不结球白菜ISSR反应条件的优化

Optimization for ISSR-PCR System in Non-heading Chinese Cabbage

本文通过单因素试验确定了Taq DNA聚合酶、dNTP、引物和Mg^(2+)4种因素在不结球白菜ISSR反应体系中的适宜浓度范围,并在此基础上利用正交试验设计,从4种因素3个水平对不结球白菜ISSR反应体系进行了优化,确立了适合不结球白菜的ISSR反应体系,并在10个不结球白菜品种中进行了验证。在20μL反应体系中,含Taq DNA聚合酶1U、dNTP 0.25 mmol/L、引物0.25μmol/L、1×PCR buffer、Mg^(2+) 2.5 mmol/L、模板DNA 30 ng。通过梯度PCR测验和总循环次数梯度处理试验,确定了适宜的退火温度和总循环数。这一体系的建立为今后利用ISSR技术进行不结球白菜种质资源分类、遗传图谱构建和基因定位奠定了技术基础。

In this paper, the suitable concentration of four factors, Tatl polymerase, dNTP, primer and Mg^2＋, were identified by single factor tests in the ISSR amplification system on non-heading Chinese cabbage （Brassic campestris L. ssp. Chinensis Makino）. And then orthogonal scheme in four factors at three levels was designed based on the result of above single factor tests to optimize ISSR amplification system. An ideally ISSR reaction system was established that included 20 μL reaction volume containing Taq polymerase 1 U, dNTP 0.25 mmol/L, primer 0.25 μmol/L, 1 ×PCR buffer, Mg^2＋ 2.5 mmol/L, and template DNA 30 ng. The optimal annealing temperature and cycling numbers for ISSR-PCR reaction were confirmed by gradient PCR and the gradient treatment of cycling numbers. This work might provide a groundwork for germplasm classification, genetic map construction and important gene localization in non-heading Chinese cabbage.