Genetic Diversity and Population Structure of
20 North European Cattle Breeds
J. Kantanen, I. Olsaker, L.-E. Holm, S. Lien, J. Vilkki, K. Brusgaard,
E. Eythorsdottir, B. Danell, and S. Adalsteinsson
Blood samples were collected from 743 animals from 15 indigenous, 2 old imported, and 3 commercial North European cattle breeds. The samples were analyzed
for 11 erythrocyte antigen systems, 8 proteins, and 10 microsatellites, and used to
assess inter- and intrabreed genetic variation and genetic population structures.
The microsatellites BoLA-DRBP1 and CSSM66 were nonneutral markers according
to the Ewens–Watterson test, suggesting some kind of selection imposed on these
loci. North European cattle breeds displayed generally similar levels of multilocus
heterozygosity and allelic diversity. However, allelic diversity has been reduced in
several breeds, which was explained by limited effective population sizes over the
course of man-directed breed development and demographic bottlenecks of indigenous breeds. A tree showing genetic relationships between breeds was constructed from a matrix of random drift-based genetic distance estimates. The breeds were
classified on the basis of the tree topology into four major breed groups, defined
as Northern indigenous breeds, Southern breeds, Ayrshire and Friesian breeds, and
Jersey. Grouping of Nordic breeds was supported by documented breed history
and geographical divisions of native breeding regions of indigenous cattle. Divergence estimates between Icelandic cattle and indigenous breeds suggested a separation time of more than 1,000 years between Icelandic cattle and Norwegian native breeds, a finding consistent with historical evidence.
From Animal Production Research, Agricultural Research Centre (MTT ), Jokioinen, Finland ( Kantanen
and Vilkki), Department of Morphology, Genetics and
Aquatic Biology, Norwegian College of Veterinary Science, Oslo, Norway (Olsaker), Department of Animal
Breeding and Genetics, Danish Institute of Agricultural
Science, Tjele, Denmark ( Holm and Brusgaard), Department of Animal Science, Agricultural University of
Norway, Ås, Norway ( Lien), Agricultural Research Institute, Keldnaholt, Reykjavik, Iceland ( Eythorsdottir),
Department of Animal Breeding and Genetics, Swedish
University of Agricultural Sciences, Uppsala, Sweden
( Danell), and the Nordic Gene Bank for Farm Animals,
Department of Animal Science, Agricultural University
of Norway, Ås, Norway (Adalsteinsson). J. Kantanen is
currently at the Department of Genetics, Trinity College, Dublin 2, Ireland. S. Adalsteinsson is currently at
Sudurgata 24, IS-101, Reykjavik, Iceland. The authors
would like to thank Nina Hovden-Sæther, Jon V. Jonmundsson, Elisabeth Koren, and Bine Melby for organizing animal sampling, Claes-Gøran Fristedt, Mogens
Kjaer, Tuula-Marjatta Nieminen, and Thorsteinn Olafsson for collection of blood samples, Valgerdur Andresdottir and Soeren Svendsen for isolation of DNA, Ole
Albert Guttersrud for technical assistance on DNA extraction and microsatellite genotyping, and Kaj Sandberg and Leif Anderson for genotyping Danish, Icelandic, Norwegian, and Swedish breeds for plasma
proteins and BoLA-DRBP1. We are indebted to Kari Elo
and Jaakko Lumme for valuable comments on an earlier draft of this manuscript. We acknowledge the financial support provided by the Nordic Council of Ministers and the Norwegian Ministry of Agriculture.
2000 The American Genetic Association 91:446–457
446
Genetic diversity of domestic cattle is typically distinguished into two components:
genetic differences between breeds and
genetic differences between individuals
within a breed. Previous studies on genetic diversity in cattle are based on data derived from typing of red cell antigens and
plasma proteins. Recently, increased preference has been given to microsatellites
that are ubiquitous throughout the cattle
genome and highly polymorphic (e.g., Machugh et al. 1997). Studies of genetic relationships between cattle breeds provide
useful information on the evolution of
breeds, gene pool development, and the
magnitude of genetic differentiation ( Baker and Manwell 1980; Blott et al. 1998;
MacHugh et al. 1997; Medjugorac et al.
1994). These studies are important for decisions concerning breed conservation.
Breeds with a unique evolutionary history
could potentially have a value in the maintenance of genetic diversity at the species
level ( Hall and Bradley 1995), but also for
breeding strategies aimed at improving
traits through heterosis (Graml and Pirchner 1984).
From the patterns of within-population
genetic variation at marker loci, it is possible to deduce demographic factors important to the conservation of domestic
cattle diversity (e.g., Cornuet and Luikart
1996; Kantanen et al. 1999). Breeds can be
observed to have lost within-population
genetic variation as a result of low effective population sizes ( Kantanen et al.
1999). At the marker loci, the level of allelic diversity is typically reduced more by
decreases in effective population sizes
(Ne ) than the multilocus heterozygosity,
establishing a situation known as allelic
deficiency within a population (Maruyama
and Fuerst 1985). Luikart et al. (1998) and
Luikart and Cornuet (1998) have presented a statistical test and a qualitative
graphical method for identifying this allele
deficiency from allele frequency data, and
have successfully applied these methods
to demonstrate an influence of demographic bottlenecks on genetic variation
in wildlife species.
A large phenotypic variation between
cattle populations from different geographical regions has been characteristic
for animal husbandry in northern Europe
( Denmark, Finland, Iceland, Norway, and
Materials and Methods
Breeds
The Danish, Finnish, Icelandic, Norwegian,
and Swedish breeds and the numbers of
animals (ranging from 11 to 49 animals per
breed, with a mean of 37 individuals per
breed, a total of 743 animals) studied
were:
Figure 1. The original breeding areas of indigenous and old imported North European cattle breeds are presented. Danish Jersey and Danish Shorthorn originated from British populations. Abbreviations of breed names
are as follows: JER, Danish Jersey; DKH, Danish Shorthorn; IS, Icelandic cattle; RDM, Red Danish 1970; SDM, Danish
Black-Pied 1965; SJM, Jutland breed; ORA, Eastern Red Polled; VRA, Western Red Polled; TM, Telemark cattle; DOL,
Doela cattle; VFJ, Western Fjord cattle; STN, Blacksided Troender and Nordland cattle; ROK, Swedish Red Polled;
SFR, Swedish Mountain cattle; WFC, Western Finncattle; EFC, Eastern Finncattle; NFC, Northern Finncattle.
Sweden). As a consequence of demands
for high-input farming systems, local cattle
breeds have become almost totally displaced by commercial red-and-white Ayrshire or black-and-white Holstein-Friesianbased breeds during the last decades.
Knowledge of the exact genetic relationships between native North European cattle breeds and their divergence from commercial cattle breeds is still lacking,
despite the risk of extinction for some of
the breeds. Icelandic cattle are assumed
to have descended from animals introduced from Norway when Iceland was colonized during the years 874–930 (Adalsteinsson 1981) and to have remained
isolated from other cattle populations.
Studies on bovine red cell antigen systems
( Brænd et al. 1962; Kidd and Cavalli-Sforza
1974) have indicated that Icelandic cattle
are closely related to the Norwegian native breeds Doela, Telemark, and Blacksided Troender and Nordland cattle. Local
Norwegian cattle populations are thought
to originate from Denmark, Sweden, and
possibly Finland ( Tuff 1951).
Within the present study, genetic diversity was estimated in 15 indigenous, 2 old
imported, and 3 commercial modern
North European cattle breeds using multilocus heterozygosity, the mean number
of alleles per locus, and the proportion of
polymorphic loci, and their genetic structure was examined. The present-day pattern of within-population genetic variation
of the North European cattle breeds was
also evaluated by adopting the methods of
Luikart et al. (1998) and Luikart and Cornuet (1998). The use of genetic markers
provides the only tool to assess population genetic structures of the most endangered indigenous cattle breeds because
they lack detailed pedigree recording data.
