in situ and genetic characterization of wild grapevine populations

12 Pages·2014·1.27 MB·English

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IN SITU AND GENETIC CHARACTERIZATION OF WILD GRAPEVINE
POPULATIONS IN THE CASTILIAN AND LEON REGION (SPAIN)
RafaelOCETERUBIO1,JoséManuelVALLE2,M.ÁngelesPÉREZIZQUIERDO1,
M.ÁngelesLÓPEZ1,OsvaldoFAILLA3,AlbaMaríaVARGAS4,JuanCarlosSANTANA5,
ElenaHIDALGO4andRosaARROYO-GARCIA4,*
1:LaboratoriodeEntomologíaAplicada,FaculdaddeBiología,UniversidaddeSevilla,Avda.ReinaMercedes6,
41012Sevilla,Spain
2:EscuelaUniversitariadeIngeniería,UniversidaddelPaísVasco,Vitoria-Gasteiz,Spain
3:DipartimentodiScienzeAgrarieeAmbientali,UniversitàdiMilano,ViaCeloria2,20133Milano,Italy
4:DepartamentodeBiotecnología,CBGP-INIA,CampusdeMontegancedo,AutovíaM-40,km38,Pozuelode
Alarcón,28223Madrid,Spain
5:DepartamentoProducciónVegetalyRecursosForestales,ETSdeIngenieriasAgrarias,Universidadde
Valladolid,Avda.Madrid44,34004Palencia,Spain
Abstract Résumé
Aim:Assessinglevelsandpatternsofpopulationgenetic Objectif:Lefaitd’évaluerlesniveauxetlestypesde
variation in combination with morphological variationsgénétiquesdespopulationsenrelationavecles
characterizationrepresentsanimportantstepforevaluating caractéristiquesmorphologiquesreprésenteunpasimportant
rareorendangeredspeciesanddeterminingappropriate dansl’évaluationdesespècesraresetendangeretla
conservationstrategies. Thisisparticularlyimportantfor déterminationdesstratégiesdeconservationappropriées.
ensuringthepreservationofvaluablegeneticvariationin Ceciestparticulièrementvalablepourassurerla
wildrelativesofcrops,whichcouldprovidebeneficial préservationd’unevariationgénétiqueimportantedansles
allelesforplantbreedingandimprovement. espècessauvages,cequipeutfournirdesallèlesbénéfiques
pourlaculturedelaplanteetsondéveloppement.
Methods and results:Asurveyoftherelictwildgrapevine
populationwascarriedoutintheprovinceofBurgos,inthe Méthodes et résultats:Uneétudeaétémenéesurunreliquat
CastilianandLeonregion(Spain).Geneticdiversity, devignesauvagedanslaprovincedeBurgos,danslarégion
inbreedingextentandpossiblehybridizationwithcultivated deCastille-et-León(Espagne).Ladiversitégénétique,
grapevinewereinvestigatedusingmolecularmarkers. l’extensiondelaconsanguinitéetunepossiblehybridation
Resultsshowedthatoverall,geneticdiversitywaslowbut avecdesvignescultivéesontfaitl’objetd’uneinvestigationau
inbreedingwasnotpresent.Privatealleleswerefoundinthe moyendemarqueursmoléculaires.Lesrésultatsontdémontré
Burgoswildgenotypes,suggestingthepotentialvalueof que,dansl’ensemble,ladiversitégénétiqueestfaiblemaisque
theseaccessions.Comparisonsofmorphologicaltraitsand laconsanguinitén’existepas.Desallèlesparticulierstrouvés
molecularfeaturesamongthewildpopulationshoweda danslesgénotypessauvagesdelaprovincedeBurgos
closegeneticrelationshipamongthemandnogenetic soulignentlavaleurdecesacquis.Descomparaisonsde
relationshiptoCastiliangrapevinecultivars. caractèresmorphologiquesetdecaractéristiquesmoléculaires
parmilespopulationssauvagesontmontréuneétroiterelation
Conclusion:Thegeneticdifferentiationobservedbetween
génétiqueentreellesetaucunerelationgénétiqueavecles
wildanddomesticatedformsofgrapevinepointsoutthe
vignescultivéesdeCastille.
interesttocharacterizeandconservetheexistingpopulations
asasourceofnovelallelesforfuturegrapevinebreeding Conclusion:Ladifférenciationgénétiqueobservéeentreles
programs.Atthesametime,theobservedenological vignessauvagesetlesvignescultivéessoulignel’intérêt
differencesbetweenwildanddomesticatedgrapevine d’évalueretdeconserverlespopulationsexistantesentant
populationscouldbeinterestingforthewineindustry. quesourcedenouveauxallèlespourlesfutursprogrammes
Finally,giventhatthesepopulationsareatsevereriskof desélectiondelavigne.Enmêmetemps,lesdifférences
extinction,werecommendthatthispopulationbeprioritized œnologiquesobservéesentrelesvignessauvagesetles
forex situ andpossiblyon-farmconservationaswellasin vignescultivéespourrontsemontrerintéressantespourla
situprotection. productionduvin.Enfin,cespopulationscourantungrand
risqued’extinction,nousrecommandonsleurconservation
Significance and impact of the study:Thisworkshowed
aussibien“exsitu”quedansdescentresspécialisés(“àla
theexistenceofnovelallelesandenologicalcharactersin
ferme”)ainsiqueleurprotection“insitu”.
naturalwildgrapevinepopulations,whichrepresentsafirst
stepinthepotentialcontributionofthesenaturalpopulations Signification et impact de l’étude:Cetteétudemontre
toviticulture,suggestingapossibleroleinfuturebreeding l’existencedenouveauxallèlesetdecaractèresœnologiques
programs. danslespopulationsdevignessauvages,cequireprésente
unpremierpasdanslacontributionpotentielledeces
Key words:Vitis vinifera L.subsp.sylvestris (Gmelin)
populationsàlaviticulture,suggérantainsiunrôlepossible
Hegi,geneticdiversity,geneticrelationship,morphological
danslesprogrammesdesélection.
characterization,pathogeninfection,conservationprograms
Mots clés:Vitis viniferaL.subsp.sylvestris(Gmelin)Hegi,
diversitégénétique,relationgénétique,infectionpathogène,
programmesdeconservation
manuscript received 5th March 2014 - revised manuscript received 9th June 2014
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*Correspondingauthor:[email protected]
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RafaelOCETERUBIOet al.
