QTL mapping of the production of wine aroma compounds by yeast
© Steyer et al.; licensee BioMed Central Ltd. 2012
Received: 29 May 2012
Accepted: 4 October 2012
Published: 30 October 2012
Wine aroma results from the combination of numerous volatile compounds, some produced by yeast and others produced in the grapes and further metabolized by yeast. However, little is known about the consequences of the genetic variation of yeast on the production of these volatile metabolites, or on the metabolic pathways involved in the metabolism of grape compounds. As a tool to decipher how wine aroma develops, we analyzed, under two experimental conditions, the production of 44 compounds by a population of 30 segregants from a cross between a laboratory strain and an industrial strain genotyped at high density.
We detected eight genomic regions explaining the diversity concerning 15 compounds, some produced de novo by yeast, such as nerolidol, ethyl esters and phenyl ethanol, and others derived from grape compounds such as citronellol, and cis-rose oxide. In three of these eight regions, we identified genes involved in the phenotype. Hemizygote comparison allowed the attribution of differences in the production of nerolidol and 2-phenyl ethanol to the PDR8 and ABZ1 genes, respectively. Deletion of a PLB2 gene confirmed its involvement in the production of ethyl esters. A comparison of allelic variants of PDR8 and ABZ1 in a set of available sequences revealed that both genes present a higher than expected number of non-synonymous mutations indicating possible balancing selection.
This study illustrates the value of QTL analysis for the analysis of metabolic traits, and in particular the production of wine aromas. It also identifies the particular role of the PDR8 gene in the production of farnesyldiphosphate derivatives, of ABZ1 in the production of numerous compounds and of PLB2 in ethyl ester synthesis. This work also provides a basis for elucidating the metabolism of various grape compounds, such as citronellol and cis-rose oxide.
The wide diversity that can be observed among individuals of the same species is one of the most remarkable aspects of life. Deciphering the mechanisms explaining this phenotypic variety is among the major aims of evolutionists and geneticists. Quantitative genetics has been applied to untangle these issues, and over the last 30 years numerous studies have illustrated the power of these genetic approaches, and in particular quantitative trait locus (QTL) mapping, with the characterization of many genomic regions linked to or containing genes responsible for quantitative variations in a phenotype. These approaches have been extensively used in plant and cattle breeding programs; they have contributed to the understanding of resistance to several diseases  and also led to a significant improvement in crop yields and cattle breeding. Surprisingly quantitative genetic approaches have been applied only recently to budding yeast, initially to elucidate various complex mechanisms, including sporulation efficiency , thermotolerance [3, 4], and drug resistance . Even more recently, this quantitative approach has been used to decipher complex traits [6, 7] at high resolution [8, 9]. It has also been applied successfully to the analysis of variations in gene expression [10, 11]. The QTL approach is now being used to study features important for the beverages industry, for example wine fermentation [12, 13], sake technological traits  and ethanol tolerance for ethanol production . All these studies have implicated defective alleles, of for example AMN1, ASP1 or ABZ1, in the diversity of the phenotypes of segregants. Strain By4741 possesses a defective allele of AMN1 which leads to faster daughter cell separation; wine strain SB possesses a defective allele of the ASP1 gene involved in asparagine catabolism; and S288C possesses a defective allele of ABZ1 that codes for an enzyme which catalyzes the synthesis of 4-amino-4- deoxychorismate from chorismate, a step in the synthesis of paraminobenzoic acid. This defective allele of ABZ1 modulates the fermentation rate by controlling nitrogen utilization .
Wine aroma is complex and results from the blending of numerous compounds synthesized by vines, some of which are transformed by yeast, together with compounds directly produced by yeast as a result of its primary metabolism [17, 18]. The metabolic pathways leading to the synthesis of these yeast volatile compounds are numerous and incompletely described. The roles of some of the key genes, such as ATF1 for acetates and EEB1 for ethyl esters, has been demonstrated [19, 20]. Nevertheless, little is known about the factors explaining large strain-to-strain differences in the production of volatile compounds [21–23]. Holistic approaches  have given new insights into the roles of various key genes in the diversity of production of some volatile compounds. Further work from the same group highlighted how a few key players, such as transcription factors, may explain some of the differences between strains .
