Genetic clustering and parentage analysis of Western Balkan grapevines (Vitis vinifera L.)

Vi is 54 (Special Issue), 67–72 (2015) Gene ic clus e ing and pa en age analysis o Wes e n Balkan g ape ines (Vi is ini e a L.) N. ŠTAJNER1), L. TOMIĆ), V. PROGAR1), T. POKORN1), T. LACOMBE2), V. LAUCOU2),J. M. BOURSIQUOT3), B. JAVORNIK1) and R. BACILIERI2) 1) Uni e si y o Ljubljana, Bio echnical Facul y, Ljubljana, Slo enia 2) UMR AGAP, Equipe Di e si é Adap a ion e Amélio a ion de la Vigne, INRA, Mon pellie , F ance 3) UMR AGAP, Equipe Di e si é Adap a ion e Amélio a ion de la Vigne, Mon pellie SupAg o, Mon pellie , F ance Co espondence o: D . R. BACILIERI, UMR AGAP, Equipe Di e si é Adap a ion e Amélio a ion de la Vigne, INRA, 2, place Pie e Viala, 34060 Mon pellie Cedex 02, F ance. E-mail: [email p o ec ed] Summa y A o al o 90 g ape ine samples collec ed in i e coun ies o he Wes e n Balkan egion we e e alua - ed o ueness- o- ype and kinship ela ions based on compa a i e analysis wi h 1,130 g ape ine geno ypes held a he INRA "Domaine de Vassal" F ench G ape Ge mplasm Reposi o y, using 14 mic osa elli e ma k- e s. In he con ex o he compa a i e analysis, wen y- ou synonyms/coun e pa s and he pu a i e pa en s o wel e Balkan accessions we e iden i ied. We disco - e ed i e pai s o homonyms, subsequen ly con i ming he iden i y o pa en age o h ee o hem. Some o he examined accessions we e iden i ied ei he on he ba- sis o he geno ypes ound in he li e a u e, o h ough pa en age ela ionships e ealed in his s udy. Fo he emaining i y accessions we we e unable o es ablish ei he hei pedig ee o o iden i y hem on he basis o SSR p o iles a ailable elsewhe e. Finally, he Balkan geno ypes ha we e no well classi ied by synonymy o pa en age analysis we e u he s udied wi h a P in- cipal Coo dina e Analysis o e eal gene ic clus e ing wi hin la ge da ase s o geno ypes. The g aphical dis- play o he indi idual and g oup dis ances showed ha abou o y accessions (85 %) a e s uc u ed wi hin a g oup o Balkan and Eas e n Eu ope geno ypes and only a mino p opo ion esul ed in admixed popula- ion assignmen . K e y w o d s : mic osa elli es; iden i y; synonyms; p inci- pal coo dina e analysis. In oduc ion In ecen decades, p og ess in genome sequencing has enabled he epo ing o hund eds o polymo phic neu al ma ke s, such as mic osa elli es, which ha e p o en o be powe ul ools o pa en al and kinship analysis, including wi h g ape ines. The la ges g ape ine pa en age analysis o da e was pe o med on a da ase o 2,344 unique geno- ypes (LAUCOU e al. 2011) (held in he INRA "Domaine de Vassal" F ench G ape Ge mplasm Reposi o y). The ull pa en age o 828 accessions was iden i ied using 20 mi- c osa elli e loci (LACOMBE e al. 2013). The second la g- es s udy explo ed he pa en age o 1,005 g ape ines and he analysis o kinship unco e ed 74 comple e pedig ees, wi h bo h pa en s iden i ied (CIPRIANI e al. 2010). How- e e , o he smalle pa en age s udies ha e been ca ied ou in se e al coun ies (e.g. MENA e al. 2014). In ou s udy, mic osa elli es we e used o in es iga e he pa en age and o igin o Balkan g ape ines collec ed in i e Wes e n Bal- kan coun ies - Slo enia (SVN), Se bia (SRB), Bosnia and He cego ina (BIH), Macedonia (MKD) and Mon eneg o (MNE). To in eg a e and e alua e he SSR da a o Bal- kan