Hybridization within Saccharomyces Genus Results in Homoeostasis and Phenotypic Novelty in

24 Pages·2015·3.17 MB·English

RESEARCHARTICLE
Saccharomyces
Hybridization within Genus
Results in Homoeostasis and Phenotypic
Novelty in Winemaking Conditions
TelmadaSilva1☯,WarrenAlbertin2,3☯,ChristineDillmann4,MarinaBely3,Stéphanela
Guerche5,ChristopheGiraud6,SylvieHuet7,DelphineSicard4,IsabelleMasneuf-
Pomarede3,8,DominiquedeVienne4,PhilippeMarullo3,9*
1 INRA,UMR0320/UMR8120GénétiqueVégétale,Gif-sur-Yvette,France,2 ENSCBP—BordeauxINP,
Pessac,France,3 UniversitédeBordeaux,ISVV,EA4577,UnitéderechercheŒnologie,Villenave
d'Ornon,France,4 UniversitéParis-Sud,UMR0320/UMR8120GénétiqueVégétale,Gif-sur-Yvette,
France,5 SARCO,Bordeaux,France,6 CMAPEcolePolytechnique,Palaiseau,France,7 INRA,MIA,
Jouy-en-Josas,France,8 BordeauxSciencesAgro,Gradignan,France,9 Biolaffort,Bordeaux,France
☯Theseauthorscontributedequallytothiswork.
* [email protected]
OPENACCESS Abstract
Citation:daSilvaT,AlbertinW,DillmannC,BelyM,
laGuercheS,GiraudC,etal.(2015)Hybridization Despiteitsbiotechnologicalinterest,hybridization,whichcanresultinhybridvigor,hasnot
withinSaccharomycesGenusResultsin commonlybeenstudiedorexploitedintheyeastgenus.Fromadialleldesignincluding55
HomoeostasisandPhenotypicNoveltyin
intra-andinterspecifichybridsbetweenSaccharomycescerevisiaeandS.uvarumgrownat
WinemakingConditions.PLoSONE10(5):
e0123834.doi:10.1371/journal.pone.0123834 twotemperaturesinenologicalconditions,weanalyzedasmanyas35fermentationtraits
withoriginalstatisticalandmodelingtools.Wefirstshowedthat,dependingonthetypesof
AcademicEditor:JosephSchacherer,Universityof
Strasbourg,FRANCE trait–kineticsparameters,life-historytraits,enologicalparametersandaromas–,the
sourcesofvariation(strain,temperatureandstrain*temperatureeffects)differedinalarge
Received:November24,2014
extent.Thenwecomparedgloballythreegroupsofhybridsandtheirparentsattwogrowth
Accepted:February21,2015
temperatures:intraspecifichybridsS.cerevisiae*S.cerevisiae,intraspecifichybridsS.
Published:May6,2015 uvarum*S.uvarumandinterspecifichybridsS.cerevisiae*S.uvarum.Wefoundthathy-
Copyright:©2015daSilvaetal.Thisisanopen bridizationcouldgeneratemulti-traitphenotypeswithimprovedoenologicalperformances
accessarticledistributedunderthetermsofthe andbetterhomeostasiswithrespecttotemperature.Theseresultscouldexplainwhyinter-
CreativeCommonsAttributionLicense,whichpermits
specifichybridizationissocommoninnaturalanddomesticatedyeast,andopenthewayto
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare applicationsforwine-making.
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Funding:SARCOandBIOLAFFORTprovided
supportintheformofsalariesforauthors(PM,SlG), Introduction
butdidnothaveanyadditionalroleinthestudy
Planthybridscommonlyharbornon-additiveinheritanceforpolygenictraits,withphenotypic
design,datacollectionandanalysis,decisionto
valuesusuallydifferentfromthemeanparentalvalues.These“monsters”producedbyhybrid-
publish,orpreparationofthemanuscript.Thespecific
rolesoftheseauthorsarearticulatedinthe‘author ization[1]haveevolutionaryimplications[2,3]andareextensivelyexploitedforproducing
contributions’section. improvedcrops.Forinstanceinmaize,F1hybridsbetweenhomozygouslinesshowheterosis
of100to400%forgrainyield[4,5],andmanyothercomplextraits,suchasheight,leafarea,
CompetingInterests:Theauthorshavethe
followinginterests.StaphanelaGuercheandPhilippe grainsize,germinationrate,rootgrowthandrootnitrogenuptake,alsodisplayheterosis[6,7].
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 1/24
HybridizationEffectsinYeast
MarulloareemployedbySARCOandBIOLAFFORT, Hybridizationaffectsnotonlythephenotypicvalues,butalsotheirstabilityoverenviron-
respectively.Thisdoesnotaltertheauthors' mentalchanges.Homeostasis,canalizationorrobustness—thetermdependsonthebiological
adherencetoallthePLOSONEpoliciesonsharing field[8–11]allowstheorganismstobuffertheeffectsofexternalperturbationsthroughmeta-
dataandmaterials.
bolic,physiologicalordevelopmentaladjustments,andthustomaintainfitnessindiversehabi-
tats.Homeostasisisusuallyhigherinintra-orinterspecifichybridsthanintheirparents,as
shownforinstanceforyield,tolerancetosoilacidityandtosoilmoisturestressinmaize[12]
ormorphometrictraitsinmice[13].
