BACKGROUND
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  <dig> years numerous studies have illustrated the power of these genetic approaches, and in particular quantitative trait locus  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  <cit>  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  <cit> , thermotolerance  <cit> , and drug resistance  <cit> .
even more recently, this quantitative approach has been used to decipher complex traits  <cit>  at high resolution  <cit> .
it has also been applied successfully to the analysis of variations in gene expression  <cit> .
the qtl approach is now being used to study features important for the beverages industry, for example wine fermentation  <cit> , sake technological traits  <cit>  and ethanol tolerance for ethanol production  <cit> .
all these studies have implicated defective alleles, of for example amn <dig> <cit> , asp <dig> <cit>  or abz <dig> <cit> , in the diversity of the phenotypes of segregants.
strain by <dig> possesses a defective allele of amn <dig> which leads to faster daughter cell separation; wine strain sb possesses a defective allele of the asp <dig> gene involved in asparagine catabolism; and s288c possesses a defective allele of abz <dig> 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 abz <dig> modulates the fermentation rate by controlling nitrogen utilization  <cit> .
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  <cit> .
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 atf <dig> for acetates and eeb <dig> for ethyl esters, has been demonstrated  <cit> .
nevertheless, little is known about the factors explaining large strain-to-strain differences in the production of volatile compounds  <cit> .
holistic approaches  <cit>  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  <cit> .
to analyze the differences in the production of wine aroma compounds linked to yeast strain diversity, we used qtl analysis with a population of  <dig> segregants arising from a cross between the laboratory strain s288c and 59a, a spore isolated from the industrial wine strain ec <dig>  this population of segregants has been genotyped with affymetrix ygs <dig> microarrays to obtain a high density genetic map and was used for the first quantitative analysis of transcriptome variations during enological fermentation  <cit> .
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.
RESULTS
each of the  <dig> segregants was tested in two experimental designs.
in the first design , 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  <cit> , the fermentations were run at 28°c in medium with a high lipid content .
the fermentation kinetics of the  <dig> segregants presented significant diversity, from typical wine fermentations to clearly sluggish as observed for s288c .
unlike the parental strains, several segregants presented a clear ability to flocculate.
this resulted in large and significant diversity in the concentrations of volatile compounds at the end of the alcoholic fermentations.
we measured a set of  <dig> compounds in experiment a, and  <dig> compounds in experiment b. we performed a principal component analysis to reduce the multidimensional data set of experiment a into three more informative dimensions .
the first three axes explained 51% of the global variance  and in this analysis the various compounds are grouped according to chemical family.
ethyl esters and medium chain fatty acids were correlated to the first axis, the various acetates correlated together with the second axis and 2-phenyl ethanol and isoamylalcohol were correlated to axis three.
the representation of individual progeny strains in the factorial plan indicated a substantial diversity in the ability to produce volatile compounds.
this was especially clear for acetates, as many strains were able to produce more acetates in the media than either of the two parents .
in addition, some strains more than others metabolized geraniol into citronellol or into the high olfactive impact compound cis-rose oxide.
this indicates that the characteristics of the yeast strain have a significant and variable impact on the grape aroma fraction.
a similar picture was obtained from the analysis of experiment b.
genetic analysis of the volatile compounds production
from experiment a, heritability was estimated to be greater than 70% for  <dig> of  <dig> compounds, which included the grape aroma compounds geraniol, linalool, citronellol and geraniol acetate.
to identify qtl for these technological features, we performed a linkage analysis with the previously reported genotypes for these progeny  <cit> .
the concentrations of most compounds obtained for the population of segregants did not follow normal distributions , so we performed linkage analysis with both parametric and non parametric models.
we identified four and six regions involved in variations in the production of different compounds in experiments a and b, respectively .
however, additional factors may contribute to wine aroma production: flocculation is one  <cit> ; and the presence of the abz1-s288c allele, which is responsible for large variations in fermentation kinetics  <cit> , may also have an indirect effect.
to overcome the potential effects of these factors, we performed a second linkage analysis taking these two factors into account as covariables in the model.
this enabled us  to improve the significance for some qtl detected after a simple scan ,  to detect a genetic effect for additional compounds  of one region already found and  to detect three and one additional genomic regions in experiments a and b, respectively, for other compounds.
the effects of flocculation and of abz <dig> allele on aroma production for each qtl are given as additional file  <dig>
regions above the  <dig>  threshold are indicated in italics.
only compounds with differences that are significant or close to the significance threshold are given.
