Gene . Sel. E ol. 40 (2008) 321–331 A ailable online a :
c
INRA, EDP Sciences, 2008 www.gse-jou nal.o g
DOI: 10.1051/gse:2008006 O iginal a icle
Gene ic diffe en ia ion be ween he Old
and New ypes o Se bian Tsigai sheep
Mi jana ´
Cinkulo 1, Miika Tapio2, Mikhail Oze o 2,
Ta yana Kiselyo a3, Nu biy Ma zano 4,I anPihle 1,
Ing id Olsake 5,Mensu Vega a6, Juha Kan anen2∗
1Depa men o Animal Science, Facul y o Ag icul u e, Uni e si y o No i Sad,
21000 No i Sad, Se bia
2Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland,
31600 Jokioinen, Finland
3All-Russian Resea ch Ins i u e o Animal Gene ics and B eeding, Russian Academy
o Ag icul u al Science, 189620 S Pe e sbu g-Pushkin, Russia
4All-Russian Resea ch Ins i u e o Animal Husband y, Russian Academy
o Ag icul u al Science, 142132 Dub o i sy, Russia
5Depa men o Basic Sciences and Aqua ic Medicine, No wegian School
o Ve e ina y Science, PO Box 8146 Dep., 0033 Oslo, No way
6No ag ic, Depa men o In e na ional En i onmen and De elopmen S udies,
No wegian Uni e si y o Li e Sciences (UMB), P.O. Box 5003, 1432 Ås, No way
(Recei ed 23 July 2007; accep ed 13 No embe 2007)
Abs ac – Two Tsigai sheep popula ions exis in Se bia: he Old ype, called ˇ
Cokan,and he
New ype. I is assumed ha he New ype esul s om upg ading Tsigai sheep wi h exo ic ge-
ne ic ma e ial. We in es iga ed gene ic di e si y and diffe en ia ion o hese ypes by analysing
23 au osomal mic osa elli es. Tes s o Ha dy-Weinbe g p opo ions, linkage equilib ium be-
ween geno ypes ac oss loci and he calcula ion o inb eeding coefficien s we e pe o med and
he de iciency in he numbe o alleles wi hin he Tsigai ypes was examined using a Wilcoxon
sign- ank es . The New ype displayed a highe le el o gene ic a iabili y han he ˇ
Cokan in
e ms o allele numbe s, bu he New Tsigai showed a pa e n o he e ozygosi y de iciency. The
posi i e alue o he ˇ
Cokan sugges s he occu ence o inb eeding in his ype. The p opo ion
o linkage disequilib ium was below ha expec ed by chance. Exclusion o wo loci in Ha dy-
Weinbe g disequilib ium did no al e ou conclusions based on he en i e da a se i.e. he wo
Tsigai ypes a e clea ly diffe en ia ed and he New Tsigai ype has been in luenced by c oss-
b eeding. The e o e, he ˇ
Cokan Tsigai should be conside ed as a dis inc endange ed b eed in
he FAO classi ica ion.
mic osa elli e /sheep /Tsigai
∗Co esponding au ho : juha.kan anen@m . i
A icle published by EDP Sciences and a ailable a h p://www.gse-jou nal.o g
o h p://dx.doi.o g/10.1051/gse:2008006
322 M. ´
Cinkulo e al.
1. INTRODUCTION
The Tsigai sheep is one o he oldes Sou heas Eu opean sheep b eeds,
used o milk, mea and wool p oduc ion and is associa ed wi h local adi-
ions and ood cul u e. The b eed migh ha e o igina ed om Tu key [18] and
subsequen ly sp ead o he Balkan egion, Hunga y, Slo akia, Czech Republic,
Molda ia and Russia. Tsigai sheep a i ed in he o me Yugosla ia in he 18 h
cen u y [11]. Du ing he 20 h cen u y, bo h officially eco ded go e nmen al
and poo ly documen ed p i a e sheep sec o s exis ed in he o me Yugosla ia.
