The genetic basis of natural variation in oenological traits in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is the main microorganism responsible for wine alcoholic fermentation. The oenological phenotypes resulting from fermentation, such as the production of acetic acid, glycerol, and residual sugar concentration are regulated by multiple genes and vary quantitatively between different strain backgrounds. With the aim of identifying the quantitative trait loci (QTLs) that regulate oenological phenotypes, we performed linkage analysis using three crosses between highly diverged S. cerevisiae strains. Segregants from each cross were used as starter cultures for 20-day fermentations, in synthetic wine must, to simulate actual winemaking conditions. Linkage analysis on phenotypes of primary industrial importance resulted in the mapping of 18 QTLs. We tested 18 candidate genes, by reciprocal hemizygosity, for their contribution to the observed phenotypic variation, and validated five genes and the chromosome II right subtelomeric region. We observed that genes involved in mitochondrial metabolism, sugar transport, nitrogen metabolism, and the uncharacterized ORF YJR030W explained most of the phenotypic variation in oenological traits. Furthermore, we experimentally validated an exceptionally strong epistatic interaction resulting in high level of succinic acid between the Sake FLX1 allele and the Wine/European MDH2 allele. Overall, our work demonstrates the complex genetic basis underlying wine traits, including natural allelic variation, antagonistic linked QTLs and complex epistatic interactions between alleles from strains with different evolutionary histories.

Figure 1. Quantitative variation in oenological phenotypes.

Seven strains were phenotyped for production of key metabolites for wine making after 20 days fermentation. Three strains (EC1118, L-1374, L-1578) are used in real wine making setting. The other strains are representative of diverged genomic clusters and used for QTL mapping. (A) Succinic acid production. (B) Acetic acid production. (C) Residual sugar. (D) Glycerol production. (E) Statistical differences in the oenological phenotypes between the parental strains. (F) Example of continuous distribution in acetic acid production of segregants from three crosses.

Figure 2. Identification of WA alleles contributing to high level of acetic acid and residual sugar.

(A) Reciprocal hemizygosity analysis of subtelomeric region of chromosome II-L (Chr II-R) and ALD6 for acetic acid production. The hybrid hemizygote strains with the WA or NA allele for Chr II-R and ALD6 are showed. (B) Acetic acid production of segregant strains sorted for Chr II-R and ALD6 genotypes. The horizontal lines represent the average phenotype value. (C) Reciprocal hemizygosity analysis of MBR1 and HAP4. The hybrid hemizygote strains with the WA or NA allele for MBR1 and HAP4 are shown. (D) Residual sugar of segregant strains sorted for Chr II-R and ALD6 genotypes. The horizontal lines represent the average of the phenotype values. (*) represents a significant statistical difference between the hemizygote strains for the same gene (ANOVA P<0.05).

Figure 3. Multiple antagonistic QTLs in the WE background.

(A, C and E) Reciprocal hemizygosity analysis of candidate genes for residual sugar, succinic acid production and glycerol production respectively. The hybrid hemizygote strains with the WE or SA allele are showed. (B, D and F) Residual sugar, succinic acid production and glycerol production of segregant strains, sorted by genotypes. The horizontal lines represent the average phenotype value. (*) represents a significant statistical difference between the hemizygotes strains (ANOVA P<0.05).

Figure 4. Higher expression levels of WA ALD6 are consistent with increased acetic acid production.

(A) Analysis of promoter region of the ALD6 gene. Residue at positions −82 and −50 are evolutionary conserved in other Saccharomyces sensu stricto species but not in the West African lineage. Nucleotide changes with respect to the reference genome (S288c strain) are shown in red. (B) Expression of ALD6 in the parental and hybrid strain at three different time points of the fermentation process.

Figure 5. Strong epistatic interaction between diverged alleles.

We measured levels of succinic acid production in parental, hybrid, and all possible single and double hemizygote combinations for FLX1 and MDH2 alleles in the WE and SA cross. (*) Indicates a significant statistical difference between the hemizygote strains (ANOVA P<0.05). (**) Indicates significant statistical difference between double hemizygote strains (ANOVA P<0.05).

Figure 6. Functional role of winemaking QTLs.

Genes that contributed to the natural variation in wine making QTLs are shown. Genes in red and green contribute to the natural variation in the NA x WA and WE x SA crosses, respectively. The green squares indicate genes showing positive epistatic interaction. ACG = α – ketoglutarate. Each arrow shows a metabolic step from one compound to another. See main text for further details.