Onetto, C. A.; Rossi, J.; Cordente, A. G.; Van Den Heuvel, S.; Borneman, A. R.

Brettanomyces bruxellensis is an industrially relevant yeast and major spoilage organism in wine, where tolerance to sulphur dioxide (SO2), the primary preservative used for its control, varies between strains. To investigate the genetic basis of SO2 tolerance in winery populations, 26 isolates from Australian wineries were phenotypically characterised and sequenced using long-read technology. Isolates showed a wide range of SO2 tolerance that correlated with phylogenetic clade and ploidy. Haplotype phasing of SSU1, a sulphite efflux pump linked to SO2 tolerance, identified nine distinct haplotypes, including the previously described high-tolerance H1 allele. Highly tolerant strains carried duplications of H1, frequently associated with retrotransposon insertions and chromosomal rearrangements at the SSU1 locus. Comparative analyses with laboratory-evolved strains confirmed that retrotransposons facilitated the acquisition of additional SSU1 copies. These findings suggest that transposon-mediated structural variation drives adaptive increases in SO2 tolerance in B. bruxellensis populations.