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Domestication is an excellent model for studying adaptation processes, constituting recent adaptation and diversification, as well as degeneration of unused functions. Geotrichum candidum is a fungus used for cheese-making and is also found in other environments such as soil and plants. By analyzing whole genome data from 98 strains, we found that all strains isolated from cheese formed a monophyletic clade. Within the cheese clade, we identified three differentiated populations and we detected footprints of recombination and hybridization. The genetic diversity in the cheese clade was the lowest but remained high compared to other domesticated fungi, indicating milder bottlenecks. Commercial starter strains were scattered across the cheese clade, not constituting a single clonal lineage. The cheese clade was phenotypically differentiated from other populations, with a slower growth on all media even cheese, a prominent production of attractive cheese flavors and a lower proteolytic activity. Furthermore, cheese populations displayed contrasted phenotypes, with one cheese population producing a typical blue cheese flavor and another one displaying denser and fluffier colonies, excluding more efficiently cheese spoiler fungi. Cheese populations lost two beta lactamase-like genes, involved in xenobiotic clearance, and displayed transposable element expansion, likely due to relaxed selection. Our findings suggest the existence of genuine domestication in G. candidum, which led to diversification into three varieties with contrasted phenotypes. Some of the traits acquired by cheese strains indicate convergence with other, distantly related fungi used for cheese maturation.

Here, we report the draft genome sequence and annotation of the yeast Candida railenensis strain CLIB 1423. The assembly consists of 57 nuclear scaffolds and 1 complete mitochondrial chromosome, for a total of 13.8 Mb ( N 50 , 0.54 Mb; L 50 , 9). The annotation contains 6,013 coding DNA sequences (CDSs) (BUSCO completeness, 99.6%).

Domestication is an excellent model for studying adaptation processes, involving recent adaptation and diversification, convergence following adaptation to similar conditions, as well as degeneration of unused functions.Geotrichum candidumis a fungus used for cheese making and is also found in other environments such as soil and plants. By analyzing whole-genome data from 98 strains, we found that all strains isolated from cheese formed a monophyletic clade. Within the cheese clade, we identified three genetically differentiated populations and we detected footprints of recombination and admixture. The genetic diversity in the cheese clade was similar as that in the wild clade, suggesting the lack of strong bottlenecks. Commercial starter strains were scattered across the cheese clade, thus not constituting a single clonal lineage. The cheese populations were phenotypically differentiated from other populations, with a slower growth on all media, even cheese, a prominent production of typical cheese volatiles and a lower proteolytic activity. One of the cheese clusters encompassed all soft goat cheese strains, suggesting an effect of cheese-making practices on differentiation. Another of the cheese populations seemed to represent a more advanced stage of domestication, with stronger phenotypic differentiation from the wild clade, harboring much lower genetic diversity, and phenotypes more typical of cheese fungi, with denser and fluffier colonies and a greater ability of excluding cheese spoiler fungi. Cheese populations lacked two beta lactamase-like genes present in the wild clade, involved in xenobiotic clearance, and displayed higher contents of transposable elements, likely due to relaxed selection. Our findings suggest the existence of genuine domestication inG. candidum, which led to diversification into different varieties with contrasted phenotypes. Some of the traits acquired by cheese strains indicate convergence with other, distantly related fungi used for cheese maturation.

Recent studies have suggested that species of the Kazachstania genus may be interesting models of yeast domestication. Among these, Kazachstania barnettii has been isolated from various microbially transformed foodstuffs such as sourdough bread and kefir. In the present work, we sequence, assemble, and annotate the complete genomes of two K. barnettii strains: CLIB 433, being one of the two reference strains for K. barnettii that was isolated as a spoilage organism in soft drink, and CLIB 1767, recently isolated from artisan bread-making sourdough. Both assemblies are of high quality with N50 statistics greater than 1.3 Mb and BUSCO score greater than 99%. An extensive comparison of the two obtained genomes revealed very few differences between the two K. barnettii strains, considering both genome structure and gene content. The proposed genome assemblies will constitute valuable references for future comparative genomic, population genomic, or transcriptomic studies of the K. barnettii species.