In Se bia, he go e nmen al a ms aised pu eb ed Tsigai sheep, which o m
he co e o he cu en Old Tsigai ype, also called he ˇ
Cokan. Du ing he same
pe iod, he p i a e sec o de eloped a New Tsigai sheep ype, which based on
pheno ypic simila i ies, could esul om undocumen ed c ossb eeding wi h
Be gamo sheep om I aly. The Old Tsigai ype has ela i ely homogeneous
pheno ypic cha ac e s while he New ype shows g ea e a ia ion among in-
di iduals [3]. Because o i s la ge body size, he New Tsigai ype has become
popula among a me s and inc eased in numbe while he popula ion size o
he Old Tsigai ype is dec easing, wi h cu en ly less han 500 b eeding e-
males [22].
In he p esen s udy, we analysed he gene ic di e si y and diffe en ia ion
o he wo Tsigai ypes in Se bia using 23 mic osa elli es. In addi ion, we ex-
amined hei gene ic diffe en ia ion in a wide con ex by including Finnish,
Russian and Uk ainian sheep b eeds in he analysis. The choice o he b eeds
selec ed o he analysis was based on demog aphic and geog aphic cha ac e -
is ics: Russian Tsigai is assumed o sha e ances y wi h he Se bian Tsigai, he
Ca pa hian Moun ain Sheep and Sokolsk o igina e om geog aphically p ox-
ima e egions and Finnsheep and Romano a e examples o b eeds ha ha e
been b ed pu e and kep isola ed om o he b eeds. I he ˇ
Cokan sheep shows
clea diffe en ia ion om he New Tsigai ype and o he sheep b eeds, i should
be gi en an FAO s a us o an endange ed-main ained b eed [21]. P e iously,
he gene ic di e gence be ween closely ela ed popula ions belonging o he
same b eed, was s udied in Me ino, Sa da Sheep and Finnsheep [6, 14, 23],
in Hols ein-F iesian ca le [8] and in he Lipizzan Ho se [1]. These molecula
s udies showed ha gene ic p o iles o a m animal b eeds do no necessa -
ily e lec he assumed demog aphic his o y and ha b eeds can show a s ong
agmen ed wi hin-b eed s uc u e as a esul o gene ic diffe en ia ion be ween
he subpopula ions o he b eed.
Gene ic di e si y in Se bian Tsigai sheep 323
2. MATERIALS AND METHODS
2.1. Animals, DNA ex ac ion and mic osa elli e analysis
Blood samples we e andomly collec ed om 50 Tsigai sheep o each ype.
Two sheep b eeds om Uk aine (Ca pa hian Moun ain and Sokolsk), wo om
Russia (Russian Tsigai and Romano ) and one om Finland (Finnsheep),
desc ibed by Tapio e al. [25, 26], we e included in he analysis o b eed
ela ionships.
DNA was ex ac ed om he whole blood using a s anda d phe-
nol/chlo o o m ex ac ion p o ocol [20]. In o al 100 Se bian Tsigai sheep
we e geno yped o 23 mic osa elli e ma ke s (BM0757,BM1314,BM1818,
BM4621,BM6506,BM6526,BM8125,CSSM31,MAF214,MAF36,MAF48,
MAF65,McM527,Oa CP20,Oa CP34,Oa CP38,Oa FCB11,Oa FCB128,
Oa FCB304,Oa FCB48,Oa HH47,Oa HH64 and Oa VH72). Indi idual mi-
c osa elli es we e geno yped by PCR ampli ica ion in a o al olume o 10 µL
con aining 10 ng DNA empla e, 1 X PCR buffe wi h 5% DMSO, 1.5 mM
MgCl2o 3.25 mM MgCl2( o BM1818 and CSSM31), 1 uni o Taq DNA
polyme ase, 0.2 mM o each dNTP and 2 pmol o each p ime , one o which
was labelled wi h luo escen dye. Ampli ica ion was pe o med using a ouch-
down p o ocol: ini ial dena u a ion a 94 ◦C, 5 min, 4 ounds o 3 cycles a
94 ◦C, 45 s and 63 ◦C, 60 ◦C, 57 ◦C and 54 ◦C espec i ely o 1 min, ollowed
by 23 o 30 cycles o 94 ◦C, 45 s, 52 ◦C, 1 min and a inal ex ension a 72 ◦C o
4 min. The allele sizes we e sco ed acco ding o he TAMRA 500 size s anda d
on an ABI p ism 377 sequence (Applied Biosys ems, Fos e Ci y, CA, USA).
