大蒜ISSR-PCR反应体系的正交优化建立

Optimization of ISSR-PCR System of Garlic Based on Orthogonal Design Method

为探寻出更适宜大蒜的ISSR反应体系,以‘弥渡紫皮’大蒜为试材,对ISSR-PCR反应体系中的5个主要因素(dNTPs、Taq酶、Mg2+、引物浓度、模板DNA)在4个不同水平上进行正交设计L16(45)优化。结果表明,含10×PCRBuffer2.5μL、2.5mmol/LMg2+、0.15mmol/LdNTPs、1.5UTaqDNA聚合酶、0.4μmol/L引物、20ng/μL模扳DNA的25μL反应体系为大蒜的最佳反应体系。利用新建立的反应体系对20个不同大蒜品种进行扩增,具有极好的稳定性和重复性。

The main factors affected ISSR reaction were tested with Miduzipi garlic as test materials to optimize ISSR amplification system. In this research, the orthogonal design was used to optimize ISSR-PCR system on pepper in for 4 levels of 5 factors （Taq DNA polymerase, primer concentration Mg2, dNTPs and DNA template） respectively. The results showed that： the orthogonal experiment design was a practicable method and in a total volume of 25 μL ISSR-PCR system, the optimum reaction system of ISSR-PCR was 10x PCR Buffer 2.5 μL, 2.5 mmol/L Mg2＋, 1.5 U Taq DNA polymerase, 0.15 mmol/L dNTPs, 0.4μmol/L primer and 20 ng template DNA. The ISSR amplifications for 20 cuhivars of common garlic were then investigated through the above reaction system. The system was completely suitable to analyze the genetic information for different garlic eultivars and thus supplied the further genetic diversity of garlic with theoretic bases.