天门冬AFLP反应体系的建立及优化

Establishment and Optimization of AFLP Analysis System in Asparagus

为探讨天门冬种质间遗传多样性奠定基础,采用单因子试验,建立并优化了天门冬AFLP反应体系,研究酶的用量、酶切时间、预扩增体系和选择性扩增等对PCR扩增的影响。酶切反应体系加入5.0U EcoR I和Mse I于37℃保温3h;连接反应体系加入1.0U T4-DNA连接酶、2.0pmol Mse I接头和2.0pmol EcoR I接头,16℃保温过夜;选择性扩增反应体系加入1.0U Taq DNA聚合酶,0.4μL EcoR I和Mse I选择性引物;并筛选得到了8对清晰、多态性高的AFLP引物。17份天门冬种质对建立的AFLP反应体系检验,证明该体系稳定可靠,可用于天门冬种质多样性的分析。

To establish and optimize AFLP reaction system forAsparagus and lay foundation to analyze its genetic diversity,the single-factor was applied for optimizing four factors in the reaction system including restriction enzyme amount,time of digestion,preamplification system and final amplification.The DNA was completely digested by 5 U enzymeEco RⅠandMse Ⅰin 37℃ water-baths for 3 h.The digested fragments were legated with 2.0 pmolEco R Ⅰ,2.0 pmolMse Ⅰ adaptors and 1 U T4-DNA ligase at 16℃ to stand overnight.The Final amplification system included 1.0 U Taq polymerase,0.4 μLEco R I andMse I primers.On this basis,8 primers were screened with stable amplification and rich polymorphism.It was proved that this system was stable and credible.This optimized AFLP system would provide the basis for the genetic analysis of Asparagus.