菊花SSR-PCR反应体系的建立和优化  

Establishment and Optimization of Chrysanthemum SSR-PCR Reaction System

作　　者：李亚慧[1,2] 黄丛林[2] 董然[1] 

机构地区：[1]吉林农业大学园艺学院,吉林长春130118 [2]北京市农林科学院北京农业生物技术研究中心,北京100097

出　　处：《北方园艺》2012年第13期127-131,共5页Northern Horticulture

基　　金：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)

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25μL:60ng模板DNA、2.0mmol/L Mg2+、0.1mmol/L dNTP、0.3μmol/L引物、1UTaq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5min;32个循环的94℃变性50s、53.1℃退火50s、72℃延伸50s;72℃延伸8min,4℃保存。该体系的建立为今后菊花SSR分析奠定了基础。In order to obtain the optimal SSR-PCR system for Chrysanthemum moriforlium,an orthogonal diagram L 16（4 5） experimental design was employed to evaluate five factors（template DNA,Mg 2＋,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 ： 60ng DNA template,2.0mmol/L Mg 2＋,0.1mmol/L dNTP,0.3 μ mol/L primer,1UTaq DNA polymerase in 25 μ L 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 5min,followed by 35 cycles at 94℃ for 50s,53.1℃ for 50s,72℃ for 50s;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.

关 键 词：菊花 SSR标记 正交设计 反应体系优化 

分 类 号：S682.11[农业科学—观赏园艺]