Tracing the genetic roots of the indigenous White Park Cattle

A. Ludwig*, L. Alderson†, E. Fandrey‡, D. Lieckfeldt*, T. K. Soederlund* and K. Froelich‡

*Department of Evolutionary Genetics, Leibniz-Institute for Zoo and Wildlife Research, 10324, Berlin, Germany. †Rare Breeds International,

4 Cleaves Avenue, Colerne, Chippenham,Wilts, SN14 8BX, UK. ‡Tierpark ArcheWarder e.V., Zentrum f€ur alte Haus- und Nutztierrassen e.V.,

Langwedeler Weg 11, 24646, Warder, Germany.

Summary The White Park Cattle (WPC) is an indigenous ancient breed from the British Isles which

has a long-standing history in heroic sagas and documents. The WPC has retained many

primitive traits, especially in their grazing behaviour and preferences. Altogether, the aura of

this breed has led to much speculation surrounding its origin. In this study, we sequenced the

mitogenomes from 27WPC and three intronic fragments of genes from the Y chromosome of

three bulls. We observed six novel mitogenomic lineages that have not been found in any

other cattle breed so far. We found no evidence that the WPC is a descendant of a particular

North or West European branch of aurochs. The WPC mitogenomes are grouped in the T3

cluster together with most other domestic breeds. Nevertheless, both molecular markers

support the primitive position of the WPC within the taurine breeds.

Keywords ancient breed, conservation, domestication, entire mitochondrial genome,

genetic diversity, USP9Y, UTY, ZFY

Introduction

The White Park Cattle (WPC) is a primitive breed which has

been retained in Britain for many centuries. The breed is

known for ease of calving, excellent foraging ability,

longevity, milkiness, high fertility and exceptional hybrid

vigour (Alderson 1997). Today, the breed is used mainly for

the production of high-quality meat and for conservation

grazing.

The supposition of an old history for the WPC is based on

a few important references to cattle with a white coat

colour, which are found in ancient Irish history starting in

the first century B.C. (Cattle Raid of Cooley – O’Rahilly

1970). The aura of an ancient phenotype of white cattle has

led to much speculation surrounding the origin of the WPC.

However, their origin is still a mystery and an open

question. Today, it is widely accepted that modern taurine

breeds are descended from the extinct aurochs, Bos prim-

igenius, but this species had several genetic lineages, which

are partly distantly related (G€otherstr€om et al. 2005; Stock

et al. 2009; Edwards et al. 2010; Lari et al. 2011). Their

contribution to the gene pool of domestic cattle breeds

has been discussed controversially (Loftus et al. 1994;

G€otherstr€om et al. 2005; Achilli et al. 2008; Stock et al.

2009; Edwards et al. 2010; Lari et al. 2011). On one hand,

the WPC could be descended from the northern (British)

aurochs, but on the other hand, they could be the north-

western extremity of migration from the Middle East in

common with many other West European cattle breeds.

Although mitochondrial sequences have been used many

times successfully for phylogenetic reconstructions of breed

origin in domestic animals during the last decade (Cieslak

et al. 2010; Groeneveld et al. 2010; Lenstra et al. 2012), the

Y chromosomal sequences were less intensively investi-

gated (Lippold et al. 2011). Recently, a novel polymorphism

at the Y chromosome was detected (Bonfiglio et al. 2012a,

b), which allows the classification of paternal haplogroups

in cattle. In this study, we trace back the genetic roots of

the WPC using entire mitochondrial genome sequences

(maternal lineages) as well as short intron sequences of the

Y chromosome (paternal lineages) for detecting haplo-

groups.

Material and methods

In total, 19 EDTA blood samples and eight hair samples

were analysed (for their origin see Table S1). DNA from

blood was extracted using the standard protocol of the

DNeasy Blood and Tissue Kit (Qiagen), whereas the hair

protocol of the All-Tissue DNA-Kit (GEN-IAL) was used for

the hair samples.

Mitogenome analysis: We used D-loop sequences for a

genetic proof of origin indentifying offspring from the same

Address for correspondence

A. Ludwig, Department of Evolutionary Genetics, Leibniz-Institute for

Zoo and Wildlife Research, 10324 Berlin, Germany.

E-mail: Ludwig@izw-berlin.de

Accepted for publication 17 December 2012

doi: 10.1111/age.12026

1© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics

matrilineage. The mitogenome analysis was conducted

following a previously published procedure (Achilli et al.

