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采用正交设计法优化适合于杂交油菜的ISSR体系,对影响ISSR-PCR的多个因素,包括Mg2+、dNTP、TaqDNA聚合酶、引物用量等进行了比较、优化;对DNA模扳浓度,退火温度进行了筛选。结果确定了杂交油菜ISSR-PCR反应体系的最佳方案为:94℃预变性5分钟,1个循环;每个循环94℃变性20s;56.2℃退火1分钟,72℃延伸80s,38个循环;72℃完全延伸6分钟;4℃保存。20μl PCR反应体系包括10×Buffer 2.5μL、模板20ng 1μl、2.5mmol/L dNTP1μl、10μmol/L Primer 1.2μl、5U TaqE0.2μl、25mmol/L Mg2+1.4μl。
Orthogonal design method was used to optimize the ISSR system for hybrid rapeseed. The factors influencing ISSR-PCR including Mg2 +, dNTP, Taq DNA polymerase and primers were optimized and optimized. The DNA template concentration, annealing temperature Screening was conducted. The results showed that the optimal protocol for ISSR-PCR reaction was as follows: pre-denaturation at 94 ℃ for 5 minutes and 1 cycle; denaturation at 94 ℃ for 20s each cycle; annealing at 56.2 ℃ for 1 minute and extension at 80 ℃ for 80s and 38 cycles; 72 ℃ complete extension of 6 minutes; 4 ℃ preservation. The 20μl PCR reaction system consisted of 2.5μl of 10 × Buffer, 1μl of template 20ng, 1μl of 2.5mmol / L dNTP, 1.2μl of 10μmol / L Primer, 0.2μl of 5U TaqE and 1.4μl of 25mmol / L Mg2 +.