Furthermore, we studied genetic relationships between existing North European
cattle breeds, based on calculated genetic
distances and dendrograms. The magnitude of genetic differentiation between the
breeds was tested and divergence times
between Icelandic cattle and indigenous
and old imported cattle breeds were estimated. Icelandic cattle were selected as a
reference population because its history is
well documented. The results are based
on 29 loci, including microsatellites, red
cell antigen systems, plasma proteins, and
milk proteins.
(1) 15 indigenous breeds: Danish [Jutland Breed (49 animals), Danish Black-Pied
1965 (27), and Red Danish 1970 (39)],
Finnish [Eastern Finncattle (31), Northern
Finncattle (26), and Western Finncattle
(40)], Icelandic [Icelandic cattle (44)], Norwegian [Blacksided Troender and Nordland cattle (34), Doela cattle (35), Eastern
Red Polled (11), Telemark cattle (46),
Western Fjord cattle (41), and Western
Red Polled (36)], and Swedish [Swedish
Mountain cattle (41), and Swedish Red
Polled (34)]. All native Nordic breeds, except Icelandic cattle, consist of fewer than
5000 reproducing females (for census sizes, see Kantanen et al. 1999).
(2) Two old imported breeds: Danish
[Danish Jersey (41) and Danish Shorthorn
(41)]. These breeds were imported in the
19th century from Great Britain. The present census size of Danish Shorthorn is
approximately 200 breeding cows, and
that of the original Danish Jersey is 10,000.
(3) Three modern commercial breeds:
Finnish [Finnish Ayrshire (46) and Finnish
Holstein-Friesian (43)], and Norwegian
[Norwegian cattle-NRF (38)]. These are
large cattle breeds with population sizes
in excess of 300,000, 100,000, and 300,000,
respectively.
Finnish Ayrshire and NRF are classified
as Ayrshire-based breeds, and Finnish Holstein-Friesian, Danish Black-Pied 1965, and
Jutland Breed are black-and-white Friesian
breeds. Danish Black-Pied 1965 and Red
Danish 1970 represent original relics of the
old Danish Black-Pied and Red Danish
breeds, before being crossed with commercial black-and-white or red breeds.
The analyzed Danish Jersey animals were
original and have not been crossbred with
North American Jersey. The map ( Figure
1) shows the original geographical breeding areas of the indigenous and old imported breeds.
Collection of blood samples and extraction of DNA has been described previously
by Lien et al. (1999).
Loci
Estimates of genetic relationships between breeds, within-population diversi-
Kantanen et al • Genetic Diversity in North European Cattle 447
ty, and population structures were based
on allelic variation at 10 microsatellite
loci, 11 red cell antigen systems, 4 milk
protein loci, and 4 plasma protein loci.
The following microsatellites were genotyped: BoLA-DRBP1, CSSM66, INRA005,
INRA035, INRA063, ILSTS005, HEL1, HEL5,
HEL9, and HEL10 ( Barendse et al. 1997;
Kappes et al. 1997). Polymerase chain reactions (PCRs) of microsatellite loci were
carried out using either fluorescent or radioactively labeled primers. The amplified
products were separated on 6% denaturing polyacrylamide gels. Genetic variants
were visualized using either the A.L.F.
DNA Sequencer (Pharmacia, Uppsala, Sweden) or by autoradiography. Milk protein
genes (␣s1-casein, ␤-casein, ␬-casein, and
␤-lactoglobulin), antigens within each red
cell antigen system (A, B, C, F, J, L, M, S,
Z, R⬘, and T ), and one plasma protein
( Transferrin) were previously analyzed
( Kantanen et al. 1999; Lien et al. 1999). Detection of polymorphisms at three other
plasma protein loci (amylase-I, posttransferrin II, and postalbumin) were performed by electrophoretic methods according to Mazumder and Spooner (1970)
and Gahne et al. (1977).
Allele frequencies of the codominant
loci (microsatellites, milk and plasma proteins, and red cell antigen systems F and
R⬘) were estimated by direct allele counting. In the case of dominant loci (the red
cell antigen systems A, B, C, J, L, M, S, Z,
and T⬘), a maximum likelihood method
was used ( Hartl and Clark 1989). Alleles of
the A system were A, AH, H, and the recessive a, and those of the S system SH⬘,
H⬘, U⬘, H⬘U1, and the recessive s. Three alleles were detected in the F system (F, V1,
and V2). Polymorphic B and C systems
were both reduced to four alleles. Alleles
of the B system were B (antigen B), BGK
(antigen complex BGK; refer to Grosclaude
et al. 1979), non-B (those other than the B
or BGK antigen complex), and b (no antigens of system B detected). Accordingly,
alleles of the C system were C1, C2, C⬙, and
non-C. This last allele indicating an absence of C1, C2, or C⬙ antigens in the phenotype of an individual is extremely rare.
Information on alleles of other red cell antigen systems are documented by Kantanen et al. (1999). Allele frequencies of the
milk proteins (for all breeds) and transferrin (for Danish Jersey, Western Finncattle,
Finnish Ayrshire, and NRF) have been reported previously ( Kantanen et al. 1999;
Lien et al. 1999).
448 The Journal of Heredity 2000:91(6)
Statistical Analysis
Within-breed genetic variation and population structures. Intrabreed genetic variation
of the microsatellites, proteins, and erythrocyte antigen systems were quantified using observed heterozygosities and mean
expected unbiased heterozygosities ( Nei
1978) (in the case of dominant loci, only
mean expected unbiased heterozygosity
was possible), the average number of alleles per locus, and the percentage of
polymorphic loci. Loci were defined as
polymorphic when the frequency of the
most common allele was less than 0.95. Estimates of intrabreed genetic variation
were derived using the BIOSYS computer
program (Swofford and Selander 1989).
An exact test was used to determine
possible deviations from Hardy–Weinberg
proportions and the existence of nonrandom associations of genotypes across
polymorphic codominant loci (Guo and
Thompson 1992; Weir 1990). Exact tests
were performed using the program GENEPOP version 3.1 (Raymond and Rousset
1995b). A Markov chain Monte Carlo method was applied to compute unbiased estimates of the exact probabilities (P values). The length of the chain was set to be
100,000 iterations. Multiple test significance was assayed using Bonferroni correction (Weir 1990).
Population subdivision was examined
with Wright’s fixation indexes, using the
variance-based method of Weir and Cockerham (1984). F statistics were computed
using the FSTAT computer program (Goudet 1995). Means and standard deviations
of the F-statistics parameters, that is, the
correlation of genes in structured populations, F, ⌰, and f were obtained for each
locus across breeds by the jackknife procedure (Weir 1990). The significance of F,
⌰, and f estimates was determined by permuting alleles within the total population
(including all 20 breeds), genotypes within
the total population, and alleles within
samples, respectively, with 5000 permutations. The null hypothesis was that the estimates were not significantly different
from zero. The level of significance (P ⬍
.05) was adjusted with the Bonferroni correction.
The pattern of present-day within-population genetic variation was further assessed on the basis of frequency data of
codominantly inherited alleles (Cornuet
and Luikart 1996). Expected unbiased heterozygosities (Hexp) estimated assuming
Hardy–Weinberg equilibrium were compared with the theoretical values (Heq) calculated on the basis of the number of sam-
pled chromosomes and alleles observed
in the sample of a breed (Cornuet and Luikart 1996; Luikart and Cornuet 1998). This
statistical test is known to be a sign test
for heterozygosity excess. The theoretical
heterozygosity at a locus under the supposition of mutation-drift equilibrium was
obtained by 1000 iterations, assuming that
the allelic states of microsatellites change
according to the stepwise mutation model
(SMM) and proteins and erythrocyte antigen systems according to the infinite allele model ( IAM) (Cornuet and Luikart
1996). In addition, the microsatellite, protein and erythrocyte antigen alleles were
classified into 10 frequency classes. The
proportion of different classes was demonstrated using the qualitative graphical
method of Luikart et al. (1998), with the
aim of determining whether the distribution of allele frequencies follow the normal
L-shaped form, where alleles with the lowest frequencies (0.001–0.1) are the most
abundant. The computer program BOTTLENECK (Piry et al. 1999) was used for
calculations.
