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以14份乌腺金丝桃为试材,采用L_(16)(4~5)正交实验设计,建立并优化乌腺金丝桃的ISSR-PCR反应体系,并利用优化的反应体系分析供试材料间的遗传多样性。结果表明:20μL ISSR-PCR反应体系中应含有Taq DNA聚合酶1.0U,dNTPs 0.2mmol·L~(-1),Mg~(2+)2.0mmol·L~(-1),模板DNA 45ng。从45条ISSR引物中筛选出11条多态性引物,利用筛选的多态引物对14份乌腺金丝桃材料进行了ISSR遗传多样性分析,共扩增出85条带,其中多态性条带57条,占67.06%;4个乌腺金丝桃居群的遗传相似系数介于0.375 1~0.725 2,平均值为0.541 6;通过UPGMA进行聚类分析表明,14份材料可分为3个类群4个亚群。乌腺金丝桃居群间遗传多样性水平明显高于居群内,其遗传距离与地理距离具有一定的相关性。
Based on the orthogonal experiment design of L_ (16) (4 ~ 5), 14 ISSR-PCR reaction systems were established and optimized, and the optimal reaction system was used to analyze Genetic diversity among test materials. The results showed that Taq DNA polymerase 1.0 U, dNTPs 0.2 mmol·L -1, Mg 2+ 2.0 mmol·L -1 and 45 ng of template DNA should be contained in 20 μL ISSR-PCR reaction system. Eleven polymorphic primers were screened from 45 ISSR primers and ISSR genetic diversity was analyzed using 14 polymorphic primers. A total of 85 bands were amplified, of which polymorphism 57 bands, accounting for 67.06%. The genetic similarity coefficients of four populations of Ungulate Hypericum were between 0.375 1 and 0.725 2 with an average of 0.541 6. Cluster analysis by UPGMA showed that 14 materials could be divided into 3 groups 4 subgroups. The genetic diversity among populations of Hypericum perforatum was significantly higher than that of populations, and the genetic distance was related to geographical distance.