摘要
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 genetic diversities of Mucuna pruriens from different habitats according to their DNA molecule and investigate genetic relationships among them.Method： Inter-simple sequence repeat（ISSR） molecular marker technique was adopted to analyze genetic diversity of M.pruriens from different habitats,ISSR-polymerase chain reaction（PCR） amplification procedure was as following： preliminary denaturation at 94 ℃ for 4min; 40 cycles each involved denaturation at 94 ℃ for 45 s,annealed at 48-54 ℃ for 1min and extended 90s at 72 ℃; extended at 72 ℃ for 7min and kept at 10 ℃.Based on single factor tests,effects of five main reaction elements（Mg2 ＋,Taq DNA polymerase,d NTPs,template DNA and primers） on ISSR-PCR were optimized by orthogonal test.Eleven samples was analyzed by Ntsys-pc and Popgene software.Result： Optimum reaction system was as following： template DNA of 20 ng,ISSR primers of 0.3μmol·L-1,d NTPs of 0.15 mmol·L-1,Mg Cl2 of 2.4 mmol·L-1,Taq DNA polymerase of 1 U with 5 U·μL-1,PCR buffer of 2μL,adding sterilized water to 20μL.Ten ISSR primers with good repeatability and clear loci were selected from40 primers and used for ISSR amplication,97 loci were amplicated, in which 25 were polymorphic loci,accounting for 25.8%.Genetic similarity coefficient of 11 samples was 0.866 0-0.969 1 and samples could be classified into three groups.Conclusion： Genetic variation of M.pruriens has a close relationship with geographical distribution,but genetic variation is small with strong genetic stability.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2015年第6期86-90,共5页
Chinese Journal of Experimental Traditional Medical Formulae
关键词
猫豆
ISSR分子标记
遗传关系
聚类分析
Mucuna pruriens
inter-simple sequence repeat molecular markers
genetic relationships
cluster analysis
