西葫芦ISSR-PCR反应体系优化及引物筛选  

Optimization of ISSR-PCR Reaction System and Selection of Primers in Cucurbita pepo

作　　者：郭秀霞[1] 李灵芝[1] 马太光 雷逢进[2] 张瑞腾 李海平[1] 

机构地区：[1]山西农业大学园艺学院,山西太谷030801 [2]山西省农业科学院棉花研究所,山西运城044000

出　　处：《山西农业科学》2017年第3期325-327,331,共4页Journal of Shanxi Agricultural Sciences

基　　金：国家农业科技成果转化资金项目(2014GB2A300017);山西省科技攻关项目(201303110084);山西省回国留学人员科研资助项目(2011050);山西农业大学引进人才博士科研启动基金项目(2013YJ23)

摘　　要：为了确定西葫芦ISSR-PCR的最佳反应体系,以西葫芦基因组DNA为模板,采用正交试验方法,对模板DNA、引物及2×Taq PCR Master Mix的用量进行了优化,并通过梯度PCR确定不同引物的最佳退火温度。结果表明,最佳反应体系为:在20μL的体系中,模板DNA(50 ng/μL)1 L,引物(10μmol/L)3 L,Master Mix 10μL;利用该体系从50条ISSR引物中筛选出18条扩增条带清晰、多态性好的引物。这一体系的建立及引物筛选为以后利用ISSR分子标记技术对西葫芦进行研究提供了科学依据。To determine the best ISSR-PCR reaction system for Cucurbita pepo, experiment with Cucurbita pepo genome DNA as the template, the concentrations of template DNA, primers and 2×Taq PCR Master Mix were optimized by an orthogonal experimental method. The optimal anneal temperature of different primers was determined by gradient PCR. The results showed that the best reaction system was as follows： template DNA（50 ng/μL）1 L, primers（10 μmol/L）3 L, Master Mix 10 μL. Using this system, 18 primers with clear and multiple polymorphic bands were selected from 50 primers. The establishment of this system and polymorphism primers could provide a scientific basis for related research using ISSR molecular marker technology in Cucurbita pepo in the future.

关 键 词：西葫芦 ISSR-PCR 正交设计 引物筛选 

分 类 号：S642.6[农业科学—蔬菜学]