J. Anim. Breed. Genet. ISSN 0931-2668 ORIGINAL ARTICLE Effects of a 10-year conservation programme on the genetic diversity of the Pottoka pony – new clues regarding their origin F. Rendo, M. Iriondo, C. Manzano & A. Estonba Department of Genetics, Physical Anthropology and Animal Physiology; Faculty of Science and Technology, University of the Basque Country, Bilbao, Spain. Keywords Summary conservation programme; European pony; Here, we present the results of a genetic analysis of 463 Pottoka ponies genetic diversity; Horse; microsatellite; corresponding to four generations, using 17 microsatellite markers. Ten Pottoka breed. years after the beginning of the Pottoka conservation programme, the Correspondence values for the genetic diversity of the breed are still high and stable, Andone Estonba, Department of Genetics, indicating the success of the programme. We found null alleles in Pot- Physical Anthropology and Animal Physiology; toka for the ASB23, HMS3 and HTG10 microsatellites. Together with Faculty of Science and Technology, University information obtained from other pony breeds from the Iberian Penin- of the Basque Country, E-48940 Bilbao, Spain. sula, this finding indicates that these microsatellites should not be used Tel.: +34 94 601 55 17; Fax: +34 94 601 for phylogenetic analyses or parentage tests, at least for these breeds. 31 45; E-mail: [email protected] The high heterozygosity exhibited by this breed in comparison to other ponies, together with its genetic proximity to the centroid of the allele Received: 4 October 2010; frequencies, suggest that Pottoka allele frequencies are close to those ini- accepted: 1 August 2011 tially exhibited by the ancestors of current European ponies. The results obtained in the current work, together with results from previous stud- ies of ponies and horses from the Iberian Peninsula, corroborate the idea of a unique origin of all ponies from the European Atlantic Area. In contrast, our results do not corroborate the idea that these are derived from a domestication event in the Iberian Peninsula, nor that they have incorporated ancient Iberian horse genes into their genetic pool to a lar- ger extent than other horse breeds. ers of Pottoka Basque Ponies – EPOFE, personal Introduction communication). According to the breed standard, Pottoka is a semi-feral pony breed which inhabits the Pottoka is a pony with a height of 1.15–1.32 m. the Basque Country (Western Pyrenees). It was cata- at the withers, with a black or dark-bay coloured logued by the Food and Agricultural Organization coat, a head with a sub-concave profile, small ears (FAO) as an endangered breed, since in 1995, there and large nasal orifices, a thick and strong neck with were only 400 females and 170 males. Moreover, long mane, sub-concave back and with a thick coat Pottoka presented a decreasing population trend. in Winter (EPOFE, http://www.pottoka.info/ The establishment of the Pottoka studbook in 1995 index.php?id=en; Figure 1). contributed to reversing this trend, with the result These animals freely reproduce within their herd, that in 2009, there were around 685 animals (610 but EPOFE certifies, within each herd, the appropri- mares and 75 studs) which were classified as being ateness of the males as well as the females which are purebred (data from the Basque Federation of Breed- to be used for reproductive ends. Each breeder can ª 2011 Blackwell Verlag GmbH • J. Anim. Breed. Genet. (2011) 1–10 doi:10.1111/j.1439-0388.2011.00955.x Genetic diversity and origin of Pottoka pony F. Rendo et al. revealing a pattern of linkage disequilibrium which is compatible with an early domestication of widespread herds of females and an intimate relation among all equine breeds worldwide (Wade et al. 2009). Regard- ing the origin of Iberian breeds, two studies of the ori- gin and history of mitochondrial DNA (mtDNA) lineages (Cieslak et al. 2010; Lira et al. 2010) have raised the possibility of the existence of an indepen- dent domestication episode in the Iberian Peninsula, or at least, the use of wild Iberian maternal lineages in a process of restocking populations. In this regard, a study employing nuclear markers (Warmuth et al. 2011) has suggested that the Iberian Peninsula as well as the steppes of Eastern Europe may have been refuges during the Holocene, from which were Figure 1 A typical purebred male Pottoka pony. derived the equine populations of the central and northern Europe. It cannot be concluded from these results that the Iberian Peninsula was an area of inde- have its own male, or EPOFE can provide its own pendent domestication; nevertheless, in keeping with males to breeders who request them. On the other the mtDNA results, these findings do indicate that the hand, there is a programme to restock males for their genetic contribution of these