Optimization of the ISSR reaction system of Elymus nutansby orthogonal design

Five factors（DNA polymerase, Mg2+, template DNA, dNTPs, primer) in ISSRPCR reaction system were carried on the optimized experiment by taking the genomic DNA of Elymus nutans as template. The optimum reaction system and reaction process of E. nutans ISSRPCR analysis were established. The results of this study showed that the volume of reaction system was 25 L. It included 2.5 L 10buffer (except Mg2+), 1.0 U TapDNA polymerase, 2.0 mmol/L Mg2+, 30－120 ng template DNA，0.25 mmol/L dNTP and 0.25 mol/L primer. Eleven primers with stable amplification and rich polymorphism were obatined based on the optimal PCR reaction system.The reaction program was devised for 2 min at 94℃ in the first circle， 1 min at 94℃, 51℃ for 1 min (depended on different primer), 72℃ for 1.5 min for 41 cycles, and 72℃ for 10 min for extending，which was obatined across the gradient PCR experiments, including temperature gradient,cycle gradient and extending time gradient experiments. Validation of the optimistic ISSRPCR reaction system and amplification program showed that the reaction system and amplification program had high stability and good reproducibility.