Samples om No dic s anda d animals we e included in all gel- uns allowing
adjus men o all allele sizes o he ag eed sizes o he No h-SheD e e ence
samples ([25], No hSheD p ojec www.lbhi.is/no hshed).
2.2. S a is ical analysis
Wi hin-popula ion gene ic a ia ion was quan i ied by calcula ing he mean
numbe o alleles pe locus, mean obse ed and mean expec ed he e ozygosi-
ies using POPGENE 1.21 [27].
De ia ions om Ha dy-Weinbe g equilib ium (HWE) we e es ed us-
ing he exac es implemen ed in GENEPOP .3.1 [17]. In HWE es s, a
MCMC me hod was applied o compu e unbiased es ima es o he exac
p obabili ies. The leng h o he chain was se a 100 000 i e a ions. Wei
and Cocke ham’s [28] wi hin popula ion ixa ion index (FIS) was calcu-
la ed using FSTAT 2.9.1 [7]. The signi icance o was de e mined by
324 M. ´
Cinkulo e al.
1000 pe mu a ions. Linkage disequilib ium be ween pai s o mic osa elli e loci
was es ed using he exac es in GENEPOP 3.1.
The occu ence o ecen gene ic bo lenecks was es ed using
BOTTLENECK [16]. In he es ing, he a io o allele numbe s and expec ed
he e ozygosi ies was compa ed o he a io expec ed unde he mu a ion-d i
equilib ium [9]. The equilib ium he e ozygosi ies we e ob ained om 1000 i -
e a ions, assuming ha he allelic s a es o mic osa elli es change acco ding
o he S epwise Mu a ion Model (SMM) and Two-phased model o mu a ions
(TPM) [4]. The de aul se ings o he p opo ion o s epwise and la ge
mu a ions and a iance o allele size change we e used [16]. A e simula ion,
he signi icances o de ia ions we e es ed wi h he Wilcoxon sign- ank es .
The magni ude o gene ic diffe en ia ion be ween he wo Tsigai sheep
ypes was calcula ed wi h Wei and Cocke ham’s he a (θ) [28] using FSTAT
.2.9.1 [7].
In o de o es ima e he likelihood o an indi idual’s mul ilocus geno ype
occu ing in a gi en popula ion, we compu ed he s a is ical ce ain y o as-
signmen o each indi idual by using he Bayesian based assignmen es in
he GENECLASS .1.0 p og am [5]. The simula ion op ions (1000 simula ed
indi iduals pe popula ion and h eshold alue o ejec popula ion was 1%)
and he di ec assignmen wi h he ‘lea e one ou ’ p ocedu e we e applied.
The b eed ela ionship analysis o he wo Se bian Tsigai ypes, Russian
Tsigai,Ca pa hian Moun ain sheep, Sokolsk,Romano and Finnsheep was
based on he DAdis ance [10]
DA=1−1/

j
mj

i
√XijYij (1)
whe e Xij and Yij e e o he equencies o he i h allele a he j h locus in
popula ions X and Y, espec i ely, mjis he numbe o alleles a he j h locus,
and is he numbe o analysed loci. A se o 15 mic osa elli es a ailable o
all b eeds ( e e o [25]) was used o he calcula ion. The analysis was done
using DISPAN [12]. The obus ness o he Neighbou -joining ee [19] was
es ed by boo s apping (5000 eplica es o e loci) and p esen ed g aphically
using TREEVIEW 1.6 6. [13].
3. RESULTS
3.1. Gene ic a ia ion and popula ion s uc u e
Geno ype da a a e a ailable upon eques om M. ´
C. All mic osa elli e loci
we e polymo phic in bo h Tsigai sheep ypes. A o al o 205 alleles we e ound.
