油橄榄ISSR-PCR反应体系优化与引物筛选  

Optimization of ISSR-PCR Reaction System and Primer Selection for Olea euyopaea

作　　者：李瑞[1] 陈少瑜[1] 宁德鲁[2] 李勇杰[2] 毛云玲[1] 

机构地区：[1]云南省林业科学院云南省森林植物培育与开发利用重点实验室国家林业局云南珍稀濒特森林植物保护和繁育重点实验室,云南昆明650204 [2]云南省林业科学院经济林研究所,云南昆明650204

出　　处：《安徽农业科学》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)

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.

关 键 词：油橄榄 基因组DNA 体系优化 引物筛选 

分 类 号：S565[农业科学—作物学] TQ644.1[化学工程—精细化工]