geno ypes in a la ge con ex , he da ase om a la ge scale pa en age s udy (LACOMBE e al. 2013) was chosen o compa ison. The da ase used in his wo k hus encom- passes 1,130 geno ypes om he INRA Domaine de Vassal g ape ine eposi o y (h p://www1.mon pellie .in a. / as- sal; LACOMBE e al. 2013) and 90 geno ypes om he Bal- kan egion (h p:// i is.a cglabs.com). Panels o synonyms/ coun e pa s and pu a i e pa en age-o sp ing ios we e ob ained, which a e p esen ed and ela ed o some exis - ing epo s. The s anda diza ion and in eg a ion p ocedu e o he wo se s o da a is desc ibed, oge he wi h issues ela ed o he linking p ocedu e. The disco e ed gene ic clus e ing in compa ison o he la ge e e ence da ase , a e discussed. Ma e ial and Me hods Plan ma e ial and DNA analysis: The i s s ep o his s udy was he iden i y and pa en age analysis o g ape ine geno ypes om wo ecen ly pub- lished s udies (LACOMBE e al. 2013, ŠTAJNER e al. 2013), using 7 mic osa elli e loci (VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VVS2) ha we e com- mon o he wo s udies. The ob ained esul s sugges ed ha 90 samples om he Balkan egion (Tab. 1) a e in e es ing o u he analysis wi h an addi ional se o loci: VVMD21, VVMD24 (BOW- ERS e al. 1999a), VVIp31, VVI 67, VVIp60, VVIn16, VVIb01 and VVIq52 (MERDINOGLU e al. 2005). S and- a diza ion o allele leng h and panel bins be ween he wo da ase s was ca ied ou using common e e ence samples 'Cabe ne -Sau ignon', 'Cha donnay', 'Me lo ', 'Pino Noi ', 'Sul anine', and 'Tou iga Na ional'. The p ocedu e o plan ma e ial sampling and DNA isola ion was as desc ibed in 68 ŠTAJNER e al. ŠTAJNER e al. (2013) as well as PCR ampli ica ions and geno yping o SSR loci. Da a analysis: Ampli ied agmen s we e an- alysed and sized wi h GeneMappe so wa e e sion 4.0 (Applied Biosys ems, Ca lsbad, Cali o nia). All he phe- og ams we e manually checked and he ela i e sizes o he ampli ied p oduc s we e de e mined. Rela i e allele sizes we e i s s anda dized using six e e ence cul i a s ha we e common o he wo da a se s; his p ocedu e ena- bled us o join ly examine da a om bo h se s o samples. To ca y ou his ask, we w o e se e al cus om R sc ip s o imp o ed managemen o inpu /ou pu da a, dealing speci ically wi h he no maliza ion o SSR da a om wo di e en se s and wi h iden i y analysis. The sc ip s, wi h ins uc ions o use a e eely a ailable om h ps://gi hub. com/ p-uni-lj/ss -link.gi . The i s s ep consis ed in he iden i ica ion o ma ching geno ypes, i.e. coun e pa s be ween he wo da ase s. The synonymous geno ypes ob ained by he R sc ip s we e also manually inspec ed. Da a we e subsequen ly analysed wi h Ce us 3.0 so wa e (MARSHALL e al. 1998; KALINOWSKI e al. 2007). Pa en age analysis was pe o med in wo s eps; 1) we an a simula- Table 1 Lis o he 90 g ape ine accessions om Wes e n Balkans and hei passpo da a Accession Name Coun y o sampling P ime Name Va ie y No. VIVC Accession Name Coun y o sampling P ime Name Va ie y No. VIVC Babić BIH Babic 844 Muska Ruža SRB Musca Rouge de Made e 8249 Bačka SRB - 21272 Muš oš Fehe SRB Mus oasa de Made a 8311 Bela Dinka SRB - 16848 Neznana Bela SVN - - Bela Zgodnja SVN Csaba Gyoengye 9166 Oh idsko Belo MKD Oh idsko Blanc 21403 Beli Medenac SRB - - Pla ac Mali BIH Pla ac Mali 9549 Belo zimsko SRB Opsimos Edessis 8789 Pla ka BIH Pla ina C na 9557 Belo ina MKD - 42060 Plo dina C na SRB - - Bena BIH - 1129 Poljšakica SVN 17712 Bla ina BIH Bla ina 1454 P okupac BIH P okupac 9734 C n Valand. D enok MKD Pa mak Ce en 8945 P okupac SRB - - Čauš MKD Chaush Che nyi 7307 P ošip BIH 24630 Čauš Bel MKD Chaouch Blanc 10196 P ošip Bijela BIH - - Dob ogos ina BIH T bjan 12619 Rado ača BIH 24598 Dolga Pen lja SVN Dolga Pe la 17675 Rado ača MNE A us Ali 122 Dolgi G ozdi SVN Pelena, Vi o ska G ganja 24459 Razaklija BIH - - D enak BIH - - Rebula SVN Ribolla Gialla 10054 D enak Beli SRB - 16564 Rebula Po alis SVN - - D enak C ni SRB Ho a 5423 Re osco SVN Re osco dal Peduncolo Rosso 9987 D enjak BIH Rude usa 10327 Re ošk SVN Re osk Te ano 12374 Elezo ka BIH - - Rezaklija BIH 24131 Fu min SRB Knippe le 6312 Ružica SRB Koe idinka 13727 Ga an SRB Gek C ni 5067 Ružica Mi isa a SRB - - Gne K as SVN - 24599 Ružica V MNE Pamid 8899 Gnje SVN Piccola Ne a 9235 Ružica VI MNE - - Godominka SRB - 16003 Sipa SVN Osipka 17705 G k BIH - - Slankamenka C ena SRB Pamid 8899 G očanka SRB 5069 Smede e ka BIH Dimya 5716 Ha sle eli SRB Ha sle elue 5314 S emska Zelenika SRB 15934 Kada ka SRB - - S anušina MKD S anusina C na 11994 Kada ka Bela SRB Kada ka Fehe 5899 S a a Žila ka BIH T bjan 12619 Kada un BIH Kada un 5900 S a i Rizling Mon eng o - - Ka čina SRB Koelne Blau 6344 Su ac Pla i BIH - - Kolana SVN - - Šipon SVN Fu min 4292 Končanka MKD - 22962 Šlji a BIH Slji a He cego acka 23206 K a ošija MNE P imi i o 9703 Tamjanika C na SRB Tamjanika C na 8057 K eaca SRB K eaca 6501 T bljan Beli SRB T bjan 12619 K i ača Bijela BIH K i alja Bijela 10050 T njak BIH - - K i aja SRB - - T njak K upni BIH Rude usa 10327 K košija BIH 16850 U ban C eni SRB U ban 12781 K košija Šupljica SRB Kujundzusa Bela 6545 V anac BIH V anac 13179 Kujundžuša BIH Kujundzusa Bela 6545 Žame o ka SVN Ka cina C na 6047 Mal azija BIH - - Žila ka BIH Zila ka 13446 Manas i sko Belo MKD 24597 Žižak MNE - - Ma bu ge SVN Neubu ge 8501 Žlozde BIH Medna 7584 Menigo ka BIH I alia 5582 Žunić SRB 15923 Gene ic clus e ing and pa en age analysis o Wes e n Balkan g ape ines 69 ion o pa en age analysis o es ima e he esol ing powe o a se ies o mic osa elli e loci and 2) pa en age analysis was pe o med o es candida e pa en s agains o sp ing and, o each o sp ing es ed, o assign he mos -likely candida e pa en wi h a p e-de e mined le el o con i- dence, o le i unassigned. LOD (loga i hm (base 10) o odds) sco es we e assigned o each possible pa en and pa - en pai . The gene ic clus e ing o Balkan samples was ob- ained wi h a P incipal Componen Analysis (PCA) using he package adegene implemen ed in R (JOMBART 2008). The in e nal s uc u e o he da a and quan i a i e a i- ables we e calcula ed o p ede e mined g oups o Vassal samples based on STRUCTURE clus e ing as epo ed by BACILIERI e al. (2013). Resul s I d e n i y a n a l y s i s : Based on he iden i y anal- ysis, wen y- ou Balkan geno ypes (Tab. 2) ound a coun- e pa in he Vassal da ase . Two pai s o nea synonyms di e ing in one allele only we e addi ionally iden i ied: 'Beli Medenac' and 'Lisz es ehe ' #2808 di e ed by 2 bp a locus VVIp60 and 'G očanka' and 'G ocanka' #3637 by 4 bp a locus VVIb01. In addi ion, he coun e pa s we e ound among published SSR da a o se en accessions: 'Ka- da ka Bela' = 'Kada