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We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of  Saccharomyces cerevisiae  strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from  Zygosaccharomyces bailii,  in the genome of  S. cerevisiae  strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28  S. cerevisiae  strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in  S. cerevisiae , strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the  Z. bailii  region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in  S. cerevisiae  represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations.

Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation