From a historical perspective, horse breeding in Bulgaria has been very well developed since the time of the Thracians (early Bronze Age c. 3000 BCE). Archaeological discoveries from this era present us with an extremely rich type diversity, including wild and local primitive horses, the prototype of heavy draft horses, and fine riding horses.The objective of this study was to investigate the genetic structure of unexamined populations of three closely related horse breeds - the Danubian Nonius Hungarian Nonius and Serbian Nonius horses. A 608 bp long fragment of the mtDNA D-loop region was amplified and sequenced. The obtained results showed completely different genetic profiles between the investigated breeds. We identified nine of the 17 haplogroups described in modern horses. Most of the obtained sequences fell into M, L, G, and O'P lineages, which reflects the genetic profiles of the ancestral mares that were probably used at the initial stages of formation of the breeds. The population of the Danubian horse was characterized by a high prevalence of Central Asian specific haplogroup G (45%), followed by Western Eurasian specific haplogroups L and M (both about 21%). In contrast to the Danubian horse, in the Nonius breed the highest frequency of Western Eurasian haplogroup M (43.5%) was found, followed by Middle Eastern haplogroups O'P (26.1%) Central Asian specific E (13.0%) and G (13.1%). The Serbian Nonius horse showed a completely different genetic profile with a prevalence of the rare for Europe haplogroup D (66.7%), followed by Central Asian specific G (16.7%). The high mitochondrial haplotype diversity (Hd = 0.886) found in the investigated samples is evidence for multiple maternal origins in all populations.In conclusion, the obtained results demonstrated a high percentage of haplogroup sharing especially in the Danubian and Hungarian Nonius horse breeds, which reflects the possible common origins of the two breeds. In contrast to these breeds, the Serbian Nonius, despite the small number of investigated animals, showed a specific genetic profile, which could be explained by different and independent origins.
Keywords: D-loop region; Nonius horse; genetic diversity; horse mitochondrial haplogroups.