江南油杉ISSR-PCR反应体系建立与引物筛选

Establishment of the ISSR-PCR Reaction System and Primers Screening in Keteleeria fortunei var. cyclolepis

为了建立江南油杉(Keteleeria fortunei var.cyclolepis)ISSR-PCR最佳反应体系,以江南油杉嫩叶为材料,采用改良CTAB法提取基因组DNA,通过单因素实验设计,进行ISSR-PCR反应体系和反应条件优化,并筛选出多态性ISSR引物。实验结果表明:江南油杉ISSR-PCR最佳反应体系为25μL体系中模板DNA 40 ng,引物浓度为0.8μmol/L,2×Taq PCR Master Mix用量为12μL。利用该体系从100条ISSR引物中筛选出扩增条带清晰、特异性好的6条引物(UBC807、UBC809、UBC811、UBC812、UBC835、UBC841)。

In order to establish the best ISSR-PCR reaction system for Keteleeria fortunei var. cyclolepis,tender leaves of K. fortunei var. cyclolepis were chosen as experimental materials. Genomic DNA was isolated by modified CTAB method to optimize the ISSR-PCR reaction system and reaction condition by the single factor experiment design,and polymorphic ISSR primers were selected by this system. The results indicated that the optimal reaction system of 25 μL included 40 ng template DNA,0.8 μmol/L primer concentration,12 μL 2 × Taq PCR Master Mix. Using the optimized reaction system, 6 of 100 ISSR primers(UBC807,UBC809,UBC811,UBC812,UBC835,UBC841) were screened out,which showed specific bands and good polymorphism.