航天搭载紫花苜蓿SSR-PCR反应体系的优化以及引物的筛选

Optimization of SSR-PCR system of spaceflight alfalfa and primers selection

航天育种的变异频率高、变异幅度大、有益变异多、稳定性强,优势明显。依据正交试验设计原理,设计了用正交试验和细调正交试验来确定航天搭载紫花苜蓿SSR-PCR反应体系中各成分的浓度,从dNTP、Taq酶、Pri mers、Mg2+4种因素对航天搭载紫花苜蓿SSR-PCR反应体系进行了优化,得到适合航天搭载紫花苜蓿SSR-PCR最佳反应体系,即25μL的反应体系中含有dNTP 0.2 mmol/L、TaqDNA聚合酶2U、引物0.7μmol/L、Mg2+2.5 mmol/L、以及10×buffer和60 ng模板DNA。在试验确定最佳反应体系基础上,对17对SSR引物进行筛选,选出6对扩增条带信号强、背景清晰的引物。

Spaceflight breeding has high variation frequency,large variation amplitude,more profitable variation and high stability.The orthogonal design was used to optimize SSR amplification system of spaceflight alfalfa.Two orthogonal experiments were conducted to investigate the amplification efficiency under different amplification conditions.Results showed that the optimum concentrations of reactants in 25 μl reaction mixture were as follows：dNTP0.2 mmol/L,Taq DNA polymerase 2U,Primers 0.7 μmol/L,Mg^2＋2.5 mmol/L,10×buffer and 60 ng template DNA.Totally 6 SSR primers were selected from 17 SSR primers by sieving which could produce DNA polymorphism.