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  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  explained between  <dig> and 73% of the metabolite variation.
one region on chromosome xii was identified in both experiments with high lod score values and explained as much as  <dig> and 72% of the variations in the production of nerolidol.
the same region was identified for other isoprenoids characterized only in design b. another region, on chromosome xiv, was also detected in both experiments and was associated with various phenotypes: ethyl hexanoate in experiment a and several compounds  in experiment b. the abz <dig> gene which maps in this region has been reported to be responsible for variations in the rate of fermentation  <cit> .
when abz <dig> polymorphism was used as a covariable, we detected other qtl for more compounds indicating that abz <dig> allelic variations may affect the production of several metabolites.
these mqtl are scattered through the genome of strain s288c such that we did not detect a major region associated with all the compounds analyzed .
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  <dig> mg/l  <cit> .
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
pdr <dig> 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  <dig> kb containing  <dig> orfs.
nerolidol, farnesene and bisabolene are all derived from farnesyl diphosphate, an intermediate in isoprenoid and ergosterol biosynthesis : 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 pdr <dig>  a transcription factor that modulates the expression of  <dig> genes  <cit>  including transporters , a gene of the ergosterol biosynthesis pathway , and enzymes involved in oxido-reduction processes .
this transcription factor was clearly a good candidate.
the nucleotide sequences of the pdr <dig> 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 pdr <dig> 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 pdr <dig> allele of 59a leading to a lower production of nerolidol .
these results are in agreement with those obtained for the whole population .
however, the parental strain s288c produced less nerolidol than most of the segregants, indicating further interactions with the genetic background.
characterization of pdr <dig> targets involved in the phenotype
to identify which of the targets of pdr <dig> explain the observed variations in nerolidol production, we measured the production of nerolidol by the corresponding  <dig> deletant strains in the by <dig> background .
the deletion of yor <dig> led to an increase of nerolidol production whereas the deletion of four other pdr <dig> target genes  led to decreases of nerolidol production similar to that observed after the deletion of pdr <dig>  the genes qdr <dig> and pdr <dig> encode transporters that may be involved in the export of nerolidol or derived compounds from the cell.
the deletion of two other genes, gph <dig> and ymr315w, resulted in a similar reductions in nerolidol production indicating other possible mechanisms.
gph <dig> is a glycogen phosphorylase required for the mobilization of glycogen, and ymr315w is an oxidoreductase enzyme thay may be involved in the reduction of farnesol .
erg <dig>  encoding mevalonate phosphate kinase, is an essential gene for isoprenoid and ergosterol biosynthesis, so it was not possible to conduct the appropriate tests with the deleted haploid strain.
deletion of only one copy of erg <dig>  in the diploid strain by <dig>  did not lead to any relevant change so we evaluated the effect of the overexpression of erg8: no significant increase of the production of nerolidol was detected .
we did not detect any variation in the expression of qdr <dig>  pdr <dig>  gph <dig> and ymr135w associated with the pdr <dig> allelic form reported in the experiment by ambroset et al.
