摘要
以苹果(Malus domestica Borkh.)品种Telamon及Telamon×Fuji的F1代为试材,采用改良的CTAB法提取苹果叶片的DNA,利用正交设计L16(45)和直观分析以及方差分析相结合,探讨了Mg2+、dNTPs、Primer、Taq聚合酶、模板DNA用量对苹果SRAP-PCR反应的影响。建立了总体积为10μL的苹果SRAP-PCR反应体系,Mg2+浓度为2.0mmol.L-1,dNTPs浓度为0.8 mmol.L-1,Primer浓度为0.2μmol.L-1,Taq DNA聚合酶含量为0.6 U,DNA含量为60 ng,并含1μL 10×buffer(Mg2+free)。应用该反应体系,用不同的引物组合对48份苹果样品DNA进行SRAP-PCR扩增,结果显示反应体系具有较高的稳定性。
Experiment was conducted with the apple cultivar Telamon and the F1 seedlings of Telamon × Fuji.The genomic DNA was extracted from the leaves by modified CTAB method.Orthogonal design was applied to optimize SRAP-PCR ampli-fication system of apple in 5 factors such as Mg2＋,dNTPs,Primer,Taq DNA polymerase and template DNA at 4 levels.The re-sult of PCR was analyzed by intuitive analysis and variance analysis.An optimal reaction system（10 μL） was established,i.e.1 μL 10×buffer（Mg2＋ free）,2.0 mmol.L-1 Mg2＋,0.8 mmol.L-1 dNTPs,0.2 mmol.L-1 Primer,0.6 U Taq DNA polymerase and 60 ng template DNA.48 F1 of Telamon × Fuji and other SRAP-PCR primer were used to test the stability of the optimized re-action system.The results indicated that the optimized reaction system was stable.
出处
《果树学报》
CAS
CSCD
北大核心
2010年第2期168-173,共6页
Journal of Fruit Science
基金
国家863项目(2006AA100108-4-12-2)
国家自然科学基金(30671455)
