The haplotype-resolved complete Tetraodon nigroviridis genome provides insights into the evolution of the most compact vertebrate genome

Genome sizes across vertebrates are remarkably variable ranging by a factor of approximately 250 from 350 Mb to over 92 Gb. Pufferfish genomes (e.g., Tetraodon nigroviridis and Takifugu rubripes ) are notable for their compact size, with T . nigroviridis possessing the smallest known vertebrate genome. Yet they all maintain a similar number of protein-coding genes. Until recently, the lack of high-quality, complete genome assemblies has hindered detailed investigations into the mechanisms of genome size evolution. Here, we present two haplotype-resolved, complete telomere-to-telomere (T2T) genome assemblies of T . nigroviridis . The two haplotype-resolved T2T assemblies each contain 21 gapless chromosomes with total lengths of 342,798,327 bp and 344,013,623 bp, respectively. This diploid genome harbors the canonical vertebrate telomeric repeat and a conserved 118 bp centromeric satellite unit shared across all chromosomes. Our analyses reveal that the compact genome of T . nigroviridis is achieved through the repression of transposable elements and the dramatic reduction of intergenic regions and intron sizes, while protein-coding regions remain highly conserved. The T. nigroviridis genome shows high GC content, especially in the small chromosomes. Comparative analyses highlight synteny conservation across vertebrates, underscoring the evolutionary stability of synteny units in vertebrate genomes despite a more than 250-fold range in genome size. In particular, chromosome 19 showed notable synteny conservation throughout teleost evolution. This work not only enhances our understanding of genome size evolution but also serves as a valuable resource for future evolutionary and comparative genomic studies.