油茶SRAP标记的PCR体系建立与优化

Establishment and optimization of SRAP-PCR system for Camellia oleifera

油茶是我国主要的木本食用油料树种,拟建立油茶新型SRAP标记(序列相关扩增多态性)的优化PCR体系。以油茶优良品种的总DNA为材料,分析了影响油茶SRAP-PCR的主要参数,包括模板DNA量、引物浓度、dNTPs、Mg2+浓度、TaqDNA聚合酶用量、退火温度6个因素的影响,建立了适合油茶SRAP-PCR的优化体系为:20μL的PCR反应体系中,2.0μL 10×PCR buffer,2.0 mmol/L Mg2+,225μmol/L dNTPs,0.6μmol/L引物,30 ng模板DNA和0.75 U Taq DNA聚合酶;最佳PCR循环条件为:94℃预变性5 min;94℃变性1 min,35℃复性1 min,72℃延伸1 min,5循环;94℃变性1 min,52℃复性1 min,72℃延伸1 min,35循环;最后72℃延伸10 min;4℃保存。本研究结果可为今后利用SRAP这一新型标记技术进行油茶分子遗传学与标记辅助选择育种研究打下基础。

Camellia oleifera is the main edible oil tree in China,in this paper,an optimized PCR system of SRAP marker（Sequence-Related Amplified Polymorphism） for Camellia oleifera was established.The total DNA extracted from excellent Camellia oleifera species were used to analyze the PCR parameters affecting SRAP-PCR of Camellia oleifera,including six factors： template DNA,primer concentration,dNTPs,Mg2＋ concentration,Taq DNA polymerase,and annealing temperature.The improved SRAP-PCR system（total 20 μL） was established as： 2.0 μL 10×PCR buffer,2.0 mmol/L Mg2＋,225 μmol/L dNTPs,20 ng template DNA,0.75 U Taq DNA polymerase;the SRAP-PCR cycle condition： 94 ℃ 5 min;94 ℃ 1 min,35 ℃ 1 min,72 ℃ 1 min,5 cycle;94 ℃ 1 min,52 ℃ 1 min,72 ℃ 1min,35 cycle;72 ℃ 10 min;4 ℃ hold.The result would lay a foundation for further research of Camellia oleifera in molecular genetics and marker assistant select breeding by new SRAP marker technology.