ka Byala' (DZHAMBAZOVA e al. 2009), 'K eaca' = 'Bana i Rizling' (GALBACS e al. 2009), 'Mus os Fehe ' = 'Mus os' (GALBACS e al. 2009), 'Tamjanika C na' = 'Muska Ruza Po ecki' (MALETIĆ e al. 1999), 'Žame o ka' = 'Schwa zg obe' (SEFC e al. 2000), 'Zimsko Belo' = 'Ka a- soba' (SEFC e al. 2000) = 'Ka a so a Naousis' (G eek Vi is Da abase) = 'Opsimos Le ko' (G eek Vi is Da abase) and 'P ošip Bijela' = 'Volo nik' (ŠTAJNER e al. 2008) = 'Vela Pe golla' (MALETIĆ e al. 1999; SEFC e al. 2001). P a e n a g e a n a l y s i s : Pa en age analysis based on 14 mic osa elli e ma ke s esul ed in a panel o po en- ially in e es ing pa en /o sp ing ios including Balkan geno ypes ha we e shown o be in ei he pa en o o - sp ing ela ionship wi h geno ypes om he Vassal collec- ion (Tab. 3). P incipal Coo dina e Analysis: Finally, he Balkan geno ypes ha we e no unco e ed by he mean- ing o synonymy we e u he analysed wi h PCA (Figu e). The PCA analysis e lec s he ela ionships among g oups om Vassal collec ion based on he STRUCTURE clus e - ing (A3 - Wes e n and Cen al Eu ope geno ypes; B3 - Fa - and Middle-Eas geno ypes and C3 - Balkan and Eas Eu- ope geno ypes) and hei dis ance o he Balkan geno ypes e e ed as D3 g oup. The PCA g ouping shows ha D3 Balkan samples a e posi ioned close o he C3 samples ha ep esen Balkan and Eas Eu ope geno ypes in he s udy o BACILIERI e al. (2013). Discussion Mos o he synonyms iden i ied in his s udy (Tab. 2) we e no epo ed in he ele an pape s published ecen ly (BEŠLIĆ e al. 2012, ZDUNIĆ e al. 2013, MARAŠ e al. 2014, SCHNEIDER e al. 2014). Among wen y- ou associa ions ound wi hin wo s udies, he highes pa o he Vassal coun e pa a e o Balkans o igin (67 %) and he es , ex- cep wo, a e o I alian o igin o which BACILIERI e al. (2013) epo ed high le el o admix u e as ega ds o ge- ne ic composi ion o geog aphic o igin. The pa en age analysis esul ed in iden i ica ion o pa en ages o wel e au hen ic Balkan geno ypes ha ing a pa en ei he om ou se o samples o om he Vassal collec ion and o ou knowledge hey ha e no ye been desc ibed in he li e a u e. All a ie ies om Vassal ha e been assigned a geog aphic o igin p esen ed in Tab. 3, whe e we can see ha e en when one pa en is iden i ied in Vassal, s ill, mos o he ime i is o Balkans o igin. The only no able excep ions a e wo pa en al accessions, one o which is 'Te ano' ha is conside ed o I alian o igin, bu is widely g own also in Slo enia and C oa ia and he second is 'Mal asia del Chian i' being pa o a Mal asia g oup de i ed om G eece. Ano he wo pa en al acces- sions o non-Balkan o igin a e 'Gouais blanc' = 'Heunisch weiss', one o he oldes a ie ies in he wo ld, used in se - e al c osses and 'Rosenmuska ' a Musca used o in oduce he musca la ou . Th ee ou o wel e o sp ing desc ibed in pa en age analysis a e able g ape ines ('G očanka', 'K i aja', and 'Šlji a') and hei candida e pa en s a e also able g ape- ines. The same indings we e epo ed by LACOMBE e al. (2013), who s a ed ha compa ison o he ui use o pa - en s o a gi en p ogeny showed a majo i y o c osses us- Table 2 Lis o iden ical g ape ine geno ypes based on ma ching allele sizes o analysed loci Balkan sample INRA sample (LACOMBE e al. 2013) Bela Zgodnja (SVN) Pe le de Csaba (#1069) (HUN) Čauš Bel (MKD) Chaouch ose (#1674) (TUR) D enak (BIH) Rosa