INTRODUCTION subspeciesbecausetheseinvasiveplantsareableto
displacefromnaturalhabitatstoautochthonousvines
Ithasbeenarguedthatgermplasmcollectionsnotonly (Terpó,1969and1974;Laguna,2003;Arrigoand
playanimportantroleintheprotectionofnative
Arnold,2007).However,geneflowdoesnotseemtobe
biodiversity(Primack,2002),butcanalsoserveas
frequentbetweennaturalizedrootstocksandwild
valuablesourcesofallelesandtraitsforongoingplant
grapevinesduetodifferentecologicalbehaviors(De
breedingandcropimprovementefforts(Hajjarand
Andréset al.,2012).Nowadays,grapevineis
Hodgkin,2007).Indeed,thegenepoolsofcrop
consideredanendangeredtaxoninEurope(Arnoldet
relativesoftenharborbeneficialalleles(Nevoand
al.,1998;WalterandGillett,1998),mainlyinthose
Chen,2010),andinthecaseofrareandendangered
countries,likeSpain,PortugalandItaly,wherethereisa
species,suchallelesareatriskofextinction.The
lackoflegalprotectionforthisplant,whereasin
availabilityofgeneticdiversityamongwildrelatives
France,Switzerland,Germany,AustriaandHungary,it
opensupopportunitiesforexploringtraitandstress
hasbeenincludedinthelistofendangeredplant
tolerancemechanismsthatdomesticationandmodern
species.Itsfuturerepresentsamajorstakein
agriculturehaveleftbehindandalsoprovidesthescope
biodiversityconservation(Terralet al.,2010)because
fornovelallelediscovery.
mostpopulationshaveasmallnumberofindividuals
TheancestorofthecultivatedgrapevineisVitis vinifera andpossiblytherecouldbeageneflowfromcultivars,
L. subsp. sylvestris (Gmelin) Hegi, a woody whichcouldmodifytheiroriginalgeneticstructure(De
heliophilousdioeciouslianagrowinguptothetopof Andréset al.,2012).
thecanopyoftheassociatedarborealvegetation(Vigier
Themainobjectiveofourworkwasthemorphological
1718).Currently,itsrelictpopulationsspreadoutfrom
characterizationandpopulationgeneticanalysisof
PortugaltotheHinduKushmountain-range(Arnold,
2002),approximatelylimitedbyparallels50North available,wild-collectedindividualsofV. vinifera
(RhineValley,Germany)and30North(OurikaValley, subsp.sylvestris fromtheCastilianandLeonregionin
Morocco)(Oceteet al.,2007).Thistaxonconstituteda Spaintoconserveandevaluatethevaluablesourcesof
plantresourcestronglylinkedtothedevelopmentof allelesandtraitsforongoingplantbreedingandcrop
severalancientcivilizationsalongitsdistributionrange. improvementefforts.Firstly,acompletein situ
Infact,fromtheNeolithicAgeuptosomedecadesago, morphologicalcharacterization,includingmainplant
berrieswereharvestedfordirectconsumption, pathogen/diseasesymptomidentificationandgrape
vinificationorvinegarmaking(RiveraandWalker, enologicalcharacterization,wasconcluded.Secondly,
1989;Gorny,1996;Ocete,2011a).InSpain,seedsof weestimatedthelevelofgeneticdiversityandthe
thisplantarerelativelyfrequentinarchaeologicalsites extentofpopulationstructureinthisrarespecies,also
belongingtotheArgarculture(BronzeAge), comparingtheresultstootherwildpopulations,inorder
meanwhiletheoldestcultivatedonesappearlaterinthe todefinetheproperstrategyforin situ andex situ
Phoeniciancoloniesfromthe7th centuryBC(Oceteet programsofgermplasmconservation.Atthesametime,
al.,2007).Recently,newevidencesofSpanish thepossiblegeneticrelationshipsbetweenwildand
viticulturedatingbacktothe1st millenniumBChave cultivatedgenotypeswereanalyzed.
beendescribedbyMartínezandMaronda(2011)and
VeraandEchevarría(2011). MATERIALS AND METHODS
Powderyanddownymildewfungaldiseasesarrivedin 1. In situcharacterization
Europeduringthe19th centuryandcausedaheavy
Thesurveyplanningwastoexploretheriverbank
impactonvineyardsandwildpopulations.Phylloxera
forestsofthemainriversandtheirtributariesinthe
(Daktulosphaira vitifoliae Fitch)probablycausedlittle
provinceofBurgos.Eachpopulationwasmappedbya
damage, because the kind of locations, soil
GPSreceiverin2005.Inthisway,weconsidered
characteristicsandclimateofwildgrapevinehabitats
arenotsuitableforthisNorthAmericanhomopteran populationsin18differentlocationswherewild
(MuñozdelCastillo1878;Oceteet al.,2006).The grapevineaccessionswereanalyzedbyin situ
negativeeffectcausedbythesepestsanddiseaseson morphologicalcharacterizationandcollected38wild
wildgrapevineswasincreasedbyhumanactivities, grapevineleafsamplesfrom7locationsforgenetic
mainlyforestexploitations,roadconstructionandriver characterization(Table1).Accessionswereassessedat
management.Furthermore,theintroductionof floweringtime(June-July)todeterminethesexofthe
Americannativespeciesandhybridcultivars,foruseas plants.Pollensamplesfromflowerswereobtainedby
Phylloxera-resistantrootstocksforEuropeangrapevine brushingthematureanthersfrom10maleand10
cultivars,representedathreatfortheEuropeanwild femalevines.GrainsweremaintainedinDPX(Fluka)
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andobservedunderopticalmicroscopeOlympusBX61 giventheknowngeneticdeterminismofsexinV.
