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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
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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
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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 polje, Podgorica Krsta$ 24 KRSTA! B. Cemovsko polje, Podgorica Krsta$ 25 KRSTA! B. Cemovsko polje, Podgorica Krsta$ 26 KRSTA! BIJELI Cemovsko polje, Podgorica Krsta$ 27 LISI!INA Brceli, Crmnica, Bar Lisi$ina 28 LISI!INA Godinje, Crmnica, Bar Lisi$ina 29 LJUTICA Ljeskopolje, Biotechnical Institute, Podgorica Krato"jia 30 MU#KA%ELA Cemovsko polje, Podgorica Muscat blanc à petits grains 31 MU#KA%ELA Cemovsko polje, Podgorica Muscat blanc à petits grains 32 MU#KA%ELA Cemovsko polje, Podgorica Muscat blanc à petits grains 33 MU#KA%ELA Nikolj Crkva Cemovsko polje, Podgorica Muscat blanc à petits grains 34 PLAVINA Herceg Novi Plavina 35 RAZAKLIJA Doljani, Podgorica Drenak crven 36 RAZAKLIJA CRNA Kuci, Podgorica Razaklija crna 37 REHULJACA Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 38 SREDNJI VRANAC Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 39 TROJKA Ljeskopolje, Biotechnical Institute, Podgorica Moscato violetto/Muscat rouge de Madere 40 TROJKA Ljeskopolje, Biotechnical Institute, Podgorica Moscato violetto/Muscat rouge de Madere 41 TROJKA Ljeskopolje, Biotechnical Institute, Podgorica Moscato violetto/Muscat rouge de Madere 42 VELJI VRANAC Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 43 VRAN Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 44 VRANAC 9 Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 45 VRANAC Ovtocic, Crmnica, Bar Vranac 46 VRANAC Ovtocic, Crmnica, Bar Vranac 47 VRANAC Ovtocic, Crmnica, Bar Vranac 48 VRANAC Limljani, Crmnica, Bar Vranac 49 VRANAC Brceli, Crmnica, Bar Vranac 50 VRANAC Cemovsko polje, Podgorica Vranac 51 VRANAC Cemovsko polje, Podgorica Vranac 52 VRANAC Cemovsko polje, Podgorica Vranac 53 VRANAC Nikolj Crkva Cemovsko polje, Podgorica Vranac 54 VRANAC Nikolj Crkva Cemovsko polje, Podgorica Vranac 55 VRANAC Nikolj Crkva Cemovsko polje, Podgorica Vranac 56 VRANAC Nikolj Crkva Cemovsko polje, Podgorica Vranac 57 VRANAC Brceli, Crmnica, Bar Vranac 58 VRANAC (clone 2/8) Crmnica, Bar Vranac 59 VRANAC (clone 6/8) Crmnica, Bar Vranac 60 VRANAC NCV 14/39 Cemovsko polje, Podgorica Vranac 61 VRANAC NCV 15/13 Cemovsko polje, Podgorica Vranac 62 VRANAC NCV 62/7 Cemovsko polje, Podgorica Vranac 63 VRANAC NCV 62/13 Cemovsko polje, Podgorica Vranac 64 VRANAC NCV 62/8 Cemovsko polje, Podgorica Vranac 65 VRANCIC Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 66 VRANCINA Ljeskopolje, Biotechnical Institute, Podgorica Krato"ija 67 &I&AK Cemovsko polje, Podgorica &i'ak 68 &I&AK Cemovsko polje, Podgorica &i'ak 69 &I&AK Cemovsko polje, Podgorica &i'ak 70 &I&AK Nikolj Crkva, Cemovsko polje, Podgorica &i'ak
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profilesof Vitis vinifera L.cultivarswas0.8036 ;the arecomplementary :Kratošijausuallyprovidesa estimatedprobabilityofidentitytodealwithunique higheramountoftotalacidsandsugars,whileVranac genotypeswas3.38E-15forunrelatedgenotypesand isricherincolouredsubstances.Bulić(1949)givesa 9.52E-6forfullsibs.Theseparametersshowedthe descriptionofKratošijawithsynonyms(Gartosija, significantreliabilityofthissetofmarkersforvarietal Grakosija,Kratkosija)fromtheninemunicipalitiesof identification. theMontenegrinseacoast.Thiscultivarisrarely foundintheDalmatiaregion,therefore,theauthor Theidentificationworkshowedthatfivegenotypes statesthatithasbeenpossiblyintroducedtoDalmatia matchwell-knowncultivars,namelyKratošija,Muscat fromMontenegro.By2001,onlyabouttwentyliving blancàpetitsgrains,MuscatrougedeMadere( alias vinesofCrljenakKaštelanskihadbeenfoundin Moscatovioletto),PlavinaandVranac.Inparticular, Dalmatia(Maletić et al. ,2009),confirmingthatthis theaccessionsnamedKratošija(plusanadjective), cultivarwasnotwidespreadinDalmatiainthe19th