甘蔗SCoT-PCR反应体系优化与多态性引物筛选及应用

Optimization of SCoT-PCR Reaction System, and Screening and Utilization of Polymorphic Primers in Sugarcane

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.

CoT（Start codon targeted polymorphism） incorporates the advantages of both ISSR and RAPD,such as simple operation,low cost,simple design of primers,good reproducibility and universality and high polymorphism of the amplification.In this paper,five impact factors of template,Mg2＋,dNTPs,primer and DNA polymerase,which were the components of PCR reaction system and affected the products of SCoT-PCR,were determined by the methods of single factor experiments.Based on the results of the single factor experiments,L16（45） orthogonal design was applied for further testing.Accordingly,a SCoT-PCR system suitable for sugarcane was established,of which the total 25 μL reaction system contained 50 ng/μL template DNA,2.0 μL 10×Ex Taq Buffer（Mg2＋Plus）,0.625 U Ex Taq DNA polymerase,and the final concentrations of dNTPs and primer were 0.22 mmol/L and 0.9 μmol/L,respectively.Furthermore,taking DNA of variety ROC22 with top cultivated area in China's Mainland as the template,16 primers were screened out from 40 tested primers based on the ability of polymorphic amplification in the above optimized system.The G＋C contents were between 50% and 67%.In addition,in order to testify the stability and the amplification ability of the primers,four out of 16 primers were randomly selected for testing on 9 sugarcane germplasms with different geographical sources and different genetic background.84 bands which including 82.14% of polymorphic bands were amplified with an average of 21 bands detected by each primer.This study provided the basis for the in-depth development and application of the functional SCoT marker in sugarcane research.