tostudythemorphologicalstructureofthegrains. vinifera L.(NegiandOlmo,1971),wildmaleplants
cannotescapefromcultivatedfieldsofhermaphrodite
Otherobservationswerecarriedouttwiceamonth
orfemalecultivarsandcannotresultfrompollination
between2005-2011toestablishanapproximate
betweenwildfemalesandcultivatedhermaphroditeor
phenologicalcalendarofthevinesfrombudbreakto
femaleplants,whereaswildfemaleplantscouldbe.
leaffall.Themainampelographicaldescriptorswere
Becauseofthepossibledifferencesintheoriginof
evaluated following the list provided by the
maleandfemalesylvestrisplants,weinitiallyanalyzed
InternationalOrganisationofVineandWine(OIV,
wildmaleplants.Asampleof38wildmaleaccessions
2009)on57femalesand100malesover2009-2010.
fromtheBurgosprovincewasanalyzed(Table1)and
Themainbotanicalsupporterswereidentifiedusing
comparedwithpublishedgenotypedata,corresponding
generalbotanicalkeysandthestudyofAizpuruet al.
to192Spanishwildgrapevinegenotypes(DeAndréset
(2003)inthefirstandsecondyearofthestudy.
al.,2012),210autochthonousgrapevinecultivarsfrom
Thedetectionofpossiblesymptomscausedbypests theCastilianandLeonregion(Santanaet al.,2010)and
anddiseaseswascarriedoutinspringandsummer 36Europeangrapevinecultivars(DeAndréset al.,
timesontheaerialorgansoftheplants(shoots,leaves 2012).
andbunches;3 maboveground)overthesevenyears
TotalgenomicDNAwasextractedfromyoungleaves
oftheinvestigation.Therootswereexcavateddownto
usingtheDNeasyTMPlantMiniKit(Qiagen).Extracted
adepthof40-50 cmtoobservepossiblesymptoms
DNAwasquantifiedandusedasa5ng/µlstock
causedbysubterraneanphytophagousandpathogens.
solution.Asetof20simplesequencerepeat(SSR)
Samplesoffinerootswereobservedunderbinocularto
microsatellitelociwasanalyzedtostudygenetic
detectpossiblePhylloxeradamage,root-knotgalls
diversity.Thegenotypeswereobtainedusingtwo
causedbynematodes,asinthecaseofMeloidogyne
independentmultiplexPCRs,labeledasAandB,as
species,androtfungi.Thedegreeofdamagescausedby
previouslydescribedbyIbáñezet al. (2009).PCRA
parasiticspecieswasevaluatedaccordingtothegrade
included11microsatellitemarkers:VVS2(Thomas
systemsproposedbyOceteet al. (2007)formitesand
andScott,1993),VVMD7,VVMD24,VVMD25
OIV(2009)formildews.Plantmaterialstakenat
(Bowerset al.,1996and,1999),VVIB01,VVIH54,
randomfromtwoplants(onemaleandonefemale)of
eachpopulation,exceptinthecaseofParadoresand VVIN73,VVIP31,VVIP60,VVIQ52(Merdinogluet
Entrambasaguas,wheretherewerenofemalevines, al.,2005)andVMC1B11(ZyprianandTopfer,2005);
wereusedtodetectthepresenceofGrapevineFanLeaf, PCRBincluded9microsatellitemarkers:VVMD5,
GrapevineLeafrollandGrapevineFleckvirusesusing VVMD21,VVMD27,VVMD28,VVMD32(Bowers
ELISAtests(Martelli,2000). et al.,1996and1999),VVIN16,VVIV37,VVIV67
(Merdinogluet al.,2005)andVMC4F3.1(DiGaspero
2. Microvinification et al.,2000).PCRamplificationswereanalyzedinan
ABI3130GeneticAnalyzer(AppliedBiosystems,
OnOctober26th,2011,6.3 kgofbuncheswere
FosterCity,CA,USA)usingGeneScan-LIZ500as
harvestedintheAnguloValleyfrom6different
internalmarker(AppliedBiosystems).Amplified
grapevines.Afterdestalking,berrieswerepressedby
fragmentsweresizedwithGeneMapper4.0software.
handand1200mlofmustwereobtained.Fermentation
withmacerationoccurredspontaneouslyfromwild Allelesizeandtotalnumberofallelesweredetermined
yeastsandlasted7days,thensomebasicenological foreachSSR.Putativealleleswereindicatedbytheir
analyseswereperformedfollowingreferencemethods estimatedsizeinbp.Privateallelesareconsidered
(OIV,2009).Moreover,asensoryanalysisofthewine
allelesthatarefoundonlyinasinglepopulationamong
wasperformedbyapanelofexperts(winemakers,
abroadercollectionofpopulations.Geneticdiversity
enologistsandconsumers)followingthefree-choice
wasestimatedusingthefollowingstatistics:numberof
profilingdescriptivemethod(Murrayet al.,2001).
alleles(Na);meannumberofallelesperlocus(MNA);
effective number of alleles (Ne); observed
3. Genetic diversity analysis
heterozygosity(Ho);expectedheterozygosity(He)
ThesamplescollectedfromtheBurgosprovincewere (Nei,1973);numberofprivatealleles;andfixation
analyzedatthemorphologicalandgeneticlevels.In index(F),alsocalledinbreedingcoefficient.These
ordertoavoidconsideringferalplants,weusedthesex statisticswerecalculatedusingGenAlexsoftware
oftheflowertodiscriminateV. vinifera subsp.sylvestris version6.0(PeakallandSmouse,2006)andtheExcel
versusV. vinifera subsp.sativa andmorphological MicrosatelliteToolkit(Park,2001).Redundant
observationtodiscriminateotherVitisssp. Furthermore, genotypeswereexcludedfromallanalyses.
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RafaelOCETERUBIOet al.
4. Population structure and genetic differentiation populationsandgeneticgroupsusingGenAlex
analysis software.PairwiseFstvalues(Slatkin,1995)amongthe
populations and subpopulations inferred by
BayesianclusteringwasappliedontheSSRgenotype
STRUCTUREwereestimatedwithArlequin3.5
datausingtheSTRUCTUREsoftwarepackage (Excoffieret al.,2005).
