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International audience; Brettanomyces bruxellensis is a unicellular fungus of increasing industrial and scientific interest over the past 15 years. Previous studies revealed high genotypic diversity amongst B. bruxellensis strains as well as strain-dependent phenotypic characteristics. Genomic assemblies revealed that some strains harbour triploid genomes and based upon prior genotyping it was inferred that a triploid population was widely dispersed across Australian wine regions. We performed an intraspecific diversity genotypic survey of 1488 B. bruxellensis isolates from 29 countries, 5 continents and 9 different fermentation niches. Using microsatellite analysis in combination with different statistical approaches, we demonstrate that the studied population is structured according to ploidy level, substrate of isolation and geographical origin of the strains, underlying the relative importance of each factor. We found that geographical origin has a different contribution to the population structure according to the substrate of origin, suggesting an anthropic influence on the spatial biodiversity of this microorganism of industrial interest. The observed clustering was correlated to variable stress response, as strains from different groups displayed variation in tolerance to the wine preservative sulfur dioxide (SO2). The potential contribution of the triploid state for adaptation to industrial fermentations and dissemination of the species B. bruxellensis is discussed.

International audience; The budding yeast is certainly the prime industrial microorganism and is related to many biotechnological applications including food fermentations, biofuel production, green chemistry, and drug production. A noteworthy characteristic of this species is the existence of subgroups well adapted to specific processes with some individuals showing optimal technological traits. In the last 20 years, many studies have established a link between quantitative traits and single-nucleotide polymorphisms found in hundreds of genes. These natural variations constitute a pool of QTNs (quantitative trait nucleotides) that modulate yeast traits of economic interest for industry. By selecting a subset of genes functionally validated, a total of 284 QTNs were inventoried. Their distribution across pan and core genome and their frequency within the 1,011 genomes were analyzed. We found that 150 of the 284 QTNs have a frequency lower than 5%, meaning that these variants would be undetectable by genome-wide association studies (GWAS). This analysis also suggests that most of the functional variants are private to a subpopulation, possibly due to their adaptive role to specific industrial environment. In this review, we provide a literature survey of their phenotypic impact and discuss the opportunities and the limits of their use for industrial strain selection.

Details of the gene deletion method. (DOCX 65 kb)

Figure S4. Linkage between translocation markers in M2xF15 and SBxGN crosses. The data represented are the number of segregant according to their genotype for the marker next to the chromosomal break point. Genotypes A strong linkage is shown with less than 10% recombinants. (PDF 4 kb)

Figure S6. Variance explained by QTL according to cross. Each dot represent a QTL. Bigger points indicate average. There is no significant difference between the two cross (Wilcoxon test pval > 0,05). (PDF 721 kb)

Figure S3. Heritability is correlated to the number of QTLs detected. The data represented are the number of QTLs identified according by cross and by trait according to the average heritability by cross and by trait among the three conditions. (PDF 21 kb)

Figure S1. Meiosis emphases phenotypic novelty. Panel A. PCA of winemaking properties of M2xF15 and SBxGN progenies and 31 CWS in M15. Panel B. Correlation circle indicating the correlation of the variables for axes 1 and 2. Panel C. Average phenotypic distance computed from M15 condition for CWS, and the two M2xF15 and SBxGN cross. (PDF 455 kb)

Figure S2. Clustering of Norm of reaction for each trait. Norm of reaction of each individual is shown in dotted line and are colored and faceted according their cluster. Solid line shown the average norm of reaction of each cluster. Number of strains within each cluster is indicated by n. (PDF 2768 kb)

