小麦秆锈菌RAPD扩增体系的正交优化

RAPD Reaction System Orthogonal Optimization for Molecular Research of Puccinia graminis f. sp. tritici

针对RAPD扩增技术在小麦秆锈菌遗传多样性的分析研究中重复性较低的不足,对小麦秆锈菌RAPD扩增体系进行优化。以小麦秆锈菌不同的生理小种为材料,采用L16(45)正交试验设计,考察模板DNA、Mg2+、TaqDNA聚合酶、dNTPs和随机引物浓度5个因素的影响,并对退火温度和循环次数进行优化。结果表明:小麦秆锈菌RAPD扩增的较优体系为25μL体系中含1×Buffer,40ng·μL-1模板DNA,1.5mmol·L-1Mg2+,2.0U TaqDNA聚合酶,0.25mmol·L-1dNTPs,0.40μmol·L-1随机引物;适宜的退火温度为36℃,适宜的循环次数为43次。该反应体系检测多态性能力强、反应系统稳定且重复性好,可以较好地应用于小麦秆锈菌的遗传多样性分析研究。

RAPD amplification technology is one of the most common analysis methods which were used in variety identification and polymorphism analysis of Puccinia grominis f. sp. tritici. This technique is rapid, inexpensive and does not require abundant quantities of high-quality DNA, but one disadvantage is poor reproducibility. In this paper, RAPD reaction system of/9. graminis f sp. tritici was optimized in order to acquire optimal PCR amplification system for researching of P.. grominis f. sp. tritici. In this study, using races of P. graminis f. sp. tritici as material, L16 （4^5） orthogonal experiment design was performed with 5 factors of the concentration of DNA, Mg〉, TaqDNA polymerase, dNTPs and random primers for the RAPD reaction system in P. grominis f. sp. tritici. Then annealing temperature and circulation times were optimized. The results indicated that the RAPD reaction system of P. graminis f. sp. tritici was lxBuffer, 40 ng·μL^-1 template DNA, 1.5 mmol·L^-1 Mg^2＋, 2.0 U TaqDNA polymerase, 0.25 mmol·L^-1 dNTPs, and 0.40 μmol·L^-1 random primer in the 25 μL reaction solution, 36℃ annealing temperature and 43 cycles. This reaction system has great ability in detecting polymorphism, stable response system and good repeatability. It could be well applied to analysis genetic diversity of R graminis f. sp. tritici.