树莓SSR反应体系的优化及应用

Optimization and application of SSR reaction system in bramble

本研究主要对树莓SSR-PCR体系进行优化,以‘来味里(Reveille)’红树莓为试材,利用L9(34)正交试验设计和两因素完全随机试验,研究各主要参数的适宜浓度,建立适合树莓SSR反应的最佳体系,并通过16个不同的树莓栽培品种对优化后的体系进行验证。结果表明:各影响因素中,引物浓度的变化对扩增结果的影响最大,优化后的最佳反应体系(25μL)中,Taq DNA聚合酶和DNA最适用量分别为1.0U和30ng;Mg2+、dNTP和引物最适浓度分别为1.5、0.2mmol/L和0.4μmol/L。利用优化后的反应体系和10对SSR引物对16个树莓品种进行PCR扩增,不同品种间扩增结果稳定,多态性位点丰富,共有54个等位位点,表明该体系稳定可靠,适合树莓的SSR分析。

This paper is concerned on optimizing the SSR-PCR reaction system on bramble.The cultivar ＇Reveille＇ of bramble was used.And the L9（34） orthogonal design and completely randomized design of two factors were used to determine the appropriate concentrations of the main factors.The best system of SSR-PCR on bramble was built and checked in 16 different bramble cultivars.The results showed that the primer concentration among the factors had highest impacts on SSR-PCR.The optimum amplification conditions of SSR-PCR were as following：Taq Polymerase 1.0 U,DNA template 30 ng,Mg2＋ 1.5 mmol/L,dNTPs 0.2 mmol/L and Primer 0.4 μmol/L in 25 μL reaction volumes.With the optimum system,the amplification of 10 SSR primer pairs in 16 bramble cultivars revealed high stability.Polymorphism with 54 alleles were obtained simultaneously.It was evident that the reaction system established in this study was stable and reliable for SSR analysis of bramble,and could be used in germplasm identification,genetic polymorphism analysis and marker assisted selection