International audience; Nature is harnessed since ancient times to fulfill human needs, and yeast culture has been mastered for bakery, brewery, or the preparation of beverages. In this context, the two recently discovered yeast species Starmerella reginensis and Starmerella kourouensis, belonging to a genus related to fermentative activities in the literature, were explored via untargeted metabolomics approaches. Ultrahigh-performance liquid chromatography hyphenated with tandem mass spectrometry and a deep investigation of molecular networks and spectral data allowed the annotation of, respectively, 439 and 513 metabolites for S. reginensis and S. kourouensis, with approximatively 30% compound annotations and 40% chemical class annotations for both yeast strains. These analyses and Fourier transform ion cyclotron resonance mass spectrometry accurate metabolic profiles unveiled a rich content of alkaloids, lipids, amino acids, and terpenoids for S. reginensis. S. kourouensis presents a similar profile with more sulfated compounds. In short, these results enrich the current knowledge about Starmerella yeast secondary metabolites and reveal their significant structural diversity of small molecules.

Nature is harnessed since ancient times to fulfill human needs, and yeast culture has been mastered for bakery, brewery, or the preparation of beverages. In this context, the two recently discovered yeast species

International audience; Natural products are a reliable source of bioactive molecules and represent an industrial and pharmaceu-tical stake. Indeed, the model yeast species Saccharomyces cerevisiae is a well-known eukaryotic organismlargely used as a biotechnological tool, but still a topical subject of study. In this work, the exploration ofSaccharomyces cerevisiae is taken further through an untargeted metabolomics workflow. The aim is toenrich databases and bring new information about the standard S. cerevisiae strain in a given medium.Analytical methods and bioinformatics tools were combined in a high-throughput methodology useableto dereplicate many types of biological extracts and cartography secondary metabolites. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analyses were carriedout and spectral data were pre-processed to build molecular networks. Annotations were attributedto compounds through comparison with databases and manual investigation of networks. Ultra-high-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) brought additionalinformation thanks to a higher dynamic range and enhanced UHPLC-MS/MS results by unveiling ambi-guities and bringing accurate molecular formulae. Therefore, accurate and reliable annotated featuresresulted from the UHPLC-MS/MS data while FTICR-MS provided an overall cartography of metabolitesthanks to van Krevelen diagrams. Various small molecules such as amino acids derivatives and indolealkaloids have been determined for the first time in this yeast. The complementarity of FTICR-MS and UHPLC-MS/MS for secondary metabolite annotation brought this new mapping of S. cerevisiae.

Two strains (DMKU-GTCP10-8 and CLIB 1740) representing a novel anamorphic yeast species were isolated from a grease sample collected from a grease trap in Thailand and from an unidentified fungus collected in French Guiana, respectively. On the basis of phylogenetic analysis based on the combined D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, Lachancea fermentati CBS 707T was the closely related species with 12.8 % sequence divergence (70 nucleotide substitutions and three gaps in 571 nucleotides) and 28.1 % sequence divergence (93 nucleotide substitutions and 90 gaps in 651 nucleotides) in the D1/D2 domain of the LSU rRNA gene and the ITS region, respectively. Phylogenetic analysis based on the concatenated sequences of the five genes including the small subunit rRNA gene, the D1/D2 domain of the LSU rRNA gene, the ITS region, translation elongation factor-1 alpha (TEF1) and RNA polymerase II subunit 2 (RPB2) genes confirmed that the two strains (DMKU-GTCP10-8 and CLIB 1740) were well-separated from other described yeast genera in Saccharomycetaceae. Hence, Savitreea pentosicarens gen. nov., sp. nov. is proposed to accommodate these two strains as members of the family Saccharomycetaceae. The holotype is S. pentosicarens DMKU-GTCP10-8T (ex-type strain TBRC 12159=PYCC 8490; MycoBank number 835044).