Gene ic di e si y in Se bian Tsigai sheep 325
Table I. Wi hin-popula ion gene ic di e si y and inb eeding es ima es in he Old and
New Se bian Tsigai ypes. In addi ion, p obabili ies ob ained om he Wilcoxon sign-
ank es in he BOTTLENECK analysis a e p esen ed.
Tsigai ype nMNA HOBS HEXP
Old 50 6.7 0.64 0.69 Gene ic di e si y
New 50 7.5 0.66 0.70
[95% CI o ]
Old 50 0.086 [0.033, 0.148] Inb eeding
New 50 0.072 [0.007, 0.150] all 23 ma ke s
Old 50 0.073 [0.02, 0.136] Inb eeding
New 50 0.040 [–0.11, 0.094] 21 ma ke s*
Wilcoxon’s sign- ank es
P- alueSMM P- alueTPM
Old 50 0.1234 0.6001 Bo leneck es ing
New 50 0.0002 0.0022 all 23 ma ke s
Old 50 0.2367 0.8855 Bo leneck es ing
New 50 0.0007 0.0057 21 ma ke s*
n=Numbe o indi iduals included in he analysis.
MNA =Mean numbe o alleles pe mic osa elli e locus.
HOBS =Mean obse ed he e ozygosi y.
HEXP =Mean expec ed he e ozygosi y.
=Wi hin-popula ion inb eeding es ima e.
95% CI o =95% con idence in e als o he es ima e o .
P- alueSMM =A p obabili y alue ob ained assuming ha mic osa elli es e ol e acco ding o
he s epwise mu a ion model.
P- alueTPM =A p obabili y alue ob ained assuming ha mic osa elli es e ol e acco ding o
he wo-phased mu a ion model.
* Es ima es a e based on 21 ma ke s, he MAF214 and Oa HH64 we e excluded om he
analysis.
The mean allele numbe pe locus was 8.7, anging om 5 (BM0757,
BM6506 and Oa CP34) o12(BM1314 and BM1818). Two loci (MAF214
and Oa HH64) showed signi ican de ia ions (P <0.001, Bon e oni co -
ec ion applied) om HWE in bo h Tsigai popula ions due o he excess o
homozygo es.
Wi hin he Tsigai sheep he mean allelic numbe pe locus was highe in he
New ype (7.5) han in he Old ype (6.7) (Tab. I). Fo y-one pe cen (85/205) o
he alleles we e no sha ed be ween he wo Tsigai ypes. Thi y- h ee alleles
we e speci ic o he Old Tsigai, while 52 alleles we e obse ed only in he
New Tsigai. The mean obse ed and expec ed he e ozygosi ies in he Old and
New Tsigai ypes a e gi en in Table I.

326 M. ´
Cinkulo e al.
The exac es o non- andom associa ion o geno ypes ac oss loci ga e
23 signi ican alues (P <0.05) om 506 compa isons (10 in he New
and13in heOldTsigai ype). The o al numbe o signi ican ly small P- alues
was he e o e less han would be expec ed by chance alone o 506 independen
es s.
Wi hin-popula ion inb eeding es ima es ( ) in bo h Tsigai ypes we e pos-
i i e and signi ican ly diffe en om ze o (P <0.001), indica ing ha pa en s
we e mo e ela ed han expec ed unde andom ma ing (Tab. I). Excluding he
ma ke s MAF214 and Oa HH64, ha displayed a s a is ically signi ican ex-
cess o homozygo es, bo h Tsigai ypes s ill showed posi i e -es ima es, bu
only he es ima e o he Old ype de ia ed signi ican ly om ze o (Tab. I).
Pe o ming he Wilcoxon sign- ank BOTTLENECK es [4] assuming ha
he mic osa elli e alleles e ol ed acco ding o SMM o TPM models did no
indica e loss o alleles in he Old Tsigai (Tab. I). Howe e , he New Tsigai dis-
played a pa e n o he e ozygosi y de iciency, i.e. he e we e oo many alleles
compa ed o he le el o gene di e si y. Excluding he wo anomalous ma ke s
did no al e he es conclusions.
