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Journal of Applied Animal Research

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Genetic polymorphism of -lactoglobulin in sheep

raised for milk production
a

Aldona Kawecka & Anna Radko

National Research Institute of Animal Production , 32-083, Krakw, Poland

Published online: 01 Jun 2011.

To cite this article: Aldona Kawecka & Anna Radko (2011) Genetic polymorphism of -lactoglobulin in sheep raised for
milk production, Journal of Applied Animal Research, 39:1, 68-71, DOI: 10.1080/09712119.2011.565223
To link to this article: http://dx.doi.org/10.1080/09712119.2011.565223

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Journal of Applied Animal Research,

Vol. 39, No. 1, March 2011, 68
71

Genetic polymorphism of b-lactoglobulin in sheep raised for milk production

Aldona Kawecka* and Anna Radko
National Research Institute of Animal Production, 32-083 Krakow, Poland

Downloaded by [95.76.154.78] at 00:16 15 November 2014

(Received 18 February 2010; final version received 22 November 2010)

To investigate b-lactoglobulin polymorphism using polymerase chain reaction
restriction fragment length
polymorphism in Polish Mountain, East Friesian, Polish Merino and Austrian Bergschaf sheep raised for milk
production, three b-LGB genotypes (AA, AB and BB) were found in all the groups, AB being the most frequent
genotype in all the groups. The frequency of b-LGB A genes was 0.66 in Friesian, 0.55 in Bergschaf, 0.5 in Polish
Mountain and 0.48 in Polish Merino sheep with no deviations from genetic equilibrium according to HardyWeinberg law. The relationship between b-lactoglobulin genotype and milk yield and milk composition was
investigated, but no statistical differences were found. The present findings provide no conclusive evidence about
the effect of different b-LGB variants on milk production traits in sheep.
Keywords: sheep; b-lactoglobulin; polymorphism; milk

Introduction
In Poland, raising sheep for milk is of regional
importance and, until recently, milk was only obtained
from Polish Mountain sheep. This production type is
now attracting growing interest in lowlands also,
where milk is obtained from Polish Merino sheep and
their crossbreds with East Friesian milk sheep. Attempts were also made to milk Bergschaf sheep
imported from Austria.
The b-lactoglobulin (b-LGB) is the main whey
protein of ruminant milk and contains 162 amino
acids. Its biological role is not completely understood, but it probably takes part in the transport of
vitamin A and other low-molecule compounds. The
b-lactoglobulin gene locus in sheep is localised on
chromosome 3 and the genetic polymorphism of
b-lactoglobulin is determined by three alleles
A, B
and C. Milk protein (b-lactoglobulin and casein)
polymorphism was described in considerable detail in
dairy cattle. In many cases, the genetically determined diversity of these proteins was shown to be
associated with the yield of milk and milk components (Litwinczuk et al. 2006). Milk protein genes
can, therefore, be viewed as quantitative trait loci
(QTL) markers in terms of dairy traits and can thus
be used in selection programmes.
The issue of polymorphism has not been adequately studied among Polish breeds of sheep raised
for milk production. Studies on Polish Merino sheep
(Mroczkowski et al. 2004) have shown that dairy
traits vary according to b-LGB genotype. Whereas,
other studies have reported no effect of this genotype
*Corresponding author. Email: akawecka@izoo.krakow.pl
ISSN 0971-2119 print/ISSN 0974-1844 online
# 2011 Taylor & Francis
DOI: 10.1080/09712119.2011.565223
http://www.informaworld.com

on milk production traits (Piwczynski et al. 2002).

The aim of the study was to characterise b-LgB
polymorphism using PCR-RFLP in sheep raised for
milk production in Poland and to determine the
relationship between the occurrence of different
genetic variants and milk production traits.

