Abstract
China harbors a rich diversity of donkey breeds, with small-sized donkeys (<110 cm) representing a largely underexplored group. Here, we present the first high-quality, chromosome-level genome assembly of a small-sized donkey, generated using PacBio HiFi sequencing (286.7 Gb), Hi-C scaffolding (240.47 Gb), and annotated with RNA-seq data. The final assembly has a total length of 2.7 Gb and comprises 32 chromosomes (including both X and Y chromosomes), in which five chromosomes were fully assembled without gaps. It possesses a scaffold N50 of 106.70 Mb and 84 contigs (contig N50 = 63.60 Mb), and captures 99.2% of BUSCO genes. The assembly achieved a consensus quality value (QV) of 77.44, corresponding to an extremely low base-level error rate, indicating exceptional nucleotide accuracy. This high-quality genome provides a valuable resource for investigating genetic variation, adaptive evolution, and domestication processes in small-sized donkeys, and will facilitate the conservation and sustainable utilization of rich donkey genetic resources in China.