(Pritchardet al.,2000)revisedversion2.1(Falushet
al.,2003).Analyseswereperformedwiththetotal RESULTS
collection(460uniquegenotypes)andwitheach
1.In situcharacterization
populationobtainedfromthisfirstanalysis.Only
accessionswithancestryvalueshigherthan0.7were Duringthissurvey,18wildgrapevinepopulations
includedineachpopulationanalysis.Admixturemodel totaling157accessionswereanalyzed(Table1).Ina
andcorrelatedallelicfrequencieswereusedtoanalyze previousstudy,onlytwopopulationsintheCastilian
thedatasetwithoutpriorpopulationinformation,as andLeonregionwererevealed(Oceteet al.,1999).
suggestedbyFalushet al. (2003).Tensimulationsper WildpopulationswereonlyfoundintheMenaand
Kvaluewereperformedforeachpopulation(K)(set AnguloValleys,locatedinNorthernBurgoscloseto
from1to10).Burn-inperiodandMonteCarloMarkov theborderswiththeCantabriaandBasqueregions
Chain(MCMC)lengthweresetupat100,000and (Figure 1).Thelistofsitesalonggalleryforestsand
300,000ineachrun,respectively.ToassessthebestK colluvialpositionsappearsinTableS1a.The
valuesupportedbythedata,wecalculatedthesecond accompanyingfloracorrespondstoRiparian-colluvial
orderchangeofthelikelihoodfunctiondividedbythe Atlanticvegetation,withcoolthermalregimeand
standarddeviationofthelikelihood(ΔK)(Evannoet annualprecipitationofabout1200mm(TableS1b).
al.,2005).However,naturalpopulationsareexpected Theapproximatephenologicalcalendarwas:bud
toshowmorecomplexrelationships,andthismay swelling:April21-May3;flowering:June16-27;
affectthemannerinwhichSTRUCTUREassigns veraison(onsetofripening):July28-August22;fruit
individualtoclusters.Forthatreason,inordertoknow ripening:November4-16;andendofleaffall:
themostlikelyKvalue afterthefirstrunofthe November11-27.Themainmorphologicaldescriptors
STRUCTUREprogram,wehavesplitthedataseton offemaleandmalevines(youngandmatureshoot,
thebaseofoptimalKtorepeattheprocedureineach youngandmatureleaf,woodycane,flower,bunch,
geneticgroup. berry,seedandphenology)demonstratedverylow
polymorphism(TableS2).Pollengrainswere
Analysisofmolecularvariance(AMOVA,Excoffieret tricolporatedinmaleandacolporatedinfemale
al.,1992)wasperformedtocharacterizethepartitionof flowers.Nosymptomsofinfestationorinfection
theobservedgeneticvariationamongandwithin attributabletoPhylloxera(Daktulosphaira vitifoliae
Figure 1. Distribution of the 18 wild grapevine populations
in the Burgos province (Spain).
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Fitch),root-knotnematodesormyceliumofrotfungi andtanninlevelsweremedium(TableS3).According
weredetected.Leafdamagescausedbymites,namely tothesensoryevaluation,thewinerevealednoticeable
theErineumstrainofColomerus vitis (Pagenstecher) fruitaromasofwildberryandacidity.
andCalepitrimerus vitis (Nalepa)(Acari,Eriophyidae),
wereveryfrequentinallpopulations.Symptomsofthe 3. Genetic diversity of the wild populations
Erysiphe necator (Schwein.)Burrielfunguswere
Thegeneticdiversitypresentinwildgrapevine
commoninthemajorityofthevines,mainlyonleaves
populationsfromtheBurgosprovincewasanalyzed
andshootaxes. Oilspots onleavestogetherwithother
damagesonshootaxesandbunchescausedbydowny usingmicrosatellitegenotypingperformedinthe
mildew,Plasmopara viticola (BerkeleyandCurtis) initiallyselected38wildmalegrapevinesamples
(BerleseanddeToni),werealsofrequent.Theintensity (TableS4).22uniquegenotypeswereidentified,the
ofinfestationorinfectionvariedamongvinesofthe remaining16samplescorrespondedtoredundant
samepopulation(Table1)butwasnotconsiderably genotypes.Theseuniquegenotypesshowedamean
differentfromthedomesticatedgrapevines.Regarding numberof5.5alleles,includinganaverageof0.5
viralinfections,alltheElisatestswerenegativeforthe privatealleles(Table2).Thegeneticcomparisons
threepathogenstested. betweenthewildgrapevinegeneticgroupsfromthe
IberianPeninsula,whichareherecalledNorthernand
2. Microvinification
SouthernSpainwildpopulations(DeAndréset al.,
Theexperimentalwineshowedloweralcoholcontentin 2012),andtheBurgosprovincepopulationsledtothe
comparisontowinesproducedwithcultivatedvarieties identificationofprivateallelesintheBurgoswild
inthearea.TotalaciditywashighandpHwaslow, populations(TableS5).Theyarenotpresentinother
giventhemedium/highmalicacidcontent;color Spanishwildpopulationsandsomehaveafrequency
intensityandanthocyaninconcentrationwereveryhigh; higherthan10 %.Thisresultpointedouttheselection
Table 1. Number and sex of the vines and incidence of pests and diseases (number of individuals affected and
intensity of infestation/infection) according to Ocete et al.(2007) and OIV (2009).
