Single nucleotide polymorphism-based diagnostic system for crop-associated Sclerotinia species.
A molecular diagnostic system using single nucleotide polymorphisms (SNPs) was developed to identify four Sclerotinia species: S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. DNAs of samples are hybridized with each of five 15-bp oligonucleotide probes containing an SNP site midsequence unique to each species. For additional verification, hybridizations were performed using diagnostic single nucleotide substitutions at a 17-bp sequence of the calmodulin locus. The accuracy of these procedures was compared to that of a restriction fragment length polymorphism (RFLP) method based on Southern hybridizations of EcoRI-digested genomic DNA probed with the ribosomal DNA-containing plasmid probe pMF2, previously shown to differentiate S. sclerotiorum, S. minor, and S. trifoliorum. The efficiency of the SNP-based assay as a diagnostic test was evaluated in a blind screening of 48 Sclerotinia isolates from agricultural and wild hosts. One isolate of Botrytis cinerea was used as a negative control. The SNP-based assay accurately identified 96% of Sclerotinia isolates and could be performed faster than RFLP profiling using pMF2. This method shows promise for accurate, high-throughput species identification.
Andrew
Marion
M
Department of Ecology and Evolutionary Biology, University of Toronto, Mississauga, Ontario L5L 1C6, Canada. marion.andrew@utoronto.ca
Kohn
Linda M
LM
D001203:Ascomycota / Q000145:classification* / Q000302:isolation & purification*; D015139:Blotting, Southern; D002147:Calmodulin / Q000235:genetics; D018556:Crops, Agricultural / Q000382:microbiology*; D016172:DNA Fingerprinting / Q000379:methods*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016533:Mycological Typing Techniques / Q000379:methods; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013997:Time Factors
D002147:Calmodulin; D004271:DNA, Fungal; D015345:Oligonucleotide Probes