2008). However, this approach produced satisfactory

results only for the blood samples. Additional primers

(Table S2) were necessary for the hair samples, shortening

the fragment lengths. Sanger Sequencing was done with the

BigDye Ready Reaction kit v.3.1 (Applied Biosystems) on a

3130xl Genetic Analyzer (Applied Biosystems) following

standard procedure.

Y chromosomal analysis: Our sample set included three

bulls for which we were able to investigate Y chromosome

variation in the intronic sequences of the following genes:

ZFY, UTY and USP9Y. We followed the procedure recently

described (Bonfiglio et al. 2012a,b). In addition to these

procedures, we decided to perform capillary sequence

analyses (ABI 3130xl) to achieve the detection of additional

sequence variants. Originally restriction analyses were

suggested, which are less sensitive but easier to handle for

large sample sets.

Phylogenetic calculations: The neighbour-joining trees

were calculated in MEGA 5.0 Tamura et al. (2011) using

p-distance values (1000 bootstrap replicates). Additionally, a

median-joining network (not shown) was calculated illus-

trating the phylogenetic relationships of the T3 cluster in

more detail. We used NETWORK 4.5.1.6 (Fluxus Technology

Ltd.) for this calculation.

Beef cattle [DQ124401]Korean cattle [DQ124375]

9856

T4

Ukrainian grey [GQ129208]Bos primigenius [BVA2] ItalyWPC21_25 [KC153977]Bos reference sequence [V00654]Pietmontese [EU177815]

Korean cattle [DQ124379]

56

67

51

[ ]Korean cattle [DQ124371]

WPC6_26 [KC153972]Holstein Friesean [DQ124406]Chianina [EU177825]Beef cattle [DQ124387]WPC24 [KC153976]

67

65

T

T3

Pettiazza [EU177832]WPC3_19 [KC153974]Chillingham cattleWPC23 [KC153975]WPC1 [KC153971]

Cabannina [EU177840]99

59

54

T

T1/2/3Cabannina [EU177850]

Redena [EU177861]Podolica [EU177843]Beef cattle [DQ124399]

Calvana [JN817306]Iraqi [EU177864]100

94

84

100

T1

T2

T5Piedmontese [EU177863]Chianina [FJ971081]Italian Red Pied. [FJ971082]Romagnola [FJ971083]Bos primigenius [JQ437479] Poland

DQ124389 FC3 P

100

99100

96

P

Q

T5

100Bos primigenius CPC98 England

Romagnola [FJ971087]Cinisara [FJ971086]Agerolese [FJ971084]

Mongolian cattle [FJ971088]Iranian cattle [EU177870]100

85

I (indicus)

R100

100

0.002

Figure 1 Phylogenetic tree calculated in MEGA-based p-distance values from the mitogenome sequences of domestic cattle breeds and aurochs

sequences. Novel White Park Cattle(WPC) mitogenomes are in bold.

© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12026

Ludwig et al.2

Results

We found six sequence variants in the WPC mitochondrial

genome. All of them were novel in Bos BLAST searches and

represent a unique mitogenomic pool for the WPC. All

mutations were compared with the bovine reference

sequence (BRS) (Table S3) and are archived in GenBank

(KC153972–KC153977). Within-group variation ranged

from one to 14 mutations. Sequence lengths differed from

16 339 bp (all other) to 16 426 bp (WPC1) and 16 448 bp

(WPC23) respectively. The differences are caused by tandem

repetition of a 22-bp motif located between positions 15 974

and 15 995 (control region) of the BRS. This repeat motif is

present five times inWPC1and six times inWPC23. Themotif

is found in different members of the bovine family, but so far

no repetition has been detected (BLAST search 08/10/2012).

The repeat formation in the WPC leads to heteroplasmatic

events, which were also found in some other bovine species

(data not shown). Phylogenetic reconstructions based on

neighbour joining, including representatives of all bovine

haplogroups, showed that all mitogenomes of the WPC

belong to the haplogroup T, main subgroup T3. However,

WPC1, WPC23 and the Chillingham cattle (Hudson et al.

2012) were grouped together with EU177840 (Cabannina

breed) representing a T1/2/3 haplotype (Achilli et al. 2008).

This group has a basal position to T3 and T4 in the

phylogenetic reconstruction (Fig. 1) and is discussed as

primitive within domestic cattle (Achilli et al. 2008).

