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 stablereaction system could amplify high levels of polymorphism, good repeatability and clear band patterns. ISSRsystem(total volume of 20 μL) established was as follows: template DNA 10 ng/μL 3 μL, dNTP 2.5 mmol/L1.5 μL, 10×PCR Buffer 3.0 μL, primer 4 μmol/L 3.5 μL, Taq polymorphism 5 U 0.2 μL. It showed that theoptimized system in this experiment could be applied in the ISSR analysis for Dendrobium. This researchprovided technical support for genetic diversity and genetic relationship research, linkage map construction,QTL location, gene mapping and gene cloning and so on.