To analyze the differences in the production of wine aroma compounds linked to yeast strain diversity, we used QTL analysis with a population of 30 segregants arising from a cross between the laboratory strain S288C and 59A, a spore isolated from the industrial wine strain EC1118. This population of segregants has been genotyped with Affymetrix YGS98 microarrays to obtain a high density genetic map and was used for the first quantitative analysis of transcriptome variations during enological fermentation . We tested this population of segregants for the production of aromatic compounds in two different experimental conditions: synthetic musts mimicking white and red wine fermentations. These analyses enabled us to detect the involvement of eight genomic regions in the production of various volatile compounds explaining 39% to 72% of the diversity. As examples, we characterized the role of two genes by hemizygote analysis and identified another candidate gene by analysis of the phenotype of a deleted mutant. Our findings provide new insights into the genetic architecture underlying the production of wine aroma by yeast.
Each of the 30 segregants was tested in two experimental designs. In the first design (experiment A), white wine fermentation was simulated by fermentation at 20°C in medium with a low lipid content, whereas in the second design, mimicking red wine fermentation , the fermentations were run at 28°C in medium with a high lipid content (experiment B).
Genetic analysis of the volatile compounds production
From experiment A, heritability was estimated to be greater than 70% for 21 of 27 compounds, which included the grape aroma compounds geraniol, linalool, citronellol and geraniol acetate.
QTL analysis of volatile compounds produced during alcoholic fermentation (experiment A, geraniol 5mg/L)
Single QTL scan
Flocculation as a covariable
Fraction of variation explained by the QTL
Chr XIV 634-687
2 phenyl ethanol
Chr VIII 422-469
Chr XII 675-704
Chr XIII 290-342
Chr I 21-55
Chr VII 47-85
Cis/trans rose oxide ratio
Chr XIV 537-589
QTL analysis of volatile compounds produced during alcoholic fermentation (experiment B, Ambroset et al. 2011)
Single QTL scan
Flocculation (1) orABZ1(2) as a covariable
Fraction of variation explained by the QTL
(coordinates in kb)
Chr XIII 255-305
Chr XIII 245-304
Chr XIII 230-290
Chr XIV 657-702
Chr VII 332-370
Chr XII 674-705
Chr VIII 423-481
Chr XIII 234-285
(E,Z)- or (Z,E)-Farnesol
Chr II 593-646
Chr XII 711-750
Chr XII 693
Chr XII 735
Chr XII 706-757
Chr XII 675-704
For several compounds we were unable to identify any QTL despite a high heritability. This were the case for instance for isoamyl-alcohol and its acetate ester. In the case of isoamyl-alcohol, this might be due to two isomeric compounds (3-methyl-1-butanol and 2-methyl-1-butanol) both being involved. Nevertheless, we detected several regions involved in the diversity of the production of various compounds in the acid, alcohols ethyl ester and isoprenoid chemical families. As a whole, these metabolic QTL (mQTL) explained between 43 and 73% of the metabolite variation.
The other regions detected for several compounds were each found in only one experiment. These regions also explained a smaller part of the variations in the production of the various volatile metabolites despite a high heritability. Possibly, the production of most of these compounds is under multigenic control and the corresponding regions cannot be detected with such a small population of segregants.
In addition to compounds produced by yeast during alcoholic fermentation, we also studied the fate of geraniol, which is present in grape must at up to 3 mg/L . We did not detect any region explaining variations in the metabolism of geraniol. However, we detected one QTL explaining 43% of the variations in the concentration of citronellol a compound produced from geraniol during alcoholic fermentation. The synthetic pathway for citronellol has not been clearly described and our results may indicate new targets to investigate. Two other QTL explained variations in the content of cis-rose oxide and in the ratio between cis and trans isomers of rose oxide. These QTLs may be technologically interesting as the two isomers of rose oxide present different olfactive thresholds.
