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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。
In this study, the genomic DNA of Marigold male sterile line 2-2 was used as experimental material to optimize the concentration of Mg2 +, dNTPs, Taq enzyme, primer and template DNA in SRAPPCR reaction system by single factor and L16 (45) To get the best SRAP-PCR reaction system. The results showed that the optimal reaction system was 2.0 mmol / L Mg2 +, 0.3 mmol / L dNTPs, 0.75 U Taq DNA polymerase, 0.2 μmol / L primer and 80 ng DNA template. The annealing temperature was optimized by using the established reaction system. The two-step annealing temperature was 35 ℃ and 50.2 ℃ respectively. Finally, the obtained PCR system was validated by nine Marigold CMS lines, which proved that the system was highly stable and reproducible, which could lay the foundation for screening specific markers linked to the male sterility gene of marigold basis.