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目标起始密码子多态性(Start condon targeted polymorphism,SCoT)分子标记是一种新型的标记,结合了ISSR标记和RAPD标记的优点.本研究针对SCoT-PCR反应体系的影响因素,以甘蔗叶片DNA为材料,在单因子实验的基础上,采用L16(45)正交实验设计,进一步探讨了模板DNA、Mg2+、dNTPs、引物及Taq酶等5个因素对甘蔗SCoT-PCR扩增效果的影响,建立了甘蔗SCoT-PCR的优化反应体系.25μL PCR反应混合液中,含50 ng DNA模板、2.0μL 10×Ex Taq Buffer(Mg2+Plus)、0.625 U Ex Taq酶,dNTP和引物的终浓度分别为0.22 mmol/L和0.9μmol/L.以我国种植面积最大的栽培品种新台糖22号为模板,应用优化体系,对40条SCoT标记引物进行测试,筛选出16条有效扩增的引物,且均为多态性引物,其GC含量在50%～67%之间.该体系的稳定性和SCoT标记引物的扩增能力,通过基于随机选择的4条引物对9份具有地理来源和遗传背景不同的甘蔗种质进行标记分析来验证,结果共扩增出84条带,其中多态性条带占82.14%,平均单条引物可扩增出21条.研究结果为在甘蔗上进一步开发和应用功能性SCoT标记奠定了基础.
SCoT molecular marker is a new type of marker that combines the advantages of ISSR and RAPD markers.This study aimed at the influencing factors of SCoT-PCR reaction system, DNA was used as the material. Based on the single factor experiment, the effects of five factors of template DNA, Mg2 +, dNTPs, primers and Taq enzyme on the amplification of SCoT-PCR of sugarcane were further studied by L16 (45) orthogonal design. , A optimized reaction system for sugarcane SCoT-PCR was established.The final concentration of 50 ng DNA template, 2.0 μL 10 × Ex Taq Buffer (Mg2 + Plus), 0.625 U Ex Taq enzyme, dNTP and primer Respectively, 0.22 mmol / L and 0.9 μmol / L. With 40 new cultivars, Xintaandu 22 with the largest planting area as the template, 40 SCoT-labeled primers were tested and 16 effective amplification primers were screened, And all of them were polymorphic primers with GC content of 50% -67%. The stability of the system and the ability of SCoT-labeled primers to amplify were determined by using four primers based on random selection of 9 primers with geographical origin and genetic Background of different sugarcane germplasm markers analysis Validation, the results amplified a total of 84 bands, of which 82.14% of the polymorphic bands, the average single primer can be amplified 21. The results provide a basis for the further development and application of functional SCoT markers on sugarcane.