Establishment of the Optimum ISSR-PCR Reaction System in Hemerocallis spp.

Inter-simple sequence repeat(ISSR) method was applied to detect inter-and intra-specific genetic variation,and better reveal genetic relationship and genetic diversity.The factors which affect the inter-simple sequence repeat(ISSR) analysis were studied through 5 daylily cultivars.The optimal ISSR-PCR reaction conditions for daylily were established as follows: in a volume of 20 μL containing 2 μL 10×buffer,0.2 mmol/L dNTP,1.5 mmol/L Mg2+,0.5 mmol/L primer,10.002 nkat Taq DNA polymerase,and 20 ng template DNA.The optimal amplified procedure was as follows: after a pre-denaturing of 5 min at 94 ℃,40 cycles were performed with denaturing of 40 s at 94 ℃,annealing of 45 s at 50 ℃,extension of 1.5 min at 72 ℃,a final extension of 10 min at 72 ℃.The clear and stable amplified bands might be obtained with the above ISSR-PCR reaction conditions.