01-MARAS_05b-tomazic 18/07/14 15:52 Page87 SSR MOLECULAR MARKER ANALYSIS OF THE GRAPEVINE GERMPLASM OF MONTENEGRO 1 1 2 2* VesnaMARAŠ ,VladanBOZOVIC ,SabrinaGIANNETTO andMannaCRESPAN 1:Departmentofdevelopment,13julPlantaže,P. RadomiraIvanovića2,Podgorica,Montenegro 2:ConsiglioperlaRicercaelasperimentazioneinAgricoltura-CentrodiRicercaperlaViticoltura(CRA-VIT), vialeXXVIIIAprile26,31015Conegliano(Treviso),Italy Abstract Résumé Aim :Giventhattheinformationabouttheorigin,genetic Objectif :Lesinformationsdisponiblessurl’origine,les relationships,anddiversityofMontenegringrapevinesis relationsgénétiquesetladiversitédesvariétésdevignedu stillpartial,weperformedadetailedanalysisofthe Monténégroétantencorepartielles,nousavonsréaliséun germplasminthiscountryusingsimplesequencerepeat inventairedugermoplasmedecepaysàl’aidedemarqueurs (SSR)markers.Ourmaingoalwastodeterminetheidentity SSR(répétitionsdeséquencessimples).Notreobjectif ofcultivarsuniquetoMontenegroandthosesharedwith principalétaitdedéterminerlesvariétésuniquesdansle othercountries,especiallytheneighbouringones. Monténégroetlesvariétéscommunesaveclesautrespays, etenparticulieravecceuxlimitrophes. Methods and results :Seventysampleswerecollectedand 14genotypeswerefound.AfterSSRprofilecomparison Méthodes et résultats :Soixante-dixéchantillonsontété withavailablemoleculardatabasesandliteraturedata,the prélevéset14génotypesontétédéterminés.L’identitéde identityofeachgenotypewasestablished.Fivewell-known chaquegénotypeaétédéfiniesuiteàlaconfrontationdes cultivarswerefound,theothersbeingminor,lesser-known profilsSSRaveclesbasesdedonnéesmoléculaires cultivars. disponiblesetlesdonnéesfourniesdanslalittérature.Cinq cépagesbienconnusontétéidentifiés,lesautressontdes Conclusion :Thisresearchprovidesanoverviewofthe variétésmineuresmoinsconnues. Montenegringrapevineassortment.Therearecultivars sharedwithothercountries,mainlytheneighbouringones, Conclusion :Cetterecherchefournitunéventailde whileothersarelikelynativetoMontenegro.TheKratošija l’assortimentvariétalviticoleduMonténégro.Certaines population( alias Primitivo,ZinfandelandCrljenak variétéssontidentiquesàcellesd’autrespays,enparticulier Kaštelanski)hasalargenumberofdifferentnamesin ceuxlimitrophes,alorsqued’autressontprobablement Montenegroandalsoawidemorphologicalvariability. indigènesduMonténégro.LapopulationdeKratošija( alias Therefore,Montenegroisthebestcandidateastheorigin Primitivo,ZinfandeletCrljenakKaštelanski)aungrand andspreadingpointofthiscultivar. nombredenomsdifférentsdansleMonténégroainsiqu’une vastevariabilitémorphologique.C’estpourcetteraisonque Significance and impact of the study :Thepresentstudy leMonténégroreprésentelemeilleurcandidatcommezone addsinformationontheidentity,origin,diffusionand d’origineetdediffusiondececépage. variabilityofsomegrapevinecultivars,allowingusto reconstructthehistoryandevolutionofnationalandtrans- Importance et impact de l’étude :Cetterechercheapporte nationalampelographicassortmentofMontenegro. denouvellesinformationssurl’identité,l’origine,la diffusionetlavariabilitédecertainesvariétésdevigne, Key words :SSRgenotyping,Kratošija,Bioka,intra- permettantainsidereconstruirel’histoireetl’évolutionde