元宝枫SSR-PCR反应体系优化及引物筛选

Optimization and Primers Screening of SSR-PCR Reaction System for Acer truncatum

本研究采用改良的CTAB法提取元宝枫基因组DNA,并使用L16(45)正交试验设计。通过5因素4水平实验,筛选Taq DNA聚合酶、dNTPs、引物浓度、Mg2+、模板DNA浓度及其用量,建立并优化元宝枫(Acer truncatum)的最佳SSR-PCR反应体系,即总体积20μL,Taq DNA聚合酶、dNTPs、引物、Mg2+和模板DNA浓度分别为1 U/20μL、0.2 mmol/L、0.6 mmol/L、1.25 mmol/L和75 ng/20μL。扩增实验结果表明,该反应体系稳定性较好,可重复性高,具有较好的分辨率。可从59对引物中筛选出14对适用于元宝枫并具有较明显的多态性的SSR引物,为进一步研究元宝枫的分子标记辅助育种、SSR遗传多样性分析和遗传图谱构建提供了良好的基础。

In this study,the modified CTAB method was used to extract the genomic DNA of Acer truncatum Five factors of 4 levels in the SSR-PCR system were selected by L16(45) orthogonal experiment.The optimal SSR-PCR system of Acer truncatum was established and optimized by screening Taq DNA polymerase,dNTPs,primer concentration,Mg2+,template DNA concentration and their dosage.The result showed that the optimized SSR-PCR for Acer truncatum was 20μL total volume including 1 U/20μL Taq DNA polymerase,0.2 mmol/L dNTPs 0.6 mmol/L forward and reverse primers,1.25 mmol/L Mg2+,and 75 ng/20μL DNA.The results of amplification experiments showed that the reaction system has good stability,high repeatability and good resolution.Using the optimized SSR-PCR system,14 pairs of SSR primers suitable for Acer truncatum with obvious polymorphism were selected from 59 pairs of primers.The result could provide a good foundation for further application in molecular marker assisted breeding,SSR genetic diversity analysis,genetic map construction and other research in Acer truncatum.