In bold, locations where the male vines were genetically analyzed
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RafaelOCETERUBIOet al.
ofallelesthatarenotpresentintheotherstudied supposethatthesegeneticgroupscomprisemorethan
Spanishpopulations.Meannumberofallelesinthe onesubpopulation.Forthatreason,inordertoknow
differentpopulationsrangedfrom5.5to9.1;effective themostlikelyKvalueafterthefirstrunofthe
numberofallelesrangedfrom2.4to4.7,withan STRUCTUREprogram,wehavesplitthedataseton
averageof2.4intheBurgoswildpopulations; thebaseofoptimalKtorepeattheprocedureineach
InformationIndexrangedfrom1to1.7;observed geneticgroup.Analyseswereperformedwith221
heterozygosityrangedfrom0.5to0.6,whereas genotypesfromthecultivatedgroupand148genotypes
expectedheterozygositywasslightlyhigher,ranging fromthewildgroup;91genotypeswerediscarded
from0.5to0.7;andFixationIndexrangedfrom0.012 becauseonlyaccessionswithancestryvalueshigher
to0.121,pointingtotheputativeexistenceof than0.7wereincludedineachpopulationanalysis.
inbreedingdepressioninsomewildgrapepopulations Thisanalysisshowedthatthemostlikelynumberof
(Table2).Ingeneral,thewildpopulationsfromthe geneticgroupswasK= 5.Thefirstcluster(wild
provinceofBurgosshowedlowervaluesinallthe grapevine)wasdividedintwosubclusters(SW1and
allelicpatternsstudied,probablyduetothesmall SW2),whichcorrespondtosouthernandnorthernwild
populationsize. grapevineaccessionsincludingthewildgenotypes
fromtheBurgosprovince;thesecondcluster
4. Population structure between wild grapevine and (cultivatedgrapevine)wasdividedinthreesubclusters
cultivated genotypes (SC1,SC2andSC3)(TableS6).SC1containedthe
majorityofnorthernSpanishcultivarsandsome
ThegeneticstructureanalysisincludedNorthernand
autochthonouscultivarsfromtheCastilianregion;SC2
SouthernSpainwildgrapevinepopulations(192
includedthemajorityofautochthonouscultivarsfrom
samples),wildaccessionsfromtheBurgosprovince(22
theCastilianregionandoneFrenchaccessions;and
samples),cultivatedgrapevinesfromSpainincluding
SC3includedautochthonousSpanishgrapevine
theCastilianandLeonregion(210samples),and
cultivarsandsomeEuropeancultivars.However,the
Europeancultivars(36samples),especiallyfrom
consistencyofthesubpopulationsdescribedby
France. The analysis was achieved using the
STRUCTURE was very weak. In fact, the
STRUCTUREprogram;thechoiceofafixedKisnot
STRUCTUREprogramwasrunseveraltimeswith
arbitrary,eachofwhichischaracterizedbyasetof
different input data, resulting in different
allelefrequenciesateachlocus.Individualsinthe
subpopulationsforthecultivatedgrapevine.Forthis
sampleareprobabilisticallyassignedtogeneticgroups
reason,weconcludedthatK= 2wasthemostlikely
orjointlytooneormoregroups.Consequently,we
numberofgeneticgroups,correspondingtowildand
performedananalysiswhereKcouldvaryfrom1to
cultivatedgrapevinegenotypes(Figure2).
10.Thisanalysisshowedalargeincreaseoflikelihood
fromK=1to2andsmallerincreasesfromK=2to10. TheoutputoftheSTRUCTUREanalysisshowedthe
UsingthemethodologyofEvannoet al. (2005),this ancestryvalue,whichisanestimationoftheproportion
resultsupportedK= 2(FigureS1)asthemostlikely ofthegenomeofanindividualthatoriginatedfroma
numberofgeneticgroups,whichcorrespondstowild givenpopulation.Theancestryvaluevariesfrom0to
andcultivatedgrapevinegenotypes(Figure 2).Taking 1.Anancestryvaluecloseto0or1inonegroup
intoconsiderationthegeneticstructureanalysisdoneby suggestsnoevidenceofintrogressionfortheindividual
DeAndréset al. (2012)reportingthegeneticstructure studied.Intermediatevaluessuggestintrogression.
intheSpanishwildpopulations,itispossibleto Usingthesedata,wecanconcludethatthewild
Table 2. Allelic patterns (means ± standard deviation)
and Fixation Index across wild grapevine populations from Spain.
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accessionsfromtheBurgosprovinceshowedthesame
ancestryvaluesasthewildaccessionsfromNorthern
Spain(TableS6).Incontrast,theautochthonous
Castiliancultivarsshowedhighancestryvalueswiththe
Frenchcultivars(TableS6).TherestoftheSpanish
cultivarsshoweddifferentlevelsofintrogressionwith
theEuropeancultivars(TableS6). Figure 2. Graphical representation of ancestry
membership coefficients of all 460 individuals.
5. Genetic differentiation among genetic groups Each individual is shown as a vertical line divided
into segments representing the estimated membership
AMOVAanalysisusingthegeneticgroupsshowedthat proportions in the two genetic clusters
(wild and cultivated grapevine genotypes)
mostoftheexistinggeneticdiversitywasdistributed
inferred with STRUCTURE.
within(85 %)ratherthanamong(15 %)populations.
TheFstvaluesamongthefivegeneticgroupsareshown
Intheseconditions,thesepopulationsareuniqueinthe
inTable3.ThehighestandmostsignificantFstvalues
IberianPeninsulaandhaveprobablydeveloped
wereobservedinpairwisecomparisonsbetweenthe
ecologicaladaptivetraitsthathavenotbeendeveloped
wildgeneticgroupandthecultivatedgeneticgroup(Fst
inotherareas.Here,wecomparedthemorphological
= 0.22).Fstvalueswerefoundtobesignificantly
andgeneticdiversitypreviouslydescribedbyDe
differentfromzero(P<0.01)betweenpopulations.In
Andréset al. (2012)inwildgrapevinepopulations
addition,weobservedamoderategeneticdifferentiation
fromSpainwiththewildgrapevinepopulationsfrom
betweensubclustersSW1andSW2andbetween
subclustersSC1,SC2andSC3.Ontheotherhands,the theBurgosprovinceintheCastilianregion,inorderto
cultivatedsubclustersSC1,SC2andSC3showedhigh conserveandevaluatethevaluablesourcesofalleles
geneticdifferentiationwiththewildsubclustersSW1 andtraits,whichcanbeusefulforthedomesticated
andSW2(Table3).Allvaluesweresignificantly grapevine.
differentfromzeroinallpairwisecomparisons
Themorphologicalcharactersresultedverysimilarto
(P<0.05).Theseresultsconfirmthelowgenetic
thoseshownbywildgrapevinepopulationsfromthe
relationshipbetweenwildgrapevinefromBurgosand
neighboringBasquecountry,mainlyinthecaseofthe
thecultivatedgrapevine.
