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采用CTAB法提取大白菜基因组DNA,利用单因素试验对InDel-PCR反应体系中的dNTPs浓度、Mg2+浓度、Taq DNA聚合酶用量、模板DNA用量、引物浓度、退火温度、三温循环时间等7个因素进行筛选和优化。结果表明,适用于大白菜InDel-PCR分析的最佳扩增条件为(20μL):dNTPs 0.1mmol/L、Mg2+1.5mmol/L、Taq DNA聚合酶0.4U、模板DNA 105ng、引物浓度0.4μmol/L、退火温度50℃、三温循环时间30s-30s-45s。该体系可用于大白菜遗传多样性分析及遗传图谱构建。
The genomic DNA of Chinese cabbage was extracted by CTAB method, and the concentration of dNTPs, the concentration of Mg2 +, the amount of Taq DNA polymerase, the amount of template DNA, the concentration of primer, the annealing temperature and the three-temperature cycle time in InDel-PCR reaction system Factors for screening and optimization. The results showed that the optimal conditions for InDel-PCR analysis were as follows: dNTPs 0.1mmol / L, Mg2 + 1.5mmol / L, Taq DNA polymerase 0.4U, template DNA 105ng, primer 0.4μmol / L, annealing temperature 50 ℃, three temperature cycle time 30s-30s-45s. The system can be used for genetic diversity analysis and genetic map construction of Chinese cabbage.