Dairy bulls have been genotyped for K-casein and P-lactoglobulin from semen samples by methodology based on a polymerase chain reaction (PCR), In this study, a previously described method for κ-casein A and B variants was extended to cover also the detection of the E variant. For β-lactoglobulin the variants A and B were genotyped by another PCR-based method. The frequencies of the κ-casein and β-lactoglobulin alleles were determined from 308 and 291 Finnish Ayrshire and 42 and 44 Finnish Friesian bulls, respectively. The bulls had been born between 1973 and 1988. There was no noticeable trend in the differences between allele frequencies over the years, the overall frequencies of κ-casein A, B and E being 0.62, 0.09 and 0.29 in the Finnish Ayrshires and 0.85, 0.14 and 0.01 in Finnish Friesians. The overall frequencies of β-lactoglobulin A and B alleles were 0.25 and 0.75 in Ayrshires and 0.56 and 0.44 in Friesian

Asko Mäki-Tanila

Esa A. Mäntysaari

Riikka Velmala

Agricultural and Food Science

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Molecular genetic polymorphism at the K-casein and (3-lactoglobulin
loci in Finnish dairy hulls
Riikka Velmala, Esa A. Mäntysaari and Asko Mäki-Tanila
Velmala, R., Mäntysaari, E. A. & Mäki-Tanila, A. 1993. Molecular genetic poly-
morphism at the K-casein and li-lactoglobulin loci in Finnish dairy bulls. Agric. Sci.
Finl. 2: 431-435. (Agric. Res, Centre of Finland, Inst. Animal Prod., FIN-31600
Jokioinen, Finland.)
Dairy bulls have been genotyped for K-casein and P-lactoglobulin from semen samples
by methodologybased on a polymerase chain reaction (PCR), In thisstudy, a previously
described method for K-casein A and B variants was extended to cover also the detection
of the E variant. For P-lactoglobulin the variants A and B were genotyped by another
PCR-based method. The frequencies of the K-casein and P-lactoglobulin alleles were
determined from 308 and 291 Finnish Ayrshire and 42 and 44 Finnish Friesian bulls,
respectively. The bulls had been bom between 1973 and 1988. There was no noticeable
trend in the differences between allele frequencies over the years, the overall frequen-
cies of K-casein A, B and E being 0.62,0.09 and 0.29 in the Finnish Ayrshires and 0.85,
0.14 and 0.01 in Finnish Friesians. The overall frequencies ofP-lactoglobulin A and B
alleles were 0.25 and 0.75 in Ayrshires and 0.56 and 0.44 in Friesians.
Key words: dairy cattle, PCR, K-casein, P-lactoglobulin, genetic variation
Introduction
The genetic polymorphism of the four major casein
proteins (asi, ctS2, P and k) and two whey proteins
(a-lactalbumin and P-lactoglobulin) in cattle is
well documented (Grosclaude 1988). It derives
both from substitutions and deletions in the amino
acid sequences of theseproteins (Eigel et al. 1984).
Protein electrophoresis has been used to identify
the different protein variants of bovine milk. This
kind of methodology for the determination of bull
genotypes requires the analysis of multiple
dam/daughter pairs, thereby considerably delaying
the results. To overcome these restrictions, DNA
analysis techniques have been applied to type an-
imals for milk protein loci (LEVÉZIEL et al. 1988,
ROGNE et al. 1989). Among DNA amplification
techniques, several PCR-based methods for geno-
typing milk protein loci have been published (Me-
drano and Aguilar-Cordova 1990a, 1990b,
David and Deutch 1992,Lien et al. 1992).
We have recently initiated a study to analyze the
associations between production traits and milk
protein genotypes in Finnish dairy cattle. The pre-
sent paper is the first phase of the work. Here we
describe some modifications to existing PCR-based
methodology and present the allele frequencies of
K-casein (CASK) and P-lactoglobulin (LGB) for
the major Finnish dairy breeds from a sample of
artificial insemination (AI) bulls.
Material and methods
Frozen semen pellets from altogether 324 Finnish
Ayrshire and 53 Finnish Friesian bulls were ob-
431
Agric. Sei. Finl. 2 (1993)
tained from five Finnish AI societies. The birth
years of the bulls ranged from 1973 to 1988. Ayr-
shire bulls were divided into three age groups ac-
cording to their birth year: 1973-81, 1982-85 and
1986-88.
The method described by Zadworny and
Kuhnlein (1990) was applied to isolateDNA from
the semen. The DNA concentration was determined
by fluorescence (Hoefer TKO 100 Fluorometer).
The yield was in the range of 10-30 |ig DNA per
semen pellet. The isolation of DNA from whole
blood was done with a rapid method described by
Kawasaki (1990).
The PCR reactions, restriction enzyme diges-
tions and gel runs for CASK A and B variants and
