LI Ya-hui,HUANG Cong-lin,DONG Ran.Establishment and Optimization of Chrysanthemum SSR-PCR Reaction System[J].Northern Horticulture,2012,36(13):127-131.
菊花SSR-PCR反应体系的建立和优化
- Title:
- Establishment and Optimization of Chrysanthemum SSR-PCR Reaction System
- 文章编号:
- 1001-0009（2012）13-0127-05
- 分类号:
- S 682.1+1
- 文献标志码:
- A
- 摘要:
- 为快速确定菊花SSR反应体系,利用正交实验设计L16（45）对菊花基因组SSR-PCR反应体系的5个因素（模板DNA、Mg2+、dNTP、引物和Taq酶）在4个水平上进行正交设计，筛选出适合菊花的最佳SSR-PCR反应体系，进一步利用单因素完全随机试验筛选各反应因素的最佳水平。结果表明：建立菊花基因组DNA SSR-PCR反应体系为25 μL：60 ng 模板DNA、2.0 mmol/L Mg2+、0.1 mmol/L dNTP、0.3 μmol/L引物、1 U Taq酶。并对菊花引物进行梯度退火试验，其最佳退火温度在53.1℃；扩增程序是：95℃预变性5 min；32个循环的94℃变性50 s、53.1℃退火50 s、72℃延伸50 s；72℃延伸8 min，4℃保存。该体系的建立为今后菊花SSR分析奠定了基础。
- Abstract:
- In order to obtain the optimal SSR-PCR system for Chrysanthemum moriforlium，an orthogonal diagram L16(45) experimental design was employed to evaluate five factors (template DNA，Mg2+，dNTP，primer and Taq DNA polymerase) at four different levels.The fully random single factor experiment was used to select the optimal level for each factor to optimize the SSR-PCR amplification system.The results showed that an optimal SSR-PCR system for chrysanthemum was obtained as following：60 ng DNA template，2.0 mmol/L Mg2+，0.1 mmol/L dNTP，0.3 μmol/L primer，1 U Taq DNA polymerase in 25 μL reaction system.The optimal annealing temperature for SSR-PCR reaction system was determined as 53.1℃ by gradient PCR.The suitable thermal cycling conditions with initial melting at 95℃ for 5 min，followed by 35 cycles at 94℃ for 50 s，53.1℃ for 50 s，72℃ for 50 s；then keep the reaction mixture at 4℃ after a final extension step of 72℃ for 8 min.The optimized system would be effective as a solid foundation for chrysanthemum SSR analysis．
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备注/Memo
第一作者简介：李亚慧(1985-)，女，硕士,现主要从事园林植物资源与种质创新等研究工作。E-mail:liyahui_12@163.com.
责任作者：董然(1966-)，女，博士，教授，现主要从事长白山野生植物引种驯化工作。E-mail:dongr999@163.com.
基金项目：北京市科委资助项目(Z09050600630906，D101105046210001)；科技部科技支撑计划资助项目(2009BADB8B04)；北京市园林绿化局花卉育种研发资助项目 (YLHH201100104)。
收稿日期：2012-03-15