绣球SSR-PCR反应体系的建立与优化

为了建立适合绣球的SSR-PCR反应体系,采用正交设计L25(56)对影响SSR-PCR反应体系的5个主要因素(Mg2+、d NTPs、引物、DNA模板和Taq聚合酶)在5个水平上进行优化,筛选出每个因素的最佳水平,建立适合绣球的SSR-PCR反应体系。结果表明,20μL的SSR-PCR反应体系中,DNA模板用量为60 ng,Mg2+浓度为1.5 mmol/L,d NTPs浓度为0.3 mmol/L,引物浓度为0.4μmol/L,Taq聚合酶用量为0.8 U。扩增程序为:94℃预变性5 min;94℃变性1 min,最佳温度退火40 s,72℃1 min,33个循环;72℃延伸10 min,4℃保存。选用10个绣球品种对建立的SSR-PCR反应体系进行验证,结果表明该体系具有较好的稳定性和通用性。建立和优化的绣球SSR-PCR反应体系,为应用SSR分子标记技术开展绣球属植物遗传育种研究提供了理论依据和技术参考。

Establishment and Optimization of SSR-PCR Reaction System in Hydrangea macrophylla

In order to establish and optimize the SSR-PCR reaction system of Hydrangea macrophylla,and the orthogonal design L25 (5 6 )was applied to optimize the five main factors(Mg2 +,dNTPs,primers,DNA template,and Taq enzyme)at five levels of SSR-PCR reaction system.The PCR result was analyzed to select the best levels of five factors and established the suitable SSR reaction system for Hydrangea macrophylla.The results showed that the op-timal SSR-PCR system of 20 μL volumes consists of 60 ng DNA template,1 .5 mmol /L Mg2 +,0.3 mmol /L dNTPs, 0.4 μmol /L primer,and 0.8 U Taq polymerase.PCR amplification procedures were pre-denaturation for 5 min at 94 ℃;followed by 33 cycles of denaturation for 1 min at 94 ℃,annealing at a suitable for primers for 40 s,exten-sion for 1 min at 72 ℃;and a final extension for 10 min at 72 ℃,then stored at 4 ℃.The optimal SSR-PCR reac-tion system was used to verify by 10 cultivars of Hydrangea macrophylla,and the results showed that it was excellent stability and repeatability for the optimal reaction system.The establishment of this reaction system provides a theo-retical basis and technical references for further research on the genus Hydrangea by SSR markers.