SHORT REPORT Open Access
Gene ic componen s o g ey ca le in Es onia as
e ealed by mic osa elli e analysis using wo
Bayesian clus e ing me hods
Meng-Hua Li
1*
, Juha Kan anen
1
, Annika Michelson
2
, U mas Saa ma
3
Abs ac
Backg ound: I was ecen ly pos ula ed ha a ew indi idual g ey ca le s ill ound in Es onia migh be a elic o
he old na i e ca le s ock. Geno ypes a 17 mic osa elli e loci om a o al o 243 ca le om No h Eu opean
b eeds and 11 g ey ca le in Es onia we e used in an a emp o cla i y he gene ic composi ion o he g ey ca le.
Findings: We cha ac e ize he gene ic componen s o 11 examples o he g ey ca le in Es onia a he popula ion
and indi idual le els. Ou esul s show ha he g ey ca le in Es onia a e mos gene ically simila o he Hols ein-
F iesian b eed and seconda ily o he Es onian Red ca le.
Conclusions: Bo h Bayesian app oaches ga e simila esul s in e ms o he iden i ica ion o numbe s o clus e s
and he es ima ion o p opo ions o gene ic componen s. This s udy sugges ed ha he Es onian g ey ca le
included in he analysis a e a gene ic composi e esul ing om c oss-b eeding o Eu opean dai y b eeds.
Backg ound
Conse a ion o a m animal gene ic esou ces is o
g ea alue o he ag icul u al, economic, social and cul-
u al sec o s [1]. This is pa icula ly ue o na i e a m
animals because he speci ic genes and gene combina-
ions hey ca y may be use ul, o example o cope wi h
he challenge o global clima e change (see [2]).
Bal ic ca le popula ions ha e been g ea ly a ec ed by
a ew p oduc i e b eeds such as Danish Red, Angeln,
and Hols ein-F iesian [1]. As a esul , only e y ew
popula ions, e.g. he Es onian Na i e, a e gene ically
cha ac e is ic o he na i e ca le ha ha e su i ed in
he Bal ic coun ies [3]. Mos o he o iginal ca le ha e
de eloped in o new ed- o black-pied b eeds [1]. How-
e e , a ecen su ey sugges ed ha he e could exis
local g ey ca le in Es onia, wi h a o al popula ion size
o ca. 60 animals, which we e pos ula ed o be a elic
o he old na i e ca le s ock [4,5]. Today, hey a e
main ained in small he ds owned by olde a me s, and
as such, he e is limi ed pedig ee in o ma ion on hese
indi iduals. Typically, hei hide is g ey, blue g ey, o
g ey, ash g ey, black and whi e (see Figu e 1).
So a he gene ic composi ion o g ey ca le ela i e
o o he exis ing b eeds in Es onia is s ill unknown. In
his s udy we use a panel o 17 mic osa elli e loci and
Bayesian-based assignmen echniques o e alua e he
ela ionship o Es onian g ey ca le o o he b eeds
occu ing in No h Eu ope.
Me hods
Ca le samples and mic osa elli e da a
Geno ypes o a o al o 254 animals om se en ca le
popula ions (G ey ca le in Es onia, n= 11, see Table 1;
Es onian Na i e, n= 40, Es onian Red, n=40,Finnish
Hols ein-F iesian, n= 43, La ian Blue, n=40,La ian
Danish Red, n= 40, La ian B own, n= 40) we e included
in he analysis. Se en een (BM2113,HEL1,BM1824,
BM1818,INRA032,INRA005,INRA035,ETH3,ILSTS006,
HEL5,INRA023,INRA063,INRA037,ETH225,ILSTS005,
CSSM66 and HEL13) o 30 mic osa elli e loci ecom-
mended o gene ic di e si y s udies in ca le h p://www.
p ojec s. oslin.ac.uk/cdi /ma ke s.h ml we e included in
hisin es iga ion.Thegeno ypeda a o hesixpa en
popula ions we e ob ained om an ea lie s udy [1].
