Abstract

Homatula variegata is a small benthic loach from the upper Yangtze and adjacent basins with aquaculture and ornamental value but no reference genome. We present a near telomere-to-telomere (T2T) chromosome-level assembly built from PacBio HiFi, Oxford Nanopore ultra-long, Illumina short reads, and Hi-C. The 641.26-Mb genome resolves 24 chromosomes as single contigs (contig N50, 24.40 Mb). Hi-C confirms chromosome-length scaffolding; we detect 24 putative centromeres and 20 terminal telomeric tracts, with 22 chromosomes gap-free and two containing one gap. Annotation identifies 24,479 protein-coding genes, 93% functionally assigned, and 27.13% repetitive content dominated by DNA transposons. Quality assessments show high completeness (BUSCO, 96.48% complete) and base-level accuracy consistent with k-mer and read-mapping metrics. To our knowledge this is the first near T2T-level reference for any loach (Cobitoidei), filling a key gap in Cypriniformes genomics. This resource will enable comparative and population genomics, illuminate adaptation to montane stream habitats, and support selective breeding, conservation, and aquaculture of this native species.