menna di acca (#1662) (ROU) D enak Beli (SRB) Coa na alba (#749) (ROU) D enak C ni (SRB) Da kaia noi (#728) (MAR) Elezo ka (BIH) Chaouch blanc (#1673) (TUR) Fu min (SRB) Knippe le (#283) (FRA) Gnje (SVN) Piccola ne a (#2387) (ITA) Ha sle elu (SRB) Ha sle elu (#1609) (HUN) K a osija (MNE, MKD) P imi i o = Zin andel (#1277) (ITA) Ma bu ge (SVN) Neubu ge (#2172) (AUT) Menigo ka (BIH) I alia = Pi o ano 65 (#926) (ITA) Muska Ruza (SRB) Musca ouge de Madè e = Mosca o iole o (#576) (ITA) Pla ka (BIH) Pla ina c na (#1843) (HRV) P okupac (BIH) P okupac (#1630) (SCG) Rado aca (MNE) Da ie de Bey ou h = A uz Ali (#634) (TUR) Re osk (SVN) Te ano (#1293) (ITA) Rezaklija’ (BIH) Razachie osie (#1887) (ROU) Ruzica (SRB) Ko idinka (#1578) (YUG) Šipon (SVN) Fu min (#25) (HUN) Smede e ka (SRB) Dimia (#1666) (BGR) S em Zelenika (SRB) Sze emi zöld (#1623) (HUN) T bljan Beli (SRB) Mos osa = Empibo e bianco (#2054) (ITA) Žila ka (BIH) Zila ka (#1637) (BIH) 70 ŠTAJNER e al. ing pa en s om he same geog aphical a ea and wi h he same g ape use. The main Balkan geni o s iden i ied in ou analysis a e 'Hep akilo' (#743), a adi ional able cul i a om G eece and 'Rosa menna di acca' (#1662), a adi- ional able cul i a om Romania. These indings a e in ag eemen wi h he his o ical iew; g ape ines a i ed in he Balkan Peninsula om Cen al Asia ia he G eek is- lands and he Romans con inued o sp ead he cul i a ion o g ape ine on he Ad ia ic islands and on he mainland. Du ing he pe iod o he O oman Empi e, he p oduc ion o able g apes inc eased and was mo e widesp ead o e he egion (SAVIĆ 2003). Se e al in e es ing hypo heses ha e a isen conce ning some pu a i e ios; in oduced cul i a s ha e o en been Table 3 Lis o ull pa en ages de ec ed o Balkan g ape ine accessions. The symbol # e e s o he accession numbe in he Vassal collec ion (h p://bioweb.ensam.in a. /collec ions_ igne/) O sp ing Fi s candida e Second candida e T io loci compa ed T io loci misma ching T io LOD sco e Dob ogos ina (BIH) = S a a Žila ka (BIH) K košija (BIH) Rosenmuska (#3550) (DEU) 14 0 20 Godominka (SRB) Musca O onel (#280) (FRA) Smede e ka (SRB) = Dimia (#1666) (BGR) 14 1 23 G očanka (SRB) Bela Zgodnja (SVN) = Pe le de Csaba (#1069) (HUN) Rado aca (MNE) = Da ie de Bey ou h = A uz Ali (#634) (TUR) 15 0 30 K i aja (SRB) D enak (BIH) = Rosa menna di acca (#1662) (ROU) Rezaklija (BIH) = Razachie osie (#1887) (ROU) 14 0 23 Manas i sko Belo (MKD) D enak (BIH) = Rosa menna di acca (#1662) (ROU) Hep akilo (#743) (GRC) 14 0 31 Polšakica (SVN) Mal asia del Chian i (#1352) (ITA) P ošip Bijela (BIH) 14 1 27 P okupac (SRB) P okupac (BIH) = P okupac (#1630) (SRB) Re ošk (SVN) =’Te ano’ (#1293) (ITA) 14 0 32 Šlji a (BIH) D enak Beli (SRB) = Coa na alba (#749) (ROU) Hep akilo (#743) (GRC) 14 0 27 Žila ka (BIH) Albaimpu o a o (#44) (ROU) Dob ogos ina (BIH) 14 0 21 Bag ina (SRB) Beli Medenac (SRB) B aghina=Dinka o os (#1670) (HUN) 13 0 17 K eaca (SRB) D enak Beli (SRB) = Coa na alba (#749) (ROU) Pla ac Mali (#3144) (HRV) 14 1 14 P ošip (BIH) D enak Beli (SRB) = Coa na alba (#749) (ROU) Gouaisblanc = Heunisch weiss (#211) (FRA) 14 0 13 Figu e: P incipal componen analysis on join SSR da a A) o non-iden i ied Balkan samples (D3 g oup) and samples om Vassal collec ion subdi ided in 3 s uc u e g oups as in BACILIERI e al. (2013): A3S, Wes