Genetic differentiation. The DA distance
was computed separately for microsatellite data (10 loci), protein-red cell antigen
system data (19 loci), and finally for a
combined dataset including all 29 loci.
Takezaki and Nei (1996) demonstrated
that genetic distances based on a model
of pure genetic drift, such as DA distance,
are suitable for constructions of phylogeny irrespective of mutation model of genetic markers (i.e., SMM, IAM, or twophase mutations). Trees were constructed
from DA distances using the neighbor-joining method of Saitou and Nei (1987). The
robustness of the phylogenies was evaluated by bootstrapping (1000 replicates)
over loci. For estimating evolutionary
time, the (␦␮)2 distance of Goldstein et al.
(1995) and the genetic distance measure
DTL of Tomiuk and Loeschcke (1991, 1995)
are typically recommended ( Takezaki and
Nei 1996; Tomiuk et al. 1998). From microsatellite data, we derived (␦␮)2 and DTL
distances between the breeds. Values for
(␦␮)2 distances were found to be less reliable compared to the documented history of Icelandic cattle, and standard errors of (␦␮)2 distance values estimated by
1000 bootstraps by resampling loci were
high (SE varied from 0.055 to 6.205). In
contrast, DTL appeared to give more reliable evolutionary relationships. Standard
deviations for DTL estimates obtained by
jackknifing based on 10 microsatellites
ranged from 0.005 to 0.040. DTL was chosen
for calculations of divergence times be-
Table 1. Mean observed (Hobs) and expected (Hexp) unbiased heterozygosities, mean number of alleles (A), and percentage of polymorphic loci (P0.95) in 20
North European breeds
Codominant protein-erythrocyte
antigen data (10 loci)
Microsatellite data (10 loci)
Whole protein-erythrocyte
antigen data (19 loci)
Breed
Hobs
Hexp
A
P0.95
Hobs
Hexp
A
P0.95
Hexp
A
Danish breeds
Danish Shorthorn
Danish Jersey
Red Danish 1970
Danish Black-Pied 1965
Jutland Breed
0.49 (0.09)
0.50 (0.07)
0.52 (0.05)
0.68 (0.05)
0.57 (0.07)
0.45 (0.08)
0.53 (0.05)
0.57 (0.03)
0.64 (0.04)
0.64 (0.05)
3.4 (0.5)
3.9 (0.4)
3.7 (0.4)
4.5 (0.5)
5.5 (0.7)
90
100
100
100
100
0.25 (0.08)
0.33 (0.06)
0.27 (0.07)
0.38 (0.06)
0.35 (0.06)
0.25 (0.07)
0.34 (0.07)
0.28 (0.07)
0.36 (0.05)
0.39 (0.06)
2.0 (0.2)
2.0 (0.2)
2.1 (0.2)
1.9 (0.1)
2.3 (0.3)
Finnish breeds
Eastern Finncattle
Northern Finncattle
Western Finncattle
Finnish Ayrshire
Finnish Holstein-Friesian
0.56 (0.06)
0.62 (0.07)
0.66 (0.05)
0.64 (0.06)
0.66 (0.05)
0.69 (0.05)
0.67 (0.06)
0.67 (0.04)
0.64 (0.05)
0.64 (0.04)
6.0 (0.8)
5.1 (0.7)
5.7 (0.7)
5.3 (0.9)
5.5 (0.8)
100
100
100
100
100
0.24 (0.06)
0.34 (0.05)
0.29 (0.06)
0.35 (0.07)
0.32 (0.05)
0.29 (0.06)
0.35 (0.05)
0.29 (0.06)
0.36 (0.07)
0.35 (0.05)
Icelandic breed
Icelandic Cattle
0.56 (0.08)
0.55 (0.07)
4.1 (0.7)
90
0.31 (0.05)
Norwegian breeds
Blacksided Troender
and Nordland cattle
Doela cattle
Eastern Red Polled
Telemark cattle
Western Fjord cattle
Western Red Polled
Norwegian cattle-NRF
0.63 (0.08)
0.64 (0.06)
0.64 (0.07)
0.54 (0.07)
0.68 (0.06)
0.57 (0.06)
0.63 (0.06)
0.61 (0.07)
0.64 (0.05)
0.65 (0.04)
0.59 (0.07)
0.67 (0.05)
0.62 (0.05)
0.62 (0.06)
5.2 (0.8)
4.7 (0.4)
4.0 (0.4)
5.5 (0.7)
6.4 (1.0)
5.1 (0.7)
5.0 (0.7)
100
100
100
90
100
100
100
Swedish breeds
Swedish Mountain cattle
Swedish Red Polled
0.64 (0.06)
0.55 (0.06)
0.65 (0.05)
0.60 (0.06)
5.2 (0.7)
5.2 (0.8)
100
100
P0.95
60
80
70
90
90
0.28 (0.06)
0.37 (0.05)
0.27 (0.05)
0.34 (0.04)
0.37 (0.04)
2.2 (0.2)
2.1 (0.2)
2.2 (0.2)
2.1 (0.1)
2.4 (0.2)
63
79
68
95
95
2.3 (0.2)
2.1 (0.1)
2.3 (0.2)
2.0 (0.2)
2.3 (0.2)
70
100
80
80
90
0.31 (0.04)
0.36 (0.04)
0.34 (0.04)
0.37 (0.04)
0.38 (0.04)
2.4 (0.2)
2.4 (0.2)
2.4 (0.1)
2.2 (0.1)
2.6 (0.2)
74
95
84
90
95
0.33 (0.05)
1.9 (0.1)
90
0.33 (0.04)
2.2 (0.2)
90
0.35 (0.05)
0.32 (0.08)
0.37 (0.07)
0.26 (0.06)
0.42 (0.05)
0.38 (0.08)
0.37 (0.05)
0.34 (0.05)
0.30 (0.07)
0.35 (0.06)
0.26 (0.06)
0.41 (0.05)
0.34 (0.06)
0.38 (0.04)
2.2 (0.1)
1.9 (0.2)
2.0 (0.1)
1.9 (0.1)
2.2 (0.1)
2.2 (0.1)
2.2 (0.1)
100
70
90
90
100
80
100
0.33 (0.05)
0.32 (0.05)
0.35 (0.04)
0.31 (0.05)
0.40 (0.04)
0.34 (0.04)
0.40 (0.03)
2.3 (0.2)
2.2 (0.2)
2.1 (0.1)
2.2 (0.1)
2.4 (0.2)
2.3 (0.2)
2.4 (0.1)
90
74
84
84
100
84
100
0.42 (0.06)
0.35 (0.07)
0.37 (0.05)
0.33 (0.07)
2.2 (0.1)
2.3 (0.2)
90
90
0.31 (0.04)
0.36 (0.05)
2.3 (0.2)
2.4 (0.1)
79
95
Standard errors are presented in parenthesis.
tween Icelandic cattle and the other Nordic indigenous and old breeds. The formula DTL ⫽ 2 ␮t ( Tomiuk et al. 1998),
where ␮ is the assumed mutation rate of
microsatellites and t indicates the time of
divergence as generations, was used. The
mutation rate was assumed to be 1.4 ⫻
10⫺4/locus/gamete according to averaged
estimates over large sets of loci in sheep
populations (Crawford and Cuthbertson
1996). Computer programs DISPAN ( T.
Ota, Pennsylvania State University), MICROSAT1.5b (Minch 1997), and POPDIST1.1.1 (Guldbrandtsen et al. 2000) were
applied for the respective calculations.
The null hypothesis, H0 ⫽ ‘‘the allelic
distribution at 20 codominant loci is identical across the breeds,’’ was tested using
an exact test for population differentiation
(Raymond and Rousset 1995a), available
within GENEPOP version 3.1 (Raymond
and Rousset 1995b). A Markov chain Monte Carlo method was applied to obtain an
unbiased estimate of exact probability as
in determination of Hardy–Weinberg equilibrium and genotypic associations. Probability estimates subsequently derived
from each locus were pooled for every
pairwise population comparison ( Fisher
1970).
