Summary
Simple sequence repeats (SSRs) are a valuable source of genetic markers because of their abundance, high rate of polymorphism, ubiquitous distribution throughout the genome, codominant inheritance, high extent of allelic diversity, and ease of assay by PCR [1,2]. SSRs are considered excellent molecular markers in studies of germplasm characterization, genetic diversity, and genetic mapping [3,4]. Recent advances in next-generation sequencing (NGS) technologies provide a cost-effective, convenient and reliable approach for sequence information acquisition in non-model species and greatly accelerated the development process for molecular markers [6,7]. RNA-Seq, which is based on NGS, is a high-throughput technology that has great advantages in obtaining a large amount of sequence data for SSR mining [8]. There is not a large quantity of SSR markers developed in centipedegrass, with the exception of a recent report on EST-SSR development from a cold-stressed transcriptome of centipedegrass [21]
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