* Co espondence: [email p o ec ed]
1
Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, FI-31600
Jokioinen, Finland
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Li e al.BMC Resea ch No es 2011, 4:37
h p://www.biomedcen al.com/1756-0500/4/37
© 2011 Li e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons
A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in
any medium, p o ided he o iginal wo k is p ope ly ci ed.
Ele en Es onian g ey ca le indi iduals om di e en
s ocks we e blood-sampled. Pa icula e o s we e
made in all cases, using bo h he limi ed pedig ee
in o ma ion (e.g. mos ly only pa en -o sp ing and ull-
sibling ela ionships) a ailable and he knowledge o
local he dsmen (e.g. he a m o illage whe e he ca -
le o igina e om and he p e ious owne s) ia he
in e iew ques ionnai e, o ensu e ha he animals
we e un ela ed and had cha ac e is ics ypical o he
popula ion [4]. Genomic DNA was ex ac ed using a
s anda d phenol/chlo o o m p o ocol [6]. PCRs we e
ca ied ou ollowing he p o ocols a ailable a he
Ca le Di e si y Da abase h p://www.p ojec s. oslin.ac.
uk/cdi /ma ke s.h ml. The size cha ac e iza ion o
PCR p oduc s was done on a MegaBACE™500 capil-
la y sequence (GE Heal hca e Li e Sciences, Li le
Chal on , UK) using he F agmen P o ile p og am
e . 1.2 (GE Heal hca e Li e Sciences). In e na ional
con ol samples we e also geno yped in o de o s an-
da dize he size o allele agmen s. Blood sampling o
he 11 G ey ca le in Es onia was aken by a e e ina -
ian in a p ocedu e acco ding o he Es onian Ve e in-
a y and Food Boa d and sa is ied all e hical conce ns.
Da a analysis
Tes s o geno ypic linkage disequilib ium (LD) o each
locus pai and es s o de ia ion om Ha dy-Weinbe g
equilib ium (HWE) we e analysed in GENEPOP e sion
3.4 [7]. The global and pai wise gene ic di e en ia ion
we e de e mined as unbiased es ima es o F
ST
[8] using
FSTAT e sion 2.9.3.2 [9]. Signi icance o he esul s
was es ablished by applying sequen ial Bon e oni co -
ec ions (see [10]).
Figu e 1 A g ey cow om he Vah amäe a m in Es onia (Pho o c edi : Imbi Jäe ma).
Table 1 Da a o he 11 g ey ca le analysed in Es onia
Sample Coun y Village Gende Colou
Le1 Läänemaa Silla ♀g ey
Le2 Lääne-Vi umaa Kä sa ♀g ey
Le3 Lääne-Vi umaa Kä sa ♀g ey
Le4 Läänemaa Kinki ♀g ey
Le5 Jõge amaa Maa dla ♀da k g ey (blackish)
Le6 Jõge amaa Maa dla ♀g ey ( eddish)
Le7 Jõge amaa Maa dla ♀da k g ey
Le8 Jõge amaa Maa dla ♀da k g ey
Le9 Ha jumaa Rooküla ♂g ey
Le10 Ha jumaa Rooküla ♂g ey
Le11 Raplamaa Laukna ♀da k g ey
Li e al.BMC Resea ch No es 2011, 4:37
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A Bayesian clus e ing me hod was i s employed o
assess popula ion s uc u e using he p og am STRUC-
TURE e sion 2.2 [11]. We pe o med 10 uns o each
K alue a 2 - 10 and an he p og am assuming a
model o admix u e and co ela ed allele equencies.
We did no use any p io in o ma ion abou he popula-
ion o igin o he animals. A bu n-in pe iod o 200 000
gene a ions and MCMC simula ions o 500 000 i e a-
ions we e used in all he abo e uns. The alues o LnP
(D) ( he log p obabili y o da a) we e es ima ed assign-
ing a p io om 2 o 10 and he op imal Kwas chosen
based on he del a K(ΔK) alue. This c i e ion was o i-
ginally desc ibed in E anno e al. [12] and was shown
o be e ec i e in la e s udies [1,13]. We hen e alua ed
he popula ion and indi idual membe ship coe icien s
(Q) o he 11 g ey ca le in Es onia o he Kin e ed
clus e s.
