Species-specicc PCR primers for the rapid identiccation of yeasts ofthe genusZygosaccharomyces

摘要

Species-specific primer pairs that produce a single band of known product sizehave been developed for members of the Zygosaccharomyces clade includingZygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis,Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomycesmellis and Zygosaccharomyces rouxii. An existing primer pair for theprovisional new species Zygosaccharomyces pseudorouxii has been confirmed asspecific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, wasused as the target gene. This housekeeping gene evolves slowly and is thus wellconserved among different isolates, but shows a significant number of basepair changes between even closely related species, sufficient for species-specificprimer design. The primers were tested on type and wild strains of the genusZygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of theD1/D2 region of rDNA was used to confirm the identification of all nonculturecollection isolates. This approach used extracted genomic DNA, but in practice, itcan be used efficiently with a rapid colony PCR protocol. The method alsosuccessfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii.The method is rapid, robust and inexpensive. It requires little expertise bythe user and is thus useful for preliminary, large-scale screens.