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目的:从DNA分子水平分析不同产地猫豆的遗传多样性,探索其相互之间的亲缘关系。方法:采用简单重复序列区间(ISSR)分子标记技术对11份不同产地猫豆进行聚类分析,ISSR-聚合酶链式反应(PCR)扩增程序为94℃预变性4min,94℃变性45s,48~54℃退火1min,72℃延伸90s,40个循环,72℃延伸7min,10℃保存。在单因素试验基础上,通过L16(45)正交试验考察ISSR-PCR的5个主要因素(Mg2+,引物,dNTPs,模板DNA及Taq聚合酶)。运用Ntsys-pc软件和Popgene软件对11份样品进行分析。结果:ISSR-PCR反应最佳体系为模板DNA20ng,0.3μmol·L-1引物,0.15mmol·L-1的dNTPs,2.4mmol·L-1MgCl2,1UTaqDNA聚合酶(5U·μL-1),PCR缓冲液2μL,加入灭菌水至20μL。从40条ISSR引物中筛选出10条扩增条带清晰且重复性良好的引物,共扩增出97个条带,其中多态性条带25条,多态性位点比率25.8%。11份猫豆资源间的遗传相似系数0.866~0.9691,聚类分为3个大类群。结论:猫豆遗传差异与地理分布有一定关系,但整体遗传变异小,遗传稳定性强。
OBJECTIVE: To analyze the genetic diversity of Datla from different habitats based on the molecular level of DNA and to explore the genetic relationship among them. METHODS: Cluster analysis was performed on 11 cats of different origins using ISSR markers. The ISSR-polymerase chain reaction (PCR) amplification procedure was pre-denaturation at 94 ° C for 4 min, denaturation at 94 ° C for 45 s, 48 ~ 54 ℃ annealing 1min, 72 ℃ extended 90s, 40 cycles, 72 ℃ extended 7min, 10 ℃ preservation. Based on the single factor experiment, five main factors of ISSR-PCR (Mg2 +, primers, dNTPs, template DNA and Taq polymerase) were investigated by L16 (45) orthogonal test. Eleven samples were analyzed using Ntsys-pc software and Popgene software. Results: The optimal system of ISSR-PCR reaction was 20ng of template DNA, 0.3μmol·L-1 primer, 0.15mmol·L-1 dNTPs, 2.4mmol·L-1MgCl2 and 1UTaq DNA polymerase (5U · μL- Liquid 2μL, add sterile water to 20μL. Ten primers were screened out from 40 ISSR primers with clear and reproducible bands. A total of 97 bands were amplified, of which 25 bands were polymorphic. The ratio of polymorphic loci was 25.8%. The genetic similarity coefficients of 11 cats were between 0.866 and 0.9691. The clustering was divided into three groups. Conclusion: There is a certain relationship between genetic diversity and geographical distribution in Catharanthus. However, the overall genetic variation is small and genetic stability is strong.