以桂花〔Osmanthus fragrans (Thunb.) Lour.〕品种‘早银桂’的总 DNA 为模板,对 SRAP - PCR 扩增体系中的模板 DNA 用量,Mg^{2 +} 、 dNTPs 和引物浓度以及 Taq DNA 聚合酶用量进行单因子实验, 确立了适合桂花总 DNA 的SRAP - PCR 反应体系:反应体系总体积 10 μL,包括 30 ng 模板 DNA、 2. 5 mmol· L ^{- 1} Mg^{2 +} 、 0. 20 mmol· L ^{- 1}dNTPs、 0. 4 μmol· L ^{- 1}上下游引物和 0. 75 U Taq DNA 聚合酶及 1 × PCR buffer。 采用 SRAP 引物组合 pm21 - em8,以 78 个桂花品种的总 DNA 为样品,对优化的 SRAP - PCR 反应体系进行初步验证,共扩增出632 条带,多态性条带百分率 75. 32% ;扩增位点 15 个,多态性位点百分率为 86. 67% 。 运用优化的 SRAP - PCR 体系,使用筛选出的 18对 SRAP 引物组合对 88 个桂花品种、1 个桂花野生种以及 2 个外类群〔柊树 O. heterophyllus (G. Don) P. S. Green和华东木犀 O. cooperi Hemsl.〕的总 DNA 进行扩增,共扩增出 296 个位点,其中多态性位点 248 个,多态性位点百分率为 83. 78% 。 实验结果显示,运用优化的 SRAP - PCR 反应体系获得的 DNA 条带清晰、扩增结果稳定、多态性较丰富;SRAP 分子标记可用于桂花遗传多样性、品种资源鉴定、亲缘关系以及系统进化等方面的研究。

 

Single factor experiments on amounts of template DNA and Taq DNA polymerase,
concentrations of Mg^{2 +} , dNTPs, primers in SRAP-PCR system were performed with total DNA from Osmanthus fragrans ‘ Zao Yingui’ to establish optimal SRAP-PCR amplification system. The 10 μL system contains 30 ng template DNA, 2. 5 mmol· L ^{- 1} Mg^{2 +} , 0. 20 mmol· L ^{- 1} dNTPs, 0. 4 μmol· L ^{- 1}each primer, 0. 75 U Taq DNA polymerase and 1 × PCR buffer. Using total DNA from seventy-eight cultivars of O. fragrans as samples, preliminary verification of the optimal SRAP-PCR system was carried using primer combination pm21-em8, and 632 bands and 15 sites are got and percentage of polymorphic bands is 75. 32% and the percentage of polymorphic sites is 86. 67% . Total DNA from eighty-eight  cultivars, one wild species of O. fragrans and two outgroups 〔O. heterophyllus (G. Don) P. S. Green and O. cooperi Hemsl. 〕 were amplified by the optimal system using selected 18 pairs of primer combinations, and 296 sites are got, in which 248 sites are polymorphic and the percentage of polymorphic sites is 83. 78% . The results indicate that the amplified DNA bands using the optimal SRAP-PCR system are clear and the polymorphism is high. The optimal SRAP-PCR amplification system can be used in researches of genetic diversity, cultivar identification, genetic relationship and phylogeny of O. fragrans.