Evaluation of the role of various candidate genes in the QTL
PDR8 is responsible for variations in nerolidol production during alcoholic fermentation
The major QTL responsible for variations in the concentration of nerolidol in experiment A and of nerolidol, farnesene and bisabolene in experiment B maps to a short region of 20 kb containing 26 ORFs. Nerolidol, farnesene and bisabolene are all derived from farnesyl diphosphate, an intermediate in isoprenoid and ergosterol biosynthesis (Figure 2): at acidic pH, the instability of the diphosphate group leads to the release of farnesol and its isomer nerolidol.
It seemed likely that the gene involved in the modulation of nerolidol, farnesene and bisabolene production is involved in ergosterol biosynthesis or in farnesol/nerolidol transport because of the size of these molecules. One of the genes mapping in this region is PDR8, a transcription factor that modulates the expression of 16 genes  including transporters (AZR1, PDR15, QDR2, YOR1), a gene of the ergosterol biosynthesis pathway (ERG8), and enzymes involved in oxido-reduction processes (CTT1, GTT2, YMR315w). This transcription factor was clearly a good candidate. The nucleotide sequences of the PDR8 genes in strains S288C and 59A show numerous single nucleotide differences. These SNPs generate five non-synonymous substitutions between the Pdr8p proteins in 59A and S288C.
To confirm the involvement of the PDR8 gene in the observed phenotype, we compared two reciprocal hemizygotes between S288C and 59A containing only one of the parental alleles. These hemizygotes presented the different phenotypes observed in the population of segregants, with the enological PDR8 allele of 59A leading to a lower production of nerolidol (Figure 3A). These results are in agreement with those obtained for the whole population (Figure 3B). However, the parental strain S288C produced less nerolidol than most of the segregants, indicating further interactions with the genetic background.
Characterization of PDR8 targets involved in the phenotype
ABZ1 allelic variations affect production of 2-phenylethanol and ethyl esters by yeast during fermentation
Effects of the different alleles of the ABZ1 gene on the concentrations of several fermentation compounds (relative units)
PLB2 allelic variations may affect ethyl ester production
Polymorphism of the various genes and adaptation
We report 13 regions linked to variations in the production of wine volatile compounds. This study is the first demonstration of the potential usefulness of QTL analysis for understanding the origin of the variations in the concentrations of wine aroma compounds and deciphering this “intricate lattice of chemical and biological interactions” . It was not possible to detect QTL for all relevant compounds, despite high heritability. Presumably, the synthesis of many of these compounds is under multigenic control, such that the small size of our segregant population prevented exploration of their complexity. Until now, few key technological traits for alcoholic fermentation have been characterized [11, 13, 14, 16].
Several of the QTL found here are related to terpenoids, which constitute a large family of compounds. They include monoterpenes, which with their corresponding alcohols present useful properties, such as fragrances (in essential oils) or variety aroma (in wines), and even antimicrobial and cancer chemopreventive properties . In yeast, these compounds are synthesized through the mevalonic acid pathway from acetyl-coA, which is converted to isopentenylpyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), the building blocks of isoprenoids. The main product of this pathway is ergosterol, and geranyldiphosphate and farnesyl diphosphate are intermediate metabolites.
We did not detect any QTL explaining variations in residual geraniol. However, one QTL explained some of the variation in the concentrations of citronellol; this QTL maps to a region of chromosome XIII containing several candidate genes. We did not find a candidate explaining the variations in the concentration of cis-rose oxide in the media or in the ratio between the cis and trans isomers. This compound is significant to wine-making because of its high odor activity  and it has been shown recently that yeast can produce cis-rose oxide in wine .
We demonstrate that the alleles of PDR8 found in S288C and 59A differently regulate the QDR2 gene responsible for the release of nerolidol into the media. Farnesol and its isomer nerolidol arise from farnesyl diphosphate instability at low pH, like that in the yeast vacuole or in the exocellular medium . Therefore, it is possible that the transporter Qdr2p is responsible of the export of either farnesyl diphosphate or of nerolidol.