Bikača,Ljutica,Rehuljaca,SrednjiVranac,Velji and20thcentury(Savić,2003).Stojanović(1929) Vranac,Vran,Vrancic,Vrancinaandawronglynamed writesthatthebestvineyardsinMontenegroareinthe Vranac(id44inTable1)allbelongtotheKratošija Skadarlakeregion(Crmnica),whichisfamousforits cultivar,aspreviouslysupposedbasedon redwinesthatcanreachahighquality,whilethe ampelographicdescriptions(Maraš,2000).Crni winesproducedalongtheMontenegrinseacoastare KrstačmatchestheKratošijaprofileandisanother similartothosefromDalmatia.Thisauthorstatesthat clearmisnomer.Thefirstremarkabledescriptionsof Plavacmali,Dobričićandsomeothersarethemain VranacandKratošijacultivarshavebeengivenby cultivarsgrowninDalmatia,whereashedoesnot PetarPlamenacintheAmpelographyofVialaand mentionKratošija. Vermorel(1910).Lateron,manyscientists (Stojanović,1929 ;Bulić,1949 ;Ulićević,1959and Ampelographicdescriptionsareavailableforall 1966)statethatVranacispresentonlyinMontenegro. KratošijaaccessionslistedinTable1andgrownatthe Ulićević(1959)writesthefollowing :“characteristic ExperimentalEstateoftheBiotechnicalInstitutein cultivarofCrmnicafromtheSkadarLakeviticultural Podgorica-Ljeskopolje(Maraš,2000).Some areaisVranac,coveringabout40 %oftheassortment examplesofampelographicvariabilityinKratošija ofgrapecultivars.Asamatteroffact,thisistheonly accessionsarereportedinTable3andFigure 1.The viticulturalareawhereitprevailsandtheonlyone nameofeachKratošijabiotypeisinconnectionwith whereitisrepresentedtoagreaterextent.Itisina someofitsparticularcharacteristics.Inparticular, propersenseaCrmnicacultivar”.Nastev(1967)states thesebiotypesshowedawiderangeofvariabilityin thatVranacisaMontenegrinautochthonousgrapevine grapeyield,mainlydependentonbudfertilityrather cultivarmostlycultivatedintheSkadarlakeregion thanclusterweight.Clusterweightwashighly (Crmnica),butalsoontheMontenegrinseacoast.This variableamongaccessionsandcorrelatedwithbunch authordeclaresthatVranachasbeenspreadinthe morphology ;thesetraitsshowedtobestablewithin 1950sinMacedonia(experimentalfieldofButel). eachbiotype(Maraš,2000).Onemothervinewas Nowadays,itrepresentstheleadingcultivarforred selectedperbiotypeforthesanitarycheck-upandall wineproductioninthiscountry(Stajner et al. ,2009). showedthepresenceofviruses.Furthermore, Ulićević(1959)statesthatKratošijaisalmostas additionalinvestigationofthesanitarystatusofnewly remarkableasVranac.Inthesamepaperhewritesthat selectedvinesofKratošijafromallviticulturalregions KratošijadominatedinMontenegrinvineyardsfor ofMontenegroshowedahighlevelofviralinfection over70yearsandthatitoftenrepresented90 %ofthe (Maraš et al. ,2014). assortment.Therefore,Kratošijaisthemainand probablytheoldestMontenegringrapevinecultivar. Byputtingtogetherdifferentpiecesofinformation Ulićević(1959)arguesthattheCalifornianZinfandel fromtheliteraturecited,andgiventheresultsofour isidenticaltoKratošijaandthatZinfandellikely study,wecanconcludethatKratošijawithallits originatesfromMontenegroandhasbeenexportedby biotypeshasbeengrowninMontenegroforcenturies. Montenegrinemigrants.Thiswassupportedbythe Givenitspresenceinalltheviticulturalregionsofthis workofCalò et al. (2008),confirmingthatKratošija littlecountry,thewidevariabilityofthepopulation, hasthesamegeneticprofileasZinfandel.Avramov theimpressivehighnumberofdifferentnames,the (1988),Pejović(1988),Cindrić(1994),Cindrić et al . closegeneticrelationshipwithVranac,anditscurrent (2000),Burić(1995),Božinovik(1996),Maraš(2000) presence(survival)inviticulturalareasin andMaraš et al. (2004)writethatVranacand Montenegro,wesuggestthatthisareabeconsidereda Kratošijaareautochthonouscultivarsfrom likelyregionfortheoriginandspreadingcentreof MontenegroandthatKratošijahasavery thiscultivar.Therefore,theplaceoforiginofthis heterogeneouspopulation.Thewinesofthesecultivars cultivarshouldbemovedfurthersouththanthe
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Figure 1 - Examples of cluster variability among Kratošija biotypes. In brackets the reference id number referred to table 1.
