RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces: a fast method for species identification and the differentiation of flor yeasts

The PCR amplification and subsequent restriction analysis of the region spa nning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA ge ne was applied to the identification of yeasts belonging to the genus Sacch aromyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application wa s extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalen sis), as well as to the differentiation of an interesting group of Saccharo myces cerevisiae strains, known as flor yeasts, which are responsible for a geing sherry wine. Among the species of the Saccharomyces sensu lato comple x, the high diversity observed, either in the length of the amplified regio n (ranged between 700 and 875 bp) or in their restriction patterns allows t he unequivocal identification of these species. With respect to the four si bling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. ba yanus x S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains ex hibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this int eresting group of strains. We demonstrate that the specific patterns exhibi ted by flor yeasts are due to the presence of a 24-bp deletion located in t he ITS1 region and that this could have originated as a consequence of a sl ipped-strand mispairing during replication or be due to an unequal crossing -over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast population s.