3.2. Gene ic diffe en ia ion
The be ween-popula ion inb eeding index, he a (θ), was 0.110 (P <0.05,
signi ican ly diffe en om ze o), indica ing ha 11% o he o al gene ic a i-
a ion was explained by diffe ences be ween he Tsigai ypes, while he emain-
ing 89% was due o diffe ences among indi iduals.
Gene ic diffe en ia ion a he indi idual le el was in es iga ed wi h a
Bayesian app oach implemen ed in he GENECLASS .1.0 p og am. Ani-
mals we e assigned o he co ec Old o New Tsigai popula ions wi h success
a es o 90% and 88%, espec i ely. The ou pu o he calcula ion is p esen ed
g aphically in Figu e 1. The plo ing o log-likelihood alues ob ained o all
samples clea ly indica es dis inc g ouping o animals in o he Old Tsigai and
New Tsigai ypes. In addi ion, g ea e uni o mi y among he sampled geno-
ypes o he Old Tsigai was sugges ed by he a e age log-likelihood alue
(25.43), while o he New ype he co esponding mean sugges ed g ea e
he e ogenei y (43.72).
We calcula ed DAgene ic dis ances among he wo Se bian Tsigai ypes and
i e Eu opean sheep b eeds (Finnsheep,Romano ,Russian Tsigai,Ca pa hian
Moun ain and Sokolsk). The gene ic dis ance be ween he wo Tsigai ypes
(DA=0.2234) was g ea e han he a e age dis ances among he i e o he
sheep b eeds (DA=0.1730) s udied he e (Tab. II). Howe e , one o he h ee
Gene ic di e si y in Se bian Tsigai sheep 327
Figu e 1. A plo o log-likelihood alues ob ained in he assignmen es o all in-
di iduals. Two clea ly dis inc g oups ( he Old Tsigai and he New Tsigai) can be
iden i ied indica ing hei gene ic diffe en ia ion.
Table II. Ma ix o DAdis ances among he se en sheep b eed and popula ions.
1234567
1. Old Tsigai –
2. New Tsigai 0.2234 –
3. Romano 0.2922 0.3192 –
4. Ca pa hian Moun ain 0.1713 0.2006 0.2328 –
5. Sokolsk 0.1928 0.2039 0.2409 0.1058 –
6. Russian Tsigai 0.1848 0.1894 0.1955 0.0881 0.0996 –
7. Finnsheep 0.2411 0.2672 0.2079 0.1937 0.1951 0.1709 –
well-suppo ed clus e s in he phylogene ic ee (Fig. 2) consis ed o he wo
Tsigai ypes. The o he wo we e a no he n sho - ailed g oup, consis ing o
he Finnsheep and Romano , and in e es ingly a sou he n Russian/Uk ainian
clus e , con aining he Russian Tsigai and he wo s udied Uk ainian b eeds,
he Ca pa hian Moun ain sheep and Sokolsk.
4. DISCUSSION
The Tsigai sheep occu s in many sou heas Eu opean coun ies and also in
sou he n Russia, and consis s o pheno ypically and geog aphically diffe en
328 M. ´
Cinkulo e al.
Romano
Finnsheep
Old Tsigai
Russian Tsigai
Sokolsk
Ca pa hian Moun ain
New Tsigai
Figu e 2. Un oo ed neighbou -joining ee cons uc ed om DAdis ances showing
he ela ionships be ween se en sheep b eeds and popula ions. Numbe s a he nodes
ep esen he pe cen age o g oup occu ence in 1000 boo s ap eplica es.
a ie ies used o mea , milk and wool p oduc ion. Two diffe en ypes o
Tsigai sheep exis in Se bia: he Old ype, called ˇ
Cokan, and he New ype.