locationsalongtheCadaguaRiver(Bizkaiaprovince)
DISCUSSION andtheNavarreregion,whichweredescribedbyOcete
et al. (2004and2011b).Itisimportanttoremarkthat
IntheprovinceofBurgos,whichbelongstothe theIberianPeninsulaconstitutedarefugeareafor
Castilian region in north-central Spain, relict grapevineduringtheIceAgeintheQuaternary
populationsofwildgrapevinesarefoundonlyinthe (AntunesandBöhm2011).About64 %ofthevines
northunderAtlanticclimate.Theenvironmentalfactors, weremales.Thiscorrespondstoafemale/maleratioof
likethecoldcontinentalclimateoftheCastilianregion,
0.57,confirmingthedataobtainedbyAnzaniet al.
restrictpotentialnaturalhabitatsforwildgrapevinestoa
(1990)andOceteet al. (1999).Alltheobservedmale
fewlow“frost-free”valleysontheborderoftheregion.
flowerscorrespondedtomorphologicaltypeI(fully
developedstamensandnogynoecium)(OIV2009),as
itoccursintherestoftheSpanishpopulations(Oceteet
Table 3. Pairwise estimates of Fst values
al.,1999).NomaleplantswithflowertypeII(fully
based on data from 20 SSR loci among the model-
based clusters inferred by STRUCTURE. developedstamensandreducedgynoecium)were
All values were significantly different from zero found,confirmingthatthislattertypeofflower,which
in all pairwise comparisons (P <0.05). couldbeinterpretedasasteptowardshermaphroditism
(Orrú,2012),isveryscarceinSpain.Severalmale
individualshadasecondaryfloweringtimeinthe
middle of July. There was also pollen grain
dimorphism:themaleonewastricolporated,similarto
thatbelongingtohermaphroditecultivars,andthe
femaleonewasanaperturateovoidsac,asdescribedin
AndalusianpopulationsbyGallardoet al. (2009).All
thefemaleplantshadredberries,likeinalmostallthe
Spanishpopulations(Oceteet al.,1999).Thisintense
SW1 and SW2, wild grapevine subclusters; SC1, colorofberriesprobablyisanadaptivestrategyto
SC2 and SC3, cultivated grapevine subclusters successfullyattractbirdsandfavorseeddispersal
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RafaelOCETERUBIOet al.
(Hidalgo,2003).However,berryripeningwasnot AlicanteHenriBouschetorSyrah,ormadewithwild
uniformandcompletewithineachbunch.Besides grapesfromotherSpanishneighboringregions,suchas
dioecism,seedmorphologywasaphenotypictrait NavarraandLaRioja(Ocete,2011a;Oceteet al.,
characteristicofthiswildtaxonincomparisonto 2011b).Furthermore,itwascomparabletowinesmade
cultivarsofV. vinifera L.subsp.sativa (DC.)Hegi withwildgrapesfromMediterraneanregionsas
(Stummer,1911).Seedsfromthewildpopulationofthe Andalusia(Oceteet al.,2007)andSardinia(Lovicuet
Burgosprovinceweresubsphericalwithasmallbeak, al.,2009).Itrevealednoticeablefruityaromasanda
closetothoseofV. viniferasubsp.sylvestrisstudiedby goodageingpotentialasaconsequenceofitstannin
Anzaniet al. (1990)inItaly,Oceteet al. (2007)in levels.Characteristicssuchasanintensecoloranda
Spain,Terralet al.(2010)inFranceandOrrú(2012)in goodtotalaciditycouldbeveryinterestinginthe
Sardinia. Considering the morphological Mediterraneanclimate.Ablendofmustsfromwildand
characteristics,vinesfromthepopulationsinvolvedin domesticgrapescouldproduceredwinesenhancedin
thissurveywerealsocomparabletotheonesfoundin colorandnotrequiringaciditycorrectionbytartaric
NorthernSpain,namelyinNavarraandLaRioja acidaddition.Thecultivationofwildgrapevinesin
(Ocete,,2011a;Oceteet al.,2011b).Regarding commercialvineyardswiththeobjectivetoimprovethe
pathogeninfestation,wepreviouslyfoundthatwild characteristicsofthewinemadefromconventional
grapevinepopulationsaresensitivetoPhylloxeraunder cultivarscouldrepresentasustainableapproachforthe
artificiallaboratoryconditions(Oceteet al.,2011b). on-farmconservationofwildgrapevines.Moreover,
Theabsenceofsymptomsonrootsinthewild thequalitativetraitsofwildgrapefruitscouldbe
ecosystemsseemstobeduetotheedaphicconditions evaluatedbybreedingprogramstodevelopnew
oftheirhabitats,wherethereisseveralmonthsof cultivarswithimprovedpolyphenolandorganicacid
floodingeachyear.Similarly,thiswouldexplainthe accumulation.Finally,thissurveyprovidedaviewof
absenceofsymptomscausedbyroot-knotnematodes, thepotentialcontributionofthesenaturalpopulations
suchasgallsandsecondaryrootlets,similartothose toviticulture,giventheoccurrenceofsucha
causedbyMeloidogyne, ascitedbyPalmandWalter geneticallyuniquepopulation;ourrecommendations
(1991). Themite Colomerus vitis wasalwayspresent includepopulationsurveysoftheCastilianregionin
onallthegrapevinepopulations,asintherestofthe theexistinggermplasmcollections.
