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为开发猕猴桃EST-SSR标记,了解28个品种猕猴桃间的遗传多样性和遗传关系,对中华猕猴桃“红阳”的转录组序列进行分析,并根据分析结果设计SSR引物.之后采用CTAB法提取28个品种猕猴桃的DNA作为SSR-PCR的扩增模板,并根据扩增结果进行聚类分析.研究中共得到包含SSR的序列21 848条,其中重复单元为单碱基、双碱基、三碱基、四碱基、五碱基和六碱基的序列分别为1 642、15 965、3 141、248、368和484条,随机选择其中46条序列设计SSR引物.根据初步的PCR扩增,筛选出32对条带较少且明亮的引物分别对28个品种猕猴桃的DNA样本进行扩增,并对引物对应的SSR序列进行定位.结果显示,32对引物对应的序列中有19条能够得到完整的所在基因、染色体以及染色体中具体位置的信息.这些引物中有26对具有多态性,共统计到等位基因120个,每对引物得到1-11个等位基因,平均3.75个.28个猕猴桃品种之间的遗传相似性系数在0.53-0.97之间,在遗传相似系数为0.72的水平上,可将它们分为5大类,分类结果与传统形态学的划分基本一致.本研究揭示的各样本间的遗传关系可为未来猕猴桃的种质改良提供依据.图4表5参33
In order to develop the EST-SSR marker of kiwifruit, the genetic diversity and genetic relationship among 28 kiwifruit cultivars were analyzed, and the transcriptome sequences of kiwifruit “Hongyang” were analyzed, and the SSR primers were designed according to the results of the analysis. The DNA of 28 cultivars of Actinidia chinensis was extracted as template for SSR-PCR amplification and cluster analysis was carried out based on the results of amplification.The total number of 21 848 SSR-containing sequences was obtained, in which the repeat units were single base, double base, Four, five, and six base sequences were 1 642, 15 965, 3141, 248, 368 and 484. The SSR primers were designed by random selection of 46 sequences.According to the preliminary PCR amplification , 32 pairs of primers with fewer and clearer bands were selected to amplify the DNA samples of 28 cultivars of kiwifruit, respectively, and the corresponding SSR sequences were located.The results showed that 19 of the 32 pairs of primers corresponded to The complete genome, chromosomes and chromosome specific information were obtained.Among 26 pairs of primers, there were 120 alleles and 1-11 alleles per pair of primers with an average of 3.75 28 varieties of kiwi The genetic similarity coefficient between 0.53-0.97, genetic similarity coefficient of 0.72 level, they can be divided into five categories, the classification results are basically the same with the traditional morphological division of the sample revealed in this study The genetic relationship may provide the basis for future kiwifruit germplasm improvement