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.假俭草（Centipedegrass，学名Eremochloa ophiuroides (Munro) Hack.）是多年生暖季型C4草本植物，在热带与亚热带地区作为低维护草坪草种具备极高应用潜力。然而该物种的遗传与基因组学信息较为匮乏，这极大阻碍了其草坪品质提升及抗生物、非生物胁迫能力改良的系统性研究。本研究采用Illumina HiSeq高通量测序技术获取假俭草转录组序列，以总计352,513条组装序列为基础检索简单序列重复（simple sequence repeat, SSR）位点，最终在47,638条含SSR的序列中检测到64,470个SSR位点。其中三核苷酸重复基元为最优势重复类型，其次依次为二核苷酸、四核苷酸、六核苷酸与五核苷酸重复基元。研究人员成功在SSR侧翼序列中设计出48,061对引物，并随机选取100对引物在4份假俭草种质材料中开展初步验证：结果显示，100对测试引物中有56对（占比56.0%）可在全部4份种质中成功扩增出等位基因，其中50对被鉴定为多态性分子标记，随后利用该50对标记对43份假俭草核心种质的遗传多样性水平进行评估。遗传多样性分析结果表明，每位点的等位基因数（number of alleles, Na）范围为3~13，观测杂合度（observed heterozygosity, Ho）介于0.17~0.83之间；该类标记的多态性信息含量（polymorphism information content, PIC）值为0.15~0.78，种质间的Nei72遗传距离范围为0.07~0.48。基于非加权组平均法（UPGMA）构建的聚类树将全部43份核心种质划分为2个主类群与6个亚类群，进一步验证了这批新开发SSR标记的有效性。综上，这些新开发的SSR标记可为假俭草（E. ophiuroides）后续的遗传与基因组学研究提供重要工具与应用潜力。