In the last decades, Italy as well as other developed countries have registered a decrease in the population size of many local horse breeds. The continuous crossbreeding has determined the dilution of genetic heritage of several native breeds. The Italian Heavy Draught Horse (IHD) is the only autochthonous Italian coldblooded horse among these breeds; therefore, it represents a resource to be preserved. In 1927, the first generation of this breed was officially created by crossing different Heavy Draught horses with local mares and recorded in a Studbook. To provide the first comprehensive overview of the genetic diversity of Italian Heavy Draught horses from Central Italy, we produced and phylogenetically analysed 52 mitochondrial DNA (mtDNA) control-region sequences. Furthermore, we evaluated data available from GenBank (N = 568) to have a more complete scenario and to understand the relationships with other European Heavy Draught horse breeds. Among the IHD samples that were analysed, we identified ten of the 17 haplogroups described in modern horses. Most of these sequences fell into L, G, and M lineages, thus showing the overall mtDNA legacy of the ancestral mares that were probably used at the initial stages of breeding selections a long time ago. The high mitochondrial haplotype diversity (Hd = 0.969) found in our samples reflected the multiple maternal origins of the horses. Our results highlighted a considerable percentage of haplotypes shared especially with Bardigiano and Hungarian Heavy Draught breeds. Furthermore, both the presence of four unique haplotypes detected in our samples and their absence among all equine mitochondrial published data demonstrate a mitochondrial peculiarity that needs to be further investigated and preserved with careful breeding practices.
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