Characterization and Development of EST-SSR Markers from a Cold-Stressed Transcriptome of Centipedegrass by Illumina Paired-End Sequencing

Centipedegrass ( Eremochloa ophiuroides ) is an important warm-season grass native to the temperate and tropical regions of China. However, only a few centipedegrass molecular markers were available, which hindered genetic studies. Simple sequence repeats (SSRs) can provide abundant genetic information but the traditional method of their development was time-consuming and labor-intensive. Now, RNA-seq platform provides a relatively fast and financially affordable approach for large-scale development of SSRs. Therefore, the transcriptome of centipedegrass under cold stress was determined using Illumina paired-end sequencing in this study. Based on the 56 million paired-end reads, 45,575 unigenes with an average of 748 nt in length were assembled. A total of 8539 SSR loci were then identified. Among them, the tri-nucleotide SSRs were the most dominant repeat type (60.80%, 5192), followed by mono- (16.56%, 1414), di- (14.28%, 1219), hexa- (3.36%, 287), penta- (2.87%, 245), and tetra-nucleotide (2.13%, 182) repeats. The most frequent repeat units from mono- to hexa-nucleotide repeats were A/T, AG/CT, CCG/CGG, AAAG/CTTT, AGGGG/CCCCT, and AGGGCG/CCCTCG, respectively. We successfully designed 3511 primer pairs, and then, randomly selected 99 pairs to evaluate their interspecific transferability and preliminarily analyze the genetic diversity of centipedegrass accessions. There were 90 primer pairs that could successfully amplify targets in at least one centipedegrass samples, and 81 of them could also function in at least three of six other Poaceae relatives and 79 showed polymorphism. Based on the 79 polymorphic markers, all centipedegrass accessions could be divided into two groups, i.e., group 1 from Guangxi province and group 2 from other regions. The EST-SSRs from this study provided a useful tool and foundation for genetic diversity, linkage mapping, comparative mapping, and molecular marker-aided breeding in centipedegrass.