Abstract:

Abstract: Based on the genomic DNA extracted from cauliflower, the factors influenced ISSR were optimized and the effect of 5 factors such as annealing temperature, Taq DNA polymerase dosage, DNA templates concentration, primer concentration and dNTPs concentration on ISSR amplification were tested using single factor experiment. A reaction system and amplified procedure suitable for cauliflower were established, that is 25μl amplification reaction system containing 1×PCR buffer（Mg2+）, 0.75Ｕ Taq DNA polymerase, 0.15mmol/L dNTPs, 0.5μmol/Lprimer, 60ng template DNA. The optimized annealing temperature is 48.6℃. The optimal amplified procedure was as follows: after a pre-denaturing of 4 min at 72℃, 35 cycles were performed with 30s for denaturing at 94℃, annealing of 45s at 48.6℃,extension of 1.5 min at 65℃, 7 minutes of extension at 65℃ in the final cycle and hold at 4℃. The extablishment of the ISSR-PCR reaction system could settle favorable foundation for identification of cauliflower cultivars, classification and analysis of the genetic diversity of cauliflower using ISSR molecular marker techniques.