基于干旱胁迫下杉木转录组序列的EST-SSR分子标记开发

摘要

The EST-SSR molecular markers of Cunninghami lanceolata were developed by using the data of Chinese fir tran-scripts under drought stress.The transgenic plants under drought stress were tested on the Solexa mRNA-Seq platform with the second generation high-throughput sequencing technique.The 75 357 contigs were obtained after splicing, the total length was 65.29 Mb.And there were 2 383 EST-SSR loci,the number of trinucleotide repeats was the largest,accounting for 41.21%of the total.The 120 EST-SSRs were randomly selected and primers were designed by Primer Premier 5.Then, eight C.lanceolata DNA samples were randomly selected as a template and 24 pairs of primers(from 120 pairs of primers) were selected by 8%non-denaturing polyacrylamide gel electrophoresis.The 24 samples of C.lanceolata were used for cap-illary electrophoresis.The result showed 23 pairs of EST-SSR primers were detected with specific peaks.The 94 alleles were detected in 24 samples of C.lanceolata,each loci had an average of 4.087 0.The number of alleles(Na)ranged from 2 to 9.The number of effective alleles(Ne)ranged from 1.086 8 to 6.914 3,and the number of effective allele was 2.089 0. Observed heterozygosity(Ho)ranged from 0.250 0 to 1.000 0.Expected heterozygosity(He)ranged from 0.082 4 to 0.896 1. Shannon index ranged from 0.173 2 to 2.035 3.The EST-SSR molecular marker of C.lanceolata provides the basis for the study on genetic diversity,population structure, DNA fingerprint database construction,and genetic information preserva-tion of C.lanceolata.%利用干旱胁迫下杉木转录组数据进行杉木 EST-SSR分子标记的开发.对干旱胁迫下的杉木组培材料在Solexa mRNA-Seq平台的二代高通量测序技术下进行了转录组测定,序列拼接后共得到75357个片段,总长度65.29 Mb,共含有EST-SSR位点2383个,其中三核苷酸重复类型数量最多,占总数的41.21 %.随机挑选120个EST-SSR,用Primer Premier 5软件设计引物,再以遗传距离较远的8份杉木DNA样品为模板,通过8%非变性聚丙烯酰胺凝胶电泳,从120对引物中初选出电泳条带较好的24对引物,用24份杉木DNA样品进行毛细管电泳检测.结果表明:在初选出的24对EST-SSR引物中有23对引物检测出特异性峰;在24份杉木样品中共检测到等位基因94个,平均每个位点4.0870个,等位基因变异数范围2~9个;有效等位基因数范围1.0868~6.9143个,平均有效等位基因2.0890个;观测杂合度范围0.2500~1.0000;期望杂合度范围0.0824~0.8961;多态性信息指数范围0.1732~2.0353.开发得到的一组杉木EST-SSR分子标记为研究杉木的遗传多样性、种群结构、DNA指纹数据库构建和遗传信息的保存提供了基础.