<cit> , probably because of the high fdr rate.
therefore, we replaced the pdr <dig> allele in strain 59a and we compared the expression of these four genes between the strains containing each of the two alleles of pdr <dig>  quantative pcr  indicated that only qdr <dig> was more strongly expressed in the strain carrying the s288c-pdr <dig> allele.
abz <dig> allelic variations affect production of 2-phenylethanol and ethyl esters by yeast during fermentation
the variations in the concentrations of 2-phenyl ethanol and of ethyl hexanoate esters were linked to another mqtl corresponding to a  <dig> kb region of chromosome xiv.
this region overlaps a region involved in differences in fermentation kinetics due to allelic variations of the abz <dig> gene  <cit> .
there are five non synonymous mutations between the s288c and 59a alleles of abz <dig>  we compared two reciprocal hemizygotes between strains s288c and 59a containing only one allele of each origin to confirm the role of this gene in 2-phenylethanol production.
the hemizygote which carried the enological allele of 59a, produced more 2-phenylethanol than the hemizygote which carried the s288c allele  .
the addition of  <dig> mg/l of p-aminobenzoic acid to the fermentation media suppressed the differences in the rates of fermentation of the two strains; it caused a reduction of only 15% of the difference in the production of 2-phenylethanol , but completely abolished the differences in 2-phenyl acetate production.
abz1p uses chorismate as a substrate, which is also one of the precursors of 2-phenylethanol synthesis.
we tested for the effects of the two alleles on the concentration of the various compounds analyzed during mqtl analysis .
we observed significant effects on the concentrations of many volatile compounds, including ethyl esters, confirming the involvement of abz <dig> in their variations.
these results also validate the use of abz <dig> as an additive covariable in the model used to search for mqtls.
compounds whose concentration varies significantly are given in bold.
mean of  <dig> triplicates +/- standard deviation.
plb <dig> allelic variations may affect ethyl ester production
a 60kb region of chromosome xiii was linked with variations in the production of ethyl esters, and we identified two candidate genes with two allelic forms in this region: plb <dig> and plb <dig>  these genes code for phospholipase b which displays transacylase activity in vitro <cit> .
plb1p in 59a presents some minor differences to that in s288c, whereas plb2p of s288c carried a p378a substitution with respect to that in 59a.
this proline residue is conserved in other saccharomyces species and the mutation was not found in other available s. cerevisiae genome sequences.
the Δplb2-by <dig> strain produced much less octanoic ethyl ester than the control , and the difference was greater than that associated with deletion of eeb <dig>  one of the key genes involved in the synthesis of decanoic ethyl ester  <cit> .
these findings are consistent with the involvement of plb <dig> in this phenotype.
deletion of plb <dig> also led to a decrease in decanoic ethyl ester production and an increase of decanoic acid production, which was not observed after the deletion of eeb <dig> <cit> .
.
polymorphism of the various genes and adaptation
the two major qtl detected in this study, pdr <dig> and abz <dig>  show substantial polymorphism with numerous differences between the allelic forms in s288c and 59a.
we investigated whether the differences between the alleles originated from the introgression from a specific lineage by comparing the corresponding alleles from other yeast genome sequences.
the phylogeny  reveals that the pdr <dig> allele of s288c is related to malaysian or asian alleles, and apparently one of the closest to its s. paradoxus ortholog; by contrast, the allele in 59a is a typical wine allele.
as polymorphism may also result from specific adaptation, we performed a mcdonald kreitman test  <cit> .
this test compares the ratio of nonsynonymous to synonymous polymorphism  to the ratio of nonsynonymous to synonymous divergence with the nearest species.
this ratio is called the neutrality index .
an ni lower than one reflects a paucity of nonsynonymous polymorphism relative to nonsynonymous divergence, and is indicative of positive selection; an ni greater than one indicates negative selection of deletorious alleles driving divergence between species or balancing selection.
this test was applied to a set of  <dig> pdr <dig> alleles from strains isolated from various substrates and ni was  <dig> , indicating a significant excess of non-neutral mutations .
this suggests that pdr <dig> is subject to the accumulation of slightly deleterious mutations that are eliminated by negative selection during speciation, or alternatively that pdr <dig> presents substantial diversity that might be associated with balanced selection resulting from specific adaptation to different niches.
in contrast with pdr <dig>  the overall phylogeny  revealed that the s288c abz <dig> sequence is related to copies from clinical isolate 322134s and bread strains ys <dig> and ys <dig>  however, the s288c allelic form of abz <dig> is located at the end of a long branch such that it appears to be the result of the accumulation of numerous mutations.
similarly, the mcdonald kreitman test with a set of  <dig> abz <dig> sequences from strains isolated from various substrates indicated an excess of non-neutral mutations .
discussion
we report  <dig> 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”  <cit> .
