燕麦EST-SSR标记的开发和利用

为拓展分子标记在燕麦种质资源分析与鉴定中的应用,利用公共数据库中的25376条EST(expressed sequence tags)序列,开展了燕麦EST-SSR功能性标记的开发和利用研究。25376条EST序列经拼接去冗余后获得了11618条序列,从中筛选出含有不同重复基元的SSR且重复次数较多、长度较长的556条EST序列进行引物设计,开发了50对燕麦EST-SSR引物,通过筛选得到40对有效的EST-SSR引物。选取其中4对引物对5个燕麦种质资源进行了PCR扩增及产物测序,结果表明扩增条带多态性是由SSR差异造成的。利用40对ESTSSR引物对15个六倍体燕麦种质资源进行遗传多样性分析,共扩增出89个等位基因,平均每对引物产生2.23个等位基因;UPGMA聚类分析表明,15个六倍体燕麦种质资源在Dice系数为0.93处聚为3支,基本上是按照不同种进行聚类的,在相同种中又根据地理来源分别聚集成支。利用40对EST-SSR引物对31个遗传背景不清的燕麦种质资源进行基因组倍性鉴定,发现这些种质中可能存在有四倍体和二倍体的燕麦新资源。本研究开发的燕麦EST-SSR功能性标记将在燕麦遗传多样性分析、遗传图谱构建及燕麦属内种间基因组鉴定等方面发挥重要作用。

Development and Application of EST-SSR Markers in Oat

To expand the application of molecular markers in the analysis and identification of oat germplasm resources, EST-SSR markers were developed and utilized using publicly available oat EST data. After splice and redundancy check, 11618 sequences were obtained, and 556 ESTs containing 595 microsatellites loci were selected and assembled from 25376 oat EST sequences. Firstly, 50 primers were designed based on 556 ESTs, and 40 primers were screened. The amplified products were tested and sequenced using four primer pairs on five oat germplasm resources. The results showed the polymorphisms of bands were caused by differences in SSR. Forty effective primer pairs were tested on the genetic diversity of 15 hexaploid oat germplasm resources, and the study of transferability was analyzed among oat species. Eighty-nine loci were successfully amplified in 15 hexaploid oat germplasm resources, with an average of 2.23 loci per primer pair. Based on genetic similarity coefficient （Dice = 0.93）, the UPGMA dendrogram grouped 15 hexaploid oat germplasm resources into three clusters. The 15 germplasm resources were clustered according to species, and were sub-clustered following geographic origin. The EST-SSR primers showed higher availability on the study of chromosome ploidy of 31 unknown oat germplasm resources. Results showed some diploids and tetraploids of oat germplasm resources were tested in the genetic analysis of 31 oat germplasm resources. This study proved that EST-SSRs are valuable for genetic analysis, linkage mapping and transferability study among oat species. The EST-SSRs also proved useful in discriminating different species and different genome ploidy in oat.