The yeast S. cerevisiae is the only species able to complete the alcoholic fermentation of grape must which is the main step of the wine-making process. Because of the high technological variability found in this species, selection work is carried out with the aim of using efficient yeasts for the industry. These strains show important differences both in their fermentation kinetics and their metabolite yield which affects the wines quality. In addition to these important phenotypic variations, the phenotypic response of yeasts varies considerably and not homogeneously against environmental variations. Understanding the genetic mechanisms that explain these differentiated responses to environmental variations is a non-trivial scientific question. This is of particular importance in oenology, where the winemaking conditions are highly variable (vintages, grape variety, terroirs, oenological practices...). In order to obtain yeasts ensuring the success of the fermentations under a wide range of conditions, we propose here to better understand these mechanisms of interaction Gene x Environment in an oenological context. The identification of genetic locus (Quantitative Trait Loci (QTL)) controlling quantitative characters is made possible by QTL mapping approaches. These approaches require the study of a large progeny in segregation that must be characterized genetically and phenotypically. The establishment of a statistical link between genotype and phenotype allows the localization of QTLs that have an impact on phenotyped characters. During this thesis a phenotyping method that allows us to follow the fermentations of several hundred individuals has been developed. Thanks to it, two progenies genotyped by whole genome sequencing have been phenotyped in different environmental conditions. This lead to the identification of many QTLs and to the estimation of their impact in strain robustness. The implication of the alleles of 3 genes showing a strong interaction with environment and pleiotropic effects linked to the SO2 metabolism has been proved molecularly. The results achieved are discussed in the context of QTL exploitation for the selection of more efficient yeast., La levure S. cerevisiae est la seule espèce capable de terminer la fermentation alcoolique du jus de raisin qui est l’étape principale de la vinification. A cause de la forte variabilité technologique retrouvée chez cette espèce, des travaux de sélection sont réalisés dans le but d’utiliser des levains performants pour l’industrie. Ces souches montrent des différences importantes à la fois dans leurs cinétiques fermentaires et leur bilans en métabolites, ce qui impacte la qualité des vins. La réponse phénotypique des levures varie également de manière considérable et non homogène face aux variations environnementales. La compréhension des mécanismes génétiques expliquant cette réponse différenciée est une question scientifique non triviale. Elle revêt une importance particulière en oenologie, où les conditions de vinifications sont très changeantes (millésimes, cépages, terroirs, conduites de vinifications…). Afin de pouvoir proposer des levains garantissant le succès des fermentations dans un large éventail de conditions, nous proposons ici de mieux comprendre ces mécanismes d’interaction Gène x Environnement dans un contexte oenologique. L’identification de locus génétiques (Quantitative Trait Loci (QTL)) contrôlant des caractères quantitatifs est rendue possible par des approches de cartographie de QTLs. Celles-ci nécessitent l’étude d’une vaste descendance en ségrégation qui doit être caractérisée sur le plan génétique et phénotypique. L’établissement d’un lien statistique entre des marqueurs génétiques et un phénotype permet la localisation de QTLs influant les caractères étudiés. Au cours de cette thèse, une méthode de phénotypage pour suivre les fermentations de plusieurs centaines d’individus a été mise au point. Grâce à elle, les performances fermentaires de deux descendances génotypées par séquençage à haut débit ont été mesurées en faisant varier les conditions de fermentations. Cela a permis l’identification de nombreux QTLs et d’estimer leur impact sur la robustesse des souches. L’implication des allèles de trois gènes qui montrent une forte interaction avec l’environnement et qui possèdent des effets pléiotropiques liés au métabolisme du SO2 a été prouvée moléculairement. Les résultats obtenus font l’objet d’une discussion générale sur l’utilisation de QTLs pour la sélection de levures plus performantes.

International audience; Non-Saccharomyces (NS) species that are either naturally present in grape must or added in mixed fermentation with S. cerevisiae may impact the wine’s chemical composition and sensory properties. NS yeasts are prevailing during prefermentation and early stages of alcoholic fermentation. However, obtaining the correct balance between S. cerevisiae and NS species is still a critical issue: if S. cerevisiae outcompetes the non-Saccharomyces, it may minimize their impact, while conversely if NS take over S. cerevisiae, it may result in stuck or sluggish fermentations. Here, we propose an original strategy to promote the non-Saccharomyces consortium during the prefermentation stage while securing fermentation completion: the use of a long lag phase S. cerevisiae. Various fermentations in a Sauvignon Blanc with near isogenic S. cerevisiae displaying short or long lag phase were compared. Fermentations were performed with or without a consortium of five non-Saccharomyces yeasts (Hanseniaspora uvarum, Candida zemplinina, Metschnikowia spp., Torulaspora delbrueckii, and Pichia kluyveri), mimicking the composition of natural NS community in grape must. The sensorial analysis highlighted the positive impact of the long lag phase on the wine fruitiness and complexity. Surprisingly, the presence of NS modified only marginally the wine composition but significantly impacted the lag phase of S. cerevisiae. The underlying mechanisms are still unclear, but it is the first time that a study suggests that the wine composition can be affected by the lag phase duration per se. Further experiments should address the suitability of the use of long lag phase S. cerevisiae in winemaking.

