甘蔗黑穗病菌SCoT-PCR反应体系优化与引物筛选

Optimization of SCoT-PCR system and screening of polymorphic primers in Sporisorium scitamineum

以甘蔗黑穗病菌(Sporisorium scitamineum)交配型菌株基因组DNA为模板,在单因素优化试验的基础上,采用L16(45)正交试验设计,对影响甘蔗黑穗病菌SCoT-PCR反应体系的Mg2+、dNTPs、引物、r Taq DNA聚合酶和模板用量5个因素进行优化,建立优化的甘蔗黑穗病菌SCoT-PCR反应体系:DNA模板12.50ng、dNTPs 0.17mmol/L、引物0.46μmol/L、Mg2+1.7 mmol/L、r Taq DNA聚合酶0.85U、1×Buffer(Mg2+free),总体积25μL。应用优化体系从30条SCoT引物中筛选扩增条带清晰且多态性丰富的10条引物,并利用这10条引物对10份地理来源和寄主来源不同的甘蔗黑穗病菌交配型菌株基因组DNA进行SCoT标记,结果共扩增出86条带,多态性比率为58.67%,平均每条引物扩增8.60条。UPGMA聚类分析结果表明:在遗传相似系数为0.75的水平上,可将10个菌株分为3类,聚类结果与菌株的地理来源有一定的相关性。

In this research,the genomic DNA of mating type isolate of Sporisorium scitamineum was used as the template,five impact factors,namely,template DNA,Mg2+,dNTPs,primer and r Taq DNA polymerase,which were the components of PCR reaction system,were optimized respectively by single factor test.Based on the results,L16(45)orthogonal design was applied for establishing the best SCoT-PCR system for Sporisorium scitamineum,of which the total 25μL reaction system contained 12.50 ng template DNA,0.17mmol/L dNTPs,0.46μmol/L primer,1.7mmol/L,0.85 Ur Taq DNA polymerase and 1×Buffer(Mg2+free).Furthermore,10 polymorphic primers were screened out of 30 tested primers based on the above optimized system,and then used for testing 10 mating type isolates of Sporisorium scitamineum with different geographical origins and different hosts.A total of 86 bands were generated from the 10 SCoT primers,of which 58.67% was polymorphic and each primer generated 8.60 bands in average.UPGMA cluster analysis showed that 10 isolates could be divided into3 groups with a coefficient of 0.75,and the genetic diversity has certain correlation with geographical origins.