Enological strains have a PDR8 allele more divergent from S. paradoxus than that of S288C. The neutrality index  we calculated for this gene is incompatible with its neutral evolution: it presented a higher number of replacement polymorphisms than expected under neutral selection. This may be the result of adaptation to different niches or the results of isolation and multiple migrations as suggested by Aa et al. for SSU1. The role of farnesol and nerolidol production by S. cerevisiae is not clear. Under the anaerobic conditions of wine fermentation ergosterol synthesis is blocked, however ERG20 expression correlates with fermentation speed  even in a fermentation medium containing ergosterol (experiment B). The synthesis of farnesol diphosphate is essential for (i) the synthesis of other compounds including dolichol which is necessary for cell wall assembly [40, 41], (ii) protein prenylation such as that of Skt5p  involved in chitin synthase activity, and (iii) ubiquinone synthesis which may be less important during fermentation. Furthermore, farnesol is a biologically active compound that at concentrations higher than 50 μM inhibits S. cerevisiae and C. albicans growth [43, 44] and at lower concentrations is involved in quorum sensing by C. albicans[45, 46].
In addition to the regions affecting terpenoid production, we also linked several other regions to variations in the concentrations of various volatiles. The ABZ1 gene in one of these regions seems to have the widest effect as its allelic variations affected 2-phenylethanol and ethyl ester synthesis in experiment B, and explained as much as 50% of the variations in the concentration of ethylhexanoate in experiment A. The lower production of 2-phenylethanol was only partially restored by the addition of p-aminobenzoic acid to the fermentation media, and this suggests that this phenotype is not solely the consequence of the substantial effect of the ABZ1-S288C allele on fermentation speed via its impact on nitrogen metabolism . The phylogeny of ABZ1 was clearly different from that of PDR8: the ABZ1-S288C allele is located at the end of a long branch whose branch point is close to that of wine strains (relative to other origins). Again, the neutrality index  calculated for this gene was significantly higher than 1. Possibly, the allelic form of ABZ1 has accumulated several deleterious mutations leading to a loss of activity. This feature may explain the very particular phenotypic behavior observed for S288C .
The two experimental sets we report generated complementary results. We observed effects of PDR8 and ABZ1 allelic variations in both experiments. Nevertheless, the impact of ABZ1 polymorphism was much less pronounced in experiment A than B, as it explained only variation for ethylhexanoate production after correction for the flocculation effect. We also detected one additional region in experiment A only, and four in experiment B only; presumably the different sets of QTL identified in the two experiments reflect the effects of the different environmental conditions. By considering genes mapping in these regions, we identified PLB2 as possibly involved in the variations of ethyl ester content in experiment B. This gene may have a role complementary to EEB1 in the synthesis of ethyl esters during alcoholic fermentation.
This study shows that linkage analysis can give valuable information about the metabolic pathways involved in production of volatile compounds in yeast, even with only a small population of segregants. We identified the involvement of the PLB2 gene in the metabolism of ethyl esters, and evidenced the role of PDR8 in the release of nerolidol into the media via the regulation of QDR2 expression. We also showed that the weak activity of SC288C Abz1p allele leads to a lower production of many metabolites, including 2-phenyl ethanol, and that this effect was only partially relieved by supplementation with paminobenzoic acid. Other candidate genes are currently being evaluated (i.e. for citronellol synthesis). However, we could not find candidate genes in all regions detected, and despite a high heritability, we did not find any regions associated with the production diversity of many of the compounds considered. Possibly, a larger number of segregants is necessary for a more exhaustive analysis. Our results identify potential new targets for a marker-aided breeding strategy in yeast for the optimization of the production of volatile compounds during fermentation.
Interestingly, our genetic analysis revealed the particular evolution of the PDR8 gene. This may reflect a specific adaptation to wine fermentation conditions, but raises questions about the role of farnesol and nerolidol for S. cerevisiae during fermentation.
Strains, growth conditions, and fermentation conditions
The two parental Saccharomyces cerevisiae strains studied were the standard strain S288c (MATa; SUC2; gal2) and a haploid derivative of the industrial wine strain EC1118 (HO/ho), herein referred to as 59A (MATa; ho). This strain is prototrophic and has fermentation properties similar to the diploid strain EC1118. The population of 30 segregants obtained from these two parental strains used for QTL analysis have been genotyped after hybridization on high density olignonucleotide microarrays Affymetrix YGS98 oligoarrays.
