铁线莲园艺品种ISSR-PCR反应体系优化与引物筛选

Optimization of ISSR-PCR System and Selection of Primers for Horticultural Varieties of Clematis

以铁线莲的园艺品种Clematis‘Gravetye Beauty’为试材,采用改良CTAB法提取其基因组DNA为模板,采用单因子试验设计,研究模板DNA含量、引物浓度、Master Mix对ISSR-PCR反应的影响,以期构建基于ISSR分子标记技术的铁线莲园艺品种遗传图谱。结果表明:25μL C.‘Gravetye Beauty’ISSR-PCR最佳反应体系的模板DNA用量5ng、引物浓度0.8μmol/L、Master Mix 12μL;利用该体系从38条ISSR引物中筛选出扩增条带清晰、多态性好的11条引物。

AClematis garden species（Clematis‘Gravetype Beauty＇）was chosen as the experiment material to built the genetic map based on the ISSR molecule marker technology.The‘Gravetye Beauty＇genomic DNA was used as the DNA model,and simple factor design of experiment was used to study the impacts of model NDA concentration,primer concentration,and Master Mix on ISSR-PCR reaction.The results showed that the optimum system was DNA model 5ng,primer concentration 0.8μmol/L,Master Mix 12μL and the whole reaction system was 25μL.Using this system,11 primers with clear stripe and good polymorphism were selected from 38 ISSR primers.