for LGB A and B variants were performed under
the conditions described by Medrano and Agui-
lar-Cordova (1990a, 1990b), with following
modifications: The amount of DNA in the reaction
was 100 ng; the reaction volume was 25 pi; the
reaction buffer did not contain gelatin; and 1.25
units of Taq DNA polymerase (Promega) were
used per reaction. The oligonucleotides used for the
amplification ofCASK were
K346A (5 ’-CATTTATGGCCATTCCACCAAAG-3’)
and
K3468 (5’-CATTTCGCCTTCTCTGTAACAG-3 ’).
These primers are otherwise the same as JKSOI
and JK3O2 (Medrano and Aguilar-Cordova
1990a) but they are 2 and 4 bases shorter. Also,
K 3468 is an inversion of the JK3O2 orientated
erroneously, as could be deduced from the K-casein
sequence (Alexander et al. 1988).For CASK the
PCR programme consisted of denaturation for 3
min at 94°C followed by 1 min at 94°C, 50 sec at
60°C, 30 sec at 72°C for 10 cycles, 1 min at 94°C,
50 sec at 60°C, 45 sec at 72°C for 20 cycles, 1 min
at 94°C, 50 sec at 60°C, 1 min at 72°C for 10cycles.
A final extension of primers for 5 min at 72°C was
included for both PCR programmes. To improve
specificity and yield of the reaction, the ’hot start’
technique was applied to start the reactions: the
nucleotides needed for amplification were added
during the first denaturation step of the PCR pro-
gramme. The amplifications were performed with a
Hybaid Thermal Reactor (Hybaid Limited, Middle-
sex, UK).
CASK E variant has been genotyped at DNA
level using the restriction enzyme Hae 111 (SCHLIE-
ben et al. 1991).We found that with the PCR-prod-
uct obtained by the method of Medrano and
Aguilar-Cordova (1990a), also a specific band-
ing pattern for E variant could be observed after
digestion with Hae111. Thus, the method for CASK
was extended to cover also the detection of the E
variant. An aliquot of 10 p.l of the PCR product was
used for all restriction enzyme digestions.
Prior to this study, 20 cows ofFinnish Ayrshire
breed with known CASK and LGB genotypes had
been tested with the above genotyping assays from
blood samples. The results deduced from the DNA
analysis were identical to those obtained at protein
level.
Results and discussion
In addition to CASK A and B variants we also
detected the E variant. The mutation in codon 155
(Ser to Gly) is known to be responsible for this
variant (Erhardt 1989). The digestion of the
CASK PCRproduct (346 bp) with Hae 111 resulted
in fragments 337 bp (and 9 bp) for non-E and 192
bp, 145 bp (and 9 bp) for the E variant. Bands were
easily distinguishable since a strong amplification
product was obtained in most cases (Figure 1). The
polymorphism at codon 148 was detected using the
procedure of Medrano and Aguilar-Cordova
(1990a) for CASK A and B variants. The polymor-
phism at codon 118 was utilized for LGB (Me-
drano and Aguilar-Gordova 1990b).
A successful amplification was obtained from
308 and 291 Ayrshire samples and from 42 and 44
Friesian samples in CASK and LGB, respectively.
The allele distributions calculated from these res-
ults are presented in Table 1. For the Finnish Ayr-
shire breed, the allelic frequencies of A, B and E at
the CASK locus were 0.61, 0.10 and 0.29, and the
frequencies ofA and B at the LGB locus were 0.25
and 0.75. For the Finnish Friesian, the allelic fre-
quencies of A, B and E at CASK were 0.85, 0.14
432
Agric. Sei. Finl. 2 (1993)
and 0.01 and those of A and B at LGB were 0.56
and 0.44.
Our results for the overall allele frequencies of
CASK for Ayrshires and Friesians are in agreement
with previous results obtained for these breeds by
protein electrophoresis (Piironen et al. 1992). In
Ayrshires, there was some variation in the fre-
quency of A and E allele between the age groups.
These differences, however, did not represent any
trend in time. There had been no drastic alterations
in selection criteria over the studied period, so that
the changes could not be explained as being result-
ing from a correlated response to selection for pro-
duction. The frequency fluctuations are most likely
due to genetic sampling involving only a small
number of parents sireing the bulls analyzed.
In LGB, there were no noticeable differences in
allele frequencies between the age groups. The fre-
quency of LGB B allele, however, was somewhat
higher in Ayrshires and lower in Friesians com-
pared to previous results for these breeds (Aal-
tonen and Antila 1987, Atroshi et al. 1982,
Piironen et al. 1992, Tervala et ai. 1983). The
differences here, too, may be due to genetic sampl-
ing. On the other hand, the present study was made
with bulls whereas the previous ones have dealt
with cows. Another factor is that in the 1980‘s there
