2024年6月25日 星期二
菊花SRAP-PCR反应体系的优化与确立
Optimization and establishment of SRAP-PCR reaction system of Dendranthema×grandiflorum
2009年 第18卷 第3期 页码[44-49]    下载全文[0.8MB]  
摘要

采用L16(45)正交实验设计,对SRAP-PCR反应体系中Mg2+、dNTPs和引物浓度以及TaqDNA聚合酶和模板DNA用量等5个因素进行了优化,并确立了适合菊花〔Dendranthema×grandiflorum (Ramat.) Kitamura〕SRAP-PCR的反应体系。菊花的SRAP-PCR最佳反应体系为:反应体系总体积20μL,含3.125 mmol.L-1Mg2+、187.5μmol.L-1dNTPs、10.0μmol.L-1引物、50 ng模板DNA、0.5 UTaqDNA聚合酶及1×PCR buffer。各因素对菊花基因组DNA SRAP-PCR扩增结果的影响程度不同,其中dNTPs浓度影响最大,TaqDNA聚合酶用量的影响最小。运用菊花品种‘奥运含笑’和‘雨花落英’及二者的F1杂交后代单株的基因组DNA对优化的SRAP-PCR反应体系进行验证,均获得了多态性丰富、条带清晰的扩增图谱,表明所确立的菊花SRAP-PCR反应体系稳定可靠。

Abstract

By the orthogonal experiment design  L16(45), five factors including Mg2+ concentration, dNTPs concentration, primer concentration, TaqDNA  polymerase amount and template DNA amount in SRAP-PCR reaction system were optimized, and the optimization SRAP-PCR reaction system suitable for genomic DNA from Dendranthema ×grandiflorum(Ramat.) Kitamura were also established. The optimal SRAP-PCR reaction system is as follows: total volume 20μL,  containing 3.125 mmol·L- 1  Mg2+,187.5μmol·L- 1dNTPs, 10.0μmol·L- 1primer, 50ng template DNA,  0.5U  Taq DNA  polymerase  and 1×PCR buffer. The effects of five factors on amplification result of SRAP-PCR are different, in which, the effect of dNTPs concentration is the greatest, but that of Taq DNA polymerase amount  is the least. The optimal SRAP-PCR reaction system is identified bymeans of genomic DNA from  `Aoyunhanxiao',  `Yuhualuoying' and their F1 progenies, and the amplification pattern with rich polymorphism and clear band is obtained. It is concluded that this SRAP-PCR  reaction system used for genomic DNA from Dendranthema×grandiflorum is steady and reliable.

关键词菊花; SRAP-PCR; 正交实验设计; 反应体系优化;
Key wordsDendranthema×grandiflorum (Ramat.) Kitamura; SRAP-PCR; orthogonal experiment design; optimization of reaction system
作者张 飞,陈发棣,房伟民,李风童,刘浦生
所在单位南京农业大学园艺学院,江苏南京210095
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基金项目教育部新世纪优秀人才支持计划(NCET-06-0489); “十一五”国家科技支撑计划项目(2006BAD01A18);