瓜蒌SSR标记开发及遗传多样性分析

Development of SSR markers and analysis on genetic diversity of Trichosanthis Fructus

目的开发SSR分子标记,分析不同产地瓜蒌Trichosanthis Fructus药材的遗传结构和进化及不同种质资源的遗传多样性。方法提取瓜蒌RNA,反转录获得cDNA,PCR富集得到cDNA文库,经高通量测序,组装后,获得高质量Unigene后,用MISA(MicroSatellite identification tool)鉴定简单重复序列(simple sequence repeat,SSR),设计两端引物,从中选取64对引物对13个来源于3个地理居群的瓜蒌资源进行群体结构和遗传多样性分析。结果所开发的SSR标记具有良好的功效,在选用的整个群体中,其多态性信息含量(polymorphic information content,PIC)、观察等位基因数(number of observed alleles,Na)、有效等位基因数(number of effective alleles,Ne)、香农信息指数(Shannon information index,I)和Nei遗传距离(Nei genetic distance,GD)依次为0.7587、3.5312、2.5883、1.0006和0.5592,3个自然群体之间,合肥和潜山自然群体的遗传相似性高,达到0.9417,与其他野生资源之间存在较大的遗传差异。经聚类分析,将13个品种资源分成2个亚群,其中2个长萼瓜蒌资源单独组成1个亚群,其余11个双边瓜蒌Trichosanthes rosthornii资源被分为另外1个亚群,而双边瓜蒌又被分为2个亚群,并且4个合肥育成品种分别在不同的亚群中,表明合肥育成品种来源较丰富。其他地区瓜蒌种质资源与安徽原产地瓜蒌遗传差异较大,能够为安徽瓜蒌育种拓宽种质资源基础。

Objective To elucidate the genetic structure and evolution of Gualou(Trichosanthis Fructus)originating from diverse regions and the genetic diversity of various germplasm resources,SSR molecular markers were designed and utilized.Methods RNA of Trichosanthis Fructus was extracted,reversed transcription to obtain cDNA,and enriched to obtain cDNA library with PCR reaction.High-quality Unigenes were obtained using high-throughput sequencing and assembly.Simple sequence repeats(SSRs)were identified using MicroSatellite identification tool(MISA),and primers were designed at both ends of the SSR.64 pairs of primers were selected to analyze the population structure and genetic diversity of 13 resources from three natural populations.Results The developed SSR markers have good efficacy.The polymorphic information content(PIC),number of observed alleles(Na),number of effective alleles(Ne),Shannon information index(I)and genetic distance(H)of the whole population were 0.7587,3.5120,2.5883,1.0006 and 0.5592,respectively.The genetic similarity between Qianshan and Hefei natural groups was highest,reaching 0.9417 between the three natural populations.13 resources were divided into two subgroups through cluster analysis.Among them,two resources of Trichosanthes laceribractea clustered into one subgroup separately,and the other eleven Trichosanthes uniflora resources were clustered into another subgroup.11 T.uniflora resources had been further divided into two subgroups.The distribution of four varieties from Hefei in different subgroups indicated that the genetic basis of Hefei varieties was relatively richness.There are significant genetic differences between Trichosanthis Fructus germplasm resources in other and Anhui ecoregions,which could broaden the germplasm resource foundation for breeding.