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  <cit> .
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  or variety aroma , and even antimicrobial and cancer chemopreventive properties  <cit> .
in yeast, these compounds are synthesized through the mevalonic acid pathway from acetyl-coa, which is converted to isopentenylpyrophosphate  and its isomer dimethylallyl pyrophosphate , 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  <cit>  and it has been shown recently that yeast can produce cis-rose oxide in wine  <cit> .
we demonstrate that the alleles of pdr <dig> found in s288c and 59a differently regulate the qdr <dig> 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  <cit> .
therefore, it is possible that the transporter qdr2p is responsible of the export of either farnesyl diphosphate or of nerolidol.
enological strains have a pdr <dig> allele more divergent from s. paradoxus than that of s288c.
the neutrality index  <cit>  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 ssu <dig> <cit> .
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 erg <dig> expression correlates with fermentation speed  <cit>  even in a fermentation medium containing ergosterol .
the synthesis of farnesol diphosphate is essential for  the synthesis of other compounds including dolichol which is necessary for cell wall assembly  <cit> ,  protein prenylation such as that of skt5p  <cit>  involved in chitin synthase activity, and  ubiquinone synthesis which may be less important during fermentation.
furthermore, farnesol is a biologically active compound that at concentrations higher than  <dig> μm inhibits s. cerevisiae and c. albicans growth  <cit>  and at lower concentrations is involved in quorum sensing by c. albicans <cit> .
in addition to the regions affecting terpenoid production, we also linked several other regions to variations in the concentrations of various volatiles.
the abz <dig> 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  <cit> .
the phylogeny of abz <dig> 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 .
again, the neutrality index  <cit>  calculated for this gene was significantly higher than  <dig>  possibly, the allelic form of abz <dig> has accumulated several deleterious mutations leading to a loss of activity.
this feature may explain the very particular phenotypic behavior observed for s288c  <cit> .
the two experimental sets we report generated complementary results.
we observed effects of pdr <dig> and abz <dig> allelic variations in both experiments.
nevertheless, the impact of abz <dig> 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 plb <dig> as possibly involved in the variations of ethyl ester content in experiment b. this gene may have a role complementary to eeb <dig> in the synthesis of ethyl esters during alcoholic fermentation.
CONCLUSIONS
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 plb <dig> gene in the metabolism of ethyl esters, and evidenced the role of pdr <dig> in the release of nerolidol into the media via the regulation of qdr <dig> 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 .
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 pdr <dig> 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.

