Verification of horse maternal lineage based on derived mitochondrial DNA sequence

Extensive pedigree records in stud books characterize many horse breeds and are a resource for scientific studies, for example testing the conservation of mitochondrial DNA (mtDNA) nucleotide sequence through extended generations. This study showed that even without access to biological material from the horses in question, analysis of mtDNA sequence in tail-female relatives can be used to derive haplotypes and test alternative dam possibilities. DNA sequence of the D-loop hypervariable region was obtained from fragments generated by polymerase chain reaction (PCR) amplification of mtDNA from peripheral blood samples. PCR primers for the hyperviable region of the D-loop between tRNA(Pro) and the large central conserved sequence block were designed based on published horse sequence. Horses tracing to the same tail-female lines had identical DNA sequences, despite separation from common maternal ancestors by more than 80 years and by pedigree loops of up to 17 generations.