紫色观赏辣椒基因组DNA提取及ISSR-PCR体系的优化

Genomic DNA Extraction and Optimization of ISSR-PCR System for Purple Ornamental Pepper

为探索紫色观赏辣椒基因组DNA的最佳提取条件和建立ISSR-PCR最优反应体系,对比了3种基因组DNA提取方法,并通过正交设计试验优化了ISSR-PCR反应体系。结果表明,采用高盐低pH法从2种观赏辣椒中提取的基因组DNA的A(260/280)值分别为1.807和1.818,DNA电泳条带明亮整齐,无拖尾;ISSR-PCR最优反应体系(20μL)为:1×PCR buffer,1.5 mmol/L Mg^2+,0.6μmol/L引物,0.2 mmol/L dNTPs,1.0 U Taq DNA聚合酶,60 ng模板DNA,扩增所得分子图谱稳定性高、重复性好;唯有高盐低pH法提取的DNA经该ISSR-PCR体系扩增能体现出2个辣椒品种间分子图谱的差异。

In order to establish a genomic DNA extraction method and ISSR-PCR system suitable for purple ornamental peppers, three DNA extraction methods were compared and the ISSR-PCR system was optimized by orthogonal design experiment. The results showed that, the A260/280 values of the genomic DNA extracted from two pepper varieties by high salt/low pH method were 1.807 and 1.818 respectively, and the DNA bands were clear without tailing. The optimal ISSR-PCR conditions were： 1 × PCR buffer, 1.5 mmol/L Mg2＋, 0.6 μmol/L primer, 0.2 mmol/L dNTPs, 1.0 U Taq DNA polymerase, 60 ng template DNA, 20 μL reaction system. The ISSR results were stable and repeatable, and two pepper varieties can be distinguished from ISSR map only using the DNA templates extracted via high salt/low pH method.