BAPS e sion 5.4 [14] was un se ing he maximum
numbe o clus e s a 20. Resul s we e based on 50
simula ions om he pos e io allele equencies. Since
he mode o he pos e io dis ibu ion o Kalmos
always p o ided an o e es ima e o K, we used he num-
be o clus e s con aining mo e han 3 indi iduals as a
poin es ima e o K, as ecommended by Tang e al.
[14]. Fo uns in which Kwas co ec ly es ima ed, we
calcula ed he a e age p obabili y (q) o assignmen o
he ‘co ec ’clus e (’co ec ’de ined as q>0.9in he
co ec clus e ). Indi iduals wi h a likelihood admix u e
a io g ea e han 3.0 we e conside ed o be signi ican ly
admixed.
Resul s
The F
ST
analysis ac oss b eeds showed ha 5.6% o he
o al gene ic a ia ion could be explained by he di e -
ence among popula ions. A low le el o gene ic di e en-
ia ion was ound be ween he g ey ca le in Es onia and
Finnish Hols ein-F iesian (F
ST
= 5.2%; esul s no
shown) as well as be ween he g ey ca le in Es onia and
he Es onia Red ca le (F
ST
= 5.6%; esul s no shown).
Nei he o he alues was s a is ically signi ican a he
0.05 le el (P> 0.05). No speci ic locus pai s showed a
consis en de ia ion om LE ha would ha e been in
each, o e en in mos , o he popula ions. De ia ions
om HWE ac oss he loci we e p esen in he popula-
ion o g ey ca le in Es onia, which is mos p obably
due o he small popula ion size. Howe e , no e idence
o signi ican de ia ion om HWE was de ec ed when
a es was pe o med ac oss all loci o all popula ions.
Based on he popula ion Q- alues, he STRUCTURE
p og am iden i ied six clus e s among he se en popula-
ions, bu could no disce n all se en popula ions
(Figu e 2A). Mo e exac ly, i ailed o di e en ia e be ween
he g ey ca le in Es onia and Finnish Hols ein-F iesian.
O e he en i e ca le popula ions, Ln P(D) inc eased om
K=2 oK= 6, a e which i began o decline, indica ing
he mos likely alue o be K= 6 ( esul s no shown).
When we used ΔK o in e he numbe o clus e s, we
ound ha K= 6 was clea ly a ou ed ( esul s no shown).
A K= 6, all he g ey ca le in Es onia we e cha ac e ized,
wi h he highes p opo ion o membe ship om he Fin-
nish Hols ein-F iesian clus e (Q
FiHF
). Fi e g ey ca le
(Le1, Le4, Le9, Le10 and Le11) showed high alues o
Q
FiHF
> 0.9 and he emaining g ey ca le a e sugges ed o
ha e la ge membe ship ac ions in mul iple clus e s o
he sampled popula ions. In pa icula , he g ey ca le Le8
ha e simila alues o Q o wo dis inc popula ions, Fin-
nish Hols ein-F iesian (Q
FiHF
= 0.480) and he Es onian
Red (Q
EsR
= 0.351; Table 1).
Wi h espec o he o e all pa e n o popula ion clus-
e ing, esul s wi h BAPS we e mos ly consis en wi h
hose ob ained wi h STRUCTURE. The analysis o popu-
la ion gene ic s uc u e ca ied ou wi h BAPS sugges ed
K= 6 o be he bes clus e ing op ion (see Figu e 2B),
wi h he six clus e s co esponding o he six sou ce
popula ions. Figu e 2B shows he p opo ions o mem-
be ship (q) o each g ey ca le indi idual in each o he
six iden i ied clus e s, while he co esponding alues a e
p esen ed in Table 2. Fi e samples (Le1, Le4, Le9, Le10
and Le11) exhibi ed q alues o 100% o he clus e o
Hols ein-F iesian and one (Le8) o Es onian Red. The
emaining samples ecei ed p opo ions o membe ship
om mul iple clus e s, while he highe a e age p opo -
ion o hei membe ship was om Finnish Hols ein-
F iesian ollowed by Es onian Red (Table 1).