Ou mic osa elli e da a e ealed a subs an ial gene ic diffe en ia ion be ween
hese Se bian Tsigai ypes. Acco ding o he esul s o F-s a is ics [28], 11%
o he o al gene ic a ia ion in he Se bian Tsigai sheep can be explained by
ype diffe ences. This signi ican diffe en ia ion exceeds ha de ec ed among
six Spanish sheep b eeds (6%, eigh ma ke s in common wi h ou s udy) [2],
among se en Bal ic b eeds (8.8%, 21 common ma ke s) [24] and e en ha p e-
sen ed o 57 Eu opean and Middle Eas e n sheep b eeds (app oxima ely on
a e age 6%, eigh common ma ke s) [15]. Mo eo e , he Bayesian assignmen
o animals o he sou ce popula ions suppo ed he clea -cu diffe en ia ion be-
ween he wo Tsigai ypes (Fig. 1). The deg ee o diffe en ia ion be ween he
wo Tsigai ypes was u he quan i ied using DAgene ic dis ance and a se o
e e ence b eeds. The analysis indica ed ha he wo ypes a e as diffe en , i
no mo e diffe en , as an a e age b eed pai . In p e ious mic osa elli e-based
s udies, a simila kind o high gene ic di e gence among popula ions wi hin he
same b eed om diffe en geog aphical loca ions has been eco ded [6,8,14],
e lec ing he impo an con ibu ion o gene ic d i o he diffe en ia ion o
domes ic animal popula ions.
Gene ic di e si y in Se bian Tsigai sheep 329
In he New ype, 19 o he 23 mic osa elli e loci showed oo many alleles
compa ed o he le el o gene di e si y, which is signi ican ly mo e equen
han ha supposed by s ochas ic effec s alone. The mos ob ious explana ion
o his obse a ion and he highe wi hin-popula ion di e si y ound in he
New ype is ha he popula ion has been in luenced by gene low om an
exo ic b eed (o b eeds), in oducing o eign a e alleles, o in he ex eme case
making he o iginal Tsigai alleles a e [9]. The c ossb eeding may also explain
he g ea e pheno ypic di e si y o he New ype han ha displayed by he Old
ype [3]. The census size o he Old Tsigai ype has dec eased d ama ically,
bu he p esen bo leneck es did no indica e loss o alleles in he popula ion
due o a se e e educ ion o effec i e popula ion size [9]. Howe e , he posi i e
alue indica es he occu ence o inb eeding in he Old ype.
The gene ic di e gence o he wo Se bian Tsigai popula ions was also in-
es iga ed in a wide con ex by pe o ming an analysis o b eed ela ionships
including Russian Tsigai, wo Uk ainian long- ailed sheep b eeds and wo
no he n Eu opean sho - ailed b eeds (Fig. 2). The b anching pa e n o he
ee was obus as indica ed by high boo s ap alues and sugges s ha he
Old and he New Se bian Tsigai ypes cons i u e a g oup o hei own. The
clea gene ic di e gence be ween he Se bian Tsigai ypes is appa en and is
e idenced by he leng h o he b anches. The Russian Tsigai ends o g oup
wi h he Uk ainian b eeds wi h sho b anch leng hs, indica ing ha he gene
pool o he Russian Tsigai may ha e been in luenced by c ossb eeding. Ou
mic osa elli e da a indica e ha he ˇ
Cokan has no been upg aded wi h exo ic
gene ic ma e ial, making he popula ion an impo an gene ic ese oi in he
conse a ion o gene ic esou ces o na i e sou heas Eu opean Tsigai sheep.
We ecommend ha an FAO s a us o endange ed sheep b eed be gi en o he
ˇ
Cokan Tsigai sheep.
ACKNOWLEDGEMENTS
The au ho s wish o exp ess hei g a i ude o owne s o Tsigai sheep o
hei help and collabo a ion in he sampling o he p esen esea ch ma e-
ial. We acknowledge he inancial suppo om he No wegian Royal Min-
is y o Fo eign Affai s and he Resea ch Council o No way. We a e hank ul
o Ole Albe Gu e s ud on echnical assis ance in mic osa elli e yping and
D . Meng-Hua Li o aluable commen s on an ea lie d a o his manusc ip .
M Jo o Kosano ic om he Cen e o Feed Technology, No way, is acknowl-
edged o collabo a ion and suppo .