A complete diploid chicken genome reveals hidden complexity of avian genomes
The inherent complexity of the avian genome, marked by numerous repetitive dot microchromosomes and the heterochromatic W sex chromosome, has impeded the creation of a complete, haplotype-resolved reference. Although recent efforts yielded the highly contiguous GGswu1 assembly, it remains technically incomplete and unphased, limiting the study of diploid genomic complexity. To overcome these challenges, we employed a comprehensive trio-sequencing approach, utilizing PacBio HiFi and ONT ultralong reads on a female chick, supplemented by parental ONT data for accurate haplotype phasing. This strategy generated the nearly complete, haplotype-resolved paternal (GGswu2-p, ~1.13 Gb) and maternal (GGswu2-m, ~1.14 Gb) diploid assemblies. GGswu2 resolved ~51-56 Mb of previously unresolved regions (PURs), predominantly within dot chromosomes, where they accounted for approximately 50% of the chromosome size. This enhanced resolution recovered 766-777 of 782 previously unmapped non-reference genes, suggesting they were simply PURs in prior assemblies. Comparative analyses of the diploid sequence revealed that dot chromosomes exhibit low nucleotide diversity but a high density of indels and structural variants between haplotypes. Furthermore, dot chromosomes display the highest de novo mutation rate, approximately 24 times greater than macrochromosomes, leading to the previous underestimation of chicken mutation rate when dot chromosomes were not included in the reference. The findings establish an essential foundation for future avian genomics, underscoring that comparative genomics must incorporate the high-resolution of these dynamic microchromosomal elements to fully understand vertebrate genome evolution and genetic complexity.