IberiansitescitedbyOceteet al.(1999)andalsointhe
Caucasianregion(Oceteet al.,2012).Itisinterestingto Precisedetectionandquantificationofgeneticvariation
notethatinsomespecimenswithalowlevelof isaprerequisiteforthesuccessfulconservationand
infestation,erineawasoccasionallyfoundontheupper exploitationofplantgeneticresources.Firstly,we
leafsurface.Symptomscausedbyanothereriophid, found16redundantgenotypesinthe38wildgrapevine
Calepitrimerus vitis,werelessfrequent.Itspresence accessions.Thisresultcanbeexplainedbecause,as
wascited,also,inpopulationsonthecoastof mentionedbefore,thewildancestorisawoodyliana
GuipúzcoaandtheEbroValley(Oceteet al.,2004)and growinguptothetopofthecanopyoftheassociated
someriverbankforestsfromAndalusia(Oceteet al., arborealvegetationandinthesamplingitiseasytoget
2007).TheconstantpresenceofColomerus vitis and thesamesample.Inaddition,wefoundhighergenetic
thefrequentoneofCalepitrimerus vitis onthesites relationshipsbetweenaccessionsfromthesame
sampledwouldsuggestthatbothspeciesofobligatory populationbecausethenumberofgrapevineplants
andmonophagousparasiteshavecohabitedwithvines foundperpopulationislow(5-8onaverage)andthat
sinceremotetimes,withoutcausinganyserious thepossibilityofhybridizationandseeddispersionis
damage.Onthecontrary,damagescausedbyNorth higherforplantsfromthesamepopulation.Atthesame
Americandownymildewareoccasionallyserious time,thisresultindicatesthatinthefuturethesampling
dependingontheseasonalweathercircumstancesand methodcouldbeoneortwoaccessionsperpopulation.
thespecificmicroclimaticconditionsoftheparticular
Theresultsofthegeneticanalysiswiththeunique
vine(Ocete,2011a). Insummary,wedidnotfindany
genotypesshowedthattheCastilianregion(Burgos)
individualsignificantresistancetopathogeninfestation
stillharborsanumberofwildgrapevineaccessions
thatcouldbeusefultothedomesticatedgrapevine.
withlowlevelsofheterozygosity.Asimilarresultshas
Ontheotherhand,itshouldbeunderlinedthatthe beenobservedinwildgrapevinepopulationsin
analyticalcharacteristicsofthewineobtainedbythe Morocco(Zinelabidineet al.,2010),Sardinia(Zeccaet
microvinificationofwildgrapesdemonstrated al.,2010),Portugal(Cunhaet al.,2007;Lopeset al.,
interestingtraitsforthewineindustrylikehighcolor 2009),France(DiVecchi-Starazet al.,2009)andItaly
intensityandtotalacidity(Ayalaet al.,2011).The (Grassiet al.,2003).However,observedheterozygosity
experimentalsamplecorrespondedtoafull-scalered wasnotsignificantlylower(P≤0.05)thanexpected
wine,similartothoseobtainedfromcultivars,suchas heterozygosityinwildBurgospopulations,inparallel
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withtheestimationofFvaluesclosetozero(Table2). correspondence,whichexcludesgenefloweventsof
SinceFvaluesareexpectedtobeclosetozerounder seedsorpollendispersionfromdomesticgrapevines
randommating,andthatFvaluesclosetozerohave towardsthewildconditions.Atthesametime,wild
beenobservedinBurgoswildpopulations,arandom grapevine samples did not show any genetic
matingpopulationstatuscouldbesupposed.Thisresult relationshipwithdomesticatedformsfromthesame
isdifferentfromtheanalysesdonebyDeAndréset al. region,asobservedintheSTRUCTUREanalyses.
(2012),whichhighlightedadeficiencyofheterozygotes Althoughafewcasesofhybridfromcultivatedtowild
(inbreeding)intheSpanishwildpopulation.Wecan populationshavebeenreported(DiVecchi-Starazet al.
explainourresultasaconsequenceofarandomgenetic 2009),ourfindingsaresupportedbythepicture
driftofallelefrequenciesintheBurgosprovince
obtainedthankstoseveralscientificreportsbasedon
populationsduetotheirsmallsizeortosomesampling
nuclearSSRwhichdemonstrateacleardifferentiation
bias(samplecomposedonlyofmaleaccessions).Atthe
betweencultivarsandwildgrapevines(Snoussiet al.
sametime,therelativelowergeneticdiversityinthis
2004;Grassiet al. 2008;DeAndréset al. 2012).
populationmaybeduetothesamplesizebecausea
However,wecannotexcludetheputativegenetic
positivecorrelationofnumberofalleleswithsample
relationshipbetweenthewildgenotypeandother
sizewasexpected(DeAndréset al. 2012).However,
domesticatedgrapevinesthathavenotbeenincludedin
theidentificationofprivateallelesinBurgoswild
thesurvey.
individualsisvaluableandconfirmstheimportanceto
establishaconservationprogramfortheBurgoswild Theanalysesofthegeneticstructurehighlighttwo
populations.Conservationisveryurgentbecausethe maingeneticgroupscorrespondingtodomesticatedand
CadaguariverbankmanagementinVillasana,the wildgermplasm.Bothgroupsshowedveryhigh
destructionofvegetationfollowingroadsandpath averageprobabilityofassignmenttotheirowncluster,
ditches,aswellasthegalleryforestmanagementofthe inagreementwiththehypothesisthattheyare
riversandcreeksofMenaandAngulohavedestroyeda geneticallydistinct.Actually,thegeneticcomparison
lotofvinesduringthelastdecades.Inaddition,thereis betweendomesticatedandwildformsofgrapevineis
alsoageneralsystematiccoppicingofthesurviving stilllimited,andthepicturearisingtodayisaclear
plantsincludedinthemarginalhedgesduringthe differentiationofdomesticatedandwildgrapesbased
maintenanceofthebordersofthepastureplots. onnuclearSSR(Snoussiet al. 2004;Grassiet al.