Consequencesofhybridizationhavebeenstudiedinalargerangeofwildaswellasdomesti-
catedspecies,buthavescarcelybeenstudiedandexploitedinindustrialeukaryoticmicro-or-
ganismssuchasyeast.Recentlysomeauthorsinvestigatedtheheterosisphenomenonwithin
naturalanddomesticatedstrainsofSaccharomycescerevisiae[14–17],butthesestudieswere
mainlyfocusedoncellgrowthinlaboratoryconditions.
IntheSaccharomycessensustrictoclade,yeastspeciesshowedaseverereproductiveisola-
tion(lessthan1%ofviablespores)[18].However,theprezygoticbarriercanbeeasilybypassed
leadingtoviableinterspecifichybrids[19].NumerousinterspecifichybridsbetweenS.cerevi-
siaeandpsychrophilicspeciesS.uvarumorS.kudriavzeviihavebeenisolatedinwineandnat-
uralenvironment[20–28].Thesenaturalhybridshavetechnologicalpropertiesdifferingfrom
thoseoftheirrespective“parental”species,withsometimesbetterrobustness[29–32].More-
oversomewinestartersempiricallyselectedprovedtobeinterspecifichybrids[25,33,34],pro-
motingtheideathatinterspecifichybridizationisagoodwayforobtainingvaluablestrainsfor
winefermentation.Howeverinthepreviousworkstheparentalstrainsofthehybridswerenot
known,soitwasnotpossibletostatedefinitelythatinterspecifichybridizationconferrednovel
phenotypesandpossiblybetterhomeostasis.Moreover,sincetheirgenesis,thesenaturalhy-
bridsmayhaveundergonegenomicmodificationsthatcandrasticallyaffecttheirphenotype
suchgrosschromosomalrearrangement[35,36],lossofheterozygosity[23,37],particular
mitotypes[38],aneuploidies[37]andintrogressions[39].
Inordertoassessrigorouslythephenotypicimpactofintra-andinterspecifichybridization,
thehybridsmustbecomparedtotheirparentalstrains.Aspreviouslyreviewed[19,40]various
laboratorieshaveproducedsuchhybridsbetweenSaccharomycesspecies[27,41–43].However,
onlyafewinterspecifichybridswerecomparedtotheirparents,andforquiteasmallnumber
oftraits[44–46].AstheSaccharomycesstrainsharborhugegeneticandphenotypicdiversity
[47,48],thebehavioroffewhybridsisnotsufficienttohaveanoverallviewontheeffects
ofhybridization.
InthisworkweexaminedtheextenttowhichhybridizationwithinandbetweenSaccharo-
mycesspeciesmodifiedalargeseriesoftraitsmeasuredduringandattheendoffermentation
attwotemperatures,withparticularattentiontohomeostasis.WefocusedonS.cerevisiaeand
S.uvarum(formerlyS.bayanusvar.uvarum)[28,49,50],tworelatedspeciesnaturallyassoci-
atedwithwinefermentations[51–53].S.cerevisiaeisthemainyeastabletoachievegrapemust
fermentation,butS.uvarumcandisplaysimilarfermentationperformance,particularlyatlow
temperature[40,54–56].Althoughthesesisterspeciessharelargesynteny[57,58],theydiffer
forseveraltechnologicaltraits.First,S.cerevisiaehasahigherresistancetohightemperature
stress(upto37°C)[30]whileS.uvarumismoretoleranttolowtemperatures[59].Second,S.
uvarumexhibitsaspecificaromaticprofilebyproducinghigheramountsofphenyl-2-ethanol
andphenyl-2-ethanolacetatethanS.cerevisiaestrains[31,41,44].Finally,althoughS.uvarum
harborsahighethanolresistance(upto15%[41]),itislessresistantthanS.cerevisiae[60].Sev-
eralnaturalhybridsbetweenthesetwospecieshavebeendescribed[26,30,61],andthepossi-
bilitytoproducesyntheticinter-specifichybrids[62]establishedS.cerevisiaeandS.uvarumas
modelsystemsforhybridizationstudies.
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 2/24
HybridizationEffectsinYeast
Measuringanunprecedentednumberoftraits,weinvestigatedthephysiologicalandtech-
nologicalpropertiesofacollectionoffourS.uvarumandsevenS.cerevisiaeparentalstrains
andtheir55possiblehybrids,namely27intraspecifichybridsand28inter-specifichybrids,
underwinemakingconditionsattwotemperatures.Weanalyzedthesourcesofphenotypic
variation—geneticand/orenvironmental—forvariouscategoriesoftraits(fermentationkinet-
ics,life-history,winecompositionandorganolepticquality),wecomparedtheintra-andinter-
specifichybridsandassessedtheextenttowhichhybridizationincreasedhomeostasisata
multi-traitlevel.