varietalbiodiversity,minorcultivars l’assortimentvariétalnationalettransnationaldu Monténégro. Mots clés :génotypageSSR,Kratošija,Bioka,biodiversité intra-variétale,variétésmineures manuscript received 3rd October 2013 - revised manuscript received 8th May 2014 J. Int. Sci. Vigne Vin , 2014, 48 , 87-97 *Correspondingauthor : [email protected] - 87 - ©Vigne et Vin Publications Internationales (Bordeaux, France) 01-MARAS_05b-tomazic 18/07/14 15:52 Page88 VesnaMARAŠ et al. INTRODUCTION context,ourworkhaddifferentpurposes.Usingsimple sequencerepeat(SSR)markers,wewantedi)to Montenegroisasmallcountry locatedinthe Balkan investigatetheidentityofalmostallgrapevinecultivars Peninsulaand overlookingthe AdriaticSea. Grapevine growninMontenegro ;ii)todeterminewhichcultivars growinginMontenegrodatesbackbeforetheRoman arelikelynativetoMontenegroandwhicharecommon period(Vuksanović,1977).Theclimatic,soiland toneighbouringcountries ;iii)toascertaintheidentity orographicconditionsofsouthernMontenegrodefine ofpresumedKratošija biotypescollected inthe twowineregions :theMontenegroSkadarLakebasin, experimentalfieldoftheBiotechnicalInstitutein encompassingthePodgorica,Crmnica,Rijecki, LjeskopoljeandcomingfromdifferentMontenegro BjelopavlickiandKatunskisubregions,andthe privatevineyards,wheretheyarecultivatedundermany Montenegrinseacoastregion,includingtheBoko- differentnames,dependingonthearea ;andiv)to Kotorski,Budvansko-Barski,UlcinjskiandGrahovsko- verifythecorrectidentityofVranacmothervines Nudolskisubregions.Ancientandprobably chosenforclonalselectionprocess. autochthonouscultivarsdominatethepresentgrapevine assortmentofthesetworegions,andvery MATERIALS AND METHODS heterogeneouspopulationsappearasaconsequenceof perennialgrowing.Furthermore,therearenumerous 1. Plant material localgrapevinecultivarslackingheterogeneityintheir Seventysampleswereselectedforgenotypingduring population,whichhavebeenregisteredas theperiodfrom2006to2010.Thevineswere autochthonouseventhoughtheywerenotdefinitively collectedfromdifferentviticulturalareasof identified.Mostofthesecultivarscanberecognized Montenegro(Table1). usingOIVcodes,butitisnotenoughfortheirreliable identification(Ulićević,1966 ;Pejović,1988 ;Maraš, 2. Microsatellite DNA analysis 2000). DNAwasextractedfromyoungleavesobtainedfrom CurrentviticultureandwineproductioninMontenegro cuttings,andgenotypingwasperformedwith11SSR isbasedmainlyonpresumedautochthonousgrapevine lociroutinelyemployedatCRA-VIT(Centrodi cultivars,suchasVranac,Kratošija,Krstač,andŽižak. RicercaperlaViticoltura,Italy)forcultivar Thedominantcultivarsforredwineproductionare identification :VVS2(ThomasandScott,1993) ; VranacandKratošija.Significantlylessprevalentis VVMD5,VVMD7,VVMD27andVVMD28(Bowers Krstač,usedforwhitewineproduction,whileno et al. ,1996and1999) ;VrZAG62andVrZAG79(Sefc commercialwineisproducedfromŽižak.Thepresence et al. , 1999) ;ISV2,ISV3andISV4(Crespan,2003) ; ofMuškaćelaandTrojkaissignificantlylower. andVMCNG4b9(Welter et al. , 2007),asdescribedby Crespan et al. (2006). The varietalidentification was TheresultsofCalò et al. (2008)showedthatKratošija achieved bycomparingthe obtained SSRprofiles with