摘要
本研究以万寿菊雄性不育系2-2基因组DNA为试验材料,采用单因素和L16(45)正交设计对SRAPPCR反应体系的Mg2+、d NTPs、Taq酶、引物和模板DNA浓度进行优化,以得到最佳的SRAP-PCR反应体系。结果表明最佳反应体系为:20μL体系中Mg2+2.0 mmol/L、d NTPs 0.3 mmol/L、Taq酶0.75 U、引物0.2μmol/L、模板DNA 80 ng。应用建立的反应体系对退火温度进行优化,得到两步退火温度分别为35℃和50.2℃。最后用9个万寿菊雄性不育两用系材料组对所得PCR体系进行验证,证明该体系具有较高的稳定性和重复性,可为筛选与万寿菊雄性不育基因连锁的特异性标记奠定基础。
To obtain an optimal SRAP-PCR system for Tagetes erecta L., single-factor and L16(45) orthogonal experimental design were applied to a male sterile line 2-2, in which concentration of Mg2+, d NTPs, Taq polymerase, primer, and DNA template were screened. Annealing temperature was optimized using the obtained optimal PCR system. As a result, the best SRAP-PCR amplification system for T. erecta L. male sterile lines was2.0 mmol/L Mg2+, 0.3 mmol/L d NTPs, 0.75 U Taq polymerase, 0.2 μmol/L primer and 80 ng DNA template in a20 μL reaction system. Annealing temperature of the first and second step was 35℃ and 50.2℃, respectively. The optimized SRAP-PCR system was then validated on 9 T. erecta L. male sterile AB lines, by which the high stability and repeatability of this system was confirmed. Result of this study is the foundation of screening specific markers that linked to male sterility gene in marigold.
出处
《分子植物育种》
CAS
CSCD
北大核心
2015年第6期1388-1395,共8页
Molecular Plant Breeding
基金
青海省园林植物研究重点实验室发展专项(2015-Z-Y17)
2014年度"西部之光"人才培养计划"万寿菊两用系苗期育性鉴定分子标记开发"共同资助
