白蜡属SSR-PCR反应体系优化及引物筛选

本研究建立了适合白蜡SSR-PCR反应的最佳体系，并利用该体系从50对白蜡引物中筛选出了3对条带清晰、多态性好的引物，用于白蜡SSR标记的进一步研究。该研究采用L16（4^5）正交设计和单因素试验对影响白蜡SSR-PCR的Taq聚合酶用量、Mg2＋浓度、DNA模板浓度、dNTP浓度和引物浓度等5个因素在4水平上进行筛选。优化后的白蜡SSR反应体系为：Mg2＋（25mmol／L）0．8μＬ、引物（10μmol／L）0．2μL、DNTP（10mmol／L）0．3μL、Taq酶（5U／μL）0．05μL、DNA模板（5-10ng／μL）2．00μL、10×PCR缓冲液1．0μL，ddH2O 5．45μL，总体积10．0μL。该结果为今后利用SSR-PCR标记技术研究分析白蜡奠定了基础。

Optimization of SSR-PCR Reaction System in Fraxinus and SSR Primer Selection

This study established and optimized SSR-PCR reaction system of Fraxinus, and used this system selected 3 primer pairs from 50 primer pairs for the SSR analysis based on their reproducible and clear banding patterns. These primer pairs could be used for further research. This experiment established through L16（4^5） orthogonal design which selected from 4 levels of 5 factors （Taq DNA polymerase, DNA, dNTPs concentration, primers concentration and Mg2＋ concentration） in SSR-PCR system and optimized through single factor experiment about the main factors of the PCR reactions. The optimal SSR-PCR reaction system consisted of Mg2＋ （25 mmol/L） 0.8 μL, containing forward- and reversed-primer （10 μmol/L） 0.2 μL respectively, dNTP （10 mmol/L） 0.3 μL, Taq DNA polymerase （5 U/μL） 0.05 μL, DNA （5-10 ng/μL） 2.00 μL, 10×PCR Buffer 1.0 μL and ddH20 5.45μL. The reaction system was the most conformable one for Fraxinus＂ SSR-PCR, and was established as the good foundation of SSR-PCR marker technique on Fraxinus.