isasynonymoftheCalifornianZinfandel,theItalian available moleculardatabases and literaturedata . PrimitivoandtheCroatianCrljenakKaštelanski,and suggestedafirst-degreerelationshipwiththe 3. Statistics on SSR data MontenegrinVranac.ContrarytoVranac,whichhas stablemorphologicaltraitsandhasundergone Toevaluatethediscriminationpowerofthe11SSRs expansionintherecentyears,Kratošijaisveryancient usedinthiswork,thefollowingparameterswere andalotofbiotypeshavebeenfoundwithinits calculated :thepolymorphicinformationcontent(PIC) population,notfullyinvestigatedyet.Becauseofits andthecombinednon-exclusionprobabilityforidentity heterogeneity,Kratošijaislesspresentaloneinthe ofunrelatedandfull-sibgenotypes.SSRdatawere vineyards,andisfoundmostlyincombinationwith computedusing1418uniqueprofilesof Vitis vinifera L. Vranac,whichdominatesintheassortmentofredgrape cultivarspresentintheCRA-VITmoleculardatabase, cultivars.TheheterogeneityofKratošijahasbeen includingthenewgenotypesfoundinthepresentwork. describedbymanyauthors(Stojanović,1929 ;Bulić, CERVUS3.0.3softwarewasusedforcalculations 1949 ;Jelaska,1954 ;Ulićević,1966 ;Avramov,1988 ; (Marshall et al. ,1998 ;Kalinowski et al. ,2007 ; Pejović,1988 ;Burić,1995 ;Bozinovik et al. ,1998 ; http://www.fieldgenetics.com ).Genotypesshowinga Maraš,2000 ;Maraš et al. ,2004).Itiswellknownthat singleallelewereconsideredashomozygous. thefeaturesofanoldgrapevinecultivarmaychange RESULTS AND DISCUSSION withtheaccumulationandfixationofmutationsinthe shootapicalmeristem,givingrisetomanybiotypes Fourteengenotypeshavebeenfoundwithinthe withinthepopulation,evenwithnoticeable 70 samples(Table2).Usingoursetof11SSR morphologicalorphenologicaldifferences.Inthis markers,themeanPICcomputedon1418unique J. Int. Sci. Vigne Vin , 2014, 48 , 87-97 ©Vigne et Vin Publications Internationales (Bordeaux, France) - 88 - 01-MARAS_05b-tomazic 18/07/14 15:52 Page89 Table 1 - Accession list (name, provenance and identity by genotyping) ID Accession name Provenance (viticultural areas, municipalities) Identity 1 BIKA!A Ljeskopolje, Biotechnical Institute, Podgorica Krato"jia 2 BIKA!A Kuci, Podgorica Krato"jia 3 BIKA!A Doljani, Podgorica Krato"jia 4 BIOKA Brceli, Crmnica, Bar Bioka 5 CRNA LOZA Doljani, Podgorica Crna Loza 6 CRNI KRSTA! Ljeskopolje, Biotechnical Institute, Podgorica Krato"jia 7 !UBRICA Kuci, Podgorica !ubrica 8 KADARUN Herceg Novi Kadarun 9 KRAT. SA DUB. UREZ. Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 10 KRATOSIJA Brceli, Crmnica, Bar Krato"ija 11 KRATO#IJA Cemovsko polje, Podgorica Krato"ija 12 KRATO#IJA Cemovsko polje, Podgorica Krato"ija 13 KRATO#IJA Cemovsko polje, Podgorica Krato"ija 14 KRATO#IJA Crmnica, Bar Krato"ija 15 KRATO#IJA Doljani, Podgorica Krato"ija 16 KRATO#IJA Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 17 KRATO#IJA Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 18 KRATOSIJA Nikolj Crkva, Cemovsko polje, Podgorica Krato"ija 19 KRATO#IJA SREDNJA Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 20 KRATO#IJA ILI VRAN Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 21 KRATO#IJA MALA Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 22 KRATO#IJA MALA Ovtocic, Crmnica, Bar Krato"ija 23 KRSTA! B. Cemovsko