辣椒ISSR-PCR体系优化

Optimization for ISSR-PCR System in Capsicum annuum L.

辣椒是一种重要的蔬菜和调味品,目前在辣椒上应用较多的是RAPD、RFLP和AFLP标记,而有关ISSR分子标记报道很少。本试验利用正交设计,以辣椒SS69为试材,从Taq酶、dNTPs、引物、Mg2+4因素3水平来优化辣椒ISSR-PCR反应体系。本实验中20μL反应体系初步确定各反应成分为:1×PCR buffer,1.5 mmol/L的Mg2+,50 ng辣椒模板DNA,1.5 UTaq酶,200μmol/L的dNTPs,0.6μmol/L的引物。该体系的建立为今后利用ISSR技术进行辣椒遗传连锁图谱的构建、基因定位、种质资源鉴定与分类和遗传多样性分析奠定了技术基础。

Pepper （ Capsicum annuum L. ） is a very important vegetable and condiment. The main molecular markers on pepper are RAPD, RFLP and AFLP, but ISSR used on pepper is seldom reported. In this experiment, orthogonal design with three levels of four factors （ Taq DNA polymerase, dNTPs, primer and Mg^2 ＋ ） was used to optimize the pepper ISSR - PCR reaction system. The results showed that a better amplification of ISSR was obtained with the reaction system containing 1 × PCR buffer, 1.5 mmol/L Mg^2＋ ,50 ng template DNA of pepper, 1.5 U Taq polymerase, 200 umol/L dNTPs, 0.6 umol/L primer in the total volume of 20 uL. It provided the basis for map construction, gene localization of important traits, germplasm identification and classification, and the analysis of genetic polymorphism in pepper with ISSR markers.