MaterialsandMethods
Parentalstrainsandcultureconditions
ThestartinggeneticmaterialoftheexperimentaldesignweresevenS.cerevisiaestrainsand
fourS.uvarumstrains,associatedtovariousfood-processes(enology,brewery,ciderfermenta-
tionanddistillery)orisolatedfromnaturalenvironment(oakexudates)(Table1).These
strainscouldnotbeusedassuchasparentsofadialleldesignbecausetheyweresuspectedto
beheterozygousatmanyloci.Monosporiccloneswereisolatedbytetraddissectionusinga
micromanipulator(SingerMSMManual;SingerInstrument,Somerset,UnitedKingdom).All
originalstrainsbutAlcotec24werehomothallic(HO/HO),thereforefullyhomozygousdiploid
strainswerespontaneouslyobtainedbyfusionofoppositematingtypecells.ForA24(ho/ho),
oneisolatedhaploidmeiosporewasdiploidizedviatransientexpressionoftheHOendonucle-
ase[63].Thesestrains,calledW1,D1,D2,E2,E3,E4andE5forS.cerevisiaeandU1,U2,U3
andU4forS.uvarum,wereusedastheparentalstrainsfortheconstructionofahalfdiallelde-
sign(Fig1).
Allstrainsweregrownat24°CinYPDmediumcontaining1%yeastextract(DifcoLabora-
tories,Detroit,MI),1%Bactopeptone(Difco),and6%glucose,supplementedornotwith2%
agar.Whennecessary,antibioticswereaddedatthefollowingconcentrations:100μg/mLfor
G418(Sigma,l’Isled’AlbeauFrance)ornourseothricin(WernerBioagent,Jena,Germany),
and300μg/mLforhygromycinB(Sigma).
Constructionofthedialleldesign
Inordertoproduceinterspecifichybrids,theelevendiploidparentalstrainsweretransformed
withacassettecontainingtheHOalleledisruptedbyageneofresistancetoeitherG418(ho::
KanR),hygromycinB(ho::HygR)ornourseothricin(ho::NatR)aspreviouslydescribed[62].
Aftertransformation,monosporiccloneswereisolated,andthemating-type(MATaor
MATα)ofantibiotic-resistantcloneswasdeterminedusingtestersofknownmating-type.
Straintransformationallowed(i)conversiontoheterothallismforthehomothallicstrains(all
butD2,seeTable1)and(ii)antibioticresistanceallowingeasyhybridselection.
Foreachhybridconstruction,parentalstrainsofoppositematingtypewereputincontact
for2to6hoursinYPDmediumatroomtemperature,andthenplatedonYPD-agarcontain-
ingtheappropriateantibiotics.The55possiblehybridsfromthe11parentalstrains,namely21
S.cerevisiaeintraspecifichybrids,6S.uvarumintraspecifichybridsand28interspecifichy-
brids,wereobtained.Foreachcross,afewindependentcolonieswerecollected.Afterrecurrent
culturesonYPD-agarcorrespondingto~80generations,thenuclearchromosomalstabilityof
thehybridswascontrolledbypulsedfieldelectrophoresis(CHEF-DRIII,Biorad,CA)aswellas
homoplasmy(onlyoneparentalmitochondrialgenome)asdetailedinAlbertinetal.[62].
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 3/24
HybridizationEffectsinYeast
Table1. Yeaststrainsusedinthisstudy.
Strain Genotype Ploidy Collection/supplier Origin Reference
Originalstrains
YSP128 HO/HO(S.cerevisiae) diploid SGRP ForestOakexudate,Pennsylvania,USA [64]
Alcotec24 ho/ho(S.cerevisiae) diploid HambletonBard Distillery,UK [65]
CLIB-294 HO/HO(S.cerevisiae) diploid CIRM-Levures Distillery,Cognac,France [65]
VL1 HO/HO(S.cerevisiae) diploid LaffortŒnologie Enology,Bordeaux,France [66]
F10 HO/HO(S.cerevisiae) diploid LaffortŒnologie Enology,Bordeaux,France [67]
VL3c HO/HO(S.cerevisiae) diploid LaffortŒnologie Enology,Bordeaux,France [68]
BO213 HO/HO(S.cerevisiae) diploid LaffortŒnologie Enology,Bordeaux,France [66]
PM12 HO/HO(S.uvarum) diploid EAOENOLOGY Grapemustfermentation,Jurançon,France [69]
PJP3 HO/HO(S.uvarum) diploid EAOENOLOGY Grapemustfermentation,Sancerre,France [69]
Br6.2 HO/HO(S.uvarum) diploid ADRIANormandie Ciderfermentation,Normandie,France [62]
RC4-15 HO/HO(S.uvarum) diploid EAOENOLOGY Grapemustfermentation,Alsace,France [69]
Homozygousdiploidparentalstrains
