A 380 bp fragment of the horse D-loop region was analysed in 42 founder mare samples of the bay-coated Asturcón pony obtained in three different, isolated and mountainous areas: branch of “La Bobia” (13), branch of “Carondio” (9) and branch of “El Aguión” (20). These sequences were compared with the information provided by 37 founder matrilines of black-coated Asturcón assigned to the “Sueve” (26) and the “out-of-Sueve” (11) founder populations. The aim of this note was to ascertain the differences in founder mtDNA diversity between the two strains of the Asturcón pony and if such differences have geographical consistency. Both haplotypic (0.027 ± 0.006) and nucleotide (0.021 ± 0.011) diversity were higher in bay-coated than in black-coated Asturcón (0.024 ± 0.005 and 0.016 ± 0.009, respectively). AMOVA analyses failed in assessing any statistically differentiation among Asturcón geographical populations or strains. Differentiation among strains or population took low and non-significant values (ST of 0.037 and 0.018, respectively). Differentiation between Asturcón pony strains using mtDNA marker would not have clear support. The two strains of the Asturcón pony breed likely derive from the same ancestral mare population.
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