大蒜SSR体系的建立与优化  

Establishment and Optimization of SSR Reaction System in Garlic

作　　者：周静[1] 陈书霞[1] 程智慧[1] 孟焕文[1] 

机构地区：[1]西北农林科技大学园艺学院,陕西杨凌712100

出　　处：《西北农业学报》2011年第11期117-122,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：唐仲英西北农林科技大学育种基金;西北农林科技大学青年学术骨干支持计划(01140303)

摘　　要：为建立适宜大蒜（Allium sativum L.）的SSR反应体系和扩增程序,应用L16（45）正交设计对影响SSR-PCR的主要参数进行优化。结果表明,适宜大蒜的SSR反应体系总体积为20μL,其中含TaqDNA聚合酶0.02 U/μL、引物为0.2μmol/L、Mg2＋2.0 mmol/L、dNTP 0.2 mmol/L、DNA 30 mg/L;PCR适宜扩增程序为：94℃预变性3 min,94℃变性30 s,55℃45 s,72℃延伸90 s,35次循环,94℃变性30 s,53℃45 s,72℃延伸1 min,10次循环的最后一个循环延伸增加为10 min。并优化引物最适合退火温度为53.5~58.5℃。利用此反应体系对40份大蒜品种进行SSR扩增并电泳检测,扩增谱带清晰且多态性较好,证明此体系稳定可靠。在此基础上,利用优化的SSR反应体系对100对大葱SSR引物进行筛选,从中筛选出1对扩增谱带清晰稳定性好的SSR引物,并对40份大蒜品种进行遗传多样性分析。这一对SSR引物共检测出3个多态性条带可将供试材料聚为2大类,大部分品种（系）都按照其地理来源和遗传背景进行聚类。Orthogonal experiment with five factors including Taq polymerase,primer,Mg2＋,dNTPs and template DNA concentration and several single factor experiments were designed and optimized in order to establish SSR reaction system in garlic.The results showed that 0.02 U/μL Taq polymerase,0.2 μmol/L primer,2.0 mmol/L Mg2＋,0.2 mmol/L dNTP,30 mg/L template DNA were the optimal conditions in the reaction of 20 μL volume.PCR amplification was as follow： 94 ℃（3 min） then 30 cycles each 94 ℃（30 s）,55 ℃（45 s）,72 ℃（1 min）,followed by 10 cycles of 94 ℃（30 s）,53 ℃（45 s）,72 ℃（1 min） and aflnal extension at 72 ℃ for 10 min.The optimal annealing temperature of primer SSR003 ranged from 53.5 ℃ to 58.5 ℃.Under optimal conditions,SSR reactions were carried out with 40 cultivars of garlic and tested with electrophoresis.The results revealed clear and high polymorphic bands which demonstrated that the reaction system established in this experiment was stable and reliable.At the same time,100 Allium primers have been used to screen for useful primers in study on genetic identification and purity assessments by SSR,from which only one useful primer pair was selected.This primer pair generated 3 polymorphic bands.Most of tested cultivars were clustered into two groups based on the SSR marker by UPGMA method;this is accordance with their geographic and breeding sources.

关 键 词：大蒜 SSR PCR 体系优化 遗传多样性 

分 类 号：S633.4[农业科学—蔬菜学]