小叶锦鸡儿SSR-PCR体系优化及应用

为建立适宜小叶锦鸡儿（Caragana microphylla）SSR PCR的反应体系，利用正交试验设计L16(45)，对模板DNA、Mg2+、dNTP、引物和Taq DNA聚合酶的浓度进行五因素四水平优化筛选，确立最佳反应体系和扩增程序。即在25 μL反应体系中包括：40 ng模板DNA，3 mmol/L Mg2+，300 μmol/L dNTP，0.6 μmol/L引物，1 U Taq DNA聚合酶，1×Buffer。扩增反应程序为：94℃10 min；94℃ 45 s，65℃ 1 min，72℃ 1 min，10个循环，每循环的复性温度递减1℃；94℃ 45 s，55℃ 1 min，72℃ 1 min，30个循环；72℃ 10 min，4℃保存。应用该优化体系对小叶锦鸡儿种质资源及不同引物进行了检验及筛选。本研究表明，该体系的建立为今后利用SSR标记对小叶锦鸡儿属遗传多样性研究、遗传图谱构建及种质资源鉴定等研究工作提供依据。

Optimization of SSR-PCR system on Caragana microphylla and its application

An orthogonal design was applied to optimize the SSR-PCR system on Caragana microphylla,which employed four levels and five factors(DNA,Mg2+,dNTP,primers,and Taq DNA polymerase,respectively).The optimum SSR reaction included 40 ng template DNA,3 mmol/L Mg2+,300 μmol/L dNTP,0.6 μmol/L primer,1 U Taq DNA polymerase,1×Buffer with total 25 μL reaction volumes.The PCR procedure was an initial step of 94℃ for 10 min,followed by 10 cycles of 94℃ for 45 s,65℃ for 1 min(decreasing 1℃ every cycle),72℃ for 1 min;30 cycles of 94℃ for 45 s,55℃ for 1 min,72℃ for 1 min,and a final extension at 72℃ for 10 min.The optimized SSR-PCR system was stable and universality,which was proved by application on C.microphylla germplasm and different SSR primers.This work could be used in analysis of genetic diversity,construction of a genetic map,and fingerprint in Caragana spp..