华仁杏ISSR-PCR反应体系的正交优化分析

Optimization of ISSR-PCR System of Armeniaca cathayana Through Orthogonal Design Method

为有效利用ISSR技术开展华仁杏种质资源与遗传多样性研究,通过L16(45)正交试验设计,对华仁杏ISSR-PCR反应体系中的5个主要因素进行了最优条件的筛选试验,借助极差分析和方差分析,建立了华仁杏最佳的ISSR-PCR反应体系(25μL):10×PCR Buffer without MgCl22.5μL、MgCl22.0 mmol/L、TaqDNA Polymerase 2.0 U、dNTPMiX 100μmol/L、ISSR Primer 0.2μmol/L、DNA2.4 ng/μL。

Armeniaca cathayana D.L.Fu et al.is a new species described in 2010 and has high values for its big and not bitter kernels.In order to research its germ plasma and genetic diversity based on ISSR technique,the orthogonal design of L16（45）was used to optimize the ISSR-PCR amplification system of A.cathayana in five major factors.Based on range analysis and variance analysis,the best ISSR-PCR reaction system（25 μL）was established：10×PCR Buffer without MgCl2 2.5 μL,MgCl2 2.0 mmol/L,Taq DNA Polymerase 2.0 U,dNTP MiX 100 μmol/L,ISSR Primer 0.2 μmol/L,DNA 2.4 ng/μL.