Tracing the Genetic Roots of the Indigenous White Park Cattle

doi: 10.1111/age.12026 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 Arche Warder e.V., Zentrum fur€ 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 27 WPC 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

Gotherstr€ om€ et al. 2005; Achilli et al. 2008; Stock et al. Introduction 2009; Edwards et al. 2010; Lari et al. 2011). On one hand, The White Park Cattle (WPC) is a primitive breed which has the WPC could be descended from the northern (British) been retained in Britain for many centuries. The breed is aurochs, but on the other hand, they could be the north- known for ease of calving, excellent foraging ability, western extremity of migration from the Middle East in longevity, milkiness, high fertility and exceptional hybrid common with many other West European cattle breeds. vigour (Alderson 1997). Today, the breed is used mainly for Although mitochondrial sequences have been used many the production of high-quality meat and for conservation times successfully for phylogenetic reconstructions of breed grazing. origin in domestic animals during the last decade (Cieslak The supposition of an old history for the WPC is based on et al. 2010; Groeneveld et al. 2010; Lenstra et al. 2012), the a few important references to cattle with a white coat Y chromosomal sequences were less intensively investi- colour, which are found in ancient Irish history starting in gated (Lippold et al. 2011). Recently, a novel polymorphism the first century B.C. (Cattle Raid of Cooley – O’Rahilly at the Y chromosome was detected (Bonfiglio et al. 2012a, 1970). The aura of an ancient phenotype of white cattle has b), which allows the classification of paternal haplogroups led to much speculation surrounding the origin of the WPC. in cattle. In this study, we trace back the genetic roots of However, their origin is still a mystery and an open the WPC using entire mitochondrial genome sequences question. Today, it is widely accepted that modern taurine (maternal lineages) as well as short intron sequences of the breeds are descended from the extinct aurochs, Bos prim- Y chromosome (paternal lineages) for detecting haplo- igenius, but this species had several genetic lineages, which groups. are partly distantly related (Gotherstr€ om€ et al. 2005; Stock et al. 2009; Edwards et al. 2010; Lari et al. 2011). Their Material and methods contribution to the gene pool of domestic cattle breeds has been discussed controversially (Loftus et al. 1994; 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 Address for correspondence DNeasy Blood and Tissue Kit (Qiagen), whereas the hair A. Ludwig, Department of Evolutionary Genetics, Leibniz-Institute for protocol of the All-Tissue DNA-Kit (GEN-IAL) was used for Zoo and Wildlife Research, 10324 Berlin, Germany. the hair samples. E-mail: [email protected] Mitogenome analysis: We used D-loop sequences for a Accepted for publication 17 December 2012 genetic proof of origin indentifying offspring from the same

matrilineage. The mitogenome analysis was conducted described (Bonﬁglio et al. 2012a,b). In addition to these following a previously published procedure (Achilli et al. procedures, we decided to perform capillary sequence 2008). However, this approach produced satisfactory analyses (ABI 3130xl) to achieve the detection of additional results only for the blood samples. Additional primers sequence variants. Originally restriction analyses were (Table S2) were necessary for the hair samples, shortening suggested, which are less sensitive but easier to handle for the fragment lengths. Sanger Sequencing was done with the large sample sets. BigDye Ready Reaction kit v.3.1 (Applied Biosystems) on a Phylogenetic calculations: The neighbour-joining trees 3130xl Genetic Analyzer (Applied Biosystems) following were calculated in MEGA 5.0 Tamura et al. (2011) using standard procedure. p-distance values (1000 bootstrap replicates). Additionally, a Y chromosomal analysis: Our sample set included three median-joining network (not shown) was calculated illus- bulls for which we were able to investigate Y chromosome trating the phylogenetic relationships of the T3 cluster in variation in the intronic sequences of the following genes: more detail. We used NETWORK 4.5.1.6 (Fluxus Technology ZFY, UTY and USP9Y. We followed the procedure recently Ltd.) for this calculation.

Beef cattle [DQ124401] 98 T4 56 Korean cattle [DQ124375] Ukrainian grey [GQ129208] 51 Bos primigenius [BVA2] Italy WPC21_25 [KC153977] 67 Bos reference sequence [V00654] Pietmontese [EU177815] Korean cattle [DQ124379][] Korean cattle [DQ124371] 67 WPC6_26 [KC153972] Holstein Friesean [DQ124406] T3 65 Chianina [EU177825] Beef cattle [DQ124387] WPC24 [KC153976] T Pettiazza [EU177832] WPC3_19 [KC153974] Chillingham cattle 59 WPC23 [KC153975] 99 WPC1 [KC153971] 54 Cabannina [EU177840] T1/2/3 Cabannina [EU177850] 94 Redena [EU177861] T2 100 Podolica [EU177843] Beef cattle [DQ124399] 84 T1 Calvana [JN817306] 100 Iraqi [EU177864] T5 96 Piedmontese [EU177863] Chianina [FJ971081] 100 100 Italian Red Pied. [FJ971082] Q 99 Romagnola [FJ971083] Bos primigenius [JQ437479] Poland 100 DQ124389 FC3 P P 85 Bos primigenius CPC98 England Romagnola [FJ971087] 100 Cinisara [FJ971086] R 100 Agerolese [FJ971084] Mongolian cattle [FJ971088] I (indicus) 100 Iranian cattle [EU177870]

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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.