Discussion
On-a e age we ound highe p opo ions o membe ship
o Finnish Hols ein-F iesian and Es onian Red ca le in
he g ey ca le. The g ey ca le ep esen a composi e o
No h Eu opean ca le.
The composi e gene ic componen s may explain hei
dis inc i eg eycolou ,whichisamix u eo colou s.
This inding is also e idenced by he ac ha a g ey
cow some imes has g ey and/o black-and-whi e cal es
in he same bi h. Al hough he g ey ca le a e cha ac-
e ized as ha ing mos o hei gene ic componen s
om he black-and-whi e dai y ca le (i.e. Hols ein-
F iesian) o Es onian Red, hey can be aluable in he
in es iga ion o he gene ics o he colou genes.
Bo h STRUCTURE and BAPS co ec ly in e ed he
numbe o clus e s in a da ase when gene ic di e en ia-
ion among popula ions was low. Howe e , i seems ha
he p opo ions o indi idual membe ship in he clus e s
es ima ed by he p og am STRUCTURE a e mo e con-
sis en wi h he b eeding his o y o he popula ions.
Fo example, La ian Danish Red, Es onian Red and La -
ian B own a e he local de i ed popula ions om he
Anglen and Danish Red ca le. This sha ed ances y is
Li e al.BMC Resea ch No es 2011, 4:37
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Page 3 o 6
e lec ed in he esul s o STRUCTURE, bu no o
BAPS. Fo he 11 g ey ca le in Es onia, bo h p og ams
ga e compa able esul s o p opo ions o indi idual
membe ship. To secu e high con idence in esul s, we
ad oca e using bo h p og ams o in e ing he numbe
o clus e s and assignmen o indi iduals o clus e s,
pa icula ly when he le el o gene ic di e en ia ion
among popula ions is low.
Finally, a g owing numbe o domes ic animal popula-
ions a e geno yped o he same panel o mic osa elli es
(see [15]), o example he ma ke s ecommended by he
FAO (Food and Ag icul u e O ganiza ion o he Uni ed
Na ions). This can help add ess simila kinds o ques ions
ongene iccomponen sand hena u eo na i eanimal
s ocks because mo e da a o po en ial e e ence and pa -
en al popula ions a e a ailable. The li es ock popula ions
Figu e 2 Popula ion s uc u e o 7 ca le popula ions using:(A) model-based STRUCTURE p og am (P i cha d e al. 2000) and (B) BAPS
p og am (Tang e al. 2009). Each animal is ep esen ed by a single e ical line di ided in o Kcolou s, whe e Kis he numbe o clus e s
assumed. The colou ed segmen shows he indi idual’s es ima ed p opo ion o membe ship (a e aged ac oss 10 uns a K= 6) in ha clus e
o he STRUCTURE p og am and indica es he a e age p obabili y o assignmen o he “co ec ”clus e o he BAPS p og am. Black lines
sepa a e he popula ions labelled abo e he igu e. The labels abo e he igu e indica e he numbe o animals analysed in each b eed and he
names o he ca le popula ions analysed a e indica ed below he igu e.
Li e al.BMC Resea ch No es 2011, 4:37
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o which he e is a high p io i y o conse a ion, in e ms
o p opo ions o hei na i e gene ic componen s (e.g.
[16-18]), can be iden i ied and, hus, need o be included
in conse a ion p og ammes in he nea u u e.
Conclusions
In conclusion, gi en he low le els o gene ic di e en ia-
ion among he popula ions in es iga ed, bo h Bayesian
app oaches ga e simila esul s in e ms o iden i ica ion
o he numbe s o clus e s and he es ima ion o p opo -
ions o gene ic componen s. Ou s udy shows ha he
Es onian g ey ca le analysed we e a gene ically admixed
popula ion, mos in luenced by he Hols ein-F iesian
and Es onian Red ca le.