The strains BY4742 (MATa; his3Δ1; leu2Δ 0; lys2Δ 0; ura3Δ 0) and BY4742Δ ABZ1 (Mata; his3Δ 1; leu2Δ 0; lys2Δ 0; ura3Δ 0; YNR033w::kanMX4), and BY4742 (MATa; his3Δ 1; leu2Δ 0; lys2Δ 0; ura3Δ 0) and BY4742Δ PDR8 (Mata; his3Δ 1; leu2Δ 0; lys2Δ 0; ura3Δ 0; YNR033w::kanMX4) were used for hemizygous constructions.
Allelic replacement at PDR8 in 59A was obtained in three steps: 1) deletion of PDR8 from 59A using the hphMX4 cassette for hygromycin resistance (pAG32). Primers for cassette amplification and verification were obtained form Euroscarf. 2) preparation of a replacement cassette containing PDR8-loxP-kanMX4-loxP by the insertion of loxP-KanMX4-loxP into the terminator of PDR8 in strain S288c (primers are given in Additional file 4: Table S4). 3) replacement of the hphMX4 cassette from 59A PDR8Δ::hph with the PDR8-loxP-kanMX4-loxP replacement cassette from S288c and selection on YPD containing G418 (200 μg.l-1). The loss of hphMX4 cassette was verified by PCR and the absence of growth on hygromycin.
YPD medium was used for precultures at 28°C for 24h in 125 mL flasks with shaking.
Differences in the two experimental designs used in this work (adapted from)
Anaerobic factors for 1L
Tween 5 μL
Tween 0.5 mL
Oleic acid 0.05 μg
Oleic acid 5 μg
Sistosterol: 15 μg/L
Ergosterol 1500 μg/L
Geraniol content (mg/L)
Number of fermentations
Fermentations were performed in 250 mL flasks equipped with airlocks to maintain anaerobiosis without stirring (design A) and in 1 L fermenters with constant stirring (design B). Small flask fermentations were weighed twice daily and stopped as soon as the daily loss was less than 1% of the expected total loss.
Volatile compounds analysis
Wine aroma compounds were analyzed by the Stir Bar Sorptive Extraction method  adapted to our laboratory conditions, with a 1 μL injection volume. The analyses were performed with an Agilent 6890N gas chomatograph equipped with an Agilent 7683 automatic liquid sampler coupled to an Agilent 5975B inert MSD (Agilent Technologies). The gas chomatograph was fitted with a DB-Wax capillary column (60 m × 0.32 mm i.d. × 0.50 μm film thickness, J&W Scientific) and helium was used as carrier gas (1 mL min-1 constant flow). The GC oven temperature was programmed without initial hold time at a rate of 2.7°C min-1 from 70°C to 235°C (hold 10 min). The injector was set to 250°C and used in pulsed splitless mode (25 psi for 0.50 min). The temperatures of the interface, MS ion source and quadrupole were 270°C, 230°C and 150°C, respectively. The mass spectrometer was operated in electron impact ionization mode (EI, 70 eV) and the masses were scanned over a m/z range of 29 – 300 amu. Agilent MSD chemStation software (G1701DA, Rev D.03.00) was used for instrument control and data processing. The mass spectra were compared with the Wiley’s library reference spectral bank
The following compounds were analyzed: isoamyl alcohol, isoamyl acetate, isobutanol, 2-3 butanediol, 2-phenylethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, ethyl myristate, ethyl palmytate, ethyl laurate, 2-phenyl ethanol, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, ethyl 9-decenoate, isoamyl octanoate, 2-phenyl ethyl hexanoate, 2-phenylethyl octanoate, 2-phenylethyl decanoate, ethyl 4 hydroxy butanoate, ethyl 3-hydroxydecanoate, ethyl 3-hydroxyoctanoate, nerolidol, farnesol, 2-3 dihydro farnesol, (E, Z)- or (Z, E)- farnesol (A), (E, E)- farnesol (A), farnesyl acetate, isoamyl octanoate, isoamyl decanoate, isoamyl dodecanoate, methyl oleate, trans-β-farnesene, (Z, E)-α-farnesene, α-bisabolene, ß-bisabolene, (E, E)-α-farnesene, α-terpineol, linalol, citronellol, geraniol, nerol, citronellyl acetate, geranyl acetate, neryl acetate, cis-rose oxide, trans-rose oxide.
