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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。
The aim of this study was to develop EST-SSR molecular markers of Gleditsia sinensis, providing a basis for the future evaluation and analysis of Gleditsia sinensis germplasm resources. First of all, 41003 Unigenes were spliced by published data of Acacia transcriptome, with a total length of 70.4 Mb, an average length of 1716 bp and an N50 of 2533 bp. Furthermore, 8494 EST-SSR loci were detected in 7009 Unigenes, of which 1200 Unigenes contained two or more SSR loci and 369 complex SSRs. 6494 specific primers were designed for all EST-SSR loci, and 60 loci were randomly selected for validation and analysis. Among them, 44 pairs of primers amplified specific fragments and 17 pairs of polymorphic fragments with PIC values in the range of 0.195 ~ 0.742, the mean was 0.501, and most of the polymorphic sites were located in the UTR region. The results showed that among the 44 pairs of effective primers developed, 9 closely related species, such as American Gleditsia, Japanese Gleditsia sativa, Gleditsia sinensis, Dioscoreaceae, South China Gleditsia, Camellia oleifera, The effective amplified fragments of 32, 31, 23, 24, 7, 40, 25, 18 and 18 pairs of pods, soap pods and American soap pods, respectively, indicated that the EST- The versatility. The results show that the EST-SSR loci excavated by transcriptome data have the advantages of stable amplification, good polymorphism and common between relatives, which is one of the effective ways to develop molecular markers for forest species.