Long-read sequencing of the Amami rabbit mitochondrial genome reveals extensive tandem repeats in the D-loop region overlooked in previous assemblies

Abstract The Amami rabbit, Pentalagus furnessi , is an endangered species endemic to Japan, inhabiting only Amami-Oshima Island and Tokunoshima Island. Molecular phylogenetic analysis suggests that this species diverged before the separation of hares ( Lepus ) and rabbits ( Oryctolagus ), and it is considered a “living fossil” based on its morphological characteristics and evolutionary position. Despite its importance for population genetics and conservation, the complete mitochondrial genome structure had not been fully characterized. In this study, we performed whole-genome long-read sequencing using Oxford Nanopore Technology and determined the complete mitochondrial genome sequence prior to nuclear genome analysis. The total length of the Amami rabbit mitochondrial genome was 17,693 bp, approximately 1,000 bp longer than the previously reported sequence. This newly identified region corresponded to a tandem repeat region within the D-loop. The Amami rabbit D-loop region contains approximately 33 copies of an 8-base short tandem repeat and 7 copies of a 143-base long tandem repeat. We confirmed heteroplasmy in tandem repeat copy numbers, while no single-nucleotide heteroplasmy was detected. These tandem repeats in the D-loop region represent a characteristic feature of lagomorph mitochondrial genomes, and our findings suggest that D-loop lengths in lagomorphs, and possibly in other mammals, could be systematically underestimated in public databases, highlighting the need for long-read validation.