Statistical and QTL analysis
Heritability was calculated according to the method of Brem et al. . Statistical analyses were performed using R software version 2.13.1 . QTL analysis was done for each phenotype of the two datasets (experiments A and B) using the genetic map of 1834 markers genotyped previously . The distribution of each phenotype was verified using a Shapiro-Wilk normality test: the normality of the distribution was rejected for 17 of the 29 compounds analyzed in experiment A and 30 of 40 in experiment B, for a threshold of 0.05
As the distribution of most phenotypes was not normal and due to the small sample size, linkage analysis was performed using both parametric and non-parametric models to evaluate the robustness of the parametric model. The parametric model consists of a linkage analysis performed using a normal model with the Haley-Knott regression method implemented in the R/qtl package [52, 53]. As the results of the two analyses were concordant, only the normal analysis is presented.
To overcome the potential effects of flocculation  and of the presence of the ABZ1-S288C allele which provokes large variations in fermentation kinetics , we performed a second linkage analysis using a normal model with the Haley-Knott regression method, first with flocculation as an interactive covariate, and then with the ABZ1 marker (Chr 14, position 689.4 kb) as an additive covariate. For these regions, a significant effect was indeed observed for both flocculation and the ABZ1-specific markers.
For the three models and the two datasets analyzed, logarithm of odds (LOD) scores were computed for each marker every 2.5cM. An interval estimate of the location of each QTL was obtained as the 1-LOD support interval. The LOD significance threshold was estimated after permutation tests that were replicated 1000 times. The percentage of variance explained by each QTL was estimated from a drop-one-term analysis of results in the global model.
Q-PCR analysis of the expression of PDR8 targets after allelic replacement
Fermentations (900 mL of MS300 medium) were performed in triplicate with strains 59A and S288C-PDR8 59A, and cells were sampled when 70% of the glucose had been fermented. RNA was extracted with trizol as described previously . cDNA was produced by reverse transcription and a 1 in 25 dilution of the resulting cDNA was used for the realtime PCR assays with gene-specific primers and Strategene’s Brilliant II SYBR Green QPCR Master Mix (Santa Clara, CA) and an ABI7300 QPCR machine. Expression levels were measured relative to those of UBC6 and SCR1, both giving similar results.
Sequence analysis and phylogeny
To infer the evolutionary history of ABZ1 and PDR8, we collected their sequences from genomes available at SGD (http://www.yeastgenome.org/). All uncompleted or frameshift-containing sequences where discarded from this set. The phylogenies were inferred with MEGA  by the Maximum Likelihood method based on the Kimura 2-parameter model . The trees with the highest log likelihood are shown. The trees are drawn to scale, with branch lengths proportional to the number of substitutions per site. The significance of the Neutrality Index  test was calculated using the http://bioinf3.uab.cat/mkt/MKT.asp website.
The list of the sequences used for the two analyses is given in supplementary data (Additional file 5).
DS: performed fermentation (experiment A), aroma and QTL analysis , candidate gene search and validation (PDR8, PLB2, …). DS wrote a first draft of the manuscript. CA produced the strain set and performed fermentations (experiment B). CB performed allelic replacement for S288c- PDR8 59A strain fermentations and QPCR expression analysis. PC, analyzed aroma compounds. PD built ABZ1 hemizygote strains and performed fermentations. IS, performed statistical analysis (QTL). JLL performed phylogenic analysis and tests. CE, BB, FK, JLL conceived the study ,designed and coordinated the research JLL wrote the manuscript. All authors analyzed the data. All authors read and approuved the final manuscript.
This work has been supported by a grant from “Comité Interprofessionnel des Vins d’Alsace”, Colmar, France and from Region Alsace, France.
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