A Saccharomyces eubayanus haploid resource for research studies.

Since its identification, Saccharomyces eubayanus has been recognized as the missing parent of the lager hybrid, S. pastorianus. This wild yeast has never been isolated from fermentation environments, thus representing an interesting candidate for evolutionary, ecological and genetic studies. However, it is imperative to develop additional molecular genetics tools to ease manipulation and thus facilitate future studies. With this in mind, we generated a collection of stable haploid strains representative of three main lineages described in S. eubayanus (PB-1, PB-2 and PB-3), by deleting the HO gene using CRISPR-Cas9 and tetrad micromanipulation. Phenotypic characterization under different conditions demonstrated that the haploid derivates were extremely similar to their parental strains. Genomic analysis in three strains highlighted a likely low frequency of off-targets, and sequencing of a single tetrad evidenced no structural variants in any of the haploid spores. Finally, we demonstrate the utilization of the haploid set by challenging the strains under mass-mating conditions. In this way, we found that S. eubayanus under liquid conditions has a preference to remain in a haploid state, unlike S. cerevisiae that mates rapidly. This haploid resource is a novel set of strains for future yeast molecular genetics studies.

Rapid selection response to ethanol in Saccharomyces eubayanus emulates the domestication process under brewing conditions.

Although the typical genomic and phenotypic changes that characterize the evolution of organisms under the human domestication syndrome represent textbook examples of rapid evolution, the molecular processes that underpin such changes are still poorly understood. Domesticated yeasts for brewing, where short generation times and large phenotypic and genomic plasticity were attained in a few generations under selection, are prime examples. To experimentally emulate the lager yeast domestication process, we created a genetically complex (panmictic) artificial population of multiple Saccharomyces eubayanus genotypes, one of the parents of lager yeast. Then, we imposed a constant selection regime under a high ethanol concentration in 10 replicated populations during 260 generations (6 months) and compared them with propagated controls exposed solely to glucose. Propagated populations exhibited a selection differential of 60% in growth rate in ethanol, mostly explained by the proliferation of a single lineage (CL248.1) that competitively displaced all other clones. Interestingly, the outcome does not require the entire time-course of adaptation, as four lineages monopolized the culture at generation 120. Sequencing demonstrated that de novo genetic variants were produced in all propagated lines, including SNPs, aneuploidies, INDELs and translocations. In addition, the different propagated populations showed correlated responses resembling the domestication syndrome: genomic rearrangements, faster fermentation rates, lower production of phenolic off-flavours and lower volatile compound complexity. Expression profiling in beer wort revealed altered expression levels of genes related to methionine metabolism, flocculation, stress tolerance and diauxic shift, likely contributing to higher ethanol and fermentation stress tolerance in the evolved populations. Our study shows that experimental evolution can rebuild the brewing domestication process in 'fast motion' in wild yeast, and also provides a powerful tool for studying the genetics of the adaptation process in complex populations.

GPD1 and ADH3 Natural Variants Underlie Glycerol Yield Differences in Wine Fermentation.

Glycerol is one of the most important by-products of alcohol fermentation, and depending on its concentration it can contribute to wine flavor intensity and aroma volatility. Here, we evaluated the potential of utilizing the natural genetic variation of non-coding regions in budding yeast to identify allelic variants that could modulate glycerol phenotype during wine fermentation. For this we utilized four Saccharomyces cerevisiae strains (WE - Wine/European, SA - Sake, NA - North American, and WA - West African), which were previously profiled for genome-wide Allele Specific Expression (ASE) levels. The glycerol yields under Synthetic Wine Must (SWM) fermentations differed significantly between strains; WA produced the highest glycerol yields while SA produced the lowest yields. Subsequently, from our ASE database, we identified two candidate genes involved in alcoholic fermentation pathways, ADH3 and GPD1, exhibiting significant expression differences between strains. A reciprocal hemizygosity assay demonstrated that hemizygotes expressing GPD1WA , GPD1SA , ADH3WA and ADH3SA alleles had significantly greater glycerol yields compared to GPD1WE and ADH3WE . We further analyzed the gene expression profiles for each GPD1 variant under SWM, demonstrating that the expression of GPD1WE occurred earlier and was greater compared to the other alleles. This result indicates that the level, timing, and condition of expression differ between regulatory regions in the various genetic backgrounds. Furthermore, promoter allele swapping demonstrated that these allele expression patterns were transposable across genetic backgrounds; however, glycerol yields did not differ between wild type and modified strains, suggesting a strong trans effect on GPD1 gene expression. In this line, Gpd1 protein levels in parental strains, particularly Gpd1pWE, did not necessarily correlate with gene expression differences, but rather with glycerol yield where low Gpd1pWE levels were detected. This suggests that GPD1WE is influenced by recessive negative post-transcriptional regulation which is absent in the other genetic backgrounds. This dissection of regulatory mechanisms in GPD1 allelic variants demonstrates the potential to exploit natural alleles to improve glycerol production in wine fermentation and highlights the difficulties of trait improvement due to alternative trans-regulation and gene-gene interactions in the different genetic background.

