4种甘草属植物EST-SSR引物开发及其亲缘关系分析

通过乌拉尔甘草表达序列标签(EST)数据库查找甘草属的SSR位点,并利用Primer 3.0软件在线设计EST-SSR引物,对来自甘草属4个种22份材料的EST-SSR指纹图谱特征和聚类结果进行分析,为探讨甘草属种间亲缘关系和疑难种的分类地位提供分子依据。结果显示:(1)去掉冗余序列后共得到441条EST序列,获得504个SSR位点,其中二核苷酸为重复单元的序列最多为350个,占69.44%,重复类型中以TC/AT、TA/AG形式的微卫星最为丰富。(2)设计的40对EST-SSR引物,均能扩增出清晰条带,其中15对引物具有多态性,在22份甘草属植物材料中共获得等位基因59个,平均每对引物检测到3.93个等位基因位点,扩增产物多态性比率为89.44%,能很好地表征甘草属种间的等位基因差异。(3)引物Primer 64对4种甘草属植物均能扩增出特异性条带,黄甘草在180和220bp 2个位点分别与光果甘草、胀果甘草基因共享,具有杂交种特征。(4)聚类分析表明,当相似性系数为0.82时,22份材料被划分为四组(与经典分类结果一致),第一组为内蒙古杭锦旗分布的乌拉尔甘草;第二组为新疆巴楚分布的光果甘草、黄甘草;第三组为新疆石河子分布的胀果甘草、黄甘草;第四组为新疆石河子分布的乌拉尔甘草;不同居群的黄甘草遗传分化较大,可能与同域分布亲本种的差异及种间的渐渗杂交有关。研究表明,开发的15对EST-SSR引物在甘草属内具有很好的适用性,可以为该属的种间亲缘关系和种内遗传分化研究及物种鉴定提供分子依据。

Development of EST-SSR Primers and Genetic Relationship Analysis in Four Glycyrrhiza L.Species

The Glycyrrhiza L.SSRs were searched in the expressed sequence tag (EST) database of Glycyrrhiza uralensis,and the EST-SSR primers were designed by Primer 3.0 online software.The EST-SSR fingerprint characteristics and cluster trees of four species were then analyzed to discuss the interspecific relationships among species and taxonomic status of the doubtful species.(1)Total 504 SSRs were identified from 441 EST sequences.There were 350 dinucleotide repeat sequences with 69.44% frequency,showing the dominant types in the SSRs.Both TC/AT and TA/AG types were abundant among dinucleotide repeat sequences.(2)The distinct bands were produced in PCR amplifications for all 40 random selected EST-SSR primers.The polymorphic bands were observed in 15 primers,a total of 59 alleles in the 22 materials of the Glycyrrhiza L.were observed,which appeared to be 89.44% polymorphic among these 15 primers with an average of 3.93 alleles per primers.(3)The primer 64 could amplify specific bands in 4 species.G.eurycarpa had the same alleles as that of G.inflata at 180 bp site and that of G.grabra at 220 bp site,showing the hybrid characteristics.(4)The cluster analysis of SSR data showed that when similarity coefficient was 0.82,22 materials from 4 species were clustered into 4 groups:G.uralensis in Hangjingqi,Neimenggu;G.eurycarpa and G.grabra in Bachu,Xinjiang;G.eurycarpa and G.inflata in Shihezi,Xinjiang;G.uralensis in Shihezi,Xinjiang.This cluster was in accord with the classical taxonomy.However,there was fairly large genetic differentiation in different populations of G.eurycarpa.It may be caused by the differentiation of sympatric parent species or introgressive hybridization.These results indicates that 15 primers is suitable for EST-SSR analysis of Glycyrrhiza L..It would be useful for species identification and study of genetic variation and species relationship of Glycyrrhiza L..