Within the Bosnian Mountain Horse Registry (BMH), some specific coat colours are permitted. Accurate determination of coat colours can be challenging as there are variations in coat colour shades and several dun dilution variants occur. The previously found single nucleotide polymorphisms (SNPs) within two coat colour loci, T-box 3 (TBX3) and Agouti Signalling Protein (ASIP), were genotyped in 313 BMH individuals. The obtained genotypes were then compared to the predicted phenotypes by using the observed coat colour types from the International Association of Bosnian Mountain Horse Breeders (IABMHB) database. It was found that the dark bay and black coat colours were the most representative coat colours both phenotypically and genotypically. The frequency of the dominant Dun (D) dilution allele is (0.09), and this frequency is higher than the previously predicted frequency recorded in the available BMH registers, as also demonstrated. Among the identified alleles, there was a discrepancy or inconsistency between the predicted coat colour based on genotypes and the observed coat colour in 73 horses (23%). The most frequent error concerned the misclassification of horses with genotypes aa and Aa at the ASIP gene, non-dun1/non-dun1 (nd1/nd1) and non-dun2/non-dun1 (nd1/nd2) at the TBX3 gene, which can be associated with the occurrence of slight dilution phenotypes in these individuals. In contrast to the Konik and Hucul breeds, no homozygosity of the D allele was found in the BMH. The D allele can be easily overlooked or not recognised in different phenotypic groups, such as dark bay and black horses. Therefore, the hypothesis that Dun dilution effects itself is not as strongly epistatic in the BMH as described in other horse breeds. This could be the result of an additional genetic modifier suppressing the phenotypic effect of the D allele. It also suggests that there has been a persistent selection pressure in favour of dark base colours, which may have contributed to the observed differences in BMH. Finally, a significant proportion (35%) of BMH individuals with genetically black coat colour were officially classified as dark bay. We hypothesise that the difficulty in visually distinguishing these two phenotypes is due to an independent locus upstream of the ASIP gene. This locus was recently identified as a factor that darkens the typical pigmentation in dark bay horses. The results of the study confirm the importance of molecular testing in accurately determining the coat colour of horses. This would help to avoid errors in coat colour descriptions in official breeding records and provide valuable information for selective breeding programmes aimed at producing specific and desired coat colours.
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