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Dalmatiancoast,unlikewhathasbeenpreviously TheredberryRazaklijaaccessionfromtheSkadar suggestedbyMaletic et al. (2004). Lakeregion(Doljani),aknownsynonymofthe SerbianDrenakcrven,matchestheSSRprofileof TheSSRprofileofMuškaćelaaccessionsmatchesthat CrvenDrenok(Vitis-WBC,Western-Balkans Vitis ofMuscatblancàpetitsgrains.Thisoutcomeaddsa Database,http://vitis.atcglabs.com).Tablegrapesin newsynonymtothelonglistofthisveryancient Montenegroonlyaccountsfor5 %,ofwhich95 %is cultivar,largelyspreadallovertheviticulturalareasin theRazaklijacultivar(Drenakcrven)fromboththe Europeandrecentlyshowedtobethemainancestor SkadarLakeandtheseacoastregion(Ulićević,1959). oftheMuscatfamily(Cipriani et al. ,2010).Trojka ManyauthorsfromYugoslaviaconsiderthatRazaklija accessionshavethesameprofileasMuscatrougede originatesfromAsiaMinor(Ulićević,1966).Itisnot Madere( alias Moscatovioletto),anotherimportant knownhowandwhenitarrivedinMontenegro, memberoftheMuscatfamily(DiVecchi-Staraz et al. , Macedoniaandothercountriesandhowitspread. 2007 ;Lacombe et al. ,2013),buttheberrycolouris different(itiswhiteinouranalyzedTrojkacultivar). CrnaLoza,Čubrica,Krstač,Lisičina,Razaklijacrna MuscatrougedeMadereisaprogenyofMuscatblanc andŽižakshowuniqueSSRprofiles.CrnaLozawas àpetitsgrainsandSciaccarello(Lacombe et al. ,2013) consideredasaKratošijasynonym,butouranalysis andhaslittleimportance.Supposedtobenativeto showsadifferentSSRprofileforthiscultivar.Based Portugal,itdoesnothaveanyagronomicrelevance. onSSRallelesharingatallanalyzedloci,CrnaLoza Actually,itisnotincludedinanyofficiallistof couldbeanotheroffspringofKratošija,butfurther grapevinecultivarsauthorizedforcultivationin24 researchshouldbedonetoconfirmthishypothesis. nationalcatalogues(Lacombe et al. ,2011).Itis consideredagrapeforenthusiasts(Galet,2000)andis Ulićević(1959)statesthatČubricaisacultivarforred grownmostlyingermplasmrepositories.Thatiswhy wineproductionandisrepresentedinverysmall itwasasurprisetofinditamongMontenegrin percentageinthevineyardsofthePodgoricasub cultivatedgrapevines.Plavinaisaprogenyof region(DoljaniandKuci).ThesampleofČubrica KratošijaandVerdeca(Cipriani et al. ,2010)andisan fromKucihasaDNAprofileconfirmingthatitisan importantcultivarinthesubregionofNorth independentcultivar. Dalmatia.Allmotherplantschosenforclonal Ulićević(1959)writesthatintheSkadarLakeregion, selectionofVranac(NCV62/13 ;NCV14/39 ;NCV inthesubregionofPodgorica(BeriandDoljani), 15/13 ;NCV62/7 ;NCV62/8)showedtobecorrectly Krstačwasdominantamongthewhitegrapevine identified. cultivarsusedforwhitewineproduction,producedin AlthoughinliteratureBiokaisconsideredasa smallamountinthatperiod.Inthesubregionof synonymofKrstačinCrmnica(Ulićević,1959),SSR Podgorica,itrepresentsonly10 %ofthevineyard datashowedthatithasadifferentmolecularprofile. area,mostlyinBeriandDoljani,andverylittleinthe Therefore,Biokaisanothercultivar.Wehave Ljesanskadistrict.Krstačisbelievedtobe discoveredthatBiokasharesthesamegenotypeasthe