Brettanomyces bruxellensis wine isolates show high geographical dispersal and long remanence in cellars

Brettanomyces bruxellensis is the main wine spoiler yeast all over the world, yet the structuration of the populations associated with winemaking remains elusive. In this work, we considered 1411 wine isolates from 21 countries that were genotyped using twelve microsatellite markers. We confirmed that B. bruxellensis isolates from wine environments show high genetic diversity, with 58 and 42% of triploid and diploid individuals respectively distributed in 5 main genetic groups. The distribution in the genetic groups varied greatly depending on the country and/or the wine-producing region. However, the two wine triploid groups showing sulfite resistance/tolerance were identified in almost all regions/countries. Genetically identical isolates were also identified. The analysis of these clone groups revealed that a given genotype could be isolated repeatedly in the same winery over decades, demonstrating unsuspected remanence ability. Besides cellar residency, a great geographic dispersal was also evidenced, with some genotypes isolated in wines from different continents. Finally, the study of old isolates and/or isolates from old vintages revealed that only the diploid groups were identified prior 1990 vintages. The triploid groups were identified in subsequent vintages, and their proportion has increased steadily these last decades, suggesting adaptation to winemaking practices such as sulfite use. A possible evolutionary scenario explaining these results is discussed.

. In non-European countries (Fig 2B), the genetic distribution of B.
146 bruxellensis was also contrasted, with the diploid wine group (darkcyan) dominant in USA, Brazil and 147 South Africa, and the red triploid wine group dominant in Australia.

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When summing-up all these regional specific distributions, some trends emerged: the Wine 2N 165 years after bottling (S1 Table).

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The genetic distribution of B. bruxellensis strains in wines older than one century, with 20-years prevalence among isolates from wines produced over the last decades.
182 Figure 3. Distribution of B. bruxellensis wine isolates from different genetic groups over 183 vintages. 20 years-intervals were used. In order to calculate confidence intervals, 100 bootstraps were 184 performed (re-sampling of the population). Error bars correspond to 95% confidence interval.

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The same B. bruxellensis genotypes can be identified in wines produced in a given cellar over 187 decades

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We then focused on B. bruxellensis clones. In this paper 'clones' will be defined as genetically

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Genetically identical isolates were designed as clone group. 42 groups gathering clones isolated from 205 the same winery over different vintages were identified, corresponding to 11 wineries from France and

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Italy. Same colors as in Fig 1 for  Since some clones were able to persist over several years in the cellar, we searched whether wine-216 producing regions were associated with specific clones. No 'signature' was identified, meaning that no 217 specific genotypes were associated with the studied regions. Instead, we found that some clone groups 218 were highly disseminated. For example, the clone group n°16 (Wine 2N, darkcyan) encompassed 96 219 isolates from Denmark, France, Portugal and USA ( Fig 5A). Another example is clone group n°67 (6 220 isolates), isolated in wines from Italy, Portugal and South Africa. In the other genetic groups also,

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several examples of dissemination were found (Fig 5B): the clone group n°24 (red Wine 3N) 222 encompassed 29 isolates from France, Italy and USA, while clone group n°35 were found in France,

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Italy and South Africa. In order to quantify the level of clonal dissemination, we computed the kilometric distance separating 230 the different isolates belonging to a given clone group (Fig 6). 88% of clone pairs were localized in the 231 same region, with kilometric distance inferior to 100km ('local clone pairs'), of which 34% had 232 distance inferior to 1km. 4% were separated by ~100-750km, usually associated with inter-country 233 distances, less than 1% were distant of ~750-1000km (intra-continental distances), and 6% were 234 separated by more than 1000km (inter-continental distances). It has to be noted that all isolates were In this work, we studied the genetic diversity and structuration of a large collection (>1400) of wine isolates. It will be interesting in a near future to identify those environmental/anthropic factors, and to 261 examine the associated phenotypic characteristics.

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The analysis of clones (genetically identical isolates for all 12 microsatellites) revealed unexpected 288 Allotriploidisation: a recent adaptation to winemaking practices?

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One of the most interesting results of this work is the fact that isolates from old vintages mostly belong 290 to a unique group, the so-called "wine diploid" (darkcyan), while, intriguingly, this group represents