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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。
Five factors of Mg2 +, primer, dNTPs, DNA and Taq DNA polymerase affecting the effect of SRAP-PCR on Agropyron chinense were optimized by single factor and L16 (45) orthogonal design using DNA from leaves of Agropyron chinense as template The effects of different annealing temperature on the amplification reaction were studied. The optimal reaction system of SRAP-PCR for Agropyron chinense was established by comprehensive comparative analysis. The results showed that the optimized SRAP-PCR was 20 mmol / L Mg2 + 1.75 mmol / L, primer 0.15 μmol / L, dNTPs 0.20 mmol / L, DNA 50 ng, Taq DNA polymerase 0.75 U and 2 μL 10 × PCR buffer The effect of Mg2 + and primer concentration on the amplification was greatest, and DNA concentration had the least effect. The system was used to validate 32 Agropyron chinense, and the amplification result was clear and stable. The system was established by using SRAP molecular marker for genetic diversity, Sex markers and other research laid the technical foundation.