百合属ISSR反应条件优化的研究

建立一个稳定性高、重现性好，适合百合ISSR遗传差异分析的PCR反应体系。设定反应体系中各因子的不同浓度并进行PCR扩增，依据稳定性高、重现性好的原则，对该反应体系进行调整与优化，最终建立稳定可靠的ISSR反应体系。适于百合ISSR-PCR反应的最佳体系(25μL)为：40 ng模板DNA，2.0 mmol/L Mg²⁺，1.5 U TaqDNA聚合酶，0.8μmol/L引物，200μmol/L dNTPs；扩增程序为：94℃预变性5 min；94℃变性1 min，51.8℃退火1 min，72℃延伸2 min，共35个循环；最后72℃延伸8 min。所建立的百合ISSR反应体系具有稳定性高、重现性好、检测多态性能力强等特点，为应用ISSR标记技术进行百合属植物遗传多样性分析和品种分子鉴别等研究奠定了技术基础。

The Optimization of ISSR Reaction Conditions of Lilium

The aim was to establish a stable, reproducible, and suitable reaction system of Lilium for ISSR analysis of genetic differences. PCR amplifications were carried out until the different concentrations of the factors in the ISSR reaction system were designed, and based on the principle of high stability and reproducibility, the stable and reliable ISSR reaction system was eventually established after a series of adjustment and optimization of the reaction system. The optimum ISSR-PCR reaction system (25μL) contained 40 ng DNA template, 2.0 mmol/L Mg²⁺, 1.5 U TaqDNA polymerase, 0.8μmol/L ISSR primer and 200μmol/L dNTPs. The reaction mixtures were pre-denatured at 94℃for 5 min and subjected to 35 cycles of 1 min at 94℃, 1 min at 51.8℃, 2 min at 72℃, and a final extension step of 8 min at 72℃. The ISSR-PCR systems showed stable reaction system, better repeatability, and reliable abundant polymorphisms. This reaction system could provide a technological base for the study on genetic polymorphism analysis, cultivar identification of Lilium at the molecular level.