月季SSR-PCR反应体系的建立和优化

Optimization and Establishment of SSR Analysis System in Roses(Rosa spp.)

采用L16（54）正交设计和二因素完全随机试验,分析了月季SSR-PCR反应体系的主要成分TaqDNA聚合酶、样本浓度、Mg2＋浓度d、NTP浓度以及引物浓度对扩增结果的影响,建立了适合月季的SSR反应体系。研究结果表明：在20μL反应体系中,TaqDNA聚合酶宜加入1.5 U,样本最适宜浓度为30-40 ng,Mg^2＋的最适浓度为1-1.5 mmol/L,dNTP最适浓度为0.2 mmol/L,单引物的最适浓度均为1μmol/L。用建成的反应体系对19对引物筛选,对12个月季品种扩增,3%的琼脂糖凝胶电泳检测,品种间DNA谱带多态性丰富,证明该体系稳定可靠。

Using orthogonal design, five factors including concentration of template, Mg^2 ＋ , dNTP, primers and Taq DNA polymerase were optimized to establish SSR analysis system in roses. The results showed that 30 -40 ng DNA template, 1 - 1.5 mmol/L Mg^2 ＋ , 0.2 mmol/L dNTP, 1 μmol/L each primer and 1.5 U Taq DNA polymerase in 20 μL reaction system were the optimal option. Through the optimized reaction system, SSR fragments of 12 rose cultivars were obtained and detected. Polymorphism between different cuhivars was abundantly detected by 3 % agarose gels, which showed that the system established in this study was suitable and stable.