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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。
In this study, the raspberry SSR-PCR system was optimized. Reveal raspberry was used as experimental material. By using L9 (34) orthogonal design and two-factor randomized trial, the main parameters The optimal system for rapeseed SSR reaction was established, and the optimized system was verified by 16 different raspberry cultivars. The results showed that the most influential factor of amplification was the change of primer concentration. Among the optimal reaction system (25μL), the best dosage of Taq DNA polymerase and DNA were 1.0U and 30ng, The optimal concentrations of dNTP and primer were 1.5, 0.2mmol / L and 0.4μmol / L, respectively. Sixteen raspberry cultivars were amplified by PCR using 10 pairs of SSR primers and the optimized reaction system. The results of amplification among different cultivars were stable and the polymorphic loci were abundant with a total of 54 alleles, indicating that the system was stable and reliable , Suitable for raspberry SSR analysis.