Chromosome-level assembly of the mustache toad genome using third-generation DNA sequencing and Hi-C analysis

The mustache toad, Vibrissaphora ailaonica, is endemic to China and belongs to the Megophryidae family. Like other mustache toad species, V. ailaonica males temporarily develop keratinized nuptial spines on their upper jaw during each breeding season, which fall off at the end of the breeding season. This feature is likely result of the reversal of sexual dimorphism in body size, with males being larger than females. A high-quality reference genome for the mustache toad would be invaluable to investigate the genetic mechanism underlying these repeatedly developing keratinized spines.To construct the mustache toad genome, we generated 225 Gb of short reads and 277 Gb of long reads using Illumina and Pacific Biosciences (PacBio) sequencing technologies, respectively. Sequencing data were assembled into a 3.53-Gb genome assembly, with a contig N50 length of 821 kb. We also used high-throughput chromosome conformation capture (Hi-C) technology to identify contacts between contigs, then assembled contigs into scaffolds and assembled a genome with 13 chromosomes and a scaffold N50 length of 412.42 Mb. Based on the 26,227 protein-coding genes annotated in the genome, we analyzed phylogenetic relationships between the mustache toad and other chordate species. The mustache toad has a relatively higher evolutionary rate and separated from a common ancestor of the marine toad, bullfrog, and Tibetan frog 206.1 million years ago. Furthermore, we identified 201 expanded gene families in the mustache toad, which were mainly enriched in immune pathway, keratin filament, and metabolic processes.Using Illumina, PacBio, and Hi-C technologies, we constructed the first high-quality chromosome-level mustache toad genome. This work not only offers a valuable reference genome for functional studies of mustache toad traits but also provides important chromosomal information for wider genome comparisons.