安徽乌菜SRAP技术体系的优化

Optimization of SRAP-PCR Reaction for Brassica campestris L. ssp. Chinese(L.) Makino var. rosularis Tsen et Lee.

以合肥黄心乌为试材,利用正交试验设计,对SRAP-PCR反应体系中的Mg2+浓度、dNTPs浓度、引物浓度、Taq聚合酶浓度和模板DNA浓度进行5因素4水平的筛选分析,用me3-em3引物组合进行PCR扩增以确定最佳反应体系。结果表明,安徽乌菜SRAP-PCR最佳反应体系为:10×PCR buffer 1μL,Mg2+ 3.0mmol·L-1,dNTPs 0.2mmol·L-1,引物各0.5mol·L-1,模板DNA 4.0ng·μL-1,Taq聚合酶0.05U·μL-1,总体积为10μL。利用此反应体系对安徽乌菜进行PCR扩增并电泳检测,其结果清晰、稳定、可靠,可用于安徽乌菜的遗传分析。

To establish SRAP system for Anhui Wucai （Brassica campestris L. ssp. Chinese （L.）Makino var. rosularis Tsen et Lee.）for the analysis of genetic diversity of Anhui Wucai,the SRAP reaction system was optimized for the factors of Mg^2＋, dNTPs, primers,Taq polymerase and template DNA at 4 levels using primer combination em3-me3. The results indicated that the appropriate SRAP-PCR reaction for Anhui Wucai in a 10 μL reaction system was 10× PCR buffer μL,Mg^2＋ 3.0 mmol. L^-1, dNTPs 0.2 retool·L^-1, primer 0.5 ttmol·L^-1, template DNA 4.0 ng·μL^-1, and Taq polymerase 0.05 U·μL^-1. Thirty seven Anhui Wucai germplasm accessions were used to test the optimized reaction condition. The results showed that this SRAP-PCR protocol produced clear, reproducible, high polymorphic bands and suitable for the genetic analysis.