W1 DerivedfromYSP128,HO/HO diploid EAOENOLOGY [54]
D2 DerivedfromAlcotec24,ho/ho diploid EAOENOLOGY [65]
D1 DerivedfromCLIB-294,HO/HO diploid EAOENOLOGY [65]
E3 DerivedfromVL1,HO/HO diploid EAOENOLOGY [65]
E4 DerivedfromF10,HO/HO diploid EAOENOLOGY [65]
E5 DerivedfromVL3c,HO/HO diploid EAOENOLOGY [54]
E2 DerivedfromBO213,HO/HO diploid EAOENOLOGY [67]
U1 DerivedfromPM12,HO/HO diploid EAOENOLOGY [54]
U2 DerivedfromPJP3,HO/HO diploid EAOENOLOGY [54]
U3 DerivedfromBr6.2,HO/HO diploid EAOENOLOGY [54]
U4 DerivedfromRC4-15,HO/HO diploid EAOENOLOGY thiswork
Monosporicclonesusedforcrosses
D1-HYG-1A ho::hygR,MATa haploid EAOENOLOGY thiswork
D1-HYG-4C ho::hygR,MATalpha haploid EAOENOLOGY thiswork
D2-3A-HYG ho::hygR,MATalpha haploid EAOENOLOGY [62]
E2-KAN-4A ho::kanR,MATalpha haploid EAOENOLOGY thiswork
E2-KAN-4D ho::kanR,MATa haploid EAOENOLOGY thiswork
E3-NAT-1C ho::natR,MATa haploid EAOENOLOGY thiswork
E3-NAT-2C ho::natR,MATalpha haploid EAOENOLOGY thiswork
E3-KAN-1B ho::kanR,MATa haploid EAOENOLOGY thiswork
E4-NAT-3A ho::natR,MATalpha haploid EAOENOLOGY thiswork
E4-NAT-3B ho::natR,MATa haploid EAOENOLOGY thiswork
E5-KAN-1C ho::kanR,MATa haploid EAOENOLOGY thiswork
E5-HYG-5B ho::hygR,MATalpha haploid EAOENOLOGY thiswork
E5-HYG-5D ho::hygR,MATa haploid EAOENOLOGY thiswork
W1-NAT-1B ho::natR,MATa haploid EAOENOLOGY [62]
W1-NAT-1C ho::natR,MATalpha haploid EAOENOLOGY thiswork
U1-KAN-4A Suho::kanR,MATalpha haploid EAOENOLOGY thiswork
U1-KAN-5D Suho::kanR,MATa haploid EAOENOLOGY thiswork
U2-KAN-2A Suho::kanR,MATa haploid EAOENOLOGY thiswork
U2-KAN-3B Suho::kanR,MATalpha haploid EAOENOLOGY [62]
U3-KAN-3A Suho::kanR,MATa haploid EAOENOLOGY [62]
U3-KAN-3B Suho::kanR,MATalpha haploid EAOENOLOGY thiswork
U4-KAN-2C Suho::kanR,MATa haploid EAOENOLOGY thiswork
(Continued)
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 4/24
HybridizationEffectsinYeast
Table1. (Continued)
Strain Genotype Ploidy Collection/supplier Origin Reference
U4-KAN-2B Suho::kanR,MATalpha haploid EAOENOLOGY thiswork
Hybridsofthedialleldesign
DD12 D1-HYG-1A*D2-3A-HYG diploid EAOENOLOGY thiswork
DE12 D1-HYG-1A*E2-KAN-4A diploid EAOENOLOGY thiswork
DE13 D1-HYG-1A*E3-NAT-2C diploid EAOENOLOGY thiswork
DE14 D1-HYG-1A*E4-NAT-3A diploid EAOENOLOGY thiswork
DE15 D1-HYG-4C*E5-KAN-1C diploid EAOENOLOGY thiswork
DE22 D2-3A-HYG*E2-KAN-4D diploid EAOENOLOGY thiswork
DE23 D2-3A-HYG*E3-NAT-1C diploid EAOENOLOGY thiswork
DE24 D2-3A-HYG*E4-NAT-3B diploid EAOENOLOGY thiswork
DE25 D2-3A-HYG*E5-KAN-1C diploid EAOENOLOGY thiswork
DU11 D1-HYG-1A*U1-KAN-4A diploid EAOENOLOGY thiswork
DU12 D1-HYG-1A*U2-KAN-3B diploid EAOENOLOGY thiswork
DU13 D1-HYG-1A*U3-KAN-3B diploid EAOENOLOGY thiswork
DU14 D1-HYG-1A*U4-KAN-2B diploid EAOENOLOGY thiswork
DU21 D2-3A-HYG*U1-KAN-5D diploid EAOENOLOGY thiswork
DU22 D2-3A-HYG*U2-KAN-2A diploid EAOENOLOGY thiswork
DU23 D2-3A-HYG*U3-KAN-3A diploid EAOENOLOGY thiswork
DU24 D2-3A-HYG*U4-KAN-2C diploid EAOENOLOGY thiswork
DW11 D1-HYG-1A*W1-NAT-1C diploid EAOENOLOGY thiswork
DW21 D2-3A-HYG*W1-NAT-1B diploid EAOENOLOGY thiswork
EE23 E2-KAN-4A*E3-NAT-1C diploid EAOENOLOGY thiswork
EE24 E2-KAN-4D*E4-NAT-3A diploid EAOENOLOGY thiswork
EE25 E2-KAN-4A*E5-HYG-5D diploid EAOENOLOGY thiswork
EE34 E3-KAN-1B*E4-NAT-3A diploid EAOENOLOGY thiswork
EE35 E3-NAT-2C*E5-KAN-1C diploid EAOENOLOGY thiswork
EE45 E4-NAT-3A*E5-KAN-1C diploid EAOENOLOGY thiswork
EU21 E2-KAN-4A*U1-KAN-5D diploid EAOENOLOGY thiswork
EU22 E2-KAN-4A*U2-KAN-2A diploid EAOENOLOGY thiswork
EU23 E2-KAN-4A*U3-KAN-3A diploid EAOENOLOGY thiswork
EU24 E2-KAN-4A*U4-KAN-2C diploid EAOENOLOGY thiswork
EU31 E3-NAT-1C*U1-KAN-4A diploid EAOENOLOGY thiswork