Y chromosome

Identical intronic sequence fragments (overall 1237 bp) of

three different genes were detected from the three bulls. In

comparison, no new variants were found with previously

published sequences. Neighbour-joining analyses resulted in

a grouping within the Bos taurus haplogroup Y2 (Fig. S1).

This group is preferentially found in primitive breeds from

southern Europe. Recently, the Y1 haplogroup was men-

tioned as ancestral (no insertion in Bison bonasus, no data

available) for the USP9Y gene (Bonfiglio et al. 2012a,b),

whereas our analyses suggest the Y2 status as ancestral for

the ZFY and UTY genes. Both haplogroups occurred with

contrasting frequencies in two studies focussing on the

extinct aurochs (G€otherstr€om et al. 2005; Bollongino et al.

2008).

Discussion

Although records have traced back the existence of cattle

with a white coat many centuries, the WPC remained a

local breed until the early 20th century, when there were

exports to Europe, and the mid-20th century, when animals

were exported to North America (Alderson 1997). During

the 20th century, the population was small, and an acute

hierarchical structure resulted in an increasing degree of

inbreeding. Currently, the global population is about 3000

animals, and within-breed diversity in Britain has been

prioritised by expansion of population size and a dedicated

breeding programme. Comparative breed studies (Royle

1986; Blott et al. 1998) indicated that the WPC is very

distinct from other breeds, both in appearance and genetic

distance, and they retain many ‘primitive’ traits especially

in their grazing behaviour and preferences. These traits may

indicate a breed that has a unique history of artificial

selection and inbreeding in a small population. An Irish

study showed unique haplotypes based on partial mito-

chondrial D-loop sequences and suggested instead its origins

may lie in the Middle East, as it identified a link to

haplotypes found in Anatolian cattle (Flynn 2009).

The outcome of our combined mitogenome analyses and

Y chromosomal studies produced no evidence that the

WPC is a descendant of a particular North or West

European branch of aurochs. The WPC mitogenomes are

grouped in the T3 cluster together with most other

domestic breeds and the mitogenome of an Italian aurochs

(Lari et al. 2011). Notably, the Italian aurochs’s lineage is

phylogenetically only distantly related to its European

counterparts from England (Edwards et al. 2010) and

Poland (Lipinski et al. 2012). The mitogenomes of Western

and Eastern Europe aurochs were clustered in the P group,

whereas the Italian aurochs is a member of the T3 group.

Considering archaeological evidence, cattle were domesti-

cated in the Fertile Crescent about 8800 to 8300 B.C.

(Ajmone-Marsan et al. 2010), and early domestic cattle

were brought to Europe by the first farmers (Troy et al.

2001). Consequently, the roots of the genetic lineages of

the WPC are most likely in the Middle East. These founder

lineages migrated together with first farmers to southern

Europe, and sometime in the past they spread to the British

Isles. We found no evidence for introgression of North

European (British) aurochs. Nevertheless, the WPC has

substantial genetic variation. Unquestionably, the WPC

has a great conservation value resulting from its unique

cultural and historical importance.

Acknowledgements

We thank the farmers who provided samples and infor-

mation. Special thanks to Uwe G. W. Hesse (Frankenberg,

Germany) and Mario Nagel (Karlsbad/Spielberg,

Germany).

References

Achilli A., Olivieri A., Pellecchia M. et al. (2008) Mitochondrial

genomes of extinct aurochs survive in domestic cattle. Current

Biology 18, R157–8.

Ajmone-Marsan P., Garcia J.F. & Lenstra J.A. (2010) On the origin

of cattle: how aurochs became cattle and colonized the world.

Evolutionary Anthropology 19, 148–57.

© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12026

Mitogenome and y-chromosome variation of WPC 3

Alderson L. (1997) A Breed of Distinction. Countrywide Livestock

Ltd, Shrewsbury.

Anderson S., de Bruijn M.H., Coulson A.R., Eperon I.C., Sanger F. &

Young I.G. (1982) Complete sequence of bovine mitochondrial

DNA. Conserved features of the mammalian mitochondrial

genome. Journal of Molecular Biology 156, 683–717.

Blott S.C., Williams J.L. & Haley C.S. (1998) Genetic relationships

among European cattle breeds. Animal Genetics 29, 273–82.

Bollongino R., Elsner J., Vigne J.-D. & Burger J. (2008) Y-SNPs do

not indicate hybridisation between European aurochs and

domestic cattle. PLoS ONE 3, e3418.

