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。

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^{- 1}dNTPs, 10.0μmol·L^{- 1}primer, 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.