EU32 E3-NAT-1C*U2-KAN-3B diploid EAOENOLOGY thiswork
EU33 E3-NAT-1C*U3-KAN-3B diploid EAOENOLOGY thiswork
EU34 E3-NAT-1C*U4-KAN-2B diploid EAOENOLOGY thiswork
EU41 E4-NAT-3B*U1-KAN-4A diploid EAOENOLOGY thiswork
EU42 E4-NAT-3B*U2-KAN-3B diploid EAOENOLOGY thiswork
EU43 E4-NAT-3B*U3-KAN-3B diploid EAOENOLOGY thiswork
EU44 E4-NAT-3B*U4-KAN-2B diploid EAOENOLOGY thiswork
EU51 E5-HYG-5D*U1-KAN-4A diploid EAOENOLOGY thiswork
EU52 E5-HYG-5D*U2-KAN-3B diploid EAOENOLOGY thiswork
EU53 E5-HYG-5D*U3-KAN-3B diploid EAOENOLOGY thiswork
EU54 E5-HYG-5D*U4-KAN-2B diploid EAOENOLOGY thiswork
EW21 E2-KAN-4A*W1-NAT-1B diploid EAOENOLOGY thiswork
EW31 E3-KAN-1B*W1-NAT-1C diploid EAOENOLOGY thiswork
EW41 E4-NAT-3A*W1-NAT-1B diploid EAOENOLOGY thiswork
EW51 E5-HYG-5B*W1-NAT-1B diploid EAOENOLOGY thiswork
(Continued)
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 5/24
HybridizationEffectsinYeast
Table1. (Continued)
Strain Genotype Ploidy Collection/supplier Origin Reference
UU12 U1-KAN-4A*U2-KAN-2A diploid EAOENOLOGY thiswork
UU13 U1-KAN-4A*U3-KAN-3A diploid EAOENOLOGY thiswork
UU14 U1-KAN-4A*U4-KAN-2C diploid EAOENOLOGY thiswork
UU23 U2-KAN-3B*U3-KAN-3A diploid EAOENOLOGY thiswork
UU24 U2-KAN-2A*U4-KAN-2B diploid EAOENOLOGY thiswork
UU34 U3-KAN-3B*U4-KAN-2C diploid EAOENOLOGY thiswork
WU11 W1-NAT-1B*U1-KAN-4A diploid EAOENOLOGY thiswork
WU12 W1-NAT-1B*U2-KAN-3B diploid EAOENOLOGY thiswork
WU13 W1-NAT-1B*U3-KAN-3B diploid EAOENOLOGY thiswork
WU14 W1-NAT-1B*U4-KAN-2B diploid EAOENOLOGY thiswork
doi:10.1371/journal.pone.0123834.t001
Yeaststraincharacterization
Inordertodiscriminaterapidlythehybridsandparentalstrains,weusedtwopolymorphicmi-
crosatellitesspecifictoS.cerevisiae(Sc-YFR038,Sc-YML091)[70]andtwospecifictoS.
uvarum(locus4and9)[69].ThesefourmarkerswereamplifiedinamultiplexPCRreaction
Fig1.Experimentaldesign.Fullyhomozygousdiploidstrainswereusedasparentalstrains.W1,D1,D2,E2,E3,E4andE5areS.cerevisiaestrains.U1,
U2,U3andU4areS.uvarumstrains.FermentationswerecarriedoutinSauvignonblancgrapejuiceandrunattwotemperatures18°Cand26°Cintriplicate
foratotalof396experiments.Thirty-fivetraitswerecollectedandgroupedintofourclasses(FermentationKineticsTraits,LifeHistoryTraits,Basic
EnologicalParametersandAromaticTraits).Fruits,plantandsugarimageswerereprintedfromCCBYlicenses,thecopyrightholdersbeingSteveHopson,
2006(bananas);UweHermann,2007(sugarcubes);SharonMollerus,2007(strawberries);StenPorse,2005(boxwood);Lebensmittelfotos,2013(grapes).
doi:10.1371/journal.pone.0123834.g001
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 6/24
HybridizationEffectsinYeast
withthelabeledprimers(S1Table).ThePCRwascarriedoutinafinalvolumeof8μLusing
thefollowingprogram:95°Cfor5minforinitialdenaturationstep;95°Cfor30s,55°Cfor90s
and72°Cfor60srepeated35times;afinalelongationstepof30minat60°C.ThePCRprod-
uctswereanalyzedonanABI3730apparatus(AppliedBiosystem,Villebon-sur-Yvette,France)
bythegenotypingfacilitiesofBordeauxUniversity.Microsatellitelengthswereanalyzedusing
thePeakScannertool(AppliedBiosystem,Villebon-sur-Yvette,France)
Alcoholicfermentationexperiments
Experimentaldesign. The66strains(11parentaland55hybrids)weregrowninthree
replicatesattwotemperatures,26°Cand18°C.The396fermentations(66strainsx2tempera-
turesx3replicates)wereperformedfollowingarandomizedexperimentaldesign.Thedesign
wasimplementedconsideringablockastwosetsof27fermentations(26fermentationsanda
controlwithoutyeasttocheckforcontamination),onecarriedoutat26°Candtheotherat
18°C.Thedistributionofthestrainswithintheblockswasrandomizedtominimizetheresidu-
alvarianceoftheestimatorsoftheStrainandTemperatureeffects.
