Abstract:

The influenced factors of ISSR-RCR reaction were optimized and the effect of 5 factors such as annealing temperature， Taq DNA polymerase dosage， DNA templates concentration， primer concentration and dNTPs concentration using single factor experiment based on the Pyrus py rifolia Nakai genomic. Its reaction system and amplified procedure were established that is 20μl amplification reaction system containing lxTaq enzyme PCR buffer，0.5 U Taq DNA polymerase， 100μmol /L dNTPs，0.7μmol/ L primer，2.25 mmol/LMgCl2, 40ng template DNA. The optimized annealing temperature is 52℃.The optimal amplified procedure was as follows: after apre-denaturing of 5 min at 94℃,40 cycles were performed with 45s for denaturing at 94℃，annealing of 45s at 52℃，extension of 1 min at 72℃，10 minutes of extension at 72℃ in the final cycle and hold at 4℃.Using primers 863 was selected to separate 16 copies Pyrus py rifolia Nakai material. 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.