基于苎麻转录组测序的SSR序列分析及EST-SSR标记开发  

SSR Sequence Analysis and EST-SSR Marker Development Based on Ramie Transcriptome Sequencing

作　　者：许岳军 马玉申[1] 董静宇 梁波 张英[1] 汪娅梅 揭雨成[1,2] 邢虎成[1,2] Xu YueJun;Ma Yushen;Dong Jingyu;Liang Bo;Zhang Ying;Wang Yamei;Jie Yucheng;Xing Hucheng(Ramie Institute of Hunan Agricultural University,Changsha,410128;Hunan Provincial Key Laboratory of Corp Germplasm Innovation and Utiliza-tion,Hunan Agricultural University,Changsha,410128)

机构地区：[1]湖南农业大学,苎麻研究所,长沙410128 [2]湖南农业大学,湖南省作物种质创新与资源利用重点实验室,长沙410128

出　　处：《分子植物育种》2020年第13期4368-4376,共9页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31371704,31872877);国家科技平台项目(2015-070,NICGR2016-072)共同资助。

摘　　要：为加快苎麻分子遗传学研究和分子标记辅助育种,本研究利用转录组测序技术开发苎麻EST-SSR标记,筛选到48547条Unigene,覆盖31 Mb的片段长度,对所筛选到的Unigene进行SSR分析后,利用Primer 3.0批量设计引物,随机挑选1214对EST-SSR引物对表型性状差异较大的8份苎麻种质资源进行PCR扩增及聚丙烯酰胺电泳检测,以期得到可利用的EST-SSR标记。结果发现,在48547条Unigene中含有SSR的共有13770条,共有19964个SSR位点,平均每2.43条EST发现一个SSR、每1.55 kb含有1个SSR。在19964个SSR位点中,主要为单、二核苷酸,占总数的83.6%,三核苷酸重复单元占14.98%,其余核苷酸重复单元占比共为1.42%;去掉单核苷酸重复单元,共设计3579对SSR引物,从中随机选择1214对EST-SSR引物对8份苎麻种质进行SSR引物筛选及多态性分析。1214对引物中有216对引物表现出良好的多态性,占总引物的17.79%;扩增条带数为2~5;Shannon指数为0.2338~1.4615;PIC值为0.1103~0.7009。通过遗传相似性分析,上述8份种质遗传相似性系数为0.6690~0.8595,在相似系数为0.72处可将8份种质分为3类。本研究开发的EST-SSR引物可为苎麻及其近源物种遗传多样性分析,遗传图谱构建及分子标记辅助育种等研究提供帮助。In order to accelerate the molecular genetic research and molecular marker-assisted breeding of ramie,this paper developed ramie EST-SSR markers by transcriptome sequencing technology,and screened 48547 Unigenes,with 31 Mb in fragment length.After analyzing the selected Unigene,primers were designed by the software Primer 3.0.In order to obtain available EST-SSR markers,1214 EST-SSR primers were randomly selected for PCR amplification and polyacrylamide electrophoresis with 8 different ramie germplasm resources.The results showed that there were 13770 SSRs in 48547 Unigenes with a total of 19964 SSR loci.An SSR was found every2.43 ESTs and 1 SSR was included per 1.55 kb on average.Among the 19964 SSR loci,single nucleotides and dinucleotides accounted for 83.6%of the total,trinucleotide repeat units accounted for 14.98%,and the remaining nucleotide repeat units accounted for a total of 1.42%.After removing single nucleotide repeating units,a total of3579 pairs of SSR primers were designed.1214 EST-SSR primers were randomly selected for SSR primer screening and polymorphism analysis by 8 ramie germplasm resources.216 pairs of primers showed good polymorphism among the 1214 primers,accounting for 17.79%of the total primers.The number of amplified bands was 2~5,the Shannon index was 0.2338~1.4615,and the PIC value was 0.1103~0.7009.Through genetic similarity analysis,the genetic similarity coefficients of the 8 germplasms were 0.6690~0.8595,and 8 germplasm could be divided into 3 clusters with a similarity coefficient of 0.72.The EST-SSR marker developed in this study will be benefit for the genetic diversity analysis,genetic map construction and molecular marker-assisted breeding of ramie and its near-source species.

关 键 词：苎麻(Boehmeria nivea) 转录组 EST-SSR 

分 类 号：S563.1[农业科学—作物学]