Results inbreeding. Currently, the global population is about 3000 animals, and within-breed diversity in Britain has been We found six sequence variants in the WPC mitochondrial prioritised by expansion of population size and a dedicated genome. All of them were novel in Bos BLAST searches and breeding programme. Comparative breed studies (Royle represent a unique mitogenomic pool for the WPC. All 1986; Blott et al. 1998) indicated that the WPC is very mutations were compared with the bovine reference distinct from other breeds, both in appearance and genetic sequence (BRS) (Table S3) and are archived in GenBank distance, and they retain many ‘primitive’ traits especially (KC153972–KC153977). Within-group variation ranged in their grazing behaviour and preferences. These traits may from one to 14 mutations. Sequence lengths differed from indicate a breed that has a unique history of artificial 16 339 bp (all other) to 16 426 bp (WPC1) and 16 448 bp selection and inbreeding in a small population. An Irish (WPC23) respectively. The differences are caused by tandem study showed unique haplotypes based on partial mito- repetition of a 22-bp motif located between positions 15 974 chondrial D-loop sequences and suggested instead its origins and 15 995 (control region) of the BRS. This repeat motif is may lie in the Middle East, as it identified a link to present five times in WPC1 and six times in WPC23. The motif haplotypes found in Anatolian cattle (Flynn 2009). is found in different members of the bovine family, but so far The outcome of our combined mitogenome analyses and no repetition has been detected (BLAST search 08/10/2012). Y chromosomal studies produced no evidence that the The repeat formation in the WPC leads to heteroplasmatic WPC is a descendant of a particular North or West events, which were also found in some other bovine species European branch of aurochs. The WPC mitogenomes are (data not shown). Phylogenetic reconstructions based on grouped in the T3 cluster together with most other neighbour joining, including representatives of all bovine domestic breeds and the mitogenome of an Italian aurochs haplogroups, showed that all mitogenomes of the WPC (Lari et al. 2011). Notably, the Italian aurochs’s lineage is belong to the haplogroup T, main subgroup T3. However, phylogenetically only distantly related to its European WPC1, WPC23 and the Chillingham cattle (Hudson et al. counterparts from England (Edwards et al. 2010) and 2012) were grouped together with EU177840 (Cabannina Poland (Lipinski et al. 2012). The mitogenomes of Western breed) representing a T1/2/3 haplotype (Achilli et al. 2008). and Eastern Europe aurochs were clustered in the P group, This group has a basal position to T3 and T4 in the whereas the Italian aurochs is a member of the T3 group. phylogenetic reconstruction (Fig. 1) and is discussed as Considering archaeological evidence, cattle were domesti- primitive within domestic cattle (Achilli et al. 2008). cated in the Fertile Crescent about 8800 to 8300 B.C. (Ajmone-Marsan et al. 2010), and early domestic cattle Y chromosome were brought to Europe by the first farmers (Troy et al. 2001). Consequently, the roots of the genetic lineages of Identical intronic sequence fragments (overall 1237 bp) of the WPC are most likely in the Middle East. These founder three different genes were detected from the three bulls. In lineages migrated together with first farmers to southern comparison, no new variants were found with previously Europe, and sometime in the past they spread to the British published sequences. Neighbour-joining analyses resulted in Isles. We found no evidence for introgression of North a grouping within the Bos taurus haplogroup Y2 (Fig. S1). European (British) aurochs. Nevertheless, the WPC has This group is preferentially found in primitive breeds from substantial genetic variation. Unquestionably, the WPC southern Europe. Recently, the Y1 haplogroup was men- has a great conservation value resulting from its unique tioned as ancestral (no insertion in Bison bonasus, no data cultural and historical importance. 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 Acknowledgements contrasting frequencies in two studies focussing on the We thank the farmers who provided samples and infor- extinct aurochs (Gotherstr€ om€ et al. 2005; Bollongino et al. mation. Special thanks to Uwe G. W. Hesse (Frankenberg, 2008). Germany) and Mario Nagel (Karlsbad/Spielberg, Germany). Discussion Although records have traced back the existence of cattle References with a white coat many centuries, the WPC remained a Achilli A., Olivieri A., Pellecchia M. et al. (2008) Mitochondrial local breed until the early 20th century, when there were genomes of extinct aurochs survive in domestic cattle. Current exports to Europe, and the mid-20th century, when animals Biology 18, R157–8. were exported to North America (Alderson 1997). During Ajmone-Marsan P., Garcia J.F. & Lenstra J.A. (2010) On the origin the 20th century, the population was small, and an acute of cattle: how aurochs became cattle and colonized the world. hierarchical structure resulted in an increasing degree of Evolutionary Anthropology 19, 148–57.