Hybridization is known to improve complex traits due to heterosis and phenotypic robustness. However, these phenomena have been rarely explained at the molecular level. Here, the genetic determinism of Saccharomyces cerevisiae fermentation performance was investigated using a QTL mapping approach on an F1-progeny population. Three main QTL were detected, with positive alleles coming from both parental strains. The heterosis effect found in the hybrid was partially explained by three loci showing pseudooverdominance and dominance effects. The molecular dissection of those QTL revealed that the adaptation to second fermentation is related to pH, lipid, or osmotic regulation. Our results suggest that the stressful conditions of second fermentation have driven the selection of rare genetic variants adapted to maintain yeast cell homeostasis and, in particular, to low pH conditions.

The yeast S. cerevisiae is the only species able to complete the alcoholic fermentation of grape must which is the main step of the wine-making process. Because of the high technological variability found in this species, selection work is carried out with the aim of using efficient yeasts for the industry. These strains show important differences both in their fermentation kinetics and their metabolite yield which affects the wines quality. In addition to these important phenotypic variations, the phenotypic response of yeasts varies considerably and not homogeneously against environmental variations. Understanding the genetic mechanisms that explain these differentiated responses to environmental variations is a non-trivial scientific question. This is of particular importance in oenology, where the winemaking conditions are highly variable (vintages, grape variety, terroirs, oenological practices...). In order to obtain yeasts ensuring the success of the fermentations under a wide range of conditions, we propose here to better understand these mechanisms of interaction Gene x Environment in an oenological context. The identification of genetic locus (Quantitative Trait Loci (QTL)) controlling quantitative characters is made possible by QTL mapping approaches. These approaches require the study of a large progeny in segregation that must be characterized genetically and phenotypically. The establishment of a statistical link between genotype and phenotype allows the localization of QTLs that have an impact on phenotyped characters. During this thesis a phenotyping method that allows us to follow the fermentations of several hundred individuals has been developed. Thanks to it, two progenies genotyped by whole genome sequencing have been phenotyped in different environmental conditions. This lead to the identification of many QTLs and to the estimation of their impact in strain robustness. The implication of the alleles of 3 genes showing a strong interaction with environment and pleiotropic effects linked to the SO2 metabolism has been proved molecularly. The results achieved are discussed in the context of QTL exploitation for the selection of more efficient yeast., La levure S. cerevisiae est la seule espèce capable de terminer la fermentation alcoolique du jus de raisin qui est l’étape principale de la vinification. A cause de la forte variabilité technologique retrouvée chez cette espèce, des travaux de sélection sont réalisés dans le but d’utiliser des levains performants pour l’industrie. Ces souches montrent des différences importantes à la fois dans leurs cinétiques fermentaires et leur bilans en métabolites, ce qui impacte la qualité des vins. La réponse phénotypique des levures varie également de manière considérable et non homogène face aux variations environnementales. La compréhension des mécanismes génétiques expliquant cette réponse différenciée est une question scientifique non triviale. Elle revêt une importance particulière en oenologie, où les conditions de vinifications sont très changeantes (millésimes, cépages, terroirs, conduites de vinifications…). Afin de pouvoir proposer des levains garantissant le succès des fermentations dans un large éventail de conditions, nous proposons ici de mieux comprendre ces mécanismes d’interaction Gène x Environnement dans un contexte oenologique. L’identification de locus génétiques (Quantitative Trait Loci (QTL)) contrôlant des caractères quantitatifs est rendue possible par des approches de cartographie de QTLs. Celles-ci nécessitent l’étude d’une vaste descendance en ségrégation qui doit être caractérisée sur le plan génétique et phénotypique. L’établissement d’un lien statistique entre des marqueurs génétiques et un phénotype permet la localisation de QTLs influant les caractères étudiés. Au cours de cette thèse, une méthode de phénotypage pour suivre les fermentations de plusieurs centaines d’individus a été mise au point. Grâce à elle, les performances fermentaires de deux descendances génotypées par séquençage à haut débit ont été mesurées en faisant varier les conditions de fermentations. Cela a permis l’identification de nombreux QTLs et d’estimer leur impact sur la robustesse des souches. L’implication des allèles de trois gènes qui montrent une forte interaction avec l’environnement et qui possèdent des effets pléiotropiques liés au métabolisme du SO2 a été prouvée moléculairement. Les résultats obtenus font l’objet d’une discussion générale sur l’utilisation de QTLs pour la sélection de levures plus performantes.

International audience