e n and Cen al Eu ope geno ypes, B3S, Fa - and Middle-Eas geno ypes, and C3S, Balkan and Eas Eu ope geno ypes) and B) o samples o D3 g oup g aphically displaying hei in- di idual dis ances shown on he same axes as in Figu e A; i p esen s he enla ged pic u e o Figu e A, including names o accessions. Gene ic clus e ing and pa en age analysis o Wes e n Balkan g ape ines 71 gi en local names and ha e e en ually been conside ed na- i e old cul i a s. Fo example 'Žila ka' was conside ed o be a e y old Bosnia and He zego ina au hen ic cul i a . The pa en age analysis by LACOMBE e al. (2013) showed one o he p ogeni o s o 'Žila ka' o be 'Fu min ' (#25) o 'Alba impu o a o' (#44) o 'Gohe ' (#2244). MALETIĆ e al. (1999) epo ed ha DNA p o iling has sugges ed a gene - ic ela ionship be ween 'Žila ka' and 'Te an Bijeli' ('P os- ecco') g own in Is ia. Ou esul s sugges ha 'Žila ka' migh be he o sp ing o an old emale Romanian geni o 'Alba impu o a o' (#44) and ano he BIH accession 'Do- b ogos ina' = 'S a a Žila ka' (Tab. 3). The e a e no o mal eco ds on b eeding and selec ion o 'Žila ka', despi e i being a e y amous g ape ine, pa icula ly in BIH, and ha ing been g own o mo e han 600 yea s in he egion o He zego ina. The Bosnian King T ko was men ioned as d inking 'Žila ka' wine in he 14 h cen u y (VUKSANOVIĆ and KOVAČINA 1984). Wine made om 'Žila ka' is economi- cally impo an and e y popula in BIH and also in some adjacen egions o C oa ia, Macedonia, Mon eneg o and Se bia, whe e i is also g own (TOMIĆ e al. 2012). In he ield o g ape ine s udies, he e is s ill an ongo- ing deba e abou he o igin o he synonymic geno ypes: Cali o nian 'Zin andel', C oa ian 'C ljenak Kaš elanski', Mon eneg ian 'K a ošija' and I alian 'P imi i o' (MALETIĆ e al. 2004; CALÒ e al. 2008; LACOMBE e al. 2007; CIPRI- ANI e al. 2010). Some da a sugges a possible i s -deg ee ela ionship be ween he cul i a s 'K a ošija' and 'V anac' (MALETIĆ e al. 2004, CALÒ e al. 2008) and a pa en -o - sp ing ela ionship o 'K a ošija' wi h 'Pla ina' and 'V anac' has been p oposed (MALETIĆ e al. 2004; CALÒ e al. 2008). O he s ha e sugges ed ha 'Pla ina' (= 'Pla ina c na') could be an o sp ing o 'K a ošija' and 'Ve deca', a whi e cul i a g own in sou he n I aly (LACOMBE e al. 2007, CIPRIANI e al. 2010, LACOMBE e al. 2013). In ou analysis, wo ac- cessions o 'K a ošija', o igina ing om Mon eneg o and Macedonia, esul ed in iden ical allelic p o iles and we e he e o e con i med as synonyms and, on he basis o com- pa ison o geno ypes o he Vassal collec ion, hey we e also con i med as synonyms o he cul i a 'P imi i o' = 'Zin andel' (#1277). In ou pa en age analysis, 'V anac' and 'Pla ina c na' (#1843) we e one o he mos likely candida e pa en pai s o 'K a ošija' ( esul s no shown) bu , in a compa ison o he io p oposed by LACOMBE e al. (2013), by which 'Pla ina c na' is an o sp ing o 'K a ošija' (= 'P imi i o' = 'Zin andel') and 'Ve deca' (= 'Lago hi'), he la e esul ed in a highe LOD sco e alue han he o me , wi h assigned LOD sco es o 22.5 and 15.2, espec i ely. 