International audience; Analysis of yeasts isolated from various biotopes in French Guiana led to the identification of two strains isolated from flowers and designated CLIB 1634(T) and CLIB 1707(T). Comparison of the D1/D2 domain of the large subunit (LSU D1/D2) rRNA gene sequences of CLIB 1634(T) and CLIB 1707(T) to those in the GenBank database revealed that these strains belong to the Starmerella Glade. Strain CLIB 1634(T) was shown to diverge from the closely related Starmerella apicola type strain CBS 2868 T with a sequence divergence of 1.34 and 1.30 %, in the LSU D1/D2 rRNA gene and internal transcribed spacer (ITS) sequences respectively. Strain CLIB 1634(T) and Candida apicola CBS 2868(T) diverged by 3.81 and 14.96 % at the level of the protein-coding gene partial sequences EF-1 alpha and RPB2, respectively. CLIB 1707(T) was found to have sequence divergence of 3.88 and 9.16 % in the LSU D1/D2 rRNA gene and ITS, respectively, from that of the most closely related species Starmerella ratchasimensis type strain CBS 10611(T). The species Starmerella reginensis f.a., sp. nov. and Starmerella kourouensis f.a., sp. nov. are proposed to accommodate strains CLIB 1634(T) (=CBS 15247(T)) and CLIB 1707(T) (=CBS 15257(T)), respectively.

Hanseniaspora, a predominant yeast genus of grape musts, includes sister species recently reported as fast evolving. The aim of this study was to investigate the genetic relationships between the four most closely related species, at the population level. A multi-locus sequence typing strategy based on five markers was applied on 107 strains, confirming the clear delineation of species H. uvarum, H. opuntiae, H. guilliermondii, and H. pseudoguilliermondii. Huge variations were observed in the level of intraspecific nucleotide diversity, and differences in heterozygosity between species indicate different life styles. No clear population structure was detected based on geographical or substrate origins. Instead, H. guilliermondii strains clustered into two distinct groups, which may reflect a recent step toward speciation. Interspecific hybrids were detected between H. opuntiae and H. pseudoguilliermondii. Their characterization using flow cytometry, karyotypes and genome sequencing showed different genome structures in different ploidy contexts: allodiploids, allotriploids, and allotetraploids. Subculturing of an allotriploid strain revealed chromosome loss equivalent to one chromosome set, followed by an auto-diploidization event, whereas another auto-diploidized tetraploid showed a segmental duplication. Altogether, these results suggest that Hanseniaspora genomes are not only fast evolving but also highly dynamic.

Hanseniaspora, a predominant yeast genus of grape musts, includes sister species recently reported as fast evolving. The aim of this study was to investigate the genetic relationships between the four most closely related species, at the population level. A multi-locus sequence typing strategy based on five markers was applied on 107 strains, confirming the clear delineation of species H. uvarum, H. opuntiae, H. guilliermondii, and H. pseudoguilliermondii. Huge variations were observed in the level of intraspecific nucleotide diversity, and differences in heterozygosity between species indicate different life styles. No clear population structure was detected based on geographical or substrate origins. Instead, H. guilliermondii strains clustered into two distinct groups, which may reflect a recent step toward speciation. Interspecific hybrids were detected between H. opuntiae and H. pseudoguilliermondii. Their characterization using flow cytometry, karyotypes and genome sequencing showed different genome structures in different ploidy contexts: allodiploids, allotriploids, and allotetraploids. Subculturing of an allotriploid strain revealed chromosome loss equivalent to one chromosome set, followed by an auto-diploidization event, whereas another auto-diploidized tetraploid showed a segmental duplication. Altogether, these results suggest that Hanseniaspora genomes are not only fast evolving but also highly dynamic.