 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 years numerous studies have illustrated the power of these genetic approaches, and in particular quantitative trait locus 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, and drug resistance. even more recently, this quantitative approach has been used to decipher complex traits at high resolution. it has also been applied successfully to the analysis of variations in gene expression. the qtl approach is now being used to study features important for the beverages industry, for example wine fermentation, sake technological traits and ethanol tolerance for ethanol production. all these studies have implicated defective alleles, of for example amn, asp or abz, in the diversity of the phenotypes of segregants. strain by possesses a defective allele of amn which leads to faster daughter cell separation; wine strain sb possesses a defective allele of the asp gene involved in asparagine catabolism; and s288c possesses a defective allele of abz 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 abz 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. 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 atf for acetates and eeb for ethyl esters, has been demonstrated. nevertheless, little is known about the factors explaining large strain-to-strain differences in the production of volatile compounds. 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 segregants arising from a cross between the laboratory strain s288c and 59a, a spore isolated from the industrial wine strain ec this population of segregants has been genotyped with affymetrix ygs 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 segregants was tested in two experimental designs. in the first design, 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. the fermentation kinetics of the segregants presented significant diversity, from typical wine fermentations to clearly sluggish as observed for s288c. unlike the parental strains, several segregants presented a clear ability to flocculate. this resulted in large and significant diversity in the concentrations of volatile compounds at the end of the alcoholic fermentations. we measured a set of compounds in experiment a, and compounds in experiment b. we performed a principal component analysis to reduce the multidimensional data set of experiment a into three more informative dimensions. the first three axes explained 51% of the global variance and in this analysis the various compounds are grouped according to chemical family. ethyl esters and medium chain fatty acids were correlated to the first axis, the various acetates correlated together with the second axis and 2-phenyl ethanol and isoamylalcohol were correlated to axis three. the representation of individual progeny strains in the factorial plan indicated a substantial diversity in the ability to produce volatile compounds. this was especially clear for acetates, as many strains were able to produce more acetates in the media than either of the two parents. in addition, some strains more than others metabolized geraniol into citronellol or into the high olfactive impact compound cis-rose oxide. this indicates that the characteristics of the yeast strain have a significant and variable impact on the grape aroma fraction. a similar picture was obtained from the analysis of experiment b. genetic analysis of the volatile compounds production from experiment a, heritability was estimated to be greater than 70% for of compounds, which included the grape aroma compounds geraniol, linalool, citronellol and geraniol acetate. to identify qtl for these technological features, we performed a linkage analysis with the previously reported genotypes for these progeny. the concentrations of most compounds obtained for the population of segregants did not follow normal distributions, so we performed linkage analysis with both parametric and non parametric models. we identified four and six regions involved in variations in the production of different compounds in experiments a and b, respectively. however, additional factors may contribute to wine aroma production: flocculation is one; and the presence of the abz1-s288c allele, which is responsible for large variations in fermentation kinetics, may also have an indirect effect. to overcome the potential effects of these factors, we performed a second linkage analysis taking these two factors into account as covariables in the model. this enabled us to improve the significance for some qtl detected after a simple scan, to detect a genetic effect for additional compounds of one region already found and to detect three and one additional genomic regions in experiments a and b, respectively, for other compounds. the effects of flocculation and of abz allele on aroma production for each qtl are given as additional file regions above the threshold are indicated in italics. only compounds with differences that are significant or close to the significance threshold are given. 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 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 explained between and 73% of the metabolite variation. one region on chromosome xii was identified in both experiments with high lod score values and explained as much as and 72% of the variations in the production of nerolidol. the same region was identified for other isoprenoids characterized only in design b. another region, on chromosome xiv, was also detected in both experiments and was associated with various phenotypes: ethyl hexanoate in experiment a and several compounds in experiment b. the abz gene which maps in this region has been reported to be responsible for variations in the rate of fermentation. when abz polymorphism was used as a covariable, we detected other qtl for more compounds indicating that abz allelic variations may affect the production of several metabolites. these mqtl are scattered through the genome of strain s288c such that we did not detect a major region associated with all the compounds analyzed. 