Genetic basis of mycotoxin susceptibility differences between budding yeast isolates.

Micophenolic acid (MPA) is an immunosuppressant mycotoxin which impairs yeast cell growth to variable degrees depending on the genetic background. Such variation could have emerged from several phenomena, including MPA gene resistance mutations and variations in copy number and localisation of resistance genes. To test this, we evaluated MPA susceptibility in four S. cerevisiae isolates and genetically dissected variation through the identification of Quantitative Trait Loci. Via linkage analysis we identified six QTLs, majority of which were located within subtelomeres and co-localised with IMD2, an inosine monophosphate dehydrogenase previously identified underlying MPA drug resistance in yeast cells. From chromosome end disruption and bioinformatics analysis, it was found that the subtelomere localisation of IMD2 within chromosome ends is variable depending on the strain, demonstrating the influence of IMD2 on the natural variation in yeast MPA susceptibility. Furthermore, GxE gene expression analysis of strains exhibiting opposite phenotypes indicated that ribosome biogenesis, RNA transport, and purine biosynthesis were impaired in strains most susceptible to MPA toxicity. Our results demonstrate that natural variation can be exploited to better understand the molecular mechanisms underlying mycotoxin susceptibility in eukaryote cells and demonstrate the role of subtelomeric regions in mediating interactions with the environment.

Identification of Nitrogen Consumption Genetic Variants in Yeast Through QTL Mapping and Bulk Segregant RNA-Seq Analyses.

Saccharomyces cerevisiae is responsible for wine must fermentation. In this process, nitrogen represents a limiting nutrient and its scarcity results in important economic losses for the wine industry. Yeast isolates use different strategies to grow in poor nitrogen environments and their genomic plasticity enables adaptation to multiple habitats through improvements in nitrogen consumption. Here, we used a highly recombinant S. cerevisiae multi-parent population (SGRP-4X) derived from the intercross of four parental strains of different origins to identify new genetic variants responsible for nitrogen consumption differences during wine fermentation. Analysis of 165 fully sequenced F12 segregants allowed us to map 26 QTL in narrow intervals for 14 amino acid sources and ammonium, the majority of which represent genomic regions previously unmapped for these traits. To complement this strategy, we performed Bulk segregant RNA-seq (BSR-seq) analysis in segregants exhibiting extremely high and low ammonium consumption levels. This identified several QTL overlapping differentially expressed genes and refined the gene candidate search. Based on these approaches, we were able to validate ARO1, PDC1, CPS1, ASI2, LYP1, and ALP1 allelic variants underlying nitrogen consumption differences between strains, providing evidence of many genes with small phenotypic effects. Altogether, these variants significantly shape yeast nitrogen consumption with important implications for evolution, ecological, and quantitative genomics.

RIM15 antagonistic pleiotropy is responsible for differences in fermentation and stress response kinetics in budding yeast.

Different natural yeast populations have faced dissimilar selective pressures due to the heterogeneous fermentation substrates available around the world; this increases the genetic and phenotypic diversity in Saccharomyces cerevisiae In this context, we expect prominent differences between isolates when exposed to a particular condition, such as wine or sake musts. To better comprehend the mechanisms underlying niche adaptation between two S. cerevisiae isolates obtained from wine and sake fermentation processes, we evaluated fermentative and fungicide resistance phenotypes and identify the molecular origin of such adaptive variation. Multiple regions were associated with fermentation rate under different nitrogen conditions and fungicide resistance, with a single QTL co-localizing in all traits. Analysis around this region identified RIM15 as the causative locus driving fungicide sensitivity, together with efficient nitrogen utilization and glycerol production in the wine strain. A null RIM15 variant confers a greater fermentation rate through the utilization of available glucose instead of its storage. However, this variant has a detrimental effect on fungicide resistance since complex sugars are not synthesized and transported into the membrane. Together, our results reveal the antagonist pleiotropic nature of a RIM15 null variant, positively affecting a series of fermentation related phenotypes, but apparently detrimental in the wild.

Natural variation in non-coding regions underlying phenotypic diversity in budding yeast.

Linkage mapping studies in model organisms have typically focused their efforts in polymorphisms within coding regions, ignoring those within regulatory regions that may contribute to gene expression variation. In this context, differences in transcript abundance are frequently proposed as a source of phenotypic diversity between individuals, however, until now, little molecular evidence has been provided. Here, we examined Allele Specific Expression (ASE) in six F1 hybrids from Saccharomyces cerevisiae derived from crosses between representative strains of the four main lineages described in yeast. ASE varied between crosses with levels ranging between 28% and 60%. Part of the variation in expression levels could be explained by differences in transcription factors binding to polymorphic cis-regulations and to differences in trans-activation depending on the allelic form of the TF. Analysis on highly expressed alleles on each background suggested ASN1 as a candidate transcript underlying nitrogen consumption differences between two strains. Further promoter allele swap analysis under fermentation conditions confirmed that coding and non-coding regions explained aspartic and glutamic acid consumption differences, likely due to a polymorphism affecting Uga3 binding. Together, we provide a new catalogue of variants to bridge the gap between genotype and phenotype.