均匀设计优化棉花ISSR-PCR反应体系

为探索适宜棉花的ISSR-PCR反应体系,采用两轮均匀设计试验,对ISSR-PCR反应体系中Mg2+、dNTPs、模板DNA、引物、Taq DNA聚合酶的浓度或用量进行优化。结果表明,棉花20μL的ISSR反应体系的最佳组分包括2μL 10×PCR Buffer、0.5 U Taq DNA聚合酶、40 ng模板DNA、0.25 mmol.L-1 dNTPs、0.5μmol.L-1引物和2.0 mmol.L-1 Mg2+。利用8个棉花品种和三条不同引物验证该反应体系,结果表明,该反应体系的稳定性和重复性较好。

The Optimization of ISSR-PCR System of Cotton by Uniform Design

In present study，ISSR-PCR reaction conditions on Gossypium spp. were optimized. The factors which affect ISSR results of Gossypium spp. were investigated by uniform designs of two times. The results showed that 0.5 U Taq polymerase，40 ng template DNA，0.25 mmol·L^-1 dNTPs，0.5 μmol·L^-1 primer and 2.0 mmol·L^-1 Mg²⁺ in 20 μL ISSR reaction system might be the best combination. To validate the ISSR-PCR reaction system above，eight cultivars of Gossypium and three ISSR primers were employed in the research. It is indicated that the ISSR reaction system would be more productive and repeatable