摘要
[目的]对油橄榄ISSR-PCR反应体系进行优化,并筛选出多态性ISSR引物。[方法]以油橄榄嫩叶片提取的基因组DNA为模板,通过单因子试验针对反应体系主要因子Mg2+、dNTPs、引物浓度及模板用量进行油橄榄ISSR-PCR反应体系优化。[结果]优化的油橄榄ISSR-PCR反应体系为:总体系20μl中含1×Taq Buffer,3.5 mmol/L Mg2+,0.4 mmol/L dNTPs,1.0μmol/L引物,1.0 U Taq DNA聚合酶,20 ng DNA模板。反应程序为:94℃预变性5 min;94℃变性30 s,52～55℃退火30 s,72℃延伸2 min,40个循环;72℃延伸10 min,4℃保存。同时利用上述反应体系和反应程序筛选出了扩增稳定、多态性高、扩增条带清晰的ISSR引物11条。[结论]为进一步油橄榄种质资源的多样性研究和品种鉴定奠定了基础。
[Objective] This study aimed to optimize the ISSR-PCR reaction system and select polymorphic ISSR primers for Olea euyopaea.[Method] Olea euyopaea genomic DNA was extracted from leaves as the template,for optimization of ISSR-PCR reaction system by single-factor experiment of the main factors including Mg2＋,dNTPs,primer concentration and template amount.[Result] The optimal ISSR-PCR reaction system for Olea euyopaea was obtained,with a total system volume of 20 μl containing 1 × of Taq buffer,3.5 mmol/L of Mg2＋,0.4 mmol/L of dNTPs,1.0 μmol/L of primers,1.0 U of Taq DNA polymerase,20 ng of DNA template.The optimal ISSR-PCR reaction program was started with predenaturing at 94 ℃ for 5 min,followed by 40 cycles of denaturing at 94 ℃ for 30 s,annealing at 52-55 ℃ for 30 s,and extension at 72 ℃ for 2 min;the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min to allow complete extension of PCR products.PCR products were stored at 4 ℃.Based on the above conditions,11 primers with high polymorphism,clear amplified bands and good repeatability were selected.[Conclusion] This study laid the foundation for further diversity research and species identification of Olea euyopaea germplasm resources.
出处
《安徽农业科学》
CAS
2012年第10期5797-5799,共3页
Journal of Anhui Agricultural Sciences
基金
云南省重点新产品开发计划项目(2009BB006)
关键词
油橄榄
基因组DNA
体系优化
引物筛选
Olea euyopaea
Genomic DNA
System optimization
Primer selection
