INTERGENIC TRANSCRIBED SPACER PCR RIBOTYPING FOR DIFFERENTIATION OF SACCHAROMYCES SPECIES AND INTERSPECIFIC HYBRIDS

The taxonomy of the genus Saccharomyces has undergone significant chan ges recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level, The sequence o f rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of Sa ccharomyces, allows unknown Saccharomyces isolates to be assigned to t he type strains, It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PC R [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of Saccharomyces and further to assess if this metho d could distinguish strains that were interspecific hybrids, Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR usi ng ITS1 and ITS4 primers, purified by liquid chromatography, and diges ted with restriction endonucleases. Ribotyping patterns using the rest riction enzymes MaeI and HaeIII could distinguish all species of Sacch aromyces from each other, as well as from Candida glabrata, Candida al bicans, and Blastomyces dermatitidis. The only exception to this was t he inability to distinguish between Saccharomyces bayanus and S. pasto rianus (S. carlsbergensis). Furthermore, interspecific hybrids resulti ng from the mating of sibling species of Saccharomyces were shown to s hare the ITS-PCR ribotyping patterns of both parental species, It shou ld now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an incre ase in our understanding of the characteristics required by these inte rspecific hybrids for their particular ecological niches.