石斛属植物ISSR扩增体系的建立与优化

旨在建立并优化石斛属植物的ISSR-PCR反应体系。通过对ISSR-PCR反应体系中主要影响因子模板DNA、dNTP、10×PCR Buffer、引物以及Taq DNA聚合酶分别进行单因素优化，最终建立一套适用于石斛属植物的扩增多态性高、稳定性强、带型清晰的ISSR最佳反应体系。20μL反应体系的适宜浓度及用量分别为：模板DNA 10 ng/μL 3μL，dNTP 2.5 mmol/L 1.5μL，10×PCR Buffer 3.0μL，引物4μmol/L 3.5μL，TaqDNA聚合酶5 U 0.2μL。这一优化体系适用于石斛ISSR分析，为今后遗传多样性与亲缘关系分析、连锁图谱构建、QTL定位、基因定位与克隆等方面的研究提供了技术支撑。

Establishment and Optimization of ISSR Reaction System for Dendrobium

The research aimed to establish and optimize a stable ISSR-PCR reaction system for Dendrobium. In this paper, the key factors of ISSR-PCR reaction system including DNA template concentration, dNTP, 10× PCR Buffer, primer concentration, DNA Taq polymorphism were investigated and optimized. The stable reaction system could amplify high levels of polymorphism, good repeatability and clear band patterns. ISSR system （total volume of 20 μL） established was as follows： template DNA 10 ng/μL 3μL, dNTP 2.5 mmol/L 1.5 μL, 10×PCR Buffer 3.0 μL, primer 4 μmol/L 3.5 μL, Taq polymorphism 5 U 0.2 μL. It showed that the optimized system in this experiment could be applied in the ISSR analysis for Dendrobium. This research provided technical support for genetic diversity and genetic relationship research, linkage map construction, QTL location, gene mapping and gene cloning and so on.