Genetic markers for the phylogeny
study should be neutral. Therefore the Ew-
ens–Watterson test for neutrality for all
loci ( Ewens 1972; Watterson 1978) was
calculated using the computer algorithm
given by Manly (1985). The observed sum
of the squared allele frequencies (observed F), that is, homozygosity, was compared with the 95% confidence intervals
for the expected sum of the squared allele
frequencies (expected F). The sums of
squared alleles were adjusted for sample
sizes and the number of alleles. The 95%
confidence intervals and standard errors
for observed F were calculated using 1000
simulated samples. The Ewens–Watterson
test for neutrality was performed for each
locus separately across all breeds.
Results
Tables of allele frequencies for 10 microsatellites, 11 red cell antigen systems, and
8 protein loci in the 20 breeds, and tables
of DA, (␦␮)2, and DTL distances are available from the Nordic Gene Bank for Farm
Animals, Agricultural University of Norway. Allele frequencies for the four milk
proteins and transferrin ( Danish Jersey,
Western Finncattle, Finnish Ayrshire, and
NRF) have been reported by Lien et al.
(1999) and Kantanen et al. (1999), respectively.
Within-Breed Genetic Variation
A total of 92 microsatellite alleles were
identified. None of the microsatellite alleles detected were predominant across
all 20 cattle breeds. At INRA035, 19 breeds
had the same predominant allele of 104
bp. A total of 14 private microsatellite alleles (unique to one breed) were detected
in samples of Finnish Ayrshire, Finnish
Holstein-Friesian, NRF, Eastern Finncattle,
Western Finncattle, Blacksided Troender
and Nordland cattle, Doela cattle, Western
Fjord cattle, Western Red Polled, and
Swedish Red Polled. Frequencies of private alleles were lower than 0.1, except
that of the ILSTS005 allele (187 bp) identified in Doela cattle with a frequency of
0.157. The high frequency of a private allele could reflect that some individuals of
the 35 Doela cattle sampled may be related.
Mean expected unbiased heterozygosities based on microsatellite data varied
from 0.45 ( Danish Shorthorn) to 0.69
( Eastern Finncattle) ( Table 1). Values
based on codominant proteins and erythrocyte antigen systems ranged from 0.25
( Danish Shorthorn) to 0.41 (Western Fjord
Cattle), and data of all proteins and erythrocyte antigen systems including both codominant and dominant loci resulted in
similar heterozygosity values, ranging
Kantanen et al • Genetic Diversity in North European Cattle 449
from 0.27 (Red Danish 1970) to 0.40 (Western Fjord Cattle and NRF). Eastern Finncattle were exceptional since considerable
differences between observed and expected values for multilocus heterozygosity
existed at the microsatellite loci.
The average number of alleles varied
from 3.7 (Red Danish 1970) to 6.4 (Western Fjord Cattle) at the 10 microsatellite
loci studied, and from 2.1 ( Danish BlackPied 1965) to 2.6 ( Finnish Holstein-Friesian) at 19 protein loci and erythrocyte antigen systems ( Table 1). All microsatellite
loci were polymorphic in the studied
breeds with the exception of Danish Shorthorn, Icelandic cattle, and Telemark cattle.
All protein loci and erythrocyte antigen
systems were polymorphic in Western
Fjord cattle and NRF.
Hardy–Weinberg Expectations and
Random Genotypic Associations
In total, 33 of 400 locus-population comparisons revealed significant (P ⬍ .05) departures from Hardy–Weinberg proportions, which was slightly more than would
be expected by chance alone. The microsatellite locus INRA035 showed deviations
from Hardy–Weinberg proportions in Eastern Finncattle, Telemark cattle, Red Danish 1970, and Danish Jersey (P ⬍ .001, adjusted by the Bonferroni correction), and
in Swedish Mountain cattle and Swedish
Red Polled (P ⬍ .05). Significant deviations
from Hardy–Weinberg proportions were
also found at ␣s1-casein in Eastern Finncattle (P ⬍ .01), and at INRA005 in Jutland
Breed and Red Danish 1970 (P ⬍ .05).
The exact test for nonrandom association of genotypes across loci gave 20 significant values depicted in nine breeds.
Significant genotypic disequilibrium between markers on chromosome 6 was
found in Jutland breed ( between ␬-casein
and postalbumin, P ⬍ .001, adjusted by
the Bonferroni correction), in Danish Jersey ( between ␬-casein and ␣s1-casein, P ⬍
.01), in Finnish Ayrshire ( between ␬-casein
and ␤-casein, P ⬍ .001), in Icelandic cattle
( between ␤-casein and postalbumin, P ⬍
.01), and in NRF ( between ␤-casein and
postalbumin, P ⬍ .05). Further nonrandom
associations were found in Jutland breed
between INRA063 and CSSM66 (chromosomes 18 and 14, respectively; P ⬍ .001),
ILSTS005 and ␬-casein (chromosomes 10
and 6; P ⬍ .05), and HEL9 and transferrin
(chromosomes 8 and 1, respectively; P ⬍
.05), in Danish Jersey between BoLADRBP1 and CSSM66 (chromosomes 23 and
14, respectively; P ⬍ .001), and in Finnish
Ayrshire between transferrin and amylase
450 The Journal of Heredity 2000:91(6)
Table 2. Results of F statistics for each codominant locus estimated by jackknifing across the breeds
Locus
F (FITa)
⌰ (FSTa)
f(FISa)
Microsatellites
BoLA-DRBP1
CSSM66
ILSTS005
INRA005
INRA035
INRA063
HEL1
HEL5
HEL9
HEL10
Mean
0.105 (0.029)*b
0.159 (0.038)*
0.089 (0.048)
0.112 (0.030)*
0.481 (0.081)*
0.135 (0.033)*
0.080 (0.032)*
0.105 (0.022)*
0.117 (0.024)*
0.075 (0.033)*
0.133 (0.026)*
0.105 (0.021)*
0.145 (0.035)*
0.094 (0.035)*
0.078 (0.016)*
0.137 (0.049)*
0.103 (0.029)*
0.096 (0.023)*
0.133 (0.020)*
0.095 (0.017)*
0.085 (0.024)*
0.107 (0.007)*
0.000 (0.019)
0.017 (0.020)
⫺0.006 (0.047)
0.037 (0.033)
0.397 (0.073)*
0.035 (0.023)
⫺0.017 (0.027)
⫺0.032 (0.024)
0.024 (0.020)
⫺0.012 (0.031)
0.029 (0.027)*
0.143 (0.064)*
0.152 (0.056)*
0.139 (0.046)*
0.089 (0.020)*
0.193 (0.092)*
0.091 (0.036)*
0.120 (0.027)*
0.044 (0.021)*
0.156 (0.040)*
0.139 (0.035)*
0.123 (0.016)*
⫺0.034 (0.052)
⫺0.049 (0.032)
0.070 (0.039)
⫺0.011 (0.067)
⫺0.014 (0.041)
0.022 (0.034)
0.041 (0.073)
⫺0.033 (0.037)
⫺0.018 (0.034)
0.024 (0.028)
0.003 (0.013)
Proteins and erythrocyte antigen systems
0.117 (0.109)*
EAF
0.112 (0.078)
EAR
0.119 (0.056)*
Amylase I
0.088 (0.064)*
Posttransferrin II
0.181 (0.095)*
Postalbumin
0.111 (0.040)*
Transferrin
␣S1-casein
0.155 (0.059)*
␤-casein
0.011 (0.036)
␬-casein
0.141 (0.053)*
␤-lactoglobulin
0.160 (0.036)*
Mean
0.125 (0.022)*
a
b
Correspondence in Wright’s terminology, refer to Weir and Cockerham (1984).
Statistical significance P ⬍ .05 from permutation tests.
Standard deviations are presented in parentheses.
I (chromosome 1 and unknown location,
respectively; P ⬍ .05). Disequilibrium was
observed between HEL9 and HEL10 (chromosomes 8 and 19, respectively; P ⬍ .001)
and between BoLA-DRBP1 and CSSM66 (P
⬍ .001) in Western Fjord cattle, and between CSSM66 and HEL9 (P ⬍ .05) and between ␤-lactoglobulin and amylase I (chromosome 11 and unknown location,
respectively; P ⬍ .05) in Western Red
Polled. Furthermore, five nonrandom associations (P ⬍ .05) between markers in
Danish Shorthorn and one (P ⬍ .05) in
Swedish Mountain cattle were detected.
