应用正交设计对大白菜纯度鉴定中EST-SSR反应体系的优化

Optimization of EST-SSR System for Cultivars Identification in Chinese Cabbage Using Orthogonal Design

以3份大白菜杂交种及其亲本为试验材料,利用改良CTAB法提取基因组DNA,采用正交设计对Mg2+、dNTPs、引物、模板DNA和Taq酶5因素4水平进行优化,结果表明,适用于大白菜纯度鉴定EST-SSR检测体系为:在10μL反应体系中包含3.0mmol·L-1MgCl2、400μmol·L-1dNTPs、0.20μmol·L-1SSR引物、4ng模板DNA和0.05UTaq酶。利用该优化的体系,对3份大白菜杂交种进行单株育苗试验,每份杂交种取单株140株进行纯度鉴定。结果显示,扩增带型清晰、稳定,纯度检测结果与田间试验结果基本吻合,可用于大白菜种子纯度分子标记的进一步研究。

Three Chinese cabbage cuhivars genornic DNA, extracted by modified CTAB method was applied to optimize EST-SSR amplification system based on orthogonal design.Five factors were considered such as Mg2＋,dNTPs,primer, template DNA and Taq DNA polymerase at four levels. A suitable EST-SSR reaction system was established. The results showed that 10μL reaction system containing 3.0 mmol. L-1 MgC12, 400 μmol. L-1 dNTPs, 0.20μmol. L-1 primer, 4 ng DNA template and 0.05 U Taq DNA polymerase. EST-SSR PCR was amplified by using the above primer pairs and optimized system, using 140 seedlings of every Chinese cabbage, the purity identification of the 3 Chinese cabbage cultivars could be done.This indicated that EST-SSR markers could be used for rapid and accurate identification of seed purity of Chinese cabbage cultivar.