Materials and methods

Samples of blood collected from 181 sheep of the
Friesian, Polish Merino, Bergschaf and Polish Mountain sheep were studied. Genomic DNA was isolated
from peripheral blood leukocytes using proteinase K,
following the method described by Kawasaki (1990).
The b-LGB genotype was determined using PCRRFLP. Isolated DNA was amplified using the polymerase chain reaction (PCR). The PCR was performed
in 25 ml of reaction mixture containing 10  PCR
Buffer, MgCl2 (15 mM), dNTP nucleotides (2 mM),
AmpliTaq Gold polymerase (5 U/m) and LGB1 primer
sequences (0.4 mM): 5?-CAA CTC AAG GTC CCT
CTC CA-3? and LGB2: 5?-CTT CAG CTC CTC CAC
GTA CA-3? (lit). The reaction mixture was subjected to
a thermal programme of 15 min initial denaturation of
genomic DNA at 958C, 31 cycles of denaturation at
948C for 45 sec, annealing at 648C for 45 sec, elongation of primers at 728C for 1 min, and final elongation
at 728C for 60 min. the PCR reaction was performed
using GeneAmp PCR System 9600 cycler (Applied
Biosystems). Digestion of 10 ml amplification product
was performed using RsaI restriction enzyme (2 U/ml)
at 378C for 2.5 h. The digestion products obtained were

69

Journal of Applied Animal Research

Downloaded by [95.76.154.78] at 00:16 15 November 2014

separated electrophoretically. The digested product

was electrophoresed in 3% agarose gel: Agarose for
Routine Use-NuSive GTG agarose (1:3). The results of
electrophoretic separation were analysed in UV light
using a transilluminator. The conformity of the actual
distribution of genotypes in the analysed sheep groups
with theoretical distribution was verified by chi-square
test using Statistica ver.7.1 (StatSoft).
Lactating ewes were recorded for milk yield using
control milkings performed at 30-day intervals.
Samples were collected from the morning milk to
determine basic composition of fat, protein, lactose,
and solids not fat. Data on milk performance of the
sheep were analysed by the least squares method with
regard to the effect of genetic variants, using SAS
software (SAS/STAT).

Results and discussion

The PCR-RFLP method enabled b-LGB polymorphism to be accurately identified in the material
analysed. A DNA product of 103 base pairs was
obtained. Restriction fragment length polymorphism
reflected differences in nucleotide sequences resulting
from point mutations within a gene. This mutation
created a restriction site for the RsaI enzyme, which
cut DNA into two fragments of 66 and 37 base pairs.
During electrophoresis, two bands of 66 and 37 base
pairs were obtained for the AA genotype and a band
of 103 base pairs for the BB genotype. All three bands
were found in heterozygotes.
Three genotypes (AA, AB and BB) were found to
occur in all the groups of sheep (Table 1). There were
no significant differences among their frequencies,
which showed that the sheep populations studied were
in genetic equilibrium according to Hardy-Weinberg
law.

The present study demonstrated that in all breeds,

animals with the b-LGB heterozygous genotype were
predominant (Table 1). A similar relationship was
observed by Piwczynski et al. (2002) in Polish Merino
and in crosses derived from Finn, Romanov and
Booroola rams, and by Cubric-Curik et al. (2002) in
Croatian Pag sheep. For native Turkish breeds, the
frequency of AB genotypes was slightly lower than that
of AA genotypes (Elmaci et al. 2006). In Indian Jalauni
sheep the AB genotype was not found (Arora et al.
2010).
In the sheep population studied, AA homozygotes
were more frequent than BB homozygotes in
Bergschaf and Friesian sheep. BB homozygotes predominated in Merino sheep, and both genotypes had
the same frequency in Polish Mountain sheep.
In the groups described, the frequency of b-LGB
BB genotypes was low and did not exceed 15%. A less
than 5% frequency of BB homozygotes was reported
by Piwczynski et al. (2002) in Finn sheep, with no
animals of this genotype observed among crossbreds
derived from Finn rams and Merino sheep. The
BB genotype was not seen in the Iranian, Russian
(Mohammadi et al. 2006) and Romanian Karakul
(Kevorkian et al. 2008).
According to Arora et al. (2010), the B allele was
more frequent in the majority of the Indian sheep
populations. Similar observations were made for
Moghani and Afshary Iranian sheep (Elyasi et al.
2004). This variant was dominant in native Spanish
breeds Latxa, Manchega and Churra (Barillet et al.
2005).
The A allele was dominant in the East Friesian
breed mentioned above, as well as in Slovakian
Valaska and Tsigai sheep (Michalcova and Krupova
2009) and in Lithuanian Native Coarsewooled and
Blackface sheep (Kucinskiene et al. 2005).

Table 1. Distribution of b-lactoglobulin genotypes and allele frequencies.