Grapemustandfermentationconditions. WhitegrapemustwasobtainedfromSauvi-
gnonblancgrapes,harvestedinvineyardsinBordeauxarea(2009vintage).Thisgrapejuice
wasobtainedbyDucourtVignoble18LeHourc,33760Ladauxthatgivestheautorizationto
usethismaterialforthestudy.Theharvestwasrealizedbytheownerofthefieldanddidnot
involveendangeredorprotectedspecies.Tartaricacidprecipitationwasstabilizedandturbidity
wasadjustedto100NTU(NephelometricTurbidityUnit)beforestorageat–20°C.Grapejuice
hadasugarconcentrationof189g.L–1,apHof3.3andanassimilablenitrogencontentof
242mgN.L–1.TheindigenousyeastpopulationwasestimatedbyYPD-platecountingafter
mustthawingandwasconsistentlylowerthan20CFU(Colony-FormingUnit)permL.
Yeastpre-cultures(20mL)wereruninhalf-dilutedmustfilteredthrougha0.45μmnitrate-
cellulosemembrane,during24h,at24°Cwithorbitalagitation(150rpm).Cellconcentration
wasquantifiedusingaflowcytometer(seebelow)andgrapemustwasinoculatedat106viable
cellspermL.Fermentationswererunin125mLglass-reactors,lockedtomaintainanaerobio-
sis,withpermanentstirring(300rpm)at18°Cor26°C.Yeaststrainimplantationingrape
mustwascheckedwhenthestationaryphasewasreached(40%ofalcoholicfermentation).The
DNAoffermentingyeastwasextractedusingFTAclonesavercards(Whatman,France),and
strainidentitywascontrolledbymicrosatelliteanalysis.
Flowcytometryanalysis. Themonitoringofpopulationgrowth,cellsizeandviabilitywas
performedusingacytometerFC500MPL(BeckmanCoulter,Villepinte,France).Collected
sampleswerefilteredbeforeflowcytometryanalysiswith10μmdisposablefilters,CellTric
(Partec,Münster,Germany).SamplesweredilutedwithMcIlvainebufferpH4(0.1Mcitric
acid,0.2Msodiumphosphatedibasic)addedwithpropidiumiodide(0.3%v/v)inorderto
staindeadcells,anddilutionwasadaptedtoreachaflowratelowerthan2500particules/sec.
Fluorescentbeads(CellCounter,BeckmanCoulter)wereusedtonormalizethequantification
ofcellularconcentration.
Multi-traitphenotypinginwinemakingconditions
Foreachalcoholicfermentation,foursetsofexperimentaldatawereobtained:fermentationki-
neticsparameters(FK),life-historytraits(LHT),basicenologicalparameters(BEP)andaro-
matictraits(AT).
Fermentationkineticsparameters(FK). TheamountofCO releasedwasmonitored
2
dailybytheweightlossofthebioreactors.
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 7/24
HybridizationEffectsinYeast
TheamountofCO released(Y )forthefermentationiattimetwasmodeledbyaWeibull
2 it
functionfasdescribedinS1SupportingInformation.Sevenkineticsparameterswerecomput-
edfromthemodel(S1Fig):
-t-lag(h)=^t0,thefermentationlag-phasethatisthetimebetweeninoculationandthebegin-
ningofCO release(whentheCO productionratewashigherthan0.05g.L–1.h–1);
2 2
-t-V (h),thetimetoreachtheinflexionpoint,outofthefermentationlagphase;
max
-t-45(h),thefermentationtimeatwhich45g.L–1ofCO wasreleased,outofthefermentation
2
lagphase;
-t-75(h),thefermentationtimeatwhich75g.L–1ofCO wasreleased,outofthefermentation
2
lagphase;
-AFtime(AlcoholicFermentationtime,h),thetimebetweent-lagandthetimeatwhichthe
CO emissionratebecamelessthan,orequalto,0.05g.L–1.h–1;
2
-V (g.L–1.h–1),thevalueofthefirstderivativeoftheWeibullfunctionf,att-Vmax(h),(f’=
max
αdb(cid:1)(t-Vmax—t0)b–1(cid:1)exp[–α(t-Vmax—t0)b]),andcorrespondedtothemaximumCO2re-
leasedrate;
-CO (g.L–1)=d^thetotalamountofCO releasedattheendofthefermentation.
2max 2
Life-historytraits(LHT). Duringthealcoholicfermentation,cellsamplesweretakenand
analyzedasdescribedinthe“flowcytometryanalysis”session.Theexperimentalmeasurement
ofthelogarithmofcellconcentrationwasmodeledbyadiscontinuousfunctionoftimeasde-
scribedinS2SupportingInformation.Thecellsizeandviabilityweremodeledusingalinear
model(W )asdescribedinS3SupportingInformation.
it
Thesemodelsallowedcomputingeightlife-historytraits(S1Fig).
