Here, we present the results of a genetic analysis of 463 Pottoka ponies corresponding to four generations, using 17 microsatellite markers. Ten years after the beginning of the Pottoka conservation programme, the values for the genetic diversity of the breed are still high and stable, indicating the success of the programme. We found null alleles in Pottoka for the ASB23, HMS3 and HTG10 microsatellites. Together with information obtained from other pony breeds from the Iberian Peninsula, this finding indicates that these microsatellites should not be used for phylogenetic analyses or parentage tests, at least for these breeds. The high heterozygosity exhibited by this breed in comparison to other ponies, together with its genetic proximity to the centroid of the allele frequencies, suggest that Pottoka allele frequencies are close to those initially exhibited by the ancestors of current European ponies. The results obtained in the current work, together with results from previous studies 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 larger extent than other horse breeds.
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