Analysis of F Statistics
Results of F statistics across all breeds using the method of Weir and Cockerham
(1984) are given in Table 2. Mean estimates for microsatellites for F, ⌰, and f
were 0.133, 0.107, and 0.029, respectively.
Estimates across proteins and erythrocyte
antigen markers were 0.125, 0.123, and
0.003, respectively. Estimates of ⌰ for each
locus were significantly (P ⬍ .05, adjusted
by the Bonferroni correction) different
from zero. Values for F were found to be
significantly different from zero, except
those for ILSTS005, EAR, and ␤-casein.
INRA035 had an exceptionally high f estimate resulting in an overall f estimate for
the microsatellite data being significantly
different from zero (P ⬍ .05). By excluding
INRA035, the microsatellite data gave
mean estimates and standard deviations
[f(P ⬎ .05) ⫽ 0.004 (standard deviation
0.008), ⌰ ⫽ 0.105 (0.008), and F ⫽ 0.109
(0.009)] corresponding to the proteinerythrocyte antigen data. Although the
present breed samples do not allow an examination of the inheritance of microsatellite alleles, it is assumed that the results
obtained for INRA035 in the analyses of
Hardy–Weinberg equilibrium and the overall f estimate are due to the presence of
undetected null alleles at the INRA035 locus.
The Sign Test and Distribution of Allele
Frequencies
Assuming SMM, the sign test showed that
Red Danish 1970 had nine microsatellite
loci with heterozygosity excess, that is,
there were too few microsatellite alleles
compared to the level of expected heterozygosities ( Table 3). This proportion of
microsatellite loci showing heterozygosity
excess in Red Danish 1970 was significantly larger (P ⫽ .03) than that supposed by
stochastic effects alone.
Using the codominant proteins and
erythrocyte antigen systems in the sign
test, a significant proportion of loci indicated a heterozygosity excess (P ⬍ .05) in
samples of Jutland breed, Danish Jersey,
Danish Black-Pied 1965, Northern Finncattle, Finnish Ayrshire, Icelandic cattle,
Western Fjord cattle, and Swedish Moun-
Table 3. Number of loci showing heterozygosity excess or deficiency, and probabilities obtained from
the sign test, assuming that microsatellites and protein-erythrocyte antigen markers evolve according to
the stepwise mutation model and the model of infinite number of mutations, respectively
Protein-erythrocyte antigen data
Microsatellite data
Breed
Exc. Hexp
Exc.
Hobs
Def.
Hobs
P
Exc.
Exc. Hexp Hobs
Def.
Hobs
P
Danish Shorthorn
Danish Jersey
Red Danish 1970
Danish Black-Pied 1965
Jutland Breed
Eastern Finncattle
Northern Finncattle
Western Finncattle
Finnish Ayrshire
Finnish Holstein-Friesian
Icelandic Cattle
Blacksided Troender and
Nordland Cattle
Doela Cattle
Eastern Red Polled
Telemark Cattle
Western Fjord Cattle
Western Red Polled
Norwegian Cattle-NRF
Swedish Mountain Cattle
Swedish Red Polled
5.04
5.60
5.69
5.76
5.75
5.77
5.74
5.78
5.76
5.75
5.09
5
3
9
8
8
5
7
4
7
5
6
4
7
1
2
2
5
3
6
3
5
3
.617
.089
.030
.131
.129
.425
.319
.204
.324
.431
.399
3.63
3.55
3.59
3.87
4.02
4.47
4.32
4.49
3.50
4.39
3.67
5
8
5
9
8
5
8
6
8
7
8
3
0
3
0
1
5
2
4
0
3
1
.266
.001
.257
.000
.008
.487
.020
.259
.001
.088
.044
5.66
5.74
5.95
5.78
5.61
5.72
5.83
5.81
5.73
6
7
7
3
4
5
6
8
5
4
3
3
7
6
5
4
2
5
.546
.318
.370
.073
.238
.438
.590
.138
.436
4.46
3.44
4.01
3.72
4.32
4.32
4.35
4.37
4.47
7
6
6
6
9
7
7
9
6
3
2
3
3
1
3
3
1
4
.096
.071
.158
.114
.003
.081
.084
.003
.253
Exc. Hexp ⫽ expected number of loci with heterozygosity excess.
Exc. Hobs ⫽ observed number of loci with heterozygosity excess.
Def. Hobs ⫽ observed number of loci with heterozygosity deficiency.
P ⫽ probability derived from the sign test.
tain cattle, when the IAM model was assumed to explain the pattern of change in
allelic states ( Table 3).
The mode of allele frequency distribution was evaluated by plotting frequency
histograms ( Figure 2). Frequency histograms of Northern Finncattle and Doela
cattle showed that the proportion of microsatellite alleles ( black bars in Figure 2D
and F) in the class between frequencies of
0.001 and 0.1 was lower than that in the
class between 0.101 and 0.2, indicating a
mode-shift distortion in the distribution of
microsatellite alleles. Distribution of codominant protein and erythrocyte antigen
alleles (open bars) into the 10 different frequency classes deviated from the normal
L-shaped mode in Icelandic cattle ( Figure
2A), Danish Black-Pied 1965 ( Figure 2B),
Danish Jersey ( Figure 2C), Northern Finncattle ( Figure 2D), Finnish Ayrshire ( Figure 2E), Doela cattle ( Figure 2F), Eastern
Red Polled ( Figure 2G), Western Fjord cattle ( Figure 2H), NRF ( Figure 2I), and Swedish Mountain cattle ( Figure 2J).
The sign test and graphical method assumes Hardy–Weinberg equilibrium and
linkage equilibrium between loci (Cornuet
and Luikart 1996; Luikart et al. 1998).
When INRA035 was omitted from the microsatellite data, the sign test under the
SMM showed a deficiency of alleles in Red
Danish 1970 (P ⫽ .048), and in Jutland
breed (P ⫽ .050), and an excess of alleles
(Hexp ⬍ Heq) in Telemark cattle (P ⫽ .040).
The distorted mode of allele frequency
distribution due to a lack of rare microsatellite alleles was observed for Doela
cattle.
The casein loci are tightly linked ( Feretti et al. 1990). The sign test and the qualitative graphical method was performed
using 8 instead of 10 markers by omitting
␣s1-casein (showed monomorphisms in
seven breeds), and either ␤-casein or ␬casein from the protein-erythrocyte antigen marker data. When ␣s1-casein and ␤casein were excluded, the data for Doela
cattle (only seven polymorphic loci) indicated a normal L mode for allele frequency
distribution instead of a distorted mode,
and the reverse for Blacksided Troender
and Nordland cattle. When ␣s1-casein and
␬-casein were excluded, the sign test and
the graphical method gave corresponding
results as the whole dataset, except for
Western Red Polled which showed a shifted frequency distribution of protein and
erythrocyte antigen alleles.
Excluding markers deviating from the
assumptions required for bottleneck testing (Cornuet and Luikart 1996; Luikart et
al. 1998) did not drastically alter the test
results. This indicates that the tests for
bottlenecks are robust.
Genetic Distances and Trees
For the microsatellite dataset, values of
the DA distance ( Nei et al. 1983) ranged
between 0.0716 ( between Eastern and
Western Finncattle) and 0.3091 ( between
Icelandic cattle and Danish Jersey). Distance for the dataset of 11 red cell antigen
systems and eight protein loci varied between 0.0204 ( between Western Finncattle
and Western Fjord cattle) and 0.1761 ( between Red Danish 1970 and Danish Jersey). Use of the combined dataset, including all 29 loci, showed that DA distances
ranged between 0.0408 ( between Eastern
and Western Finncattle) and 0.2153 ( between Red Danish 1970 and Danish Jersey).
