Retrotransposon-mediated duplication of SSU1 in high SO₂ tolerant Brettanomyces bruxellensis winery isolates

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.