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以13个链格孢属小孢子种菌株为试材,采用正交实验设计的方法,研究Mg2+、dNTPs、Taq DNA聚合酶和引物4个因素在4个水平上对链格孢属小孢子种的ISSR和RAPD反应体系的影响。结果表明:链格孢属小孢子种ISSR和RAPD的最佳反应体系为25μL的ISSR-PCR反应体系中,10×PCR Buffer 2.50μL,Mg2+浓度为2.50mmol/L,dNTPs浓度为0.10mmol/L,引物浓度为0.30μmol/L,Taq DNA聚合酶用量为1.25U;在RAPD-PCR反应体系中,10×PCR Buffer 2.50μL,Mg2+浓度为2.00mmol/L,dNTPs浓度为0.15mmol/L,引物浓度为0.60μmol/L,Taq DNA聚合酶用量为1.25U;在此基础上,对最佳反应体系的退火温度进行了筛选确定;在确定退火温度后,采用最佳反应体系,用ISSR引物UBC808和RAPD引物OPE07进行稳定性和通用性验证,结果表明该优化反应体系具有较好的稳定性和通用性。
Thirteen strains of Alternaria microspora were used as materials to study the effects of Mg2 +, dNTPs, Taq DNA polymerase and primers on the microspore growth of Alternaria The impact of ISSR and RAPD reaction systems. The results showed that the optimal reaction system of ISSR and RAPD was 25μL ISSR-PCR reaction system, 10 × PCR Buffer 2.50μL, Mg2 + concentration 2.50mmol / L, dNTPs concentration 0.10mmol / L , The primer concentration was 0.30μmol / L and the amount of Taq DNA polymerase was 1.25U. In the RAPD-PCR reaction system, 10 × PCR Buffer 2.50μL, Mg2 + concentration 2.00mmol / L, dNTPs 0.15mmol / L, Concentration of 0.60μmol / L, the amount of Taq DNA polymerase 1.25U; On this basis, the annealing temperature of the optimal reaction system were screened to determine; after the annealing temperature was determined, using the best reaction system, using ISSR primer UBC808 And RAPD primer OPE07 for stability and versatility verification. The results show that the optimized reaction system has good stability and versatility.