were substantial semen contributions by Holstein
bulls into the Friesian population, which may have
changed the allele distribution in Friesian bulls.
Several studies have investigated the associa-
tion between milk protein variants and various
economically important milk traits, including
manufacturing properties (e.g. AALTONEN and AN-
TILA 1987, Ng-Kwai-Hang et al. 1990). Current
molecular genetic techniques allow a thorough
screening of genetic variation among Al-bulls for
milk protein loci. The analyses presented here
clearly indicate that a routine and inexpensive test-
ing for CASK and LGB is feasible. However, a
more direct and detailed analysis of the association
between the economic traits and milk protein vari-
ants is required. In that context, the CASK locus
should be analyzed together with other closely
linked casein loci (Lien et al. 1993).
Acknowledgements. We wish to thank the Al societies for
providing the required semen samples. We are grateful to
Peter Bredbacka, Juha Kantanen and Johanna Viikki for
technical support and useful discussions, and to the Finnish
Ministry ofAgriculture for part of the funding.
Table 1.Number of samples and allele frequencies of K-casein and B-lactoglobulin loci in Al bulls by breed
and age group.
K-casein P-lactoglobulin
~~Ä B E Ä B
Finnish Ayrshire No. No.
1973-81 82 0.71 0.07 0.23 75 0.26 0.74
1982-85 85 0.52 0.11 0.36 81 0.28 0.72
1986-88 141 0.62 0.10 0.27 135 0.22 0.78
total 308 0.62 0.10 0.29 291 0.25 0.75
Finnish Friesian
1975-88 42 0.85 0.14 0.01 44 0.56 0.44
No-number of animals analyzed.
Fig. 1. Discrimination of K-casein genotypes as detected by
agarose gel electrophoresis of Hinfl (lanes 1,3,5) and Hae 111
digested (lanes 2,4, 6) PCR products. The three samples
presented here were typed to be DNA genotypes EE (lanes 1
and 2), AE (lanes 3 and 4) and AB (lanes 5 and 6) of the
CASK gene. (Photo: R. Velmala).
433
Agric. Sei. Fint. 2 (1993)
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Manuscript received May 1993
Riikka Velmala
Esa A. Mäntysaari
Asko Mäki-Tanila
Agricultural Research Centre of Finland
Institute of Animal Production
Section of Animal Breeding
FIN-31600 Jokioinen, Finland
434
Agric. Sei. Fint. 2 (1993)
SELOSTUS
K-kaseiinin ja (3-laktoglobuliinin varianttien molekyyligeneettinen tunnistaminen ja
muuntelu suomalaisissa lypsykarjasonneissa
Riikka Velmala, Esa A. Mäntysaari ja Asko Mäki-Tanila
Maatalouden tutkimuskeskus
Kotieläintuotannon tutkimuslaitoksen eläinjalostusyksikössä
on aloitettu tutkimus maitoproteiinivarianttien yhteyksistä
eläinten tuotanto-ominaisuuksiin. Tässä julkaisussa esitetään
työn ensimmäinen vaihe, jossa molekyyligeneettistä menetel-
mää sovellettiin keinosiemennyssonnien K-kaseiinin ja |l-lak-
toglohuliinin genotyypitykseen. Tuloksista laskettiin näiden
lokusten alleelifrekvenssit ayrshire- ja friisiläisroduille.
DNA-monimuotoisuutta analysoitiin entsymaattisen mo-
nistamisen (PCR) ja DNA:ta spesifisesti pilkkovien entsyy-
mien avulla. K-kaseiinin variantit A, B jaE sekä fJ-laktoglobu-
liinin variantit A ja B määritettiin spermanäytteistä. Yhteensä
308/291 ayrshire-ja 42/44 friisiläisnäytettä analysoitiin K-ka-
seiinin/p-laktoglobuliinin suhteen. K-lokuksen A-, B- ja E-al-
leelien frekvenssit olivat ayrshire-sonneilla 0,61,0,10 ja 0,29
ja friisiläissonneilla 0,85, 0,14 ja 0,01. p-laktoglobuliinilo-
kuksessa olivat A- ja B-alleelien frekvenssit ayrshirella 0,25
ja 0,75 ja friisiläisillä vastaavasti 0,56 ja 0,44. Allee-
lifrekvenssejä tutkittiin ayrshirellä myös ikäryhmittäin sonni-
en syntymävuosien (1973-88) mukaan. Frekvensseissä ei ha-
vaittu systemaattista muutosta tutkittuna ajanjaksona. Näiden
rotujen K-lokuksen alleelifrekvenssit vastasivat hyvin jo ai-
emmin maidosta proteiinielektroforeesilla määritettyjä frek-
venssejä. (f-lakloglobuliinin alleelifrekvensseissä havaittiin
vähäisiä eroja aikaisempiin tutkimuksiin verrattuna. Erot saat-
tavat johtua analysoidun aineiston koosta. Lisäksi aikaisem-
mat tutkimukset on tehty lehmillä kun taas tässä tutkimukses-
sa käytettiin sonneja.
Maitoproteiinialleelien ja taloudellisten ominaisuuksien
välisistä yhteyksistä tarvitaan yksityiskohtaisempaa analyy-
siä. Tällaisessa analyysissä K-kaseiinilokus pitäisi analysoida
yhdessämuiden geneettisesti kytkeytyneiden kaseiinilokusten
(etsi-, «S2- ja fi-kaseiinin> kanssa.
435
Agric. Sei. Fint. 2 (1993)


Molecular genetic polymorphism at the κ-casein and β-lactoglobulin loci in Finnish dairy hulls

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Molecular genetic polymorphism at the κ-casein and β-lactoglobulin loci in Finnish dairy hulls

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