紫薇SSR-PCR反应体系优化和引物筛选

Optimization of SSR-PCR Reaction System and Primers Screening of Lagerstroemia indica L.

为加强紫薇分子遗传学研究,以紫薇叶片DNA为模板,采用4因素(模板浓度、dNTP、引物浓度、酶浓度)3水平的L9(34)正交设计,优化紫薇的SSR-PCR反应体系,并对24对紫薇SSR引物进行了筛选。结果表明:紫薇10μLSSR-PCR的最优反应体系为:Taq酶0.2U、Mg2+2.5mmol·L-1、dNTP 0.15mmol·L-1、Fprimer 0.25mmol·L-1、R-primer 0.25mmol·L-1、DNA 60ng,ddH2O补至10μL;24对EST-SSR引物筛选出可扩增出条带的引物23对,其中16对具有多态性,共扩增出清晰条带68条,差异条带39条。

In order to further research the molecular markers of Lagerstroemia indica L. ,taking DNA of La- gerstroemia indica L. as template,several important parameters influencing SSR-PCR amplification were stud- ied with an orthogonal design by four factors and three levels, as to establish the optimum SSR-PCR reaction system,and the primers were screened from 24 pairs of SSR primers. The results showed that the optimal SSR- PCR reaction system was established as a total volume of 10 μL including Taq DNA polymerase 0.2 U,Mg2＋ 2.5 mmol.L^-11 ,dNTPs 0.15 mmol· L-1 ,0.25 mmol·L-1 of each primer and template DNA 60 ng,deionized water to 10μL. 23 pairs of available primers were screened from 24 pairs of EST-SSR primers,there were 16 pairs of polymorphism primers amplified out 68 clear bands,39 bands with differences.