送春与多花兰杂种ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for the Hybrid of Cymbidium cyperifolim var.szechuanicum and C.floribundum

[目的]建立与优化送春与多花兰杂种ISSR-PCR的反应体系。[方法]用CTAB法提取送春、多花兰和送春×多花兰杂种的基因组DNA,针对ISSR-PCR扩增的体系中的Taq酶浓度、Mg2+浓度、dNTP浓度和引物的用量4个因素,进行L9(43)正交试验,用引物UBC827扩增两亲本的混合DNA,所得PCR产物在琼脂糖凝胶上检测,同时针对去离子甲酰胺对反应的影响进行初步对照试验。[结果]根据PCR产物的琼脂糖凝胶检测结果,由试验得到的最佳反应体系为:1×buffer、2.5 mmol/L Mg2+、0.6μmol/L引物0、.25 mmol/L dNTP、1 UTaq酶、0.4μl去离子甲酰胺5、0 ng模板DNA,总体积为20μl。[结论]该反应体系的建立为利用ISSR技术进行兰花遗传连锁图谱构建、基因定位、种质资源鉴定与分类等提供参考。

[ Objective ] The aim of the study was to establish and optimize ISSR-PCR reaction system for hybrid of Cymbidium cyperifolim var. szechuanicum and C. floribundum. [Method] The method of CrAB was applied to extract the genome DNA of C. cyperifolim, C. floribundum and their hybrids. A L9（4^3） orthogonal test was designed to study the effect of Taq polymerase, Mg^2＋ , dNTP and primer with different levels in ISSR-PCR reaction system. The mixture of two parents genome DNA was amplified by using primer UBC827, and the PCR products were examined on agarese gel. The inoact of deionized fonnamide on ISSR-PCR reaction was evaluated through the primary control test. [Result] Based on the result of PCR products tested on the agarose gel, an optimum reaction system obtained in the test was as follows： 1 × buffer, 2.5 mmol/L Mg^2＋ , 0.6μmol/L primer, 0.25 mmol/L dNTP, 1 U Taq polymerase, 0.4 μl deionized formamide, 50 ng DNA template, total volume of reaction system being 20 μl. [ Conclusion] The establishment of ISSR-PCR reaction system provides the reference for orchid genetic linkage mapping, gene mapping, germplasm identification and classification etc. by using ISSR technique.