autochthonousofMontenegroandprobably ItalianFrancavidda,acultivarregisteredintheItalian originatedinBeri(nearPodgorica) ;itsnamecomes CatalogueandgrowninApulia,theItalianregion fromthelookandshapeofthebunchthatresemblesa facingMontenegroontheothersideoftheAdriatic cross(VialaandVermorel,1910 ;Vujović,1956). Sea(http://catalogoviti.politicheagricole.it).Moreover, Žižak(orŽižakbijeliafterBulić,1949)isconsidered BiokahasthesameSSRprofileastheCroatian anotherautochthonouscultivarofMontenegroandits ZlataricaVrgorska(Zdunić et al. ,2013).Therefore, originisunknown.Individualspecimenscanbefound thesethreecultivarsrepresentanewcaseof nearbyPodgorica,butitismostlygrownonthe previouslyunknownsynonymsgrownindifferent Montenegrinseacoast(Boko-Kotorskisubregion), countriesfacingtheshoresoftheAdriaticSea whereitisusedfordessertwine(Ulićević,1959). (DalmatiaandApulia). AboutLisičina,Stojanović(1929)reportstheuseof ComparingourdatawiththeEuropean Vitis database thiscultivarforwhitewineproductioninMontenegro. (http://www.eu-vitis.de/index.php),wefoundone Bulić(1949)andUlićević(1959)foundLisičina matchatallsevencommonSSRlocibetweenthe (synonymsareLisica,Ružica,Sjemerava,Sjeruša)in MontenegrinKadarunandaKadarunaccessionfrom MontenegroaroundBar,Ulcinj,Podgoricaand Croatia(HRV041-SA#11-95)lackingthetrue-to-type Virpazar(Crmnica).TheVIVCcataloguereportsthat validation.Inthe Vitis InternationalVarietyCatalogue LisičinaisasynonymoftheCroatianLisica. (VIVC,http://www.vivc.de),Kadarunisreportedasa AccordingtoNastev(1967),Lisičinaisawrong Turkishcultivar. synonymofthePlovdina(Pamid)cultivar.
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Accordingly,theSSRprofileofourLisičinasampleis ontheiridentityandthepresentresearchcontributes differentfromthatoftheBulgarianPamid(Hvarleva toshedsomelightonthistopic. et al. ,2004 ;Dzhambazova et al. ,2009). Acknowledgments :Thisresearchwassupportedbythe Razaklijacrna(black),havingadarkbluecoloured followingprojects :“VariabilityofKratošijacultivarin skin,wasfoundonlyinKuci(subregionof Montenegrinviticulturalregions”and“Clonalselectionof Podgorica)inoldvineyardsandunderthisname.It Kratošijacultivar”fundedbytheMinistryofScienceof hastobenotedthatthisnameindicatesatablegrape Montenegro ;“Identificationandcharacterizationof cultivaroriginatedbycrossingRazaklijaRossaand Montenegrinautochthonousgrapevinecultivars”and Vranac(performedbyUlićevićM. attheBiotechnical “Clonalselectionofautochthonousgrapevinecultivars” InstituteinPodgorica).BasedonSSRallelesharingat fundedbyCompany13julPlantazeandtheMinistryof AgricultureofMontenegro ;and“IDENTIVIT/ASER” allanalyzedloci,Razaklijacrnacouldreallybea fundedbytheItalianMinistryofAgricultural,Alimentary progenyofDrenakCrven,butourSSRdataruleout andForestryPolicies.SpecialthankstoDordjijeRajkovic Vranac.Furtherdataarerequiredtovalidatethis forusefulsuggestionsandsupportandtoAngelo partialpedigree. Costacurtaforinitiatingustocultivarcharacterizationand CONCLUSION