Despite the increasing importance of palm wines as a key element in Ivorian socio-cultural practices, and the high involment of yeasts in the production of these beverages, no studies have been devoted to aroma production of yeasts and their potential uses in biotechnological applications. In this work, aroma profiles of 20 non pathogenic yeast strains (11 from raffia and 9 from ron, representing 13 species) among the 21 yeasts isolated were established using HS–SPME–GC/MS analysis. A total of 50 Volatile Organic Compounds (VOCs) produced by these yeasts were identified and grouped into five main families, namely higher alcohols, aldehydes, ketones, organic acids and esters. Among these VOCs, esters were the most abundant (28 compounds). Some strains produced specific compounds of technological interest which could be used for diverse applications, such as beverages, ice creams, baked goods and desserts and synthetic flavouring ingredient. Among them, Hanseniaspora jakobsenii CLIB 3083 produced the highest number of VOCs.

International audience; Aims To investigate the genotypic diversity and enzymatic activity of yeast flora isolated from spontaneous fermenting saps of various palm trees (Borassus aethiopum, Raphia hookeri, Elaeis guineensis) tapped for palm wines. Methods and Results PCR-restriction fragment length polymorphism of ITS-5.8S rDNA combined to 26S rRNA gene and/or the partial ACT1 gene sequencing were applied for yeast characterization, and their enzymatic profiles assessed by using API ZYM kits. Thirteen genera and 23 species were identified, with the highest diversity (14 species) in raffia wine. Saccharomyces cerevisiae was dominant and common to all palm wines. Some potentially pathogenic yeasts were also isolated. The majority of tested strains displayed high amylo-peptidase, phosphatase, beta-glucosidase and alpha-glucosidase activities and esterase activity. Conclusions Diverse yeast species colonized palm wines, among which some were related to a specific type of wine and the majority of them have the ability to digest starch, sugar, protein or lipid. Significance and Impact of the Study This study is a first step in understanding the significance of indigenous yeast flora of palm wines from Cote d'Ivoire. This knowledge is important as a tool for establishing new indigenous yeast collection; which could be used for the product quality improvement and as enzyme sources for biotechnological purposes.

In order to assess the genetic diversity and population structure of indigenous S. cerevisiae from Côte d'Ivoire, a total of 170 strains were isolated from four traditional alcoholic beverages through nine regions. Microsatellite analysis performed at 12 loci revealed that strains of palm oil and raffia wine were genetically related, unlike those of tchapalo and ron wine which formed two s from palm oil wine and raffia wine were clearly inbred. In comparison with the European, North American, Asian and others West African populations, Ivorian population was well defined, although most of these strains were admixed. Among these strains, only isolates from raffia wine appeared to have alleles in common to all populations.

International audience; Twelve strains of a novel yeast species were isolated from rotting wood, mushrooms and fruit samples in Brazil and French Guiana. Analysis of the sequences of the internal transcribed spacer region and the D1/D2 domains of the large subunit rRNA gene showed that the novel species belongs to the Kurtzmaniella Clade. The novel species differed from its closest relative, Candida natalensis, by 12 substitutions in the D1/D2 sequences. The novel species could be distinguished from C. natalensis by its inability to assimilate cellobiose and salicin, and growth at 50 % (w/w) glucose. The name Kurtzmaniella hittingeri f.a., sp. nov. is proposed for the novel species. The type strain of K. hittingeri sp. nov. is CBS 13469(T) (=UFMG CM-Y272(T)). The MycoBank number is 827183. We also propose the transfer of Candida fragi, Candida quercitrusa and Candida natalensis to the genus Kurtzmaniella as new combinations.