DTL distance estimates calculated from
microsatellite data varied from 0.040 ( between Blacksided Troender and Nordland
cattle, and Western Fjord cattle) to 0.236
( between Danish Jersey and Red Danish
1970) ( Table 4). DA ( based on 29 loci) and
DTL ( based on microsatellites; Table 4) distance estimates showed that Icelandic cattle were genetically closest to Blacksided
Troender and Nordland cattle, Swedish
Mountain cattle, Northern Finncattle, Eastern Finncattle, and Western Finncattle.
Unrooted neighbor-joining trees (Saitou
and Nei 1987) were constructed from DA
distances based on (1) 10 microsatellites,
(2) 19 proteins and red cell antigen systems, and (3) the combined dataset of 29
loci. The last is presented in Figure 3. Only
seven nodes were supported by bootstrap
values higher than 50%. In this dendrogram, Northern Finncattle, Swedish Mountain cattle, Icelandic cattle, Blacksided
Troender and Nordland cattle, Eastern
Finncattle, and Western Finncattle tended
to form one group. The nodes grouping
the three Friesian breeds were among the
most strongly supported. The phylogeny
(tree not presented) was reconstructed
using 29 loci without the three commercial
breeds of Finnish Ayrshire, Finnish Holstein-Friesian, and NRF, which have been
partially generated by crossbreeding ( Kantanen et al. 1999). The branching pattern of
the indigenous and old imported breeds
was unchanged from that in Figure 3.
Lower bootstrap values were observed
when the microsatellite tree and proteinred cell antigen tree were constructed separately (not presented) than for the total
data. The microsatellite tree showed a
similar topology to that of the dendrogram
based on all 29 loci. Nodes grouping Eastern Red Polled/Danish Jersey, Western
Fjord cattle/Western Red Polled, Red Danish 1970/Danish Shorthorn, and Northern
Kantanen et al • Genetic Diversity in North European Cattle 451
Figure 2. Distribution of allele frequencies in 20 North European cattle breeds. Black bars represent the proportion of microsatellite alleles; white bars the proportion of
protein and erythrocyte antigen alleles in different frequency classes. The x axes represents the 10 frequency classes, and the y axes the proportion of alleles. Allele frequency
distributions suggest bottlenecks (distorted shape) in breeds A–J, whereas K–T tend to indicate normal L-shaped form.
Table 4. Divergence time estimates of Icelandic cattle from other indigenous and old breeds based on
DTL genetic distance estimates (Tomiuk and Loeschcke 1991, 1995) for 10 microsatellite loci
Breeds
DTL
Divergence
times
(generations)
Icelandic cattle versus
Blacksided Troender and Nordland cattle
Swedish Mountain cattle
Northern Finncattle
Western Finncattle
Eastern Finncattle
Western Fjord cattle
Swedish Red Polled
Doela cattle
Jylland Breed
Telemark cattle
Western Red Polled
Red Danish 1970
Danish Shorthorn
Eastern Red Polled
Danish Black-Pied 1965
Danish Jersey
0.062
0.084
0.094
0.105
0.105
0.116
0.135
0.137
0.165
0.172
0.182
0.206
0.220
0.236
0.237
0.240
221
300
336
375
375
414
482
489
589
614
650
736
786
843
846
857
95% CI indicate 95% confidence intervals for divergence times.
452 The Journal of Heredity 2000:91(6)
95% CI
[206, 237]
[280, 320]
[311, 360]
[355, 395]
[357, 393]
[388, 441]
[453, 511]
[465, 514]
[554, 625]
[572, 656]
[603, 696]
[687, 784]
[730, 841]
[794, 891]
[787, 906]
[793, 921]
Finncattle/Swedish Mountain cattle were
supported by bootstrap values of 86%,
73%, 63%, and 71%, respectively. The occurrence of the Danish Black-Pied 1965/
Jutland breed grouping was 80%, and the
node grouping Finnish Holstein-Friesian
and two other Friesian breeds had an occurrence of 75%.
In the protein-red cell antigen tree (not
presented), only three nodes ( Telemark
cattle/Danish Shorthorn, Danish BlackPied 1965/Jutland breed, and the node
grouping Swedish Red Polled and Eastern
Red Polled/Red Danish 1970) were supported by bootstrap values exceeding
50%. Some differences in topology compared to the two other trees were observed. For example, Finnish Ayrshire and
Norwegian cattle were in a different cluster, and Finnish Holstein-Friesian were
grouped independently of other Friesian
breeds.
erations (1105–1326 years), but for an appreciably longer time period from other
Norwegian indigenous breeds.
Population Differentiation
The exact test for population differentiation (Raymond and Rousset 1995a) showed
that combined probabilities in every pairwise breed comparison were less than
0.01 (in the case of four comparisons P ⬍
.01, other comparisons gave P ⬍ .001), indicating that allelic distributions are significantly different between breeds.
Figure 3. Unrooted neighbor-joining tree constructed from DA distances showing the relationships between 20
North European cattle breeds. Genetic distances are based on pooled data of 29 loci. Numbers at the nodes
represent the percentage of group occurrence in 1000 bootstrap replicates.
hand with Danish Jersey occurred 857
generations ago (95% confidence intervals
were 793–921 generations). Assuming a
generation interval of 5 or 6 years ( Kantanen et al. 1999), a divergence time of
some 4200–5100 years was estimated (95%
confidence intervals give 4000–5500 years).
The results indicate that Icelandic cattle
has been separated from Blacksided
Troender and Nordland cattle for 221 gen-
Divergence Times
Divergence times between Icelandic cattle
and other North European indigenous and
old breeds based on the microsatellite
data and estimates of DTL distances are
given in Table 4. The magnitude of the DTL
genetic distance between Icelandic cattle
and Danish Jersey suggests that separation of the lineages ending on one hand
with Icelandic cattle, and on the other
Table 5. The Ewens–Watterson test for neutrality
Locus
Obs F
SE
95% CI for Exp F
BoLA-DRBP1
CSSM66
Amylase I
0.1317
0.1671
0.5069
0.0911
0.0205
0.0260
[0.1749, 0.7227]
[0.1771, 0.7297]
[0.5066, 0.9986]
Locus
Breed
Obs F
SE
95% CI for Exp F
BoLA-DRBP1
Blacksided Troender
Danish Shorthorn
Doela Cattle
Eastern Finncattle
Northern Finncattle
Blacksided Troender
Eastern Red Polled
Finnish Ayrshire
Finnish Holstein-Friesian
NRF
0.1946
0.2195
0.1804
0.1738
0.1649
0.1375
0.2686
0.1609
0.1696
0.1745
0.0144
0.0175
0.0138
0.0105
0.0087
0.0066
0.0144
0.0093
0.0124
0.0191
[0.1977, 0.6631]
[0.2296, 0.7365]
[0.1967, 0.6522]
[0.1738, 0.5869]
[0.1746, 0.5473]
[0.1440, 0.4567]
[0.2851, 0.7521]
[0.1656, 0.5458]
[0.1858, 0.6171]
[0.2199, 0.7379]
CSSM66
Observed F (Obs F) values for BoLA-DRBP1, CSSM66, and amylase I are given. Standard errors (SE) for Obs F and
95% confidence intervals (95% CI for Exp F) were calculated using 1000 simulated samples. Values for BoLA-DRBP1
and CSSM66 within individual breeds are presented.
Test for Neutrality
The overall test for neutrality (pooled
across breeds) showed that observed F
values of two microsatellite loci, BoLADRBP1 and CSSM66, outlied the lower and
upper boundaries of the 95% confidence
region, and the observed F value for amylase I approached the minimum expected
F value ( Table 5). The Ewens–Watterson
test within each breed indicated that the
observed F value for BoLA-DRBP1 was outside the 95% confidence region of expected F in the individual samples of Eastern
Finncattle, Northern Finncattle, Doela cattle, Blacksided Troender and Nordland
cattle, and Danish Shorthorn. Similar findings were attained for CSSM66 in samples
of Finnish Ayrshire, Finnish Holstein-Friesian, Eastern Red Polled, Blacksided
Troender and Nordland cattle, and Norwegian cattle ( Table 5).