-t-N (h)=^tN,thegrowthlag-phase(timebetweeninoculationandthebeginningofpopula-
0
tiongrowth);
-t-Nmax(h)=^tNmaxthetimeatwhichthecarryingcapacityKwasreached;
-r(logarithmofthenumberofcelldivisionspermLperhour),theintrinsicgrowthrate;
-K(log[cells/mL]),wasthecarryingcapacitycomputedas:K=I+r(t-N –t-lag)+C.
max
-J (g.h–1.10–8cell–1)wasthemaximumvalueoftheestimatedCO productionratedivided
max 2
bytheestimatedcellconcentration.
-Size-t-Nmax(μm),theaveragecellsizeatt-N
max
-Viability.t-N (%),thepercentageoflivingcellsatt-N
max max
-Viability.t-75(%),thepercentageoflivingcellsatt-75.
BasicEnologicalParameters(BEP). Attheendofthefermentation,sixBasicEnological
Parameters(BEP)werequantified:ResidualSugar(g.L–1),Ethanol(%vol),Sugar/EthanolYield
(g.L–1.%vol–1)(ratiobetweentheamountofmetabolizedsugarandtheamountofreleased
ethanol),Aceticacid(g.L–1H SO ),TotalSO andFreeSO (mg.L–1).ResidualSugarandEtha-
2 4 2 2
nolweremeasuredbyinfraredreflectanceusinganInfra-Analyzer450(Technicon,Plaisir,
France).Forsomestrains,ResidualSugarwasbelowthethresholdofdetection.Inthese
cases,insteadofinferringthevalue“0”,whichisnotbiologicallyrealistic,weusedthevalue:
(x/1.05)+y,wherexisthelowestvaluemeasuredinthewholedataandyisavaluedrawnina
uniformdistribution~U(0,0.001).Aceticacidwasquantifiedbycolorimetry(A460)in
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 8/24
HybridizationEffectsinYeast
continuousflux(Sanimat,Montauban,France).TotalSO andFreeSO wereassayedbyPara-
2 2
rosanilinetitration[71].
AromaticTraits(AT). Thearomaticprofileoffermentingyeastwasestimatedbyquanti-
fying14aromatictraits(AT).Themainvolatilecompoundsweremeasuredattheendofthe
alcoholicfermentationbyGC-MS.Foresters,higheralcoholsandvolatileacids,HSSEHead-
SpaceSorptiveExtractionfollowedbyGC-MSanalysiswasusedaccordingtoWeldegergis
etal.[72].Forvolatilethiols,aspecificextractionwasperformedaccordingtoTominagaetal.
[73].Theseanalyticalmethodsallowedustodetectupto22compoundsintheanalyzedsam-
ples(S2Table).Howeveronly13ofthemwerequantifiedinasufficientnumberofsamples
andwereretainedafterstatisticalanalysis.Thesecompoundswere:twohigheralcohols(Phe-
nyl-2-ethanolandHexanol,mg.L–1),sevenesters(Phenyl-2-ethanolacetate,Isoamylacetate,
Ethyl-propanoate,Ethyl-butanoate,Ethyl-hexanoate,Ethyl-octanoateandEthyl-decanoate,
mg.L–1),threemediumchainfattyacids(Hexanoicacid,OctanoicacidandDecanoicacid,mg.
L–1)andonevolatilethiol(4-methyl-4-mercaptopentan-2-one,or4MMP,ng.L–1).ForEthyl-
decanoateandEthyl-octanoate,whichweresometimesbelowthethresholdofdetection,we
proceededasdescribedaboveforResidualSugar.FinallytheAcetateratio,ratiobetweenPhe-
nyl-2-ethanolacetateandPhenyl-2-ethanol,wascomputed.Thisparameterrepresentstheacet-
ylationratioofhigheralcohols.
Dataanalyses
Single-traitanalyses. Foreachofthe35traitscollected,theeffectsofthestrain,ofthe
temperatureandofthestrain-by-temperatureinteraction,aswellastherandomblockeffect,
wereestimatedthroughthefollowingmixedmodelofanalysisofvariance(Rprogram,lme4
package):
Y ¼mþsiþtemp þðs(cid:1)tempÞ þW þE
ijk j ij k ijk
whereY wasthevalueofthetraitforstraini(i=1,...,66)attemperaturej(j=1,2)obtained
ijk
theweekk(k=1,...,22),mwastheoverallmean,s wasthefixedstraineffect,temp wasthe
i j
(cid:1)
fixedtemperatureeffect,(s temp) wastheinteractioneffectbetweentemperatureandstrain,
ij
W wastherandomblockeffectandE theresidualerror.Foreachtrait,normalityofresidual
k ijk
distributionsandthehomogeneityofthevarianceswerechecked.Someofthemdisplayedhet-
eroscedasticity,whichdecreasedthepoweroftheANOVA.Thiswasduetostrainswithweak
fermentationabilities(t-lag>40h,t-V >20h,CO <88g.L-1and/ort-75>110h).
max (cid:2) 2max
ThepredictedmeansY^ ¼m^ þs^þtedmp þ ^s (cid:1)tdempÞ werecomputedfromthemodel's
ij i j ij
parameters,aswellastheirstandarddeviations.Formanytraitsasignificantblockeffectwas
found(α=0.05).Therefore,thedecompositionofthetotalphenotypicvarianceofeachtrait
intoitsgeneticandenvironmentalcomponentswascomputedaftercorrectionfortherandom
blockeffects.Multiplenonparametriccomparisons(CampbellandSkillingsanalysis)werecar-
riedoutusingnparcomppackageoftheRprogramwithadjustedp-values[74].
