State Key Laboratory of Grassland Agro-ecosystems, Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, 730020, China.
Sci Rep. 2017 Dec 20;7(1):17959. doi: 10.1038/s41598-017-18049-8.
Melilotus is one of the most important legume forages, but the lack of molecular markers has limited the development and utilization of Melilotus germplasm resources. In the present study, 151 M clean reads were generated from various genotypes of Melilotus albus using Illumina sequencing. A total of 19,263 potential EST-SSRs were identified from 104,358 unigene sequences. Moreover, 18,182 primer pairs were successfully designed, and 550 primer pairs were selected using criteria of base repeat type, fragment length and annealing temperature. In addition, 550 primer pairs were screened by using PCR amplification products and used to assess polymorphisms in 15 M. albus accessions. A total of 114 primer pairs were detected as being highly polymorphic, and the average polymorphism information content (PIC) value was 0.79. Furthermore, those 114 polymorphic primer pairs were used to evaluate the transferability to 18 species of the genus Melilotus, and 70 EST-SSR markers were found to be transferable among the 18 Melilotus species. According to the UPGMA dendrogram and STRUCTURE analysis, the 18 Melilotus species were classified into three clusters. This study offers a valuable resource for the genetic diversity and molecular assisted breeding of germplasm resources in the genus Melilotus.
草木樨是最重要的豆科牧草之一，但缺乏分子标记限制了草木樨种质资源的开发和利用。本研究利用 Illumina 测序技术对不同基因型的白花草木樨进行测序，共获得 151 M 条高质量 clean reads。从 104,358 条 unigene 序列中鉴定出 19,263 个潜在的 EST-SSR。此外，成功设计了 18,182 对引物，根据碱基重复类型、片段长度和退火温度筛选出 550 对引物。然后，使用 PCR 扩增产物筛选 550 对引物，并用于评估 15 个白花草木樨品系的多态性。共检测到 114 对高度多态性引物，平均多态性信息含量（PIC）值为 0.79。此外，将这 114 对多态性引物用于评价对 18 种草木樨属植物的可转移性，在 18 种草木樨属植物中发现 70 个 EST-SSR 标记是可转移的。根据 UPGMA 聚类和 STRUCTURE 分析，18 种草木樨属植物分为 3 个聚类。本研究为草木樨属种质资源的遗传多样性和分子辅助育种提供了有价值的资源。