wild populations to local subsequent selection and use as reproducers. In the domestic stocks may have been considerable. first place, at 3 years of age, EPOFE morphologically Regarding genetic studies of the Pottoka breed, grades all the future studs and carries out a paternity Can˜ o´ n et al. (2000) showed using microsatellite test to verify their genealogy. Those animals which markers that this breed grouped with the other pony present a higher morphological rating and whose breeds from the northern Iberian Peninsula, genealogy is corroborated by the genetic paternity although with a confusing population structure. tests are selected as future candidate studs. At approx- These authors suggested that this was owing to a imately 5 years of age, animals with the highest grad- common origin of the analysed pony breeds which, ing based on morphological evaluation are selected as together with a similar morphology, would have the studs offered by EPOFE to breeders who request permitted in the past a reciprocal introgression them. Thus, the principal differences with respect to between these breeds. On the other hand, the results breeding prior to the publication of the studbook in obtained by Royo et al. (2005) upon analysing the 1995 consist of herds being formed exclusively by mtDNA of Iberian Peninsula breeds, did not manage purebred individuals and of the use of certified repro- to differentiate Pottoka from the other pony breeds; ductive males in those herds which require them. neither did they support the traditional classification The domestication of the horse, which began over of native Iberian breeds into northern and southern 6000 years ago (Outram et al. 2009), seems to have breeds. Finally, Solis et al. (2005) analysed the Pot- been a complex process. In present-day horses, high toka breed within the context of other autochtho- matrilineal diversity has been reported (Vila` et al. nous breeds in the western Pyrenees region (such as 2001; Jansen et al. 2002; Cieslak et al. 2010), which the Jaca Navarra pony, and the ‘Euskal Herriko Men- has been attributed to their multiple origins, an extre- diko Zaldia’ and Burguete heavy horses). The Pottoka mely large number of female founders and ⁄ or a breed was found to be situated at one of the ends of large-scale introgression of local lineages into the the spectrum of regional genetic variability, and the domestic stock (Cieslak et al. 2010). In contrast, very Jaca Navarra was found to be in a position between low levels of Y-chromosome variability have been Pottoka and the heavy horses. The within breed found (Lindgren et al. 2004). Additionally, the genetic variability detected for Pottoka and Jaca extensive mobility of the horse, favoured by its Navarra was found to be very similar to that found domestic roles, is also responsible for the progressive for these same breeds by Can˜ o´ n et al. (2000), being obscuring of the genetic structure of the species dur- slightly higher than that reported for other European ing the migrations subsequent to its domestication or American horse breeds (Solı´s et al., 2005). (Hill et al. 2002). Recently, a high quality draft of the In the light of the above, the objective of the cur- sequence of the equine genome has been published, rent study was double: on the one hand, and follow- 2 ª 2011 Blackwell Verlag GmbH • J. Anim. Breed. Genet. (2011) 1–10 F. Rendo et al. Genetic diversity and origin of Pottoka pony ing more than 10 years of implantation of the Pottoka N 6 7 4 4 7 101318184147424450545048 conservation programme, we aimed to characterize (a) 14 12 the current genetic status of the breed, thereby evalu- 10 8 ating the management to which it is being subjected. 6 On the other hand, we aimed to enhance our under- MNA 4 2 standing of the origin of this breed, by providing new 0 data regarding its genetic status within the context of other breeds of Iberian ponies in particular, and of (b) 12 10 European ponies in general. 8 6 AR 4 2 Materials and methods 0 Samples (c) 1.0 0.9 Blood samples were taken from a total of 463 pure- 0.8 o 0.7 bred Pottoka ponies, from the Gipuzkoa province, H 0.6 born between 1984 and 2005. Sampling of the horses 0.5 was generally carried out at 3 years of age, when 0.4 morphological and genetic grading of the individuals is performed. Seventeen age subgroups were estab- (d) 1.0 0.9 lished: one per year from 1990 to 2005, both inclu- 0.8 e 0.7 sive, with the exception of those born from 1984 to H 0.6 1989, which were grouped together owing to the low 0.5 number of available individuals. The sample size of 0.4 each subgroup is indicated in Figure 2. The 376 indi- 1990 1993 1994 1996 1997 2003 2004 2005 viduals born after 1998 represent 76.9% of the 489 1991 1992 1995 1998 1999 2000 2001 2002 animals currently recorded in the Gipuzkoa studbook.