Multi-traitanalyses
PrincipalComponentAnalysis(PCA)wasperformedontheANOVApredictedmeansfor
eachtemperature-straincombination(Rprogram,ade4package[75]).Theparentalstrains
wereaddedassupplementaryindividuals.TheentiredatasetusedwasgiveninS1Dataset.
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 9/24
HybridizationEffectsinYeast
Results
Large-scalephenotypingofahalfyeastdiallelunderwinemaking
conditions
Elevenparentalstrains(sevenstrainsofS.cerevisiaeandfourstrainsofS.uvarum,Table1)
andtheir55intra-andinter-specifichybridswerephenotypedunderenologicalconditions,at
twotemperatures(26°C,favorableforS.cerevisiae,and18°C,favorableforS.uvarum),inthree
replicates(Fig1andS1Fig).Atotalof396alcoholicfermentationswereperformed(66strains
(cid:1) (cid:1)
2temperatures 3replicates).Afewfermentations(26)werediscardedduetoincompleteor
absentimplantationoftheexpectedstrain.Thealcoholicfermentationswerecharacterizedin
(cid:1)
depththrough35phenotypictraits,leadingtoalmost13000numericaldata(35 370)forall
thefermentations.Thetraitswereclassifiedintofourcategories:(i)Sevenfermentationkinetics
(FK)parameters:t-lag,t-V ,t-45t-45,t-75,AFtime,V ,CO ;(ii)Eightlife-historytraits
max max 2max
(LHT):t-N ,t-N ,r,K,J (growthtraits),Sizet-N ,Viabilityt-N ,Viabilityt-75(size
0 max max max max
andviabilitytraits);(iii)Sixbasicenologicalparameters(BEP):ResidualSugar,Ethanol,Sugar/
EthanolYield,Aceticacid,TotalSO2andFreeSO2;(iv)Fourteenaromatictraits(AT):Phenyl-
2-ethanol,Hexanol,Phenyl-2-ethanolacetate,Isoamylacetate,Ethyl-propanoate,Ethyl-butano-
ate,Ethyl-hexanoate,Ethyl-octanoate,Ethyl-decanoate,Hexanoicacid,Octanoicacid,Decanoic
acid,4MMP(4-methyl-4-mercaptopentan-2-one)andAcetateratio,theacetylationrateof
higheralcohols.
Thesourcesofphenotypicvariationdifferaccordingtotraitcategories
Thesourcesofvariationofeachphenotypictraitwerestudiedbyanalysesofvariance
(cid:1)
(ANOVA)toestimatetheStrain,Temperature,andStrain Temperatureinteractioneffects
(Table2).Thepartofphenotypicvariationexplainedbythemodel(blockeffectremoved)de-
pendedonthetraitcategory,withFermentationKineticsparameters(FK)showingthehighest
R2values(0.60to0.92)andAromaticTraits(AT)thesmallest(0.09to0.66).Allthetraitsbut
three(Isoamylacetate,Ethyl-butanoateandEthyl-octanoate)displayedasignificantStrainef-
fect,accountingfor11to67%ofthevarianceexplained(p-value<0.05).Thetemperaturehad
contrastedeffectsaccordingtothetraitcategory:thetentraitsforwhichtemperatureexplained
atleast10%ofthemodelvarianceweremainlyfoundintheFermentationKinetics(FK)and
Life-historyTraits(LHT)categories,withR2valuesupto79%:t-45,AFtime,V ,t-75andt-
max
lag(FK),r,J andt-N (LHT),Aceticacid(BEP),andHexanol(AT).Finallyhighlysignifi-
max max
(cid:1)
cantStrain TemperatureinteractionswerefoundforCO ,t-lagandt-V (FK),t-N ,K,
2max max 0
Size.t-N andViability.t-75(LHT),Ethanol,ResidualSugarandSugar/EthanolYield(BEP)
max
andAcetateratio(AT).
Overall,FKandLHTtraitsdisplayedStraineffectsandlargeTemperatureeffects,andina
lesserextentStrain(cid:1)temperatureinteractions(exceptforCO withR2=0.32),BEPtraits
2max
(cid:1)
hadbothStrainandStrain temperatureeffectswithalmostnoeffectoftemperature,andfinal-
lyATtraitshadalmostexclusivelyStraineffects.
Straintypesdifferformanytraitsofbiotechnologicalinterest
(cid:1)
Foreachtrait temperaturecombination,wecomparedthemeansoftheS.cerevisiaestrains
(parentsandintraspecifichybrids),oftheS.uvarumstrains(parentsandintraspecifichybrids)
andoftheinterspecifichybrids,usingnon-parametriccomparisontests(α=0.05).In42cases
outof70(2temperaturesx35traits),atleastonemeanwassignificantlydifferentfromtheoth-
ers(S2andS3Figs).For12traits,adifferencewasobservedatbothtemperatures,for5traitsat
18°Conlyandfor12traitsat26°Conly.Forsometraitsofbiotechnologicalinterestthethree
PLOSONE|DOI:10.1371/journal.pone.0123834 May6,2015 10/24