Fragmentationofwildgrapevinehabitatshasahigh
2008;DeAndréset al.2012).Inadditiontothemajor
influenceoneffectivegeneexchangesbetween
partitioninwildanddomesticatedforms,thegenetic
populations,leadingtotheisolationofthepopulations
structureanalysisidentifiedfivegeneticgroups:
(Arnoldet al. 2005;Grassiet al. 2006).Therefore,
clustersSW1andSW2,whichincludeallthewild
accordingtoFullenwarth(1997),effectivepracticesfor
accessions,andthreeotherclusters,SC1,SC2andSC3,
conservingthenaturalbiodiversitymaybetocollect
whichincludethemajorityoftheanalyzedcultivars.
seedsfromwildgrapevines,toproduceplantsin
Theexistenceoftwogeneticgroupswithinthewild
nurseries,andtoafforesttheriverbankswithwild
accessionscouldsuggestsomelevelofisolationamong
grapevines.ItisnecessarytoremarkthattheBurgos
thosegeneticlineages(Fst= 0.10)asithasbeen
provincespreadsout14.300squarekilometerswherea
suggestedbyDeAndréset al. (2012).Thegenetic
totalofabout100vineswerefounddistributedamong
structureofthedomesticatedgrapevinesamplesisvery
severalpopulations,sothisgrapevinesubspecies
weakbecausethevinifera cultivarsrepresentalarge
constitutesarareplant.Inouropinion,thereisanurgent
complexpedigreeresultingfromanumberof
needtodevelopastrategyforitsin situ andex situ
spontaneousandinter-generationcrossesbetween
protection.Forthispurpose,aprogramofex situ
cultivarsthathavebeenvegetativelypropagatedfor
conservationandpropagationisinprogressthanksto
centuries(Myleset al. 2011;Bacilieriet al. 2013;
collaborationbetweentheLaboratoryofApplied
Lacombeet al.2013).Infact,thethreedifferentgenetic
EntomologyoftheUniversityofSevillaandthenursery
clustersdetectedwithintheanalyzedcultivarsshowed
oftheFondoForestalIbérico.
verylowgeneticdifferentiation(Table3).Thislow
Thegeneticrelationshipbetweenthewildgenotypes geneticdifferentiationwouldresultfromthehighlevel
fromBurgosandthewildgenotypespreviously ofhybridizationbetweengrapevinecultivars.These
describedshowedhighgeneticsimilaritywiththe resultssuggestedthatclosegeneticrelationshipswithin
NorthernSpainpopulations,supportingthegenetic eachclusterareconsistentwithprevioushistorical,
structurebetweennorthernandsouthernwildgrapevine viticulturalandgeneticinformation.Theclose
populationsfromSpain(DeAndréset al. 2012).The relatednessofcultivarsfromCastilianandFrench
genotypecomparisonbetweenwildBurgosindividuals varietiesalsosupportstheputativeintroductionofthese
and grapevine cultivars did not show any cultivarsalongthepilgrimageroutetoSantiagode
J. Int. Sci. Vigne Vin, 2014, 48, 111-122
- 119- ©Vigne et Vin Publications Internationales (Bordeaux, France)
01-ocete_05b-tomazic 24/09/14 19:07 Page120
RafaelOCETERUBIOet al.
Compostela.Putatively,Castilianvarietiescouldhave subsp.silvestris)intheRhineValley?Biodivers.
localoriginsorresultfromcrossesbetweenintroduced Conserv.,14,1507-1523.
andlocalvarieties(Santanaet al. 2010).Theseresults ArrigoN.andArnoldC.,2007.NaturalisedVitisrootstocks
provideaviewofhowthedomesticatedgrapevine inEuropeandconsequencestonativewildgrapevine.
geneticpoolisstructuredinaviticulturalregion, PLoS ONE,2,e521.doi:10.1371/journal.pone.
includinggermplasmoflocalandforeignoriginsand 0000521.
crossesbetweenthem.
AyalaM.C.,OceteRubioR.,GarcíaMorenoM.P.and
ArmendárizGonzálezI.,2011.Vinificacióncon
CONCLUSIONS
poblacionesdevidsilvestredelValledeMena
(Burgos)ydelValledeAmbroz(Cáceres).In:XXXIII
Thegeneticdifferentiationobservedbetweenwildand
Jornadas de Viticultura y Enología de la Tierra de
domesticatedformsofgrapevineinSpainpointsoutthe
Barros,CulturalSantaAna,Almendralejo,pp.143-
interesttocharacterizeandconservetheexisting
156.
westernpopulationsasasourceofnovelallelesforthe
futureunderstandingandimprovementofthegenetics BacilieriR.,LacombeT.,LeCunffL.,DiVecchi-StarazM.,
LaucouV.,GennaB.,PérosJ.P.,ThisP.and
ofgrapevinedomesticatedforms.Atthesametime,
BoursiquotJ.M.,2013.Geneticstructureincultivated
withtherecentlossofsuitablehabitatsresultingfrom
grapevinesislinkedtogeographyandhumanselection.
directandindirecthumanimpact,V. vinifera L.subsp.
BMC Plant Biol.,13,25.
sylvestris isnowendangeredthroughitsdistribution
range.Asaconsequence,populationsaregenerally BowersJ.E.,DanglG.S.,VignaniR.andMeredithC.P.,
smallanddispersedandthismeansasignificantriskof 1996. Isolation and characterization of new
polymorphicsimplesequencerepeatlociingrape(Vitis
extinctionsandpotentialinbreedingdepressionofwild
viniferaL.).Genome,39,628-633.
grapevine.Finally,givenourfindingsaswellasthe
observedgeneticandenologicaldifferencesbetween BowersJ.,BoursiquotJ.M.,ThisP.,ChuK.,JohanssonH.
wildanddomesticatedpopulations,werecommendthat andMeredithC.,1999.Historicalgenetics:the
thispopulationbeprioritizedforex situ andpossibly parentageofChardonnay,Gamay,andotherwine
on-farmconservationaswellasin situprotection. grapesofNortheasternFrance.Science,285,1562-
1565.
Acknowledgements:Jointpublicationoftheproject
CunhaJ.,Baleiras-CoutoM.,CunhaJ.P.,BanzaJ.,
RTA2011-00029-C02-01andtheCOSTActionFA1003 Soveral A.,CarneiroL.C.andEiras-DiasJ.E.,2007.
“East-WestCollaborationforGrapevineDiversity CharacterizationofPortuguesepopulationsofVitis
ExplorationandMobilizationofAdaptiveTraitsfor vinifera L.ssp.sylvestris (Gmelin)Hegi.Genet.
Breeding”. Resour.Crop Evol.,54,981-988.
DeAndrésM.T.,BenitoA.,Pérez-RiveraG.,OceteR.,
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