Acknowledgemen s
We wish o hank Kaia Lepik om he Es onian Fund o Na u e o he
gene ous help and p o esso Haldja Viinalass a he Es onian Uni e si y o
Li e Sciences (Ta u, Es onia) o he commen s. This s udy was suppo ed by
a g an om he Es onian Science Founda ion, a ge inancing p ojec
SF0180122s08 om he Es onian Minis y o Educa ion and Sciences; and
om he Eu opean Union h ough he Eu opean Regional De elopmen
Fund (Cen e o Excellence FIBIR).
Au ho de ails
1
Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, FI-31600
Jokioinen, Finland.
2
HAMK Uni e si y o Applied Sciences, FI-31310 Mus iala,
Finland.
3
Depa men o Zoology, Ins i u e o Ecology and Ea h Sciences,
Uni e si y o Ta u, 46 Vanemuise S ee , EE-51014 Ta u, Es onia.
Au ho s’con ibu ions
MHL designed he s udy, pe o med he da a analysis and w o e he
manusc ip . JK planned and coo dina ed he whole s udy, and con ibu ed
o he manusc ip w i ing. AM collec ed in o ma ion abou Es onian g ey
ca le, con ac ed ca le owne s and in e iewed hem. US pa icipa ed in
s udy design, sample collec ion o g ey ca le in Es onia and he p elimina y
da a analysis. All he au ho s ead and app o ed he inal manusc ip .
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Recei ed: 6 July 2010 Accep ed: 11 Feb ua y 2011
Published: 11 Feb ua y 2011
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Table 2 Membe ship p opo ions (Q) o he 11 g ey ca le in Es onia o he 6 gene ic clus e s
Animals Clus e 1
(La ian B own)
a
Clus e 2
(La ian Blue)
Clus e 3
(La ian Danish Red)
Clus e 4
(Es onian Na i e)
Clus e 5
(Es onian Red)
Clus e 6
(Finnish Hols ein-F iesian)
Le1 0.015(0.00)
b
0.003(0.00) 0.006(0.00) 0.005(0.00) 0.008(0.00) 0.963(1.00)
c
Le2 0.015(0.01) 0.015(0.04) 0.081(0.40) 0.015(0.03) 0.145(0.05) 0.729(0.47)
Le3 0.025(0.00) 0.017(0.06) 0.072(0.12) 0.008(0.00) 0.027(0.15) 0.852(0.67)
Le4 0.009(0.00) 0.019(0.00) 0.008(0.00) 0.003(0.00) 0.008(0.00) 0.953(1.00)
Le5 0.012(0.01) 0.023(0.15) 0.038(0.12) 0.016(0.02) 0.047(0.25) 0.864(0.45)
Le6 0.013(0.02) 0.008(0.00) 0.032(0.08) 0.029(0.25) 0.169(0.34) 0.749(0.31)
Le7 0.021(0.12) 0.036(0.10) 0.054(0.12) 0.009(0.10) 0.241(0.24) 0.639(0.32)
Le8 0.006(0.00) 0.007(0.00) 0.351(0.00) 0.009(0.00) 0.147(1.00) 0.480(0.00)
Le9 0.033(0.00) 0.009(0.00) 0.008(0.00) 0.012(0.00) 0.014(0.00) 0.925(1.00)
Le10 0.009(0.00) 0.005(0.00) 0.007(0.00) 0.010(0.00) 0.014(0.00) 0.954(1.00)
Le11 0.010(0.00) 0.035(0.00) 0.010(0.00) 0.014(0.00) 0.026(0.00) 0.905(1.00)
a
The gene ic clus e s in e ed using he STRUCTURE p og am.
b
The a e age p obabili y (q, in he pa en heses) o assignmen o he “co ec ”clus e ("co ec ”de ined as q> 0.9 in he co ec clus e ) by using he BAPS
p og am.
c
Values o Q> 0.9 o q> 0.9 a e in bold.
Li e al.BMC Resea ch No es 2011, 4:37
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doi:10.1186/1756-0500-4-37
Ci e his a icle as: Li e al.: Gene ic componen s o g ey ca le in
Es onia as e ealed by mic osa elli e analysis using wo Bayesian
clus e ing me hods. BMC Resea ch No es 2011 4:37.
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