identification. SeventyaccessionsbelongingtotheMontenegrin REFERENCES grapevinegermplasmwereanalyzedusingSSR AvramovL.,1988. Savremeno Gajenje Vinove Loze markers.Fourteengenotypeshavebeenfound,three (Contemporarygrapevinecultivation).Nolit, ofthembeingnativetoMontenegro(i.e.,Vranac, Beograd. KrstačandŽižak).Kratošijahasbeenrecently BowersJ.E.,DanglG.S.,VignaniR.andMeredithC.P., recognizedasaninternationalcultivar,beinga 1996.Isolationandcharacterizationofnew synonymofItalianPrimitivo,CalifornianZinfandel polymorphicsimplesequencerepeatlociingrape andCroatianCrljenakKaštelanski,whoseoriginhas (Vitis vinifera L.). Genome , 39 ,628-633. beenwidelyinvestigated.Giventhelargenumberof BowersJ.E.,DanglG.S.andMeredithC.P.,1999. differentnamesusedinMontenegroforKratošijaand Developmentandcharacterizationofadditional thewidemorphologicalvariabilityofKratošija microsatelliteDNAmarkersforgrape. Am. J. Enol. biotypes,weproposehere,forthefirsttime, Vitic. , 50 ,243-246. Montenegroasthebestcandidatecountryforthe BožinovikZ.,1996. Ampelografija (Ampelography). originandspreadingcentreofthisgroupofsynonyms. AgencijaAkademik,Skopje. CrnaLoza,Čubrica,LisičinaandRazaklijacrnaare cultivarslikelynativetoMontenegro.Muškaćelaand BožinovikZ.,PetkovM.,BeleskiK.andBoškovK.,1998. Trojka,consideredautochthonousofMontenegro,are Proizvodneitehnološkeosobinenekihvarietetasorte clearlyofdifferentorigin.Wediscoveredthat KratošijauRepubliciMakedoniji(Theproductionand MontenegrinBioka,ItalianFrancaviddaandCroatian technologicalfeaturesofsomeKratošijavarietiesin RepublicMacedonia).In : Proceedings of the XII ZlataricaVrgorskarepresentanewgroupof Conference of Viticulturists and Winemakers of synonyms. Serbia .Papersanthology,Niškabanja,pp41-44. Overall,ourgenotypingstudygaveustheopportunity Bulić S.,1949. Dalmatinska Ampelografija (Dalmatian tochecktheavailableinformationbasedon ampelography). PoljoprivredniNakladniZavod, Tisak ampelographicdescriptionsandcomparisons, NakladnogHrvatskogZavoda,Zagreb. providingamorestringentandsometimesnew Burić D.,1995. Savremeno Vinogradarstvo (Contemporary perspectiveontheoriginsandrelatednessofgrapevine viticulture).Nolit,Beograd. cultivarsinMontenegro. CalòA.,CostacurtaA.,Maraš V.,MeneghettiS.and Furtheranalysesshouldbedoneinordertoclarifythe CrespanM.,2008.MolecularcorrelationofZinfandel (Primitivo)withAustrian,CroatianandHungarian geneticrelationshipsamongMontenegringrapevine cultivarsandKratošija,anadditionalsynonym. Am. cultivarsaswellasthedegreeofrelatednessand J. Enol.Vitic. , 59 ,205-209. originofcommerciallyimportantMontenegrin cultivars. Cindrić P.,1994. Sorte Vinove Loze (Grapevinevarieties). NoviSad. Thesharing ofcommoncultivarsamongneighbouring Cindrić P.,KovačV. andKorać N.,2000. Sorte Vinove countries wasexpected asanatural consequenceof Loze .Poljoprivrednifakultet :Prometej(Grapevine easilyvegetativepropagation ofgrapevine , but only varieties.FacultyofAgriculture :Prometej).Novi recentlywehavestartedtogetaccurateinformation Sad.
