偃麦草SRAP-PCR反应体系优化

Optimization of SRAP-PCR System on Elytrigia repens

以偃麦草叶片DNA为模板,利用单因子和L16(45)正交实验设计对影响偃麦草SRAP-PCR反应效果的Mg2+、引物、dNTPs、DNA、Taq DNA聚合酶5种因素进行优化,并比较了不同退火温度对扩增反应的影响,通过综合比较分析建立偃麦草SRAP-PCR的优化反应体系。结果表明:优化的偃麦草SRAP-PCR总体系20μL中,Mg2+1.75 mmol/L,引物0.15μmol/L,dNTPs 0.20mmol/L,DNA 50ng,Taq DNA聚合酶0.75U,2μL 10×PCR buffer;Mg2+和引物浓度对扩增效果影响最大,DNA浓度影响最小;采用该体系对32份偃麦草进行验证,扩增结果清晰稳定,此体系的建立为利用SRAP分子标记进行偃麦草遗传多样性、抗性标记等研究奠定了技术基础。

With genetic DNA of the Elytrigia repens as template, single factor and orthogonal design were applied to optimize the concentration of Mg^2＋ , primer, dNTPs, DNA, Taq DNA polymerase for SRAP-PCR system of Elytrigia repens ,the effect of different annealing temperature on reaction was studied by comprehensive analysis, the optimal system of SRAP-PCR was established. The results showed that the optimal system was Mg^2＋ 1.75 mmol/L, primer 0. 15 μmol/L,dNTPs 0. 20 mmol/L, DNA 50 ng/20μL, Taq DNA polymerase 0. 75 U, 2 μL 10 × PCR in 20 μL, concemrations of Mg^2＋ and primer had the highest effect on reaction,DNA concentrations had the least effect. The test of optimal system on 32 kinds of Elytrigia repens were done,the result was steady and clear,the establishing of optimal system made the base for the studies including genetic diversity and resistance marker and so on.