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. The s udy was concei ed wi hin he
amewo k o FA COST Ac ion FA1003 "Eas -Wes Collabo a-
ion o G ape ine Di e si y Explo a ion and Mobiliza ion o
Adap i e T ai s o B eeding".
Re e ences
BACILIERI, R.; LACOMBE, T.; LE CUNFF, L.; DI VECCHI-STARAZ, M.; LAUCOU,
V.; GENNA, B.; PÉROS, J. P.; THIS, P.; BOURSIQUOT, J. M.; 2013: Ge-
ne ic s uc u e in cul i a ed g ape ines is linked o geog aphy and
human selec ion. BMC Plan Biol. 13, 25.
BEŠLIĆ, Z.; TODIĆ, S.; KORAĆ, N.; LORENZI, S.; EMANUELLI, F.; GRANDO,
M. S.; 2012: Gene ic cha ac e iza ion and ela ionships o adi ion-
al g ape cul i a s om Se bia. Vi is 51,183-189.
BOWERS, J. E.; BOURSIQUOT, J. M.; THIS P.; CHU, K.; JOHANSSON, H.; ME-
REDITH, C.; 1999: His o ical gene ics: he pa en age o Cha donnay,
Gamay, and he wine g apes o No heas e n F ance. Science 285,
1562-1565.
CALÒ, A.; COSTACURTA, A.; MARAS, V.; MENEGHETTI, S.; CRESPAN, M.;
2008: Molecula co ela ion o Zin andel (P imi i o) wi h Aus ian,
C oa ian, and Hunga ian cul i a s and K a osija, an addi ional syno-
nym. Am. J. Enol Vi ic. 59, 2205-209.
CIPRIANI, G.; SPADOTTO, A.; JURMAN, I.; DI GASPERO, G.; CRESPAN, M.;
MENEGHETTI, S.; FRARE, E.; VIGNANI, R.; CRESTI, M.; MORGANTE, M.;
PEZZOTTI, M.; PE, E.; POLICRITI, A.; TESTOLIN R.; 2010: The SSR
based molecula p o ile o 1005 g ape ine (Vi is ini e a L.) acces-
sions unco e s new synonymy and pa en ages, and e eals a la ge
admix u e amongs a ie ies o di e en geog aphic o igin. Theo .
Appl. Gene . 121, 1569-1585.
DZHAMBAZOVA, T.; TSVETKOV, I.; ATANASSOV, I.; RUSANOV, K.; MARTINEZ-
ZAPATER, J. M.; ATANASSOV, A.; HVARLEVA, T.; 2009: Gene ic di-
e si y in na i e Bulga ian g ape ine ge mplasm (Vi is ini e a L.)
based on nuclea and chlo oplas mic osa elli e polymo phism. Vi is
48, 115-121.
GALBACS, Z.; MOLNAR, S.; HALASZ, G.; KOZMA, P.; HOFFMANN, S.;
KOVACS, L.; VERES, A.; GALLI, A.; SZÖKE, A.; HESZKY, L.; KISS, E.;
2009: Iden i ica ion o g ape ine cul i a s using mic osa elli e-
based DNA ba codes. Vi is 48, 17-24.
JOMBART, T.; 2008: adegene : a R package o he mul i a ia e analysis o
gene ic ma ke s. Bioin o ma ics 24,1403-1405.
KALINOWSKI, S. T.; TAPER, M. L.; MARSHALL, T. C.; 2007: Re ising how he
compu e p og am CERVUS accommoda es geno yping e o in-
c eases success in pa e ni y assignmen . Mol Ecol. 16, 1099-1106.
LACOMBE, T.; BOURSIQUOT, J. M.; LAUCOU V.; DI VECCHI-STARAZ M.; PÉROS,
J. P.; THIS, P.; 2013: La ge-scale pa en age analysis in an ex ended
se o g ape ine cul i a s (Vi is ini e a L.). Theo . Appl. Gene .
126, 401-414.
LACOMBE, T.; BOURSIQUOT, J. M.; LAUCOU, V.; DECHESNE, F.; VARES, D.;
THIS, P.; 2007: Rela ionships and gene ic di e si y wi hin he acces-
sions ela ed o Mal asia held in he Domaine de Vassal g ape ge m-
plasm eposi o y. Am. J. Enol Vi ic. 58, 124-131.
LAUCOU, V.; LACOMBE, T.; DECHESNE, F.; SIRET, R.; BRUNO, J. P.; DESSUP, M.;
DESSUP, T.; ORTIGOSA, P.; PARRA, P.; ROUX, C.; SANTONI, S.; VARES,
D.; PEROS, J. P.; BOURSIQUOT, J. M.; THIS P.; 2011: High h oughpu
analysis o g ape gene ic di e si y as a oll o ge mplasm collec ion
managemen . Theo . Appl. Gene . 122, 1233-1245.
MALETIĆ, E.; PEJIĆ, I.; KAROGLAN KONTIĆ, J.; PILJAC, J.; DANGL, G. S.;
VOKURTA, A.; LACOMBE, T.; MIROŠEVIĆ, N.; MEREDITH, C. P.; 2004:
Zin andel, Dob ičić, and Pla ac mali: The gene ic ela ionship
among h ee cul i a s o he Dalma ian coas o C oa ia. Am. J.
Enol. Vi ic. 55, 174-180.
