葡萄iPBS-PCR反应体系的优化

Optimization of iPBS-PCR system in grapevine

【目的】iPBS标记是一种新型、快速、高效的分子标记方法,试验筛选并优化了葡萄的iPBS-PCR反应体系,以建立葡萄种质资源评价和品种鉴定的可靠方法。【方法】采用L16(45)正交试验,对影响葡萄iPBS-PCR反应的模板浓度、Mg2+浓度、dNTPs浓度、Taq酶用量、引物用量及退火温度等因素进行了优化,运用Bandscan和正交设计助手等软件对电泳结果条带进行统计分析。【结果】优化后的葡萄iPBS-PCR扩增的最佳反应条件为:DNA模板20 ng、Taq酶1.00 U、Mg2+2.40 mmol·L-1、引物0.50μmol·L-1、dNTPs 0.20 mmol·L-1,总体积20μL。退火温度53.8℃。【结论】建立了适合葡萄iPBS-PCR分析的优化反应体系,为葡萄种质遗传多样性的评价和鉴定提供了新的技术手段,为其他植物的相关研究也提供了参考。

【Objective】 iPBS is a new, rapid and efficient method of molecular markers. This study aimed to explore and optimize a suitable iPBS-PCR reaction system for grapevine, then establish and provide a reliable method for grape germplasm evaluation and cultivar identification. 【Method】 The main components of the iPBS-PCR reaction system such as the constructions of Mg2+, dNTPs, primer, template DNA, Taq DNA polymerase and annealing temperature were optimized with L16(45)orthogonal test and the results were analyzed with Bandscan and Orthogonal Experimental Design software. 【Result】 The results showed that the optimum conditions of iPBS-PCR amplification of grapevine were as follows: DNA template 20ng, Taq DNA polymerase 1.00 U, Mg2+2.40 mmol·L-1, primer 0.50 μmol·L-1and dNTPs 0.20 mmol·L-1in total 20 μL volume and annealing temperature 53.8 ℃. 【Conclusion】 The established system provided a new efficient method for genetic diversity evaluation and cultivar genotyping of grapevine, also a reference for other plants.