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 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 pdr 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 kb containing orfs. nerolidol, farnesene and bisabolene are all derived from farnesyl diphosphate, an intermediate in isoprenoid and ergosterol biosynthesis: 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 pdr a transcription factor that modulates the expression of genes including transporters, a gene of the ergosterol biosynthesis pathway, and enzymes involved in oxido-reduction processes. this transcription factor was clearly a good candidate. the nucleotide sequences of the pdr 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 pdr 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 pdr allele of 59a leading to a lower production of nerolidol. these results are in agreement with those obtained for the whole population. however, the parental strain s288c produced less nerolidol than most of the segregants, indicating further interactions with the genetic background. characterization of pdr targets involved in the phenotype to identify which of the targets of pdr explain the observed variations in nerolidol production, we measured the production of nerolidol by the corresponding deletant strains in the by background. the deletion of yor led to an increase of nerolidol production whereas the deletion of four other pdr target genes led to decreases of nerolidol production similar to that observed after the deletion of pdr the genes qdr and pdr encode transporters that may be involved in the export of nerolidol or derived compounds from the cell. the deletion of two other genes, gph and ymr315w, resulted in a similar reductions in nerolidol production indicating other possible mechanisms. gph is a glycogen phosphorylase required for the mobilization of glycogen, and ymr315w is an oxidoreductase enzyme thay may be involved in the reduction of farnesol. erg encoding mevalonate phosphate kinase, is an essential gene for isoprenoid and ergosterol biosynthesis, so it was not possible to conduct the appropriate tests with the deleted haploid strain. deletion of only one copy of erg in the diploid strain by did not lead to any relevant change so we evaluated the effect of the overexpression of erg8: no significant increase of the production of nerolidol was detected. we did not detect any variation in the expression of qdr pdr gph and ymr135w associated with the pdr allelic form reported in the experiment by ambroset et al., probably because of the high fdr rate. therefore, we replaced the pdr allele in strain 59a and we compared the expression of these four genes between the strains containing each of the two alleles of pdr quantative pcr indicated that only qdr was more strongly expressed in the strain carrying the s288c-pdr allele. abz allelic variations affect production of 2-phenylethanol and ethyl esters by yeast during fermentation the variations in the concentrations of 2-phenyl ethanol and of ethyl hexanoate esters were linked to another mqtl corresponding to a kb region of chromosome xiv. this region overlaps a region involved in differences in fermentation kinetics due to allelic variations of the abz gene. there are five non synonymous mutations between the s288c and 59a alleles of abz we compared two reciprocal hemizygotes between strains s288c and 59a containing only one allele of each origin to confirm the role of this gene in 2-phenylethanol production. the hemizygote which carried the enological allele of 59a, produced more 2-phenylethanol than the hemizygote which carried the s288c allele. the addition of mg/l of p-aminobenzoic acid to the fermentation media suppressed the differences in the rates of fermentation of the two strains; it caused a reduction of only 15% of the difference in the production of 2-phenylethanol, but completely abolished the differences in 2-phenyl acetate production. abz1p uses chorismate as a substrate, which is also one of the precursors of 2-phenylethanol synthesis. we tested for the effects of the two alleles on the concentration of the various compounds analyzed during mqtl analysis. we observed significant effects on the concentrations of many volatile compounds, including ethyl esters, confirming the involvement of abz in their variations. these results also validate the use of abz as an additive covariable in the model used to search for mqtls. compounds whose concentration varies significantly are given in bold. mean of triplicates +/- standard deviation. plb allelic variations may affect ethyl ester production a 60kb region of chromosome xiii was linked with variations in the production of ethyl esters, and we identified two candidate genes with two allelic forms in this region: plb and plb these genes code for phospholipase b which displays transacylase activity in vitro. plb1p in 59a presents some minor differences to that in s288c, whereas plb2p of s288c carried a p378a substitution with respect to that in 59a. this proline residue is conserved in other saccharomyces species and the mutation was not found in other available s. cerevisiae genome sequences. the Δplb2-by strain produced much less octanoic ethyl ester than the control, and the difference was greater than that associated with deletion of eeb one of the key genes involved in the synthesis of decanoic ethyl ester. these findings are consistent with the involvement of plb in this phenotype. deletion of plb also led to a decrease in decanoic ethyl ester production and an increase of decanoic acid production, which was not observed after the deletion of eeb.. polymorphism of the various genes and adaptation the two major qtl detected in this study, pdr and abz show substantial polymorphism with numerous differences between the allelic forms in s288c and 59a. we investigated whether the differences between the alleles