桂花SRAP-PCR体系的确立及验证

Establishment and verification of SRAP-PCR amplification system for Osmanthus fragrans

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,Mg2+、dNTPs和引物浓度以及TaqDNA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10μL,包括30 ng模板DNA、2.5 mmol.L-1Mg2+、0.20 mmol.L-1dNTPs、0.4μmol.L-1上下游引物和0.75 UTaqDNA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.cooperiHemsl.〕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 Osmanthusfragrans ＇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.