Objective: To establish optimal inter-simple sequence repeat-polymerase chain reaction(ISSR-PCR) reaction system for Gentiana straminea and provide a reference for medicinal germplasm identification and genetic diversity research. Method: Influencing factors of ISSR-RCR reaction system(template DNA

Mg2+

dNTPs

Taq DNA polymerase

primer and annealing temperature) were selected and optimized by single factor tests and orthogonal test. Result: Optimum reaction system(20 μL) contained template DNA of 1 μL(36 mg · L-1)

Mg2+ (10×PCR buffer) of 1.75 μL(20 mmol · L-1)

dNTPs of 0.8 μL(10 mmol · L-1)

Taq DNA polymerase of 0.1 μL(0.5%) and primers of 0.6 μL(10 mmol · L-1).The suitable annealing temperature of 12 primers was determined

which had rich polymorphism and stable amplification.The best annealing temperature of UBC-809 primer was 55.5 ℃

there were 6 polymorphism bands in amplification of 7 bands

the percentage of polymorphism loci reached 85.71%. Conclusion: This established ISSR reaction system is stable and credible.The polymorphic bands

which is located at 500 bp of strip

can be used as the species characteristic band.