甜菜SSR—PCR反应体系优化及基于DNA混合池的SSR引物快速筛选

Optimization of SSR- PCR reaction system in sugar beet and SSR primer selection based on multiplex PCR technique

应用L16（4^4）正交设计对影响甜菜SSR—PCR的主要参数进行优化，建立适于甜菜的SSR反应体系和扩增程序。20ul反应体系中含有模板70ngDNA，150umol／LdNTP，0．7umol／LSSR引物，0．5TaqDNA聚合酶／U，10×PCRBuffer（含Mg^2＋）2．0ul。以选择的87对SSR引物为对象，对10个甜菜基因组DNA样品等量混合，进行PCR扩增筛选引物，结果表明，与常规筛选方法相比，DNA混合池方法大幅度缩短了实验周期，显著减少了实验资源的消耗，可用于大量甜菜SSR引物的快速高效筛选。

The SSR- PCR reaction system of sugarbeet has been established by optimizing the main factors the PCR reactions. An optimal 20ul volumes would consisted of 70ng DNA template, 150umol/L dNTP,0.7umol/L SSR primer,0.5U Taq DNA polymerase, 10 × PCR Buffer（ including Mg^2＋ ）2.0ulo The PCR amplification was carried out by predenaturation for 4 min at 94℃ ,followed by 35 cycles of denaturation for I rain at 94℃, extension for 1.5rain at 72℃, the amplification was completed after extension for 10rain at 72℃, then stored at 4℃. The optimal annealing temperature for SSR- PCR reaction system would be 50.5 -59.5℃ determined by gradient PCR. DNA pooling constructed by equivalently mixing 10 sugarbeet genomic DNA templates was used to screen 87 pairs of SSR primers. The analytical results show that comparing with conventional screening method, the genomic DNA pooling method can greatly shorten experimental period and significantly reduce the consumption of genomic DNA, suggesting that this method is a fast and efficient method for screening substantive SSR primers used in sugarbeet.