Allele frequency
Breed
Bergschaf N  68
Friesian N  31
Polish Merino N  48
Polish Mountain sheep
N 34

Genotype Observed number Expected number

AA
AB
BB
AA
AB
BB
AA
AB
BB
AA
AB
BB

19
46
3
10
20
1
8
30
10
8
18
8

23.8
36.3
13.8
12.9
14.1
3.9
11.0
23.9
13.0
8.5
17
8.5

Genotype
frequency
0.257
0.621
0.122
0.323
0.645
0.32
0.167
0.625
0.208
0.235
0.53
0.235

Value of
x2 test

5.2504

0.55

0.45

5.188

0.66

0.34

3.0524

0.48

0.52

0.1176

0.50

0.50

70

A. Kawecka and A. Radko

Table 2. Effect of b-lactoglobulin genotype on milk production traits.

Downloaded by [95.76.154.78] at 00:16 15 November 2014

b
lactoglobulin genotype
Trait

AA
N  19

AB
N  46

BB
N 9

Bergschaf sheep
Mean daily milk yield (ml)
Fat (%)
Protein (%)
Lactose (%)
Solids not fat (%)

518.5
5.82
5.23
4.87
10.75

482.7
6.47
5.13
4.82
10.63

422.2
6.24
5.32
5.03
10.94

Friesian sheep
Mean daily milk yield (ml)
Fat (%)
Protein (%)
Lactose (%)
Solids not fat (%)

784.6
6.84
5.59
4.73
10.96

658.3
6.94
5.48
4.68
10.79

730.0
6.63
5.92
4.75
11.42

Polish Mountain sheep

Mean daily milk yield (ml)
Fat (%)
Protein (%)
Lactose (%)
Solids not fat (%)

320.77
5.52
5.04
4.9
10.37

337.25
6.06
5.44
4.9
11.2

267.69
5.75
5.62
4.6
11.13

Polish Merino sheep

Mean daily milk yield (ml)
Fat (%)
Protein (%)
Lactose (%)
Solids not fat (%)

376.63
6.6
6.56
4.67
11.88

392.24
6.94
6.42
4.69
11.94

488.0
6.24
6.28
4.79
11.97

Note: Means denoted with different letters differ statistically: a, b, c: PB0.05.

In our study we found no statistically significant

differences in milk yield according to genotype
(Table 2). No association among b-LGB polymorphism and amount of milk was found in Fresian sheep
(Staiger et al. 2010) and in Slovakian breeds (Michalcova and Krupova 2009). Ramos et al. (2002) observed
higher milk yield in AB heterozygotes (significant
differences) when investigating the effect of genotype
on milk yield traits of Portuguese breeds of sheep
raised for milk (Merino and Serra da Estrela).
In the present experiment, the effect of b-LGB on
the milk composition was not confirmed. Piwczynski
et al. (2002), in studies on Polish Merino sheep and
their crosses with prolific breeds, also found no close
relationship between the genetic polymorphism of
lactoglobulin and milk quality traits.
The milk fat content was higher in Portuguese
breeds with AB genotype of b-LGB than in the other
groups. Higher fat content was observed in milk of the
Italian Altamurana and Leccese sheep with AA and
AB genotypes (Dario et al. 2005; Dario et al. 2008)
and in Awassi sheep with BB genotype of lactoglobulin (Celik and Ozdemir 2006). When analysing the
effect of the b-LGB genotype on milk production
traits in Polish Merino sheep, Mroczkowski et al.
(2004) found that BB homozygotes had an advantage

over heterozygotes in protein content. Also, Celik and

Ozdemir (2006) showed these relationships in the
native Turkish breed. Michalcova and Krupova
(2009) did not find statistically significant differences
for Slovakian breeds of sheep.
The results obtained showed the distribution of
genotypes and alleles in the population of sheep used
for milk production. They provide no conclusive
evidence (as in cattle) about the effect of different
b-LGB variants on milk production traits in sheep.
As in the studies by other authors, the results showed
considerable variation.
The present findings may increase our knowledge
of the possibility of using milk protein genes as
potential markers of milk yield traits, but a larger
population would have to be studied. Under Polish
conditions, this particularly refers to the Polish
Mountain sheep whose milk is processed in the
mountain region.

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