海南油茶SRAP-PCR体系优化及有效引物筛选

海南岛为中国油茶资源分布的最南缘,海南油茶资源丰富,特色显著。本研究以海南油茶基因组DNA为模板,采用单因素试验和正交试验相结合的方法,分析DNA浓度、dNTPs浓度、Taq DNA聚合酶用量和引物浓度对海南油茶SRAP-PCR扩增结果的影响,构建海南油茶SRAP-PCR体系,对多态性引物组合进行筛选,为SRAP分子标记在海南油茶资源遗传多样性评价和鉴定提供条件。单因素试验结果表明:在本试验中,海南油茶基因组DNA浓度高低对扩增效率影响不大,低浓度dNTPs有利于获得较好的扩增产物,而中高浓度的Taq酶和引物可提高扩增效果。正交试验结果表明:在适宜浓度范围内,各因素对海南油茶SRAP-PCR扩增影响大小依次为:引物>dNTPs>Taq DNA聚合酶>模板DNA;总体系为20μL时,最佳反应体系中模板DNA用量为5 ng,dNTPs浓度为0.20 mmol/L,引物浓度为0.60μmol/L以及Taq DNA聚合酶用量为4.00 U。采用稳定SRAP-PCR体系,对400对SRAP引物进行筛选,获得32对多态性好、条带清晰的有效引物,可用于海南油茶遗传多样性分析和种质资源鉴定等研究。

Optimization of SRAP-PCR System and Screening of Effective Primers for Tea-oil Camellia Resources of Hainan Island

Hainan Island is the southernmost distribution for tea-oil camellia resources in China,with the rich and specificity of tea-oil camellia resources.In this study,The effects of the concentration of template DNA,dNTPs,Taq DNA polymerase and primers on the SRAP-PCR system of Hainan tea-oil camellia resources were analysed by single factor test and orthogonal test,then the polymorphic primers were selected,which can be to provide the conditions for SRAP molecular marker to evaluate and identify genetic diversity of Hainan tea-oil camellia resources.The single factor test results showed that the concentration of genomic DNA for Hainan tea-oil camellia resources had little influence on the amplification efficiency,the low concentration of dNTPs and the medium-high concentration of Taq DNA polymerase and primers could be increased the amplification efficiency The orthogonal test results showed that within the appropriate concentration range,the influence of each factor on SRAP-PCR amplification of Hainan tea-oil camellia resources was as follows:primer>dNTPs>Taq DNA polymerase>template DNA,and an optimal 20μL SRAP-PCR reaction system for Hainan tea-oil camellia resources was established,including 5 ng DNA,0.20 mmol/L dNTPs,0.60μmol/L primer and 4.00 U Taq DNA polymerase.32 primers were selected for SRAP-PCR analysis for Hainan tea-oil camellia resources using the optimized amplification system above,from 400 pair SRAP primers.Which can be employed for the analysis the genetic diversity and identification in Hainan tea-oil camellia resources.