MALETIĆ, E.; SEFC, K. M.; STEINKELLNER, H.; KAROGLAN-KONTIĆ, J.;
PEJIĆ, I.; 1999: Gene ic cha ac e iza ion o C oa ian g ape ine cul i-
a s and de ec ion o synonymous cul i a s in neighbou ing egions.
Vi is 38, 79-83.
MARAŠ, V.; MILUTINOVIC, M.; PEJOVIĆ, L.; 2003: Va iabili y in he au och-
honous ine a ie y K a osija. Ac a Ho ic. 640, 613-619.
MARAŠ, V.; PEJOVIĆ, L.; TARAILO, R.; MARTINOVIC, A.; 2004: Vine p o-
duc i i y in au och honous a ie y o K a osija in Mon eneg o. Ac a
Ho ic. 652, 213-217.
MARAŠ, V.; BOZOVIC, V.; GIANNETTO, S.; CRESPAN, M.; 2014: SSR molecu-
la ma ke analysis o he g ape ine ge mplasm o Mon eneg o. J.
In . Sci. Vigne Vin 48, 87-97.
MARSHALL, T. C.; SLATE, J.; KRUUK, L. E. B.; PEMBERTON, J. M.; 1998:
S a is ical con idence o likelihood-based pa e ni y in e ence in
na u al popula ions. Mol. Ecol. 7, 639-655.
MENA, A.; MARTÍNEZ, J.; FERNÁNDEZ-GONZÁLEZ, M.; 2014: Reco e y,
iden i ica ion and ela ionships by mic osa elli e analysis o ancien
g ape ine cul i a s om Cas illa-La Mancha: he la ges wine g ow-
ing egion in he wo ld. Gene . Res. C op E ol. 61, 625-637.
MERDINOGLU, D.; BUTTERLIN, G.; BEVILACQUA, L.; CHIQUET, V.; ADAM-
BLONDON, A. F.; DECROOCQ, S.; 2005: De elopmen and cha ac e i-
za ion o a la ge se o mic osa elli e ma ke s in g ape ine (Vi is
ini e a L.) sui able o mul iplex PCR. Mol B eed. 15, 349-366.
PEJOVIĆ, L.; 1988: Ampelog a ska p ouča anja a ije e a K a ošije. Ju-
goslo ensko inog ada s o i ina s o [Ampelog aphic in es iga-
ions on K a ošija bio ypes]. Jugosla ian Vi ic. Enol. 3-4, Belg ade.
R CORE TEAM; 2012: R: A Language and En i onmen o S a is ical Com-
pu ing. R Founda ion o S a is ical Compu ing, Vienna, Aus ia;
h p://www.R-p ojec .o g WebCi e
SAVIĆ, S.; 2003: Ekološki uslo i i au oh one so e ino e loze u C noj
Go i. Holding Ag okombina . 13. jul. AD Plan aže, Podgo ica.
SCHNEIDER, A.; RAIMONDI, S.; PIROLO, C. S.; TORELLO MARINONI, D.;
RUFFA, P.; VENERITO, P.; LA NOTTE, P.; 2014: Gene ic cha ac e iza ion
o g ape cul i a s om Apulia (Sou he n I aly) and synonymies in
o he medi e anean egions. Am. J. Enol. Vi ic. 65, 2.
SCHUELKE, M.; 2000: An economic me hod o he luo escen labelling o
PCR agmen s. Na . Bio echnol. 18, 233-234.
SEFC, K. M.; LOPES, M. S.; LEFORT, F.; BOTTA, R.; ROUBELAKIS-ANGELAKIS,
K. A.; IBÁÑE Z. J.; PEJIĆ, I.; WAGNER, H. W.; GLÖSSL, J.; STEINKELL-
NER, H.; 2000: Mic osa elli e a iabili y in g ape ine cul i a s om
di e en Eu opean egions and e alua ion o assignmen es ing o
assess he geog aphic o igin o cul i a s. Theo . Appl. Gene . 100,
498-505.
ŠTAJNER, N.; RUSJAN, D.; KOROŠEC-KORUZA, Z.; JAVORNIK, B.; 2008: Mi-
c osa elli e geno yping o old Slo enian g ape ine a ie ies (Vi is
ini e a L.) o he P imo je (coas al) wine g owing egion. Vi is 47,
201-204.
TOMIĆ, L.; ŠTAJNER, N.; JOVANOVIĆ CVETKOVIĆ, T.; 2012. Collec ion and
gene ic cha ac e iza ion o Vi is ini e a 'Žila ka' by mic osa elli es
and AFLP ma ke s. Ac a Ag ic. Slo enica 99, 121-173.
ULIĆEVIĆ, M.; 1966: P ilog p ouča anju osobina naj ažnijih so a a ino e
loze gajenih u SR C noj Go i. A hi za poljop i edne nauke [Con i-
bu ion o cha ac e iza ion o he mos impo an g ape ine a ie ies
g own in Republic o Mon eneg o]. A ch. Ag ic. Sci. 23, 1-100.
ZDUNIĆ, G.; PEJIC, I. ; KONTIC, J. K. ; VUKICEVIC, D. ; VOKURKA, A. ; PEZO, I.;
MALETIC, E.; 2008: Compa ison o gene ic and mo phological da a
o in e ing simila i y among na i e Dalma ian (C oa ia) g ape ine
cul i a s (Vi is ini e a L). J. Food Ag ic. En i on. 6, 333-336.