International audience; Six yeast strains representing two novel Wickerhamiella species were isolated from plants and insects collected in Costa Rica, Brazil, and French Guiana. They belong to a subclade containing Wickerhamiella domercqiae and Wickerhamiella bombiphila, and differ by approximately 12 % in the D1 /D2 sequences of the large subunit rRNA gene from these species. The intergenic spacer (ITS) regions of the two novel species differ by around 19 and 27%, respectively, from those of W. domercqiae. The novel species exhibit 5 % divergence in the D1 /D2 sequences among them (around 4 % in the ITS). The names Wickerhamiella dianesei f.a., sp. nov. and Wickerhamiella kurtzmanii f.a., sp. nov. are proposed to accommodate these species, for which a sexual cycle has not been observed. Wickerhamiella dianesei was isolated from the stingless bee, Trigona fulviventris, collected in an Asteraceae flower in Costa Rica, and from leaves of Sabicea brasiliensis (Rubiaceae) and a flower of Byrsonima crassifolia (Malpighiaceae) in Brazil. Wickerhamiellsa kurtzmanii was isolated from a flower of Ipomoea batatoides (Convolvulaceae) in Costa Rica, the surface of a fruit of B. crassifolia in Brazil, and flowers in French Guiana. The type strains are Wickerhamiella dianesei UWOPS 00-107.1(T) (=CBS 14185=NRRL Y-63789; Mycobank number MB 827008) and Wickerhamiella kurtzmanii UWOPS 00-192.1(T) (=CBS 15383=NRRL Y-63979; MB 827011).

cis- and trans-acting factors affect gene expression and responses to environmental conditions. However, for most plant systems, we lack a comprehensive map of these factors and their interaction with environmental variation. Here, we examined allele-specific expression (ASE) in an F1 hybrid to study how alleles from two Arabidopsis thaliana accessions affect gene expression. To investigate the effect of the environment, we used drought stress and developed a variance component model to estimate the combined genetic contributions of cis- and trans-regulatory polymorphisms, environmental factors, and their interactions. We quantified ASE for 11,003 genes, identifying 3318 genes with consistent ASE in control and stress conditions, demonstrating that cis-acting genetic effects are essentially robust to changes in the environment. Moreover, we found 1618 genes with genotype x environment (GxE) interactions, mostly cis x E interactions with magnitude changes in ASE. We found fewer trans x E interactions, but these effects were relatively less robust across conditions, showing more changes in the direction of the effect between environments; this confirms that trans-regulation plays an important role in the response to environmental conditions. Our data provide a detailed map of cis- and trans-regulation and GxE interactions in A. thaliana, laying the ground for mechanistic investigations and studies in other plants and environments.

International audience; A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

Sulfur is an essential macronutrient for all living organisms. Plants take up inorganic sulfate from the soil, reduce it, and assimilate it into bioorganic compounds, but part of this sulfate is stored in the vacuoles. In our first attempt to identify genes involved in the control of sulfate content in the leaves, we reported that a quantitative trait locus (QTL) for sulfate content in Arabidopsis (Arabidopsis thaliana) was underlain by the APR2 isoform of the key enzyme of sulfate assimilation, adenosine 5'-phosphosulfate reductase. To increase the knowledge of the control of this trait, we cloned a second QTL from the same analysis. Surprisingly, the gene underlying this QTL encodes the ATPS1 isoform of the enzyme ATP sulfurylase, which precedes adenosine 5'-phosphosulfate reductase in the sulfate assimilation pathway. Plants with the Bay allele of ATPS1 accumulate lower steady-state levels of ATPS1 transcript than those with the Sha allele, which leads to lower enzyme activity and, ultimately, the accumulation of sulfate. Our results show that the transcript variation is controlled in cis. Examination of ATPS1 sequences of Bay-0 and Shahdara identified two deletions in the first intron and immediately downstream the gene in Bay-0 shared with multiple other Arabidopsis accessions. The average ATPS1 transcript levels are lower in these accessions than in those without the deletions, while sulfate levels are significantly higher. Thus, sulfate content in Arabidopsis is controlled by two genes encoding subsequent enzymes in the sulfate assimilation pathway but using different mechanisms, variation in amino acid sequence and variation in expression levels.

