鹰嘴豆 SSR-PCR 反应体系优化

摘要

为了进行鹰嘴豆（Cicer arietinum L．）遗传多样性分析，以鹰嘴豆叶片基因组 DNA 为模版，通过 Mg2＋、DNA 聚合酶、模版 DNA、dNTPs 以及引物的用量，设计5因素4水平的正交试验对 SSR-PCR反应体系进行优化，并利用梯度 PCR 对退火温度进行选择。结果表明：优化的 SSR-PCR 扩增10μL 体系为1．5μL 1×PCR Buffer、1．5 mmol/L Mg2＋、1．5 U TaqDNA 聚合酶、0．75μmol/L SSR 引物、15 ng 模版DNA、250μmol/L dNTPs。扩增程序：95℃预变性5 min；94℃变性30 s，52．3℃退火30 s，72℃延伸90 s，共35个循环；最后72℃延伸5 min。所有 SSR 引物在优化的反应体系中基本能有效扩增，得到特异性。%The SSR-PCR reaction system of chickpea was optimized by studying the optimum concentration of Mg2+ ,TaqDNA polymerase, template DNA, dNTPs and prime to analyze genetic diversity of chickpea,and the annealing temperature was selected by using gradient PCR.The results showed that the optimized SSR-PCR system for 10μL amplification included 1×PCR Buffer,1.5 mmol/L Mg2+ ,1.5 U TaqDNA polymerase,0.75 μmol/L SSR primer,15 ng template DNA and 250 μmol/L dNTPs,and the amplification procedure included 35 circulations of pre-denature at 95℃ for 5min,at denature at 94℃ for 30s,annealing at 52.3℃ for30s and extension at 72℃ for 90s and final extension at 72℃ for 5min.All SSR primers all can be effectively amplified under the optimized system basically.