椰子SSR反应体系的建立和优化

Optimization and Establishment of SSR Analysis Method in Coconut(Cocos nucifera L.)

为摸索适宜椰子的SSR反应体系,分析了PCR反应体系中的Mg2＋浓度、引物浓度、dNTP浓度、TaqDNA聚合酶浓度、模板浓度以及退火温度对扩增结果的影响,建立了适合椰子的SSR反应体系。研究结果表明：在20μL反应体系中,Mg^2＋、引物和dNTP的最适浓度分别为2.5 mmol/L、0.3μmol/L、0.2 mmol/L;TaqDNA聚合酶的最佳使用量为1 U,模板DNA应加入50 ng,引物最佳退火温度比Tm值较小者低2-3℃。在参数优化的基础上,应用标记分析该反应体系对24个海南不同地区高种椰子样品进行SSR分析,不同样品间DNA谱带多态性丰富,本研究建立的分析体系将为今后椰子资源的SSR分析奠定良好的研究基础。

Coconut （Cocos nucifera L. ） is one of the key plantation crops in the tropic areas. In this study, parameters of SSR reactions including concentration of Mg^2＋, primer, template and Taq polymerase were optimized. The results showed that 2.5 mmol/L Mg^2＋ ,0. 3μmol/L primer,0. 2 mmol/L dNTP, 1 U Taq polymerase, 50 ng template DNA and the optimal annealing temperature of the primer of the least Tm- 2-3℃ in 20 μL SSR reaction system were the optimal option. Through the optimized reaction system, SSR fragments of 24 coconut cultivars were obtained and detected. Polymorphism between different cultivars was abundantly detected by 2 % agarose gels, which showed this system was suitable and stable.