淫羊藿ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System of Epimedium brevicornu Maxim.

本研究采用均匀设计和单因素试验相结合的方法,探寻淫羊藿ISSR-PCR的各组分(即引物, 2×Taq Master Mix,模板DNA)的最佳用量及退火温度对ISSR-PCR扩增的影响,为进一步使用ISSR分子标记分析淫羊藿的遗传多样性提供科学依据。结果表明,筛选的最佳体系为:在20μL的体系中,模板DNA的量为30ng,2×Taq Master Mix的量为9.8μL,引物为0.325μmol/L。此外,筛选出7条多态性较好、条带稳定的引物(UBC-808, UBC814, UBC826, UBC827, UBC840, UBC846及UBC856),并对其进行温度梯度PCR,结果表明,所选引物的最佳退火温度介于46.8℃~65℃之间。在此基础上,对13份淫羊藿种质资源进行ISSR扩增验证,结果表明建立的最佳反应体系扩增效果较好,稳定性强,对淫羊藿的遗传多样性分析、鉴定等具有较好的应用价值。

In this study, uniform design and single factor screening test were used to investigate the effect of primers, 2×Taq Master Mix and DNA template on ISSR-PCR reaction system of Epimedium brevicornu Maxim. and the effect of annealing temperature on ISSR-PCR amplication. The optimized ISSR reaction system will laid theoretical foundation for the futher genetic diversity study of Epimedium brevicornu Maxim.. The results showed that the optimal ISSR-PCR reaction system was as follows: the total 20 μL volume included 9.8 μL 2×Taq Master Mix, 30 ng DNA template, and 0.325 μmol/L primer. In addition, 7 primers(UBC808, UBC814, UBC826, UBC8-27, UBC840, UBC846 and UBC856) with stable polymorphism and abundant polymorphism were screened. The screened primers were further subjected to temperature of primers fell between 46.8℃ ~65℃. On this basis, the ISSR amplification detection of 13 Epimedium brevicornu Maxim. germplasms demonstrated the reliability of this system. The results showed that the optimum reaction system has good amplification effect and good stability,indicating it could be applied to identify Dendrobium huoshanense and Epimedium brevicornu Maxim..