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TThe<br />
PCR pproducts<br />
werre<br />
digested<br />
eendonuclease<br />
RRsa<br />
I (Segenetic)<br />
in the to<br />
µ µl at 37 ºC ovvernight<br />
accoording<br />
to the<br />
innstructions.<br />
TThe<br />
productss<br />
of restrictio<br />
separated<br />
eleectrophoreticcally<br />
on 4<br />
(MMerck)<br />
and were docummented<br />
by D<br />
(BBioRad).<br />
Inn<br />
the case oof<br />
presence oof<br />
allele A, w<br />
ddetection<br />
of aallele<br />
C as a subtype of<br />
wwas<br />
used thee<br />
method off<br />
RFLP-PCR<br />
ccomposition<br />
of reaction mixture as<br />
ddetection<br />
off<br />
alleles A and B,<br />
eexchange(figuure<br />
1). The pprimers<br />
used<br />
aallele<br />
C were:<br />
fo forward primer:<br />
(5'- TCAGGACCCCG<br />
AAAC-3')<br />
reverse<br />
primeer:<br />
( 5'- CCTTCCAGCTG<br />
AAAG-3')<br />
TThe<br />
temperature<br />
profile of the PC<br />
stated<br />
the samme,<br />
besides tthe<br />
temperatu<br />
thhat<br />
was maiintained<br />
at 663<br />
ºC. PCR<br />
ddigested<br />
by restriction eendonuclease<br />
tootal<br />
volume of 20 µl at 337<br />
ºC overni<br />
thhe<br />
manufactuurer’s<br />
instruuctions<br />
and s<br />
aagarose<br />
gel.<br />
FFrom<br />
the obbtained<br />
results,<br />
the freq<br />
ggenotype<br />
annd<br />
allelic ppresence<br />
w<br />
FFrequency<br />
hoomogeneity<br />
iin<br />
the individ<br />
ppopulations<br />
wwas<br />
evaluateed<br />
with χ<br />
FFalconer<br />
et MMackay<br />
(1996)<br />
SStatistical<br />
annalysis<br />
were<br />
SSTATISTICA<br />
6.0 (StatS<br />
aalgorithms,<br />
whhich<br />
are avai<br />
mmilk<br />
protein ccomposition<br />
w<br />
thhe<br />
least squaares<br />
method G<br />
2<br />
with restricction<br />
otal volume oof<br />
20<br />
e manufacturrer’s<br />
on analysis wwere<br />
% agarose gel<br />
DigiDoc Sysstem<br />
we have madde<br />
a<br />
f allele A. Thhere<br />
R with the same<br />
in the casee<br />
of<br />
with primmers<br />
for detection<br />
of<br />
GGAGGTGGGAC<br />
GGTCGGGTTTG<br />
R analysis was<br />
ure of anneaaling<br />
fragments wwere<br />
e Msp I in the<br />
ght accordinng<br />
to<br />
separated in 4 %<br />
quencies of the<br />
were calculaated.<br />
dually examiined<br />
test accordinng<br />
to<br />
).<br />
e carried oout<br />
in proggram<br />
Soft, Inc. PPL,<br />
2001) used<br />
ilable on requuest.<br />
The dataa<br />
on<br />
were analysedd<br />
statistically with<br />
GLM (Generaal<br />
Linear Model),<br />
TTable<br />
1: Distrribution<br />
of β-Lg<br />
genotypess<br />
and gene frrequencies<br />
in Improved Vaalachian,<br />
Cziggaia<br />
and East-Friesian<br />
sheeep<br />
bbreeds.<br />
Allele<br />
Genoty ypic frequeencies<br />
fr requency<br />
bbreed<br />
n AA AB BB χ22<br />
test A B<br />
obs. 22 34 19<br />
IImproved<br />
VValachian<br />
sheep 75 exp. 20.28 37.44 17.28 0. .633 0.5 52 0.48<br />
nn=number<br />
of ssamples,<br />
χ2 teest=chi-squareed<br />
value, obs. =observed fre equencies, expp.=expected<br />
ffrequencies<br />
ca alculated on thhe<br />
bbasis<br />
of Hardyy-Weinberg<br />
laww<br />
TThe<br />
genotypicc<br />
frequencies at the β-Lg loocus<br />
estimated<br />
by<br />
RRFLP-PCR,<br />
ddetected<br />
for thhe<br />
Improved Valachian shheep<br />
wwere<br />
0.29 (AAA);<br />
0.45 (AB) ); 0.26 (BB). In our study,<br />
the<br />
vvariant<br />
A of ββ-Lg<br />
was moore<br />
frequent in the exammined<br />
ppopulation<br />
of sheep breed tthan<br />
variant BB.<br />
It corresponnded<br />
wwith<br />
the findinngs<br />
of severaal<br />
authors tessting<br />
the diffeerent<br />
oovine<br />
breeds aas<br />
Altamuranaa<br />
(Dario et all.,<br />
2005), Masssese<br />
(RRampolli<br />
et al.,1997), Litthuanian<br />
Natiive<br />
Coarsewoooled<br />
aand<br />
Lithuaniaan<br />
Blackface sheep (Kuččinskiene<br />
et al.,<br />
22005),<br />
Hungarrian<br />
merino ett<br />
British milkk<br />
sheep (Antoon<br />
et<br />
aal.,<br />
1997), Maanchega<br />
breedd<br />
(Lopez-Gallvez<br />
et al., 1994)<br />
aand<br />
the majority<br />
of studied oovine<br />
breeds.<br />
ročník 4<br />
Pootravinárst<br />
tvo<br />
60<br />
taki ing into accouunt<br />
the effects<br />
of genotype es of ß-LG annd<br />
sam mpling. The ddifferences<br />
weere<br />
tested by y using Schefffe<br />
mul ltiple range teest,<br />
LSD test aand<br />
Student te est for all traiits<br />
stud died.<br />
M – 25 bp markeer,<br />
1 – AA geenotype,<br />
2 – AB A genotype, 3<br />
– BB<br />
genotype, 4 – PCR prodduct.<br />
Figure<br />
1: The rrepresentativee<br />
results of the t RFLP-PCCR<br />
anal lysis.<br />
RE ESULTS ANND<br />
DISCUSSSION<br />
We estimated thhe<br />
distributionn<br />
of β-Lg ge enotypes in thhe<br />
pop pulation of thee<br />
Slovak sheepp<br />
breed Impro oved Valachiaan.<br />
The e alleles A annd<br />
B were preesent<br />
in exam mined breed, oon<br />
the contrary, alllele<br />
C was nnot<br />
found. The T allelic annd<br />
gen notypic frequeencies<br />
in the exon 2 of β-Lg gene aare<br />
show wn in table 1.<br />
In the t tested bre<br />
diff ference in χ<br />
obs<br />
dist<br />
con<br />
equ<br />
The<br />
sign<br />
dist<br />
only<br />
case<br />
vs<br />
usin<br />
2<br />
eed, there wass<br />
found out th he insignificaant<br />
2<br />
test calculaated<br />
between expected annd<br />
erved genotyype<br />
frequenciies.<br />
Accordin ng to an ideeal<br />
tribution of thhe<br />
genotypes in the popu ulation, we caan<br />
nfirm that thhe<br />
populationn<br />
was in Hardy-Weinber<br />
H<br />
rg<br />
uilibrium.<br />
e Improved VValachian<br />
sheeep<br />
showed no statistically<br />
nificant effectt<br />
of the geneetic<br />
variant fo or the nitrogeen<br />
tribution in thhe<br />
proteins (taable<br />
2). We have h found thhe<br />
y statistical ddifference<br />
(P<
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