Discussion
The results support the usefulness of microsatellites as markers to examine the
evolutionary relationships between cattle
breeds. The protein-red cell antigen tree
appears to correspond less strongly with
known recent breed histories than the microsatellite and all the 29 loci-based phylogenies. As the average mutation rate of
microsatellites exceeds that of proteins,
and different genotyping techniques are
applied, the average number of observed
alleles at microsatellite loci is higher than
for protein loci (Crawford and Cuthbertson 1996; Kimura and Ohta 1975; Maruyama and Fuerst 1985; Ohta and Kimura
1973). Estimates of population genetic diversity, based on markers with a large
number of alleles, have been suggested to
be less biased than those based on lowpolymorphic markers ( Bowcock et al.
1994).
The current data ( Figure 3) suggest a division of the North European breeds into
four major genetic groups as follows:
Kantanen et al • Genetic Diversity in North European Cattle 453
Northern indigenous breeds, Southern
breeds, Ayrshire and Friesian breeds, and
the Jersey breed. The robustness of breed
classification based solely on genetic analysis appeared to be weakened by previous
or recent admixtures and insufficient numbers of analyzed microsatellite markers.
The branching pattern of the tree, DTL genetic distances between Icelandic cattle
and other indigenous and old breeds, together with historical evidence, suggest
that the Northern indigenous breed group
includes Icelandic cattle, Blacksided
Troender and Nordland cattle, Swedish
Mountain cattle, Northern Finncattle, Eastern Finncattle, and Western Finncattle.
The original breeding area of these animals has been the northernmost part of
Northern Europe ( Figure 1). Icelandic cattle are probably descendants from animals
of Norwegian origin brought into Iceland
between the years 874–930 (Adalsteinsson
1981). Previous studies on blood groups
have tended to confirm this ancestry
( Brænd et al. 1962; Kantanen et al. 1999;
Kidd and Cavalli-Sforza 1974). Current results ( Figure 3; Table 4) indicate that animals transported to Iceland may have
originated from breeding areas of the present-day Blacksided Troender and Nordland cattle rather than areas of Doela cattle and Telemark cattle as suggested by
Brænd et al. (1962) and Kidd and CavalliSforza (1974). It is possible to estimate
that the separation of Icelandic cattle from
Troender cattle occurred 1100–1300 years
ago, a finding consistent with the documented history of Icelandic cattle. Another explanation could be provided if it is
assumed that the cattle populations further south in Norway have been influenced to a greater extent by gene flow
from other geographic regions after separation from Icelandic cattle than the population resident in the north. The results
do not suggest clear allocation of Western
Fjord cattle, Doela cattle, and Western Red
Polled into any of the present breed
groups ( Figure 3), suggesting that these
breeds are admixtures of different groups
(MacHugh et al. 1997). Divergence between Icelandic cattle and Eastern and
Western Finncattle, the easternmost
breeds of the Northern breed group, is
proposed to have occurred approximately
2000 years ago.
Danish, Norwegian, and Swedish indigenous breeds were divided between more
than one cluster in the tree ( Figure 3),
whereas Finnish indigenous breeds were
clustered together in the Northern indigenous breed group. This implies that dif-
454 The Journal of Heredity 2000:91(6)
ferent native cattle breeds in Denmark,
Norway, and Sweden have their origins in
different evolutionary lineages, whereas
indigenous cattle in Finland appear to descend from one lineage. A common historic origin of the breeds classified into the
Northern indigenous group is obvious.
Medjugorac et al. (1994) studied genetic
relationships between 14 Balkan and
crossbred Balkan-Alpine cattle breeds, estimating genetic distance from the typings
of erythrocyte antigens, milk, and plasma
proteins. The patterns of genetic distances observed between these breeds reflected the historical spread of cattle populations in Europe. Similar regular patterns
can also be found in North Europe between Northern indigenous breeds. Genetic distance estimates of DA ( based on the
whole data) and DTL ( based on the microsatellite data) between Icelandic cattle
and other breeds in the Northern group
increased regularly in accordance with
geographical distribution from northwest
to southeast. As a result, similar regular
increases in divergence time estimates of
Icelandic cattle from the other Northern
indigenous breeds were observed ( Table
4). The proposed common origin of the
Northern indigenous breeds is based on
nuclear genetic marker analysis and it
would be useful to examine if mitochondrial and Y-chromosome markers would
be suitable tools to test this result (see
Bradley et al. 1994, 1996).
The original breeding area for Eastern
Red Polled, Swedish Red Polled, Telemark
cattle, and Red Danish 1970 has been confined to southern Scandinavia, but these
breeds do not form a homogeneous group.
The dendrogram ( Figure 3) indicates
grouping together of Eastern Red Polled
and Swedish Red Polled, but the grouping
of Telemark cattle with the two Red Polled
breeds is less strongly supported than
that with Red Danish 1970 and Danish
Shorthorn. The branching pattern ( Figure
3) implies clustering of Red Danish 1970
and the old imported breed, Danish Shorthorn, but the topology is not strongly supported. To conclude, we suggest that the
Southern breed group could be divided
into two subgroups: indigenous Red Polled
breeds ( Eastern Red Polled and Swedish
Red Polled), and indigenous and old imported Southern Scandinavian breeds
( Telemark cattle, Red Danish 1970, and
Danish Shorthorn).
Based on the present results on genetic
variation at nuclear genetic markers ( Figure 3) and the known history of the Friesian breeds, the three commercial
breeds—Finnish Ayrshire, Finnish Holstein-Friesian, and NRF—together with the
native Jutland breed and Danish BlackPied 1965 are suggested to form an Ayrshire and Friesian group. A similar observation concerning the grouping of Ayrshire
and Friesian breeds has recently been reported ( Blott et al. 1998). Grouping of Ayrshire-based breeds in the same cluster as
Friesian breeds indicates that these
breeds have a common ancestry.
The Danish Jersey appears to have no
close genetic relationships with the other
breeds in the study. This finding is consistent with other studies (e.g., Baker and
Manwell 1980; Medjugorac et al. 1994;
Blott et al. 1998), and it is suggested that
the Jersey form a fourth breed group ( Figure 3).
Genetic relationships between the Nordic breeds can partly explain the level of
present-day intrabreed genetic variation in
some of the breeds. The values of the
mean expected heterozygosities calculated from microsatellite data, and the mean
numbers of microsatellite and codominant
protein and erythrocyte antigen alleles decreased regularly in accordance with the
geographical distribution of the Northern
indigenous breeds from the southeast
( Eastern Finncattle, Western Finncattle,
Northern Finncattle) to the northwest
(Swedish Mountain cattle, Blacksided
Troender and Nordland cattle, Icelandic
cattle), suggesting the existence of a cline
with microsatellite-based heterozygosity
and allele numbers from the southeast toward the northwest of Northern Europe.
However, differences between estimates of
intrabreed genetic variation do not approach statistical significance as indicated
by the Student–Newman–Keuls test (results not presented). The relatively high
genetic diversity within the Western Fjord
cattle breed supports the assumption that
Western Fjord cattle have evolved through
an admixture of breeds possibly descending from different breed groups. In general,
both microsatellite data and red cell antigen and protein data indicate that the
breeds show similar levels of within-population genetic variation, estimated by
multilocus heterozygosity and allelic diversity ( Table 1), although recent demographic and evolutionary histories of Nordic cattle breeds differ considerably.
Eastern Finncattle had an unusual population structure since the breed had extraordinarily large deviations between observed and expected heterozygosities.
This indicates a subdivided population
structure for Eastern Finncattle (Wah-
lund’s effect), which is in accordance with
the fact that most of the Eastern Finncattle
animals saved for conservation originate
from genetically isolated inbred herds.
Several North European cattle breeds
showed the pattern of allelic deficiency at
the protein and erythrocyte antigen marker loci ( Table 3; Figure 2). When a population undergoes a reduction in effective
population size (Ne) it usually loses rare
alleles, but multilocus heterozygosity can
remain higher than expected, when expressed on the basis of the number of remaining alleles ( Luikart and Cornuet 1998;
Maruyama and Fuerst 1985). Thus both
the excess of heterozygosity ( Table 3) and
a distorted allele frequency distribution
( Figure 2) suggest a loss of allelic diversity
due to lowered Ne in several North European cattle breeds.
