摘要
菜薹原产中国,是华南地区重要的特产蔬菜之一。目前,分子标记技术已广泛应用于多种作物研究,但在菜薹上的应用研究刚刚起步。ISSR主要是由单一引物且以重复序列为主要引物序列的PCR标记,其反应条件受模板DNA、Taq DNA聚合酶、Mg2+、dNTP和引物等因素的影响。正交试验设计能明确各因素间的互作效应,建立最优化的反应体系。本研究利用正交试验设计,从Taq DNA聚合酶、dNTP、引物、Mg2+4种因素3个水平对菜薹ISSR反应体系进行了优化,确立了适合菜薹的ISSR反应体系并在7个菜薹品种中进行了验证。在20μl反应体系中,含Taq DNA聚合酶1U、dNTP250μmol/L、引物0.25μmol/L、1×PCRbuffer、Mg2+2.5mmol/L、模板DNA30ng。通过梯度PCR测验,确定了适宜的退火温度。这一体系的建立为今后利用ISSR技术进行菜薹种质资源分类、遗传图谱构建和基因定位奠定了技术基础。
Brassica campestris L. ssp. Chinensis var. utilis Tsen et Lee is native to China and is one of important vegetables mainly grown in the area of South China. Currently, the technology of molecular marker has been widely used in researching work in a variety of crops but such a work in Brassica campestris L. ssp. Chinensis var. utilis Tsen et Lee has just initiated. ISSR is a molecular marker taking repeat sequences as major primer sequence based on PCR, which is affected by template DNA, Taq DNA polymerase, Mg2+, dNTP and primer. Orthogonal design can determinate interactions of each factor, therefore best reaction systems can be established. In this study, orthogonal design was used to optimize ISSR amplification system on Brassica campestris L. ssp. Chinensis var. utilis Tsen et Lee in four factors (Taq DNA polymerase, dNTP, primer, Mg2+) at three levels respectively. A suitable ISSR reaction system was established, namely 20μl reaction system containing Taq DNA polymerase 1U, dNTP 250μmol/L, primer 0.25μmol/L, 1×PCR buffer, Mg2+ 2.5mmol/L, template DNA 30ng. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. It proved the basis for the analysis of diversity, map construction and gene localization of important traits in Brassica campestris L. ssp. Chinensis var. utilis Tsen et Lee, with ISSR markers.
出处
《分子植物育种》
CAS
CSCD
2006年第S2期137-141,共5页
Molecular Plant Breeding