J. Int. Sci. Vigne Vin , 2014, 48 , 87-97 - 95 - ©Vigne et Vin Publications Internationales (Bordeaux, France) 01-MARAS_05b-tomazic 18/07/14 15:52 Page96
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CiprianiG.,SpadottoA.,JurmanI.,DiGasperoG., Maleti ć E.,Peji ć I.,Konti ć J.K.,PiljacJ.,DanglG.S., CrespanM.,MeneghettiS.,FrareE.,VignaniR., VokurkaA.,LacombeT.,Mirosevi ć N. andMeredith CrestiM.,MorganteM.,PezzottiM.,PèE.,Policriti C.P.,2004.Zinfandel,Dobricic,andPlavacmali :the A.andTestolinR.,2010.TheSSR-basedmolecular geneticrelationshipamongthreecultivarsofthe profileof1005grapevine( Vitis vinifera L.)accessions DalmatiancoastofCroatia. Am. J. Enol. Vitic. , 55 , uncoversnewsynonymyandparentages,andrevealsa 174-180. largeadmixtureamongstvarietiesofdifferent Maletić E.,PejićI. andKaroglanKontić J.,2009. Plavac geographicorigin. Theor. Appl. Genet. , 121 ,1569- Mali - Hrvatska Sorta za Velika Vina (Plavacmali-A 1585. Croatiangrapeforgreatwines).GrozdPlavacmali CrespanM.,2003.TheparentageofMuscatofHamburg. d.o.o.,Zagreb. Vitis , 42 ,193-197. Maraš V.,2000.Ampelografskekarakteristikevarijeteta CrespanM.,CabelloF.,GiannettoS.,IbáñezJ.,Kontić sortevinoveloze.Poljoprivrednifakultet J.K.,Maletić E.,Pejić I.,Rodriguez-TorresI.and (Ampelographicfeaturesofgrapevinesvarieties. AntonacciD.,2006.MalvasiadelleLipari,Malvasia FacultyofAgriculture). Doctoral dissertation , diSardegna,GrecodiGerace,MalvasiadeSitgesand Zemun-Beograd. Malvasiadubrovačka–synonymsofanoldand Maraš V.,MilutinovićM. andPejović L.,2004.Variability famousgrapecultivar. Vitis , 45 ,69-73. intheautochthonousvinevarietyKratošija. Acta DiVecchi-StarazM.,BandinelliR.,BoselliM.,ThisP., Hortic. , 640 ,237-241. BoursiquotJ.M.,LaucouV.,LacombeT.andVarès Maraš V.,Tomić M.,GazivodaA.,Raičević J.,ŠućurS., D.,2007.Geneticstructuringandparentageanalysis KodžulovićV. andPavićević A.,2014.Researchon forevolutionarystudiesingrapevine :kingroupand originandgeneticsanitaryevaluationof originofthecultivarSangioveserevealed. J. Am. Soc. autochthonousgrapevinevarietiesinMontenegro.In : Hort. Sci. , 132 ,514-524. Proceedings of the International Symposium for DzhambazovaT.,TsvetkovI.,AtanassovI.,RusanovK., Agriculture and Food ,Skopje,Republicof MartínezZapaterJ.M.,AtanassovA.andHvarlevaT., Macedonia,2012(inpress). 2009.GeneticdiversityinnativeBulgariangrapevine MarshallT.C.,SlateJ.,KruukL.E.B.andPembertonJ.M., germplasm( Vitis vinifera L.)basedonnuclearand 1998.Statisticalconfidenceforlikelihood-based chloroplastmicrosatellitepolymorphisms. Vitis , 48 , paternityinferenceinnaturalpopulations. Mol. Ecol. , 115-121. 7,639-655. GaletP.,2000. Dictionnaire Encyclopédique des Cépages . NastevD.,1967. Specijano Lozarstvo (Particular Hachette,Paris. viticulture).IzdanienaUniversitetotvoSkopje, HvarlevaT.,RusanovK.,LefortF.,TsvetkovI.,Atanassov Skopje. A.andAtanassovI.,2004.GenotypingofBulgarian Pejović L.,1988. Ampelografska Proučavanja Varijeteta Vitis vinifera L.cultivarsbymicrosatelliteanalysis. Kratošije. Jugoslovensko Vinogradarstvo i Vinarstvo Vitis , 43 ,27-34. (AmpelographicinvestigationsofKratosijavarieties. Jelaska М. ,1954. Osnovi Vinogradarstva na Primorju Yugoslavviticultureandwinemaking).N. 