Bonfiglio S., De Gaetano A., Tesfaye K., Grugni V., Semino O. &

Ferretti L. (2012a) A novel USP9Y polymorphism allowing a

rapid and unambiguous classification of Bos taurus Y chromo-

somes into haplogroups. Animal Genetics, 43, 611–3.

Bonfiglio S., Ginja C., De Gaetano A. et al. (2012b) Origin and

spread of Bos taurus: New clues from mitochondrial genomes

belonging to haplogroup T1. PLoS ONE 7, e38601.

Cieslak M., Pruvost M., Benecke N., Hofreiter M., Morales A.,

Reissmann M. & Ludwig A. (2010) Origin and history of

mitochondrial DNA lineages in domestic horses. PLoS ONE 5,

e15311.

Edwards C.J., Magee D.A., Park S.D. et al. (2010) A complete

mitochondrial genome sequence from a mesolithic wild aurochs

(Bos primigenius). PLoS ONE 5, e9255.

Flynn P. (2009) Characterisation of rare Irish cattle breeds by

comparative molecular studies using nuclear and mitochondrial

DNA markers. MSc thesis, National University of Ireland, Dublin,

Ireland.

G€otherstr€om A., Anderung C., Hellborg L., Galil R., Smith E.C.,

Bradley D.G. & Ellegren H. (2005) Cattle domestication in the

Near East was followed by hybridization with aurochs bulls in

Europe. Proceedings of the Royal Society B 272, 2345–51.

Groeneveld L.F., Lenstra J.A., Eding H. et al. (2010) Genetic

diversity in farm animals – a review. Animal Genetics 41, 6–31.

Hudson G., Wilson I., Payne B.I.A., Elson J., Samuels D.C.,

Santibanez-Korev M., Hall S.J.G. & Chinnery P.F. (2012) Unique

mitochondrial DNA in highly inbred feral cattle.Mitochondrion 12,

438–40.

Lari M., Rizzi E., Mona S. et al. (2011) The complete mitochondrial

genome of an 11,450-year-old aurochsen (Bos primigenius) from

central Italy. BMC Evolutionary Biology 11, 32.

Lenstra J.A., Groeneveld L.F., Eding H. et al. (2012) Molecular tools

and analytical approaches for the characterization of farm

animal genetic diversity. Animal Genetics 43, 483–502.

Lipinski D., Zeyland J., Nowacka A., Szalata M., Dzieduszycki A.M.,

Ryba M.S., Przystalowska H. & Slomski R. (2012): Bos primige-

nius mitochondrion, complete genome (JQ437479). Unpublished

sequence from GenBank.

Lippold S., Knapp M., Kuznetsova T. et al. (2011) Discovery of lost

diversity of paternal horse lineages using ancient DNA. Nature

Communications 2, 450.

Loftus R.T., MacHugh D.E., Bradley D.G., Sharp P.M. & Cunning-

ham P. (1994) Evidence for two independent domestications of

cattle. Proceedings of the National Academy of Sciences USA 91,

2757–61.

O’Rahilly C. (1970) T�ain B�o C�ualgne. Dublin Institute for Advanced

Studies, Dublin, Ireland.

Royle N.J. (1986) New C-band polymorphism in the White Park

Cattle of Great Britain. Journal of Heredity 77, 366–7.

Stock F., Edwards C.J., Bollongino R., Finlay E.K., Burger J. &

Bradley D.G. (2009) Cytochrome b sequences of ancient cattle and

wild ox support phylogenetic complexity in the ancient and

modern bovine populations. Animal Genetics 40, 694–700.

Tamura K., Peterson D., Peterson N., Stecher G., Nei M. & Kumar S.

(2011) MEGA5: molecular evolutionary genetics analysis using

maximum likelihood, evolutionary distance, and maximum

parsimony methods. Molecular Biology and Evolution 28, 2731–9.

Troy C.S., MacHugh D.E., Bailey J.F. et al. (2001) Genetic evidence

for Near-Eastern origins of European cattle.Nature 410, 1088–91.

Supporting information

Additional supporting information may be found in the

online version of this article.

Figure S1. Phylogenetic analyses based on three intronic

Y chromosomal sequences.

Table S1. Origin and pedigree of White Park Cattle (WPC)

that were analysed in this study.

Table S2. Additional primers that were used for the hair

samples DNA.

Table S3. Mitogenome haplotype definition.

© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12026

Ludwig et al.4