'K a ošija' is an old Mon eneg in g ape accession ha , ac- co ding o he li e a u e, exis ed in hese egions be o e 'V anac', al hough 'V anac' oday p edomina es in Mon- eneg o and is o en cul i a ed oge he wi h 'K a ošija' (ULIĆEVIĆ 1966, PEJOVIĆ 1988, MARAŠ e al. 2003, 2004, 2014). MARAŠ e al. (2014) e idenced ha ‘K a ošija’ wi h all i s bio ypes has been g own in Mon eneg o o cen u ies and sugges ed ha his egion be conside ed a likely egion o he o igin and sp eading cen e o 'K a ošija'. Acco d- ing o STRUCTURE clus e ing epo ed by BACILIERI e al. (2013) cul i a s 'K a ošija' and 'V anac' we e conside ed as admixed, wi h 61 % and 72 % o i s genome belonged o S-3.3 clus e (Balkan and Eas e n Eu ope g oup), e- spec i ely. Du ing he i s in en o y o Balkan samples, we ound ha 'P okupac' ob ained om he S emski Ka - lo ci (Se bia) collec ion ineya d (whe e his cul i a is also b ed) esul ed in a dis inc allelic p o ile as 'P okupac' om Da is collec ion, (USA), bu hey sha ed 73 % o al- leles analysed a 22 loci (ŠTAJNER e al. 2013). The p esen analysis simila ly con i med ha ‘P okupac’ om Se bia is di e en om 'P okupac' (#1630) om he Vassal col- lec ion. In con as , ano he 'P okupac' om BIH esul ed in a geno ype iden ical o P okupac om Da is collec- ion and P okupac om Vassal collec ion. The pa en age analysis e ealed ha 'P okupac' om he S emski Ka - lo ci collec ion ineya d migh be a p ogeny o " ue- o- ype" 'P okupac' and 'Te ano', showing high LOD sco es (31.6) and no allele misma ch. A possible explana ion o his misnome is ha p ogenies o a pa icula pa en we e some imes ea ed he same as he pa en cul i a due o misleading documen a ion o e en s o possible mislabel- ling. The PCA analysis shows ha Balkan samples (D3, Figu e A) o e lapped wi h he geno ypes om Balkan and Eas e n Eu ope (C3S g oup). Hence, he geno ypes g aphi- cally displayed inside he D3 ellipse migh be cha ac e ized as ypically au och honous Balkan accessions (Figu e B). On he o he hand, he accessions ha a e ou o ellipse and a e classi ied be ween wo di e en g oups, e.g. 'Bla ina' and 'P osip Bijela' p obably a e ecen c osses be ween one able and one Wes e n geno ype, wi h one pa en in he A3 and he o he in he B3 g oup. Few o he analysed Balkan accessions a e shown o be nea o ei he A3 g oup ('Bena', 'Sipa', 'Re osco') o B3 g oup ('Manas i sko Belo', 'K i ača Bijela', Figu e B), which e lec s hei admixed o igin and co esponds o a ious b eeding ac i i ies in he Wes e n Balkan egion. Howe e , hese non-Balkan accessions ep esen a mino p opo ion o he da ase , while abou o y (85 %) analysed cul i a s a e conside ed ue au och- honous Balkan ge mplasm. Conclusions Pa en age analysis and PCA showed o be e y alu- able ools o iden i ica ion o ela ionships. Many o he pa en ages e ealed in his s udy we e p e iously no known and a e o conside able his o ical in e es . The PCA showed ha 44 accessions (85 %) a e s uc u ed wi hin a g oup o Balkan and Eas e n geno ypes and only a mino p opo ion esul ed in admixed popula ion assignmen (Figu e B). Howe e , he iden i y o app oxima ely o y Balkan accessions emains only pa ially explained so a . Some o hese a e p esumably old adi ional g ape ines, wi h a pu a i e Balkan o igin, and a e he e o e wo h ex- plo ing o hei o igin and iden i y. Acknowledgemen s The esea ch was unded by he Eu opean Commission’s Join Resea ch P ojec o he Join Call o he SEE-ERA.NET 72 ŠTAJNER e al. PLUS, g an no. ERA 155/01 and by he Slo enian Resea ch Agency, g an no. P4-0077. 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