An integrated physiological and proteomic approach was used to investigate the effects of high gellan gum concentration in the medium during maturation of somatic embryos (SE) of hybrid larch, by comparing embryos incubated in media with a high gellan gum concentration (8 g l(-1) ) and the standard concentration (4 g l(-1) ) after 1, 3, 6 and 8 weeks of maturation. Because of the reduced availability of water in the 8 g l(-1) medium, the cultured embryos had a lower osmotic water potential (Ψπ) and water contents, but higher dry weights (DWs), at 8 weeks compared with embryos cultured on the standard medium. The high gellan gum concentration induced a desiccation that is characteristic in zygotic embryo maturation. Total soluble proteins were extracted from SE with trichloroacetic acid (TCA)-acetone after 1 and 8 weeks of maturation on media with 4 and 8 g l(-1) of gellan gum, and separated by two-dimensional gel electrophoresis (2-DE) at pH 4-7. More than 1100 proteins were reproducibly detected on each gel. At 1 and 8 weeks respectively, the abundances of 62 and 49 spots detected in analyses of embryos matured at the two gellan gum concentrations, significantly differed. Among 62 significantly differing spots at 1 week of maturation, the corresponding proteins of 56 were reliably identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and were found to be mainly involved in 'carbohydrate metabolism', 'genetic information processing' or 'environmental information processing' according to kegg taxonomy. Both physiological parameters and the proteins identified suggested that the embryos were stressed when they were cultured on 4 g l(-1) of gellan gum.

International audience; Development of clonal propagation method, such as somatic embryogenesis, has numerous applications such as massproduction of genetically improved plants. Since the 90’s, researchers at INRA have engaged research on SE in Larix species (Larix eurolepis, Larix marschlinsii), resulting in a controlled protocol (Lelu and Label 1994). If the positive effect of high gellan gum concentration on somatic embryo maturation is well-known in pine species (Lelu-Walter et al., 2006, 2008), but had not been previously demonstrated with hybrid larches. Maturation of somatic embryos was greatly improved when embryonal masses were placed on medium supplemented with 0.2M sucrose, 60μM ABA containing high concentration of gellan gum (8gl-1). Increasing the gellan gum concentration improved both the quantity and quality of the somatic embryos by reducing EM proliferation and giving rise to well-shaped somatic embryos which subsequently germinated and developed into plantlets. Somatic embryos maturated in presence of high gellan gum, presented reduced moisture content, increase of their dry weight that could resulted from an increase of their protein content (soluble and total part). Gels 2D were realised at 8 weeks, following by LC-MS/MS analysis to identify proteins. In conclusion, hybrid larch SE should influence breeding strategies by offering an alternative tool for accelerated production of large quantities of plants for clonal tests. The improved protocol was applied for the propagation of the new hybrid variety REVE-VERT. Integration of SE into the breeding programme is now undertaken (Lelu-Walter and Pâques 2009).

Somatic embryogenesis represents an important clonal propagation method for conifers (Lelu-Walter and Pâques 2009). Desiccation treatments applied to hybrid larch somatic embryos promote plantlet development (Lelu et al. 1995). Water availability could also be modulated via the gelling agents of the culture medium. We investigated the effect of high gellan gum concentration in the medium during somatic embryo maturation of hydrid larch. High gellan gum concentration of 8 gL-1 was compared to the normal concentration of 4 gL-1 at different stages of maturation. The decrease of water availability at 8 weeks on 8 gL-1 impacted osmotic potential, water content and dry weight in the embryos. The proteomic study at 1 and 8 weeks on both culture media, indicated that 62 differing proteins at 1 week corresponded to “Carbohydrate metabolism”, “Genetic information processing” or "Environmental information processing”. The physiological parameters as well as the identified proteins suggested a stress status of the embryos when they were cultivated on 4 gL-1.We report results that describe for the first time 2-DE proteomic analysis somatic embryo maturation in larch. The protein expression profiles will provide novel insights into the process of hybrid larch somatic embryo maturation, and an outline for practical application.