【摘要】 本研究旨在开发皂荚EST-SSR分子标记,为今后皂荚种质资源评价与分析提供基础。首先通过对已公布的皂荚转录组数据进行拼接得到41003个Unigenes,总长度70.4 Mb,平均长度1716 bp,N50为2533 bp。进一步在7009个Unigenes中检测到8494个EST-SSR位点,其中1200条Unigenes含有2个及以上的SSR位点,复合型SSRs有369个。针对所有EST-SSR位点设计得到6494条特异性引物,随机挑选60个位点进行试验验证与分析,其中44对引物可扩增出特异性片段,17对具有多态性,PIC值范围为0.195~0.742,均值为0.501,且大部分多态性位点位于UTR区域。通过对皂荚近缘种进行跨种PCR扩增试验,结果显示在开发的44对有效引物中,9个近缘种美国皂荚、日本皂荚、绒毛皂荚、滇皂荚、华南皂荚、小果皂荚、野皂荚、肥皂荚和美国肥皂荚分别有32、31、23、24、7、40、25、18和18对引物可获得有效扩增片段,说明EST-SSR标记在皂荚近缘种之间具有良好的通用性。研究表明利用转录组数据挖掘的EST-SSR位点具有扩增稳定、多态性良好、近缘种间通用等优点,是林木物种开发分子标记的有效途径之一。更多还原

【Abstract】 The objective of this study is to develop EST-SSR markers for further molecular evaluation of genetic resource for G.sinensis.Based on the transcriptome RNA-seq data from NCBI,a total of 41003 Unigenes were obtained by assembling the RNA-seq data of G.sinensis.The total length,average length and N50 of the Unigenes were 70.4 Mb,1716 bp and 2533 bp,respectively.8494 EST-SSR loci were detected in 7009 Unigenes,among which 1200 Unigenes contained two or more SSRs and 369 Unigenes contained compound SSRs.For all ESTSSR loci,6494 primers were designed and 60 from them were randomly selected to validate their effectiveness.Consequently,44 primer pairs could amplify target products,among which 17 primer pairs showed polymorphism and most polymorphism markers were identified to be located in UTR region.Furthermore,the PIC value ranged from0.195 to 0.742,with an average of 0.501.Finally,we validated whether the primers could amplify target products in relatives of G.sinensis,among the 44 effective primers,32,31,23,24,7,40,25,18,and 18 primer pairs could amplify target products in G.tricanthos,G.japonica Miq.,G.japonica var.velutina L.C.Li.,G.japonica var.delavayi(Franch),G.fera(Lour.) Merr.,G.australis Hemsl.,G.microphylla D.A.Gordon ex Y.T.Lee,Gymnocladus chinensis Baill.,Gymnocladus dioica(L.) K.Koch,indicating the high transferability of our developed primers.The results demonstrated that mining EST-SSR loci using transcriptome data is one of the efficient ways for developing molecularmarkers in tree species,with the advantages of steady amplification,high polymorphism and high transferability.更多还原