originated from the introgression from a specific lineage by comparing the corresponding alleles from other yeast genome sequences. the phylogeny reveals that the pdr allele of s288c is related to malaysian or asian alleles, and apparently one of the closest to its s. paradoxus ortholog; by contrast, the allele in 59a is a typical wine allele. as polymorphism may also result from specific adaptation, we performed a mcdonald kreitman test. this test compares the ratio of nonsynonymous to synonymous polymorphism to the ratio of nonsynonymous to synonymous divergence with the nearest species. this ratio is called the neutrality index. an ni lower than one reflects a paucity of nonsynonymous polymorphism relative to nonsynonymous divergence, and is indicative of positive selection; an ni greater than one indicates negative selection of deletorious alleles driving divergence between species or balancing selection. this test was applied to a set of pdr alleles from strains isolated from various substrates and ni was, indicating a significant excess of non-neutral mutations. this suggests that pdr is subject to the accumulation of slightly deleterious mutations that are eliminated by negative selection during speciation, or alternatively that pdr presents substantial diversity that might be associated with balanced selection resulting from specific adaptation to different niches. in contrast with pdr the overall phylogeny revealed that the s288c abz sequence is related to copies from clinical isolate 322134s and bread strains ys and ys however, the s288c allelic form of abz is located at the end of a long branch such that it appears to be the result of the accumulation of numerous mutations. similarly, the mcdonald kreitman test with a set of abz sequences from strains isolated from various substrates indicated an excess of non-neutral mutations. discussion we report 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. 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 or variety aroma, 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 and its isomer dimethylallyl pyrophosphate, 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 pdr found in s288c and 59a differently regulate the qdr 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 pdr 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 ssu. 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 erg expression correlates with fermentation speed even in a fermentation medium containing ergosterol. the synthesis of farnesol diphosphate is essential for the synthesis of other compounds including dolichol which is necessary for cell wall assembly, protein prenylation such as that of skt5p involved in chitin synthase activity, and ubiquinone synthesis which may be less important during fermentation. furthermore, farnesol is a biologically active compound that at concentrations higher than μm inhibits s. cerevisiae and c. albicans growth and at lower concentrations is involved in quorum sensing by c. albicans. in addition to the regions affecting terpenoid production, we also linked several other regions to variations in the concentrations of various volatiles. the abz 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 abz 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. again, the neutrality index calculated for this gene was significantly higher than possibly, the allelic form of abz 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 pdr and abz allelic variations in both experiments. nevertheless, the impact of abz 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 plb as possibly involved in the variations of ethyl ester content in experiment b. this gene may have a role complementary to eeb 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 plb gene in the metabolism of ethyl esters, and evidenced the role of pdr in the release of nerolidol into the media via the regulation of qdr 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. 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 pdr 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. 
we detected eight genomic regions explaining the diversity concerning 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. deletion of a plb gene confirmed its involvement in the production of ethyl esters. saccharomyces cerevisiaeqtl mappingwine aromacitronellolrose oxidenerolidolfarneseneethyl esters2-phenyl ethanolpdr8abz1plb2qdr2 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. as a tool to decipher how wine aroma develops, we analyzed, under two experimental conditions, the production of compounds by a population of segregants from a cross between a laboratory strain and an industrial strain genotyped at high density. 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 pdr gene in the production of farnesyldiphosphate derivatives, of abz in the production of numerous compounds and of plb in ethyl ester synthesis. 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. this work also provides a basis for elucidating the metabolism of various grape compounds, such as citronellol and cis-rose oxide. hemizygote comparison allowed the attribution of differences in the production of nerolidol and 2-phenyl ethanol to the pdr and abz genes, respectively.
 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 compounds by a population of 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 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 pdr and abz genes, respectively. deletion of a plb gene confirmed its involvement in the production of ethyl esters. a comparison of allelic variants of pdr and abz 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 pdr gene in the production of farnesyldiphosphate derivatives, of abz in the production of numerous compounds and of plb 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. saccharomyces cerevisiaeqtl mappingwine aromacitronellolrose oxidenerolidolfarneseneethyl esters2-phenyl ethanolpdr8abz1plb2qdr2 