牡丹ISSR反应体系的优化研究+正交试验设计
通过L16（45）正交试验设计，对牡丹ISSR反应体系中Mg2+、dNTPs、Taq DNA聚合酶、引物和模板DNA浓度5因素进行了优化，并筛选合适引物。
牡丹的ISSR最佳反应体系为：反应体系总体积20 uL，含10×PCR buffer，Mg2+ 3.0 mmol•L-1、dNTPs 400 μmol•L-1、引物1.0 μmol•L-1、模板DNA 20 ng、Taq DNA聚合酶0.5 U。
关键词：牡丹；ISSR反应体系优化；引物筛选4203
Optimization of ISSR Reaction System of Tree Peony
Abstract: Paeonia suffruticosa Andr. is China's traditional flower and known as the "king of flowers" because of its big size and colorful flower [1]. With the development of the study of biology, molecular breeding in the applyment of Peony attracting much attention. Molecular marker-assisted breeding has the advantages of fast, accurate and without environmental conditions, we can take advantage of the target traits closely linked molecular markers to select new varity in the seedling stage. Thus greatly improving the breeding rate and speed up the breeding process. The key of Marker-assisted selection breeding technology is the identification of DNA molecular markers. Tightly linked with the important trait. In this paper, we create and optimize Peony ISSR reaction system using orthogonal design; and provide theoretical foundation for the polymorphism analysis of Peony F1 generation.
In the L16（45）orthogonal design, the 5 factors in Peony ISSR reaction system, Mg2+, dNTPs, Taq DNA polymerase, primers and template DNA concentration were optimized .
The ISSR optimal reaction system of Peony is:, the total volume of 20 uL contain 10 × PCR buffer, Mg2+3.0 mmol • L-1, dNTPs 400 μmol • L-1, cited material 1.0 μmol • L-1, template DNA 20 ng, Taq DNA polymerase 0.5 U.
Keywords: Paeonia suffruticosa Andr.; ISSR; Reaction system optimization
目录
1绪论 1
1.1 背景和意义 1
1.1.1 牡丹背景介绍 1
1.1.2 研究目的和意义 1
1.2 常用分子标记 1
1.2.1 分子标记的简介 1
1.2.2 分子标记的分类 2
1.3 分子标记在牡丹研究中的应用 3
1.3.1 RAPD标记 3
1.3.2 SSR标记 3
1.3.3 ISSR标记 3
1.3.4 AFLP标记 4
1.3.5 SRAP标记 4
1.4 分子标记在牡丹研究中的应用前景与展望 5
1.4.1 亲缘关系分析 5
1.4.2 品种分类鉴定 5
1.4.3 遗传图谱构建 5
1.4.4 分子标记辅助育种 5
2 材料与方法 7
2.1 材料 7
2.2 牡丹基因组DNA的提取与检测 9
2.3 ISSR-PCR反应体系的正交试验 9