Retrotransposon‐Mediated Duplication of <i>SSU1</i> in High <i>S</i> <i>O</i> ₂ ‐Tolerant <i>Brettanomyces bruxellensis</i> Winery Isolates

Background and Aims Brettanomyces bruxellensis is an industrially relevant yeast and a major spoilage organism in wine, where tolerance to sulphur dioxide ( S O 2 ), the primary preservative used for its control, varies among strains. Although laboratory evolution experiments have shown that polyploid B. bruxellensis can rapidly acquire increased S O 2 tolerance through genomic variation and SSU1 duplication, it remains unclear whether similar genetic adaptations drive S O 2 tolerance in winery populations. To address this question, B. bruxellensis strains were isolated from wineries across Australia and subjected to phenotypic characterisation of S O 2 tolerance and detailed genomic analyses to investigate the genetic basis of S O 2 tolerance in industry isolates. Methods and Results Twenty‐six isolates from Australian wineries were phenotypically characterised and sequenced using long‐read technology. Isolates showed a wide range of S O 2 tolerance that correlated with phylogenetic clade and ploidy. Haplotype phasing of SSU1 , a sulphite efflux pump linked to S O 2 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. Conclusions Structural variation at the SSU1 locus, particularly duplication of the high‐tolerance H1 haplotype, is associated with increased S O 2 tolerance in winery isolates of B . bruxellensis . The similarities between industry strains and laboratory‐evolved strains suggest that comparable adaptive mechanisms operate in winery environments. These results improve our understanding of how this spoilage yeast evolves sulphite tolerance and persists in wineries.