Alderson L. (1997) A Breed of Distinction. Countrywide Livestock Lenstra J.A., Groeneveld L.F., Eding H. et al. (2012) Molecular tools Ltd, Shrewsbury. and analytical approaches for the characterization of farm Anderson S., de Bruijn M.H., Coulson A.R., Eperon I.C., Sanger F. & animal genetic diversity. Animal Genetics 43, 483–502. Young I.G. (1982) Complete sequence of bovine mitochondrial Lipinski D., Zeyland J., Nowacka A., Szalata M., Dzieduszycki A.M., DNA. Conserved features of the mammalian mitochondrial Ryba M.S., Przystalowska H. & Slomski R. (2012): Bos primige- genome. Journal of Molecular Biology 156, 683–717. nius mitochondrion, complete genome (JQ437479). Unpublished Blott S.C., Williams J.L. & Haley C.S. (1998) Genetic relationships sequence from GenBank. among European cattle breeds. Animal Genetics 29, 273–82. Lippold S., Knapp M., Kuznetsova T. et al. (2011) Discovery of lost Bollongino R., Elsner J., Vigne J.-D. & Burger J. (2008) Y-SNPs do diversity of paternal horse lineages using ancient DNA. Nature not indicate hybridisation between European aurochs and Communications 2, 450. domestic cattle. PLoS ONE 3, e3418. Loftus R.T., MacHugh D.E., Bradley D.G., Sharp P.M. & Cunning- Bonfiglio S., De Gaetano A., Tesfaye K., Grugni V., Semino O. & ham P. (1994) Evidence for two independent domestications of Ferretti L. (2012a) A novel USP9Y polymorphism allowing a cattle. Proceedings of the National Academy of Sciences USA 91, rapid and unambiguous classification of Bos taurus Y chromo- 2757–61. somes into haplogroups. Animal Genetics, 43, 611–3. O’Rahilly C. (1970) Tain BoCualgne . Dublin Institute for Advanced Bonfiglio S., Ginja C., De Gaetano A. et al. (2012b) Origin and Studies, Dublin, Ireland. spread of Bos taurus: New clues from mitochondrial genomes Royle N.J. (1986) New C-band polymorphism in the White Park belonging to haplogroup T1. PLoS ONE 7, e38601. Cattle of Great Britain. Journal of Heredity 77, 366–7. Cieslak M., Pruvost M., Benecke N., Hofreiter M., Morales A., Stock F., Edwards C.J., Bollongino R., Finlay E.K., Burger J. & Reissmann M. & Ludwig A. (2010) Origin and history of Bradley D.G. (2009) Cytochrome b sequences of ancient cattle and mitochondrial DNA lineages in domestic horses. PLoS ONE 5, wild ox support phylogenetic complexity in the ancient and e15311. modern bovine populations. Animal Genetics 40, 694–700. Edwards C.J., Magee D.A., Park S.D. et al. (2010) A complete Tamura K., Peterson D., Peterson N., Stecher G., Nei M. & Kumar S. mitochondrial genome sequence from a mesolithic wild aurochs (2011) MEGA5: molecular evolutionary genetics analysis using (Bos primigenius). PLoS ONE 5, e9255. maximum likelihood, evolutionary distance, and maximum Flynn P. (2009) Characterisation of rare Irish cattle breeds by parsimony methods. Molecular Biology and Evolution 28, 2731–9. comparative molecular studies using nuclear and mitochondrial Troy C.S., MacHugh D.E., Bailey J.F. et al. (2001) Genetic evidence DNA markers. MSc thesis, National University of Ireland, Dublin, for Near-Eastern origins of European cattle. Nature 410, 1088–91. Ireland. Gotherstr€ om€ A., Anderung C., Hellborg L., Galil R., Smith E.C., Bradley D.G. & Ellegren H. (2005) Cattle domestication in the Supporting information Near East was followed by hybridization with aurochs bulls in Additional supporting information may be found in the – Europe. Proceedings of the Royal Society B 272, 2345 51. online version of this article. Groeneveld L.F., Lenstra J.A., Eding H. et al. (2010) Genetic Figure S1. Phylogenetic analyses based on three intronic diversity in farm animals – a review. Animal Genetics 41,6–31. Y chromosomal sequences. 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 Table S1. Origin and pedigree of White Park Cattle (WPC) mitochondrial DNA in highly inbred feral cattle. Mitochondrion 12, that were analysed in this study. 438–40. Table S2. Additional primers that were used for the hair Lari M., Rizzi E., Mona S. et al. (2011) The complete mitochondrial samples DNA. genome of an 11,450-year-old aurochsen (Bos primigenius) from Table S3. Mitogenome haplotype definition. central Italy. BMC Evolutionary Biology 11, 32.