The probability of detecting an allelic
deficiency in the present-day pattern of
within-population genetic variation depends on the duration of time since the
reduction of Ne was initiated and the
length of this reduction (Cornuet and Luikart 1996; Luikart et al. 1998; Maruyama
and Fuerst 1985). The Danish Black-Pied
1965, Red Danish 1970, Northern Finncattle, Eastern Red Polled, and Western Fjord
cattle have experienced a decrease in population size corresponding to the bottlenecks of wild animal populations in the examples of Luikart et al. (1998) and Luikart
and Cornuet (1998). This reduction of census size to less than 200 individuals may
have induced allele losses. In contrast, Icelandic cattle, Danish Jersey, Finnish Ayrshire, NRF, and Swedish Mountain cattle
have not experienced similar severe demographic bottlenecks, although their
census sizes are also in decline. Island
breeds and breeds originating from island
breeds—such as Icelandic cattle, Danish
Jersey, Danish Shorthorn and Red Danish
1970, and breeds on the British Isles
(MacHugh et al. 1997)—appear typically
to display a lower level of intrabreed genetic variation than continental breeds.
This reflects limitations in founder populations and a more restricted gene flow for
island breeds. If the original number of alleles was small due to a small founder
population, the allelic deficiency may not
be detectable after the reduction of Ne
(Maruyama and Fuerst 1985). This could
explain the equilibrium condition presently observed in the endangered Danish
Shorthorn. The detected deficiency of alleles in Icelandic cattle and Danish Jersey
implies a more pronounced reduction of
effective size during the demographic his-
tory of these breeds. In a recent report,
the variance Ne of Danish Jersey was observed to have decreased over a 40-year
period ( Kantanen et al. 1999). The assumption of a limited founder size may
not exclusively explain the lack of alleles
in Nordic cattle breeds. Therefore it appears reasonable that the deficiency in the
number of protein and erythrocyte antigen alleles can be explained by restricted
Ne induced by breeding strategies applied
over the course of several generations as
a consequence of a low number of males
needed for breeding. Furthermore, in
breeding strategies there is also a need for
groups of related individuals within a
breed in order to obtain a reliable evaluation of animal breeding values. The highest ranking animals will contribute proportionally more to genetic variation of
the next generation than their contemporaries. Despite continuous importation of
genes via semen or breeding animals, allelic deficiency appears to be present in
the Finnish Ayrshire and NRF breeds. Only
Northern Finncattle and Doela cattle
showed a lack of both microsatellite and
protein/erythrocyte antigen alleles ( Figure
2D and F; Table 3, protein and erythrocyte
antigen data), while the Red Danish 1970
only showed the deficiency in microsatellite alleles ( Table 3). Both Northern Finncattle and Doela cattle were close to extinction during the 1970s, suggesting that
these breeds have experienced a more severe reduction of Ne than other Nordic
breeds.
In this study, 20 significant nonrandom
genotypic associations were observed in
nine breeds, the Jutland breed, Danish Jersey, Danish Shorthorn, Finnish Ayrshire,
Icelandic cattle, Western Fjord cattle,
Western Red Polled, NRF, and Swedish
Mountain cattle. In cattle, albumin and the
tightly linked three casein loci locate in
the same linkage group on chromosome 6
( Barendse et al. 1997). Genomic scans for
locating production-associated quantitative trait loci (QTL) in cattle have yielded
evidence for the existence of one (Georges
et al. 1995) or two QTL ( Velmala et al.
1999) on chromosome 6 affecting milk production. The linkage disequilibrium observed on this chromosome could be explained by the physical linkage of casein
loci, as well as selection and hitchhiking
effects connected to the QTL on this linkage group. The nonrandom genotypic association of markers in different linkage
groups found in Nordic breeds could be
explained by limited and nonexpanding Ne
during their demographic history (Slatkin
1994). Especially in Norwegian breeds,
amalgamation between different lineages
may also explain the deviation from equilibrium (see Slatkin 1994).
The Ewens–Watterson test for neutrality
suggested that the BoLA-DRBP1 and
CSSM66 microsatellites are not neutral
markers ( Table 5). The BoLA microsatellite
is located within the highly polymorphic
bovine major histocompatibility complex
(MHC) on chromosome 23. Extraordinary
diversity of the MHC region in vertebrates
may be explained by some form of balancing selection ( Edwards and Potts 1996).
CSSM66 is located on chromosome 14, and
recently a close genetic linkage between
CSSM66 and a QTL affecting milk yield,
milk fat, and protein composition has
been detected in Holstein-Friesian populations (Coppieters et al. 1998). It is possible that selection for these milk production traits has affected the CSSM66 allele
frequencies due to association with production trait alleles in the commercial
dairy cattle breeds Finnish Holstein-Friesian, Finnish Ayrshire, and Norwegian cattle, as well as in the native breeds Blacksided Troender and Nordland cattle and
Eastern Red Polled. Omitting BoLA-DRBP1
and CSSM66 from the phylogeny study,
and reconstructing the tree using 27 rather than 29 loci, resulted in a similar
branching pattern but with lower bootstrap values.
In summary, genetic divergence of the
Nordic cattle breeds in addition to withinpopulation genetic diversity is a result of
the combined effects of breed origin, the
extent of admixture occurring during
breed foundation and development, and
random genetic drift ( limited Ne during
breed foundation or recently). A few microsatellite alleles observed in only one
breed may have arisen from recent mutations after breed divergence. Some form of
selection was observed to impose on two
markers. North European breeds are significantly differentiated, indicated by the
exact test for population differentiation,
and thus their gene pools are developed
through a breed-specific evolution. The ⌰
estimates obtained by the permutation
procedure asserted the statistical significance of breed differentiation ( Table 2).
Depending on the set of markers used, 11–
12% of the total genetic variation can be
explained by differences between cattle
breeds ( Table 2). Thus the present-day
subdivision of the North European cattle
into discrete breeds is moderate, and
comparable to the extent of genetic differentiation between European cattle breeds
Kantanen et al • Genetic Diversity in North European Cattle 455
presented in the microsatellite study of
MacHugh et al. (1998).
Presently all indigenous and old North
European cattle breeds are rare or endangered, and only Icelandic cattle have a
larger census size of 30,000 breeding females. The old original Danish Jersey is
also declining due to continuous crossbreeding with American Jersey. Only the
modern Ayrshire and Holstein-Friesian
breeds have populations of relatively large
size, and therefore in Northern Europe the
Ayrshire and Friesian breed group is the
only one not endangered. It appears that
genetic variation in domestic cattle is not
only threatened by extinction of individual
indigenous breeds, but whole breed
groups are also in danger. Our study
showed that the level of effective population size of Nordic commercial cattle
breeds appears to be too low to prevent
loss of genetic variation within the species.
Based on phylogenetic analyses, the
Danish Jersey, Red Danish 1970, Danish
Shorthorn, and the group comprised of
Icelandic cattle, Swedish Mountain cattle,
and Northern Finncattle were identified as
genetically differentiated breeds. However,
the Danish breeds and Icelandic cattle
showed a reduced level of heterozygosity
and allelic diversity ( Table 1). This indicates that the effects of genetic drift occurring over the course of breed foundation and/or development have lead to an
increased divergence from other related
breeds. Thus in addition to phylogenetic
distinctness and between-population genetic variation, within-population variation and gene losses should be considered
when breeds are selected for conservation
purposes. Not only are single breeds important, but representative populations
from whole breed groups could also be
conserved in order to maintain the maximum amount of diversity of domestic cattle. Breed history may also provide a foundation for conservation. Icelandic cattle
are known to have experienced isolation
for more than 1000 years and are scientifically, historically, and culturally unique.
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Received April 22, 1999
Accepted July 17, 2000
Corresponding Editor: James Womack
Kantanen et al • Genetic Diversity in North European Cattle 457