杂交油菜ISSR-PCR反应体系的建立和优化

利用正交试验设计的方法，对影响ISSR-PCR反应的Mg²⁺、dNTP、引物和Taq DNA聚合酶4个因素进行优化试验，以期建立其最佳反应条件。并在此基础上，对DNA模板浓度和ISSR PCR反应程序中的退火温度进行梯度筛选。结果表明：杂交油菜20 μL ISSR-PCR最佳反应体系包括1.50 mmol·L^-1 Mg²⁺、0.125 mmol·L^-1 dNTP、2.00 μmol·L^-1 primer、0.50 U Taq DNA聚合酶、2.5 μL 10×buffer和40 ng DNA模板；引物UBC891适宜的退火温度为54.2℃。该体系在青杂3号及其父本不同个体中能够扩增出条带清晰、稳定性好的条带。ISSR-PCR反应体系的建立为利用分子标记技术研究杂交油菜品种纯度和真实性鉴定奠定了良好基础。

Establishment and Optimization of ISSR-PCR System for Hybrid Rapeseed

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.