3-4. (Fundamentalsofviticultureatseacoast).Zagreb. Beograd. KalinowskiS.T.,TaperM.L.andMarshallT.C.,2007. Savić S.,2003. Ekološki Uslovi i Autohtone Sorte Vinove RevisinghowthecomputerprogramCERVUS Loze u Crnoj Gori (Ecologicconditionsand accommodatesgenotypingerrorincreasessuccessin autochthonousgrapecultivarsinMontenegro). paternityassignment. Mol. Ecol. , 16 ,1099-1106. HoldingAgrokombinat“13.jul”AD“Plantaže”, LacombeT.,AudeguinL.,BoselliM.,BucchettiB., Podgorica. CabelloF.,ChateletP.,CrespanM.,D’OnofrioC., SefcK.M.,RegnerF.,TuretschekE.,GlösslJ.and EirasDiasJ.,ErcisliS.,GardimanM.,GrandoM.S., SteinkellnerH.,1999.Identificationofmicrosatellite ImazioS.,JandurovaO.,JungA.,KissE.,KozmaP., sequencesin Vitis riparia andtheirapplicabilityfor MaulE.,MaghradzeD.,MartinezM.C.,MuñozG., genotypingofdifferent Vitis species. Genome , 42 , PátkováJ.K.,PejicI.,PeterlungerE.,PitsoliD., 367-373. PreinerD.,RaimondiS.,RegnerF.,SavinG., SavvidesS.,SchneiderA.,SpringJ.L.,SzokeA., StajnerN.,AngelovaE.,BozinovicZ.,PetkovM.and VeresA.,BoursiquotJ.M.,BacilieriR.andThisP., JavornikB.,2009.Microsatellitemarkeranalysisof 2011.GrapevineEuropeanCatalogue :towardsa Macedoniangrapevines( Vitis vinifera L.)compared comprehensivelist. Vitis , 50 ,65-68 . toBulgarianandGreekcultivars. J. Int. Sci. Vigne Vin , 43 ,29-34. LacombeT.,BoursiquotJ.M.,LaucouV.,DiVecchi-Staraz M.,PérosJ.P.andThisP.,2013.Large-scale Stojanović M.,1929. Novo Vinogradarstvo (New parentageanalysisinanextendedsetofgrapevine viticulture). Beograd. cultivars( Vitis vinifera L.). Theor. Appl. Genet ., 126 , ThomasM.R.andScottN.S.,1993.Microsatelliterepeats 401-414. ingrapevinerevealDNApolymorphismswhen
J. Int. Sci. Vigne Vin , 2014, 48 , 87-97 ©Vigne et Vin Publications Internationales (Bordeaux, France) - 96 - 01-MARAS_05b-tomazic 18/07/14 15:52 Page97
analysedassequence-taggedsites(STSs). Theor. Vuksanović P.,1977. Rejonizacija Vinogradarstva Crne Appl. Genet. , 86 ,985-990. Gore (ViticulturezoninginMontenegro).University Ulićević M.,1959. Prilog Rejonizaciji Vinogradarstva u “VeljkoVlahović”AgricultureInstitute,Titograd. Crnoj Gori (Contributiontothezoningofviticulture WelterL.J.,Göktütk-BaydarN.,AkkurtM.,MaulE., inMontenegro).Nasapoljoprivredaisumarstvo, EibachR.,TöpferR.andZyprianE.M.,2007.Genetic num.2/V.Titograd. mappingandlocalizationofquantitativetraitloci Ulićević M.,1966. Prilog Proučavanju Osobina affectingfungaldiseaseresistanceandleaf Najvažnijih Sorata Vinove Loze Gajenih u SR Crnoj morphologyingrapevine( Vitis vinifera L.). Mol. Gori (Contributiontotheresearchofcharacteristicsof Breed. , 20 ,359-374. themostimportantgrapevinevarietiescultivatedin Zdunić G.,PreeceJ.E.,DanglG.S.,KoehmstedtA.,Mucalo federalRepublicMontenegro).Archiveof A.,MaletićE. andPejić I.,2013.Genetic AgriculturalSciences,yearX,sv.231-100. characterizationofgrapevinecultivarscollected VialaP.andVermorelV.,1901-1910. Ampélographie I-IV . throughouttheDalmatianregion. Am. J. Enol. Vitic. , MassonetCie,Paris. 64 ,285-290. Vujović M.,1956. Vrste Domaćih Loza (Domestic grapevinevarieties).Nasapoljoprivredanum.1/II. Titograd.
J. Int. Sci. Vigne Vin , 2014, 48 , 87-97 - 97 - ©Vigne et Vin Publications Internationales (Bordeaux, France)