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The aim of this study was to verify the accuracy of the pedigree data of Old Kladruber dam lines using mtDNA analysis. The upstream part of the mtDNA D-loop hypervariable region (384 bp, nt15450 - nt15834) was sequenced. DNA was extracted from deep-frozen whole blood and fresh saliva samples of 170 Old Kladruber horses representing 23 dam lines from Kladruby n/L stud and some private farms. A sequencing analysis of a 293 bp long fragment of the upstream part of the D-loop region revealed 48 polymorphic sites created 16 distinct mitochondrial haplotypes, into which 23 maternal lines were grouped. The Old Kladruber sequences differ each other from one to twelve loci. Nine lines have their own distinct haplotypes. Eight lines are clustered in 3 haplotypes. Line Almerina according to pedigree data is splitted into 6 sublines, but mtDNA analysis showed in fact two distinct haplotypes only (subline Almerina-Aluta and the rest of the sublines), though differing only in one locus. A comparison of Old Kladruber haplotypes with 23 maternal lines (according to the pedigrees) showed a disagreement of biological parentage with pedigree data for 3 horses (i.e. 1,76%) of the sample set.
