花椰菜最适ISSR反应体系的建立及优化

Establishment and Optimization of ISSR Reaction System in Cauliflower

以花椰菜9901A×9908杂交种为材料,用高盐低pH值法提取的基因组DNA为模板,对ISSR-PCR反应体系中的镁离子浓度、dNTP浓度、TaqDNA聚合酶量、引物用量以及最适退火温度等影响因子进行了优化和筛选,建立了适合花椰菜的ISSR反应体系:20μLPCR反应体系,含10×PCR反应缓冲液2μL,2.0mmol/LMgCl2,4×dNTP混合物0.2mmol/L,引物0.5μmol/L,Taq酶1.5U,基因组DNA约30ng,最适退火温度为52.8℃。

The effect of Mg^2＋, dNTP, primer, Taq DNA polymerase and annealing temperature on ISSR-PCR amplification reaction were studied using the genomic DNA as template, extracted with low pH, high-salt method in cauliflower. The results showed that the conditions suitable for ISSR-PCR were as follows： 2 μL 10× Taq buffer, 2.0 mmol/L MgCl2, 0.2 mmol/L 4× dNTP, 0.5 μmol/L primers, 1.5 U Taq DNA polymerase and 30 ng template DNA in total 20 ttL reaction volume, and the optimum annealing temperature was 52.8℃.