甜樱桃品种SSR-PCR反应体系的优化

以甜樱桃品种那翁为试材,研究了樱桃SSR技术中PCR反应体系的主要成分对SSR扩增结果的影响,并比较了采用聚丙稀酰胺凝胶及琼脂糖电泳检测扩增产物多态性的差异.结果表明:在PCR反应体系中,DNA最适浓度30～45 ng;Mg2+的最适浓度范围为1.5～3.0 mmol/L;dNTP最适浓度为0.2～0.3 mmol/L;引物的最适浓度为0.3～0.4 μmol/L;Taq聚合酶在20 μl反应体系中宜加入0.5 U.利用此反应体系,对24份樱桃代表资源进行了SSR反应,用6%的非变性聚丙稀酰胺凝胶电泳检测,扩增产物在100～250 bp之间,不同品种间DNA谱带多态性丰富.琼脂糖电泳检测的DNA多态性不如聚丙稀酰胺凝胶丰富.

Optimization of SSR System in Sweet Cherry Cultivars

The factors which affected on the SSR results of Napolon and comparative analysis of polymorphism of SSR detected on two gel electrophoresis systems were studied. The results showed that the optimized content of DNA is 30～45 ng; the optimized content of Mg~(2+) is 1. 5 ～ 3. 0 mmol/L; the optimized content of dNTP is 0. 2～0. 3 mmol/L ; the optimized content of primer is 0. 3 ～0. 4 μmol/L; the optimized content of Taq polymerase is 0. 5 U/20 μ1 in the PCR system. Through above PCR system,SSR fragments of 24 cultivars of cherry were obtained and detected. There were significantly different between two gel systems. Polyacrylamide gel(6% )had higher ability than agarose gel(2% )in discriminating amplified fragments. Polymorphism between different cultivars was abundant detected by Polyacrylamide gel.