狗牙根ISSR-PCR反应体系的优化

Optimization of ISSR-PCR in Cynodon dactylon

利用正交设计试验,对影响ISSR-PCR的Mg2+、dNTPs、引物、TaqDNA聚合酶和模板DNA5个因素进行优化试验。结果表明:25μLISSR-PCR反应体系中各因素的最佳条件为Mg2+2.5mmol/L、dNTPs0.25mmol/L、引物0.2μmol/L、TaqDNA聚合酶1.0U,模板DNA40ng。通过对狗牙根ISSR-PCR最佳反应体系进行梯度退火试验,得到引物UBC881的最佳退火温度为50.5℃。该体系在24个狗牙根种质中获得较好的扩增结果,该优化体系为今后利用ISSR技术进行狗牙根属种质资源的遗传多样性分析奠定了技术基础。

The concentrations of Mg^2＋, dNTPs, Taq DNA polymerase, primers and DNA template which affect the ISSR-PCR reactions were optimized by using an orthogonal design with 5 factors at 4 levels. Results showed that the optimum system for 25 μL ISSR-PCR reaction was Mg^2＋ 2.5 mmol/L, dNTPs 0.25 mmol/L, primer 0.2 μmol/L, Taq DNA polymerase 1.0 U, template DNA 40 ng and that the optimal annealing temperature of primer UBC881 was 50.5 ℃. Under this optimum system amplifications carried out on 24 genotypes of Cynodon dactylon （Linnaeus） Persoon gave better results. The system was steady and reliable and could be applied to molecular genetic research of C. dactylon.