Abstract: An orthogonal design was used to optimize the ISSR-PCR system for hybrid rapeseed at three levels of four factors (Mg²⁺, dNTP, primer and Taq DNA polymerase). Then, based on the optimal ISSRPCR amplification system, the concentration of DNA template and annealing temperature were selected. As a result, the optimal PCR (20 μL) mixture contained 1.50 mmol·L-1 Mg²⁺, 0.125 mmol·L^-1 dNTP, 2.00 μmol·L^-1 primer, 0.50 U Taq DNA polymerase, 2.5 μL 10×buffer and 40ng template DNA. The suitable annealing temperature of primer UBC891 was 54.2℃. With the optimal system of ISSR-PCR reaction, clear and steady bands were obtained in different individuals of Qingza No.3 and its male parent. The establishment of ISSR-PCR system could favor the studies on the purity and genuineness of hybrid rapeseed varieties by using molecular marker techniques.

Key words: Hybrid Rapeseed, ISSR-PCR, reaction conditions, orthogonal design