兴安落叶松SSR反应体系的建立与优化

Optimization of SSR-PCR Reaction System for Larix gmelinii

采用L16(4^5)正交试验设计,对影响兴安落叶松SSR-PCR反应体系的主要因素(模板DNA、d NTPs、Mg^2+、Taq聚合酶浓度及引物浓度)进行了试验分析,其结果表明:兴安落叶松SSR-PCR体积为20μL的最佳反应体系是模板DNA用量50 ng、正反向引物浓度均为5μmol·L^-1、d NTPs浓度为2.5 mmol·L^-1、Mg^2+的浓度为2.5 mmol·L^-1、Taq聚合酶用量为1.0 U、最佳退火温度为60℃。试验所建立的反应体系可进一步用于兴安落叶松SSR遗传图谱构建、多样性分析、良种选育等方面的研究中。

The important basis of the researches on SSR marker assisting selective breeding of Larix gmelinii is to establish an SSR-PCR reaction system and get some polymorphism primers. In order to lay a foundation for the researches on applying SSR marker technology to establishment of genetic diversity and molecular breeding, some main impact factors in the Larix gmelinii SSR-PCR reaction system were analyzed by using combination method of L1 6(4^5) orthogonal test and single factor tests, including DNA templates,primer, dNTPs, Mg^2+, Taq polymerase, and annealing temperature. The results showed that the optimized PCR reaction mixture was 20 μL total volume including 50 ng template DNA, 5 μmol/L forward and reverse primers, 2.5 mmol/L dNTPs, 2.5 mmol/L Mg^2+ and 1.0 U Taq DNA polymerase, and the optimum annealing temperature was 60℃. This result indicated that the optimized SSR-PCR reaction system could provide a vital theoretic and technical support for genetic maps establishment and molecular marker assisted breeding in Larix gmelinii.