Rhodamine-pink as a genetic marker for yeast populations in wine fermentation.

Winemaking with selected yeasts requires simple techniques to monitor the inoculated yeast. New high-concentration rhodamine-resistant mutants and low-concentration rhodamine-pink mutants, easy to detect by replica-plate assay, were obtained from selected wine yeasts. The rhodamine-pink mutations were dominant and were located at the pdr5 locus that encodes for the Pdr5 ATP-binding cassette multidrug resistance transporter. The mutants were genetically stable but had lost the killer phenotype of the parent yeast strain. They were genetically improved by elimination of recessive growth-retarding alleles followed by crossing with selected killer wine yeasts. Several spore-clones were selected according to their must fermentation kinetics and the organoleptic quality of the wine. Some spore-clones were tested in industrial winemaking, and they were easily monitored during must fermentation using a simple color-plate assay. They accounted for >96% of the total yeasts in the must, and the resulting wine had as good a quality as those made with standard commercial wine yeasts. The rhodamine-pink yeasts may also be detected by direct seeding onto rhodamine agar or by observation under fluorescence microscopy. These possibilities greatly reduce the time of analysis and make the monitoring procedure for rhodamine-pink yeasts faster, easier, and cheaper than for the genetically marked wine yeasts obtained previously.

Rapid asymmetrical evolution of Saccharomyces cerevisiae wine yeasts.

Genetic instability causes very rapid asymmetrical loss of heterozygosity (LOH) at the cyh2 locus and loss of killer K2 phenotype in some wine yeasts under the usual laboratory propagation conditions or after long freeze-storage. The direction of this asymmetrical evolution in heterozygous cyh2(R)/CYH2(S) hybrids is determined by the mechanism of asymmetrical LOH. However, the speed of the process is affected by the differences in cell viability between the new homozygous yeasts and the original heterozygous hybrid cells. The concomitant loss of ScV-M2 virus in the LOH process may increase cell viability of cyh2(R)/cyh2(R) yeasts and so favour asymmetrical evolution. The presence of active killer K2 toxin, however, abolishes the asymmetrical evolution of the hybrid populations. This phenomenon may cause important sudden phenotype changes in industrial and pathogenic yeasts.

Genetic instability of heterozygous, hybrid, natural wine yeasts.

We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2(R)/cyh2(R) mutants resistant to high levels of cycloheximide can be directly isolated from cyh2(S)/cyh2(S) wine yeasts. Heterozygous cyh2(R)/cyh2(S) hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2(R)/cyh2(R) homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.