烟草靶斑病菌(Rhizoctonia solani)SRAP-PCR体系建立及优化

Establishment and optimization of SRAP-PCR reaction system for Rhizoctonia solani from tobacco target spot

采用烟草靶斑病菌YC-9,LJT-8和QYS-7为DNA模板,初步筛选SRAP引物组合;采用L16(45)正交试验设计,对烟草靶斑病菌的SRAP-PCR反应体系中的Mg2+、dNTPs,Taq DNA聚合酶、引物和DNA模板浓度等5个因素进行优化试验。结果表明:共筛出13对扩增条带清晰且多态性好的引物组合;烟草靶斑病菌的最佳SRAP反应体系为Mg2+浓度2.0 mmol/L、dNTP浓度200μmol/L、Taq DNA聚合酶0.8 U、引物浓度140 mmol/L、模板DNA 20 ng及1×PCR buffer,反应总体积为20μL;各因素对SRAP-PCR扩增反应结果影响的差异较大,依次为Taq DNA聚合酶>引物>Mg2+>dNTPs=模板DNA。

SRAP primer pairs were screened for polymorphism using DNA of Rhizoctonia solaniisolates YC-9, LJT-8 and QYS-7 as templates. An orthogonal design of L16（45） was used to optimize SRAP-PCR reaction system forR. solaniof tobacco with 5 factors, namely Mg^2＋, dNTPs, primers, Taq DNA polymerase and template DNA. Results showed that a total of 13 polymorphic SRAP primer pairs were screened out of 100 SRAP primer pairs, and a suitable SRAP-PCR reaction system for Rhizoctonia solanifrom tobacco target spot was 2.0 mmol·L-^1 Mg^2＋, 200 mmol.L-1 dNTPs, 0.8U Taq DNA polymerase, 140 mmol·L-^1 primer pairs, 20 ng template DNA and 1×PCR buffer. In addition, each factor in SRAP-PCR reaction system had different effects on amplified patterns in descending order of Taq DNA polymerase〉 primer〉Mg^2〉 dNTPs=DNA.