采用正交设计法优化梨SSR-PCR体系

Establishment and Optimization of the Pear SSR System of by Orthogonal Design

为了建立适宜梨树SSR-PCR的反应体系和扩增程序,采用正交设计L16(45)对影响梨树SSR-PCR反应体系的5个因素(Taq酶、Mg2+、模板DNA、dNTP、引物)在4个水平上进行优化,PCR结果用DPS数据处理软件分析,筛选出各反应因素的最佳水平,建立了适用于梨树的SSR反应体系。在10μL的反应体系中,模板DNA的用量为50.0 ng,TaqDNA聚合酶的用量为1.4 U,Mg2+的浓度为2.0 mmol/L,dNTPs浓度为0.1 mmol/L,引物的浓度为0.5μmol/L。扩增程序为:94℃预变性3 min;94℃变性1 min,57℃(依引物不同而改变)退火45 s,72℃1 min,35个循环;72℃延伸10 min。4℃保存。选用24个梨品种对该体系进行稳定性验证,结果证明该体系可用于梨树SSR标记的研究。

In order to establish and optimize the SSR-PCR amplification system for pear total DNA,an orthogonal design experiment of 5 elements（Taq enzyme,Mg2＋,template DNA,dNTP,primers）with 4 levels L16（45）was conducted.The PCR result was analyzed by the software DPS to select the best levels of responsive factors and established the optimal SSR reaction system.The optimal SSR-PCR system of 10 μL volumes consists of template DNA 30.0 ng,Taq DNA polymerase 1.0 U,each primer 0.2 μmol/L,Mg2＋ 2.0 mmol/L,and dNTPs 0.4 mmol/L.PCR amplification procedures was ：pre-denaturation for 4 min at 94℃,followed by 30 cycles of denaturation for 45 s at 94℃,anneal for 30 s at 48℃,extension for 30 s at 72℃,the amplification was completed after extension for 10 min at 72℃,then stored at 4℃.Total DNA of 24 pear varieties was tested to amplify SSR markers,which verified the robustness of the system.