Abstract
BACKGROUND: Potato (Solanum tuberosum) breeding is severely hindered by its highly heterozygous autotetraploid genome, where complex allelic interactions impede precise trait selection. Reconstructing complete haplotype-resolved assemblies is crucial for genome-assisted breeding. However, current assembly methods for autopolyploids often generate fragmented sequences, haplotype-switch errors, and gaps in complex regions such as centromeres. RESULTS: To address these challenges, we develop PHap, a haplotype assembly pipeline tailored for autopolyploids, using only standard sequencing data, including long-reads and Hi-C. Applying PHap to the autotetraploid potato cultivar HuaShu4, we generate a haplotype-resolved, near telomere-to-telomere assembly of 3.12 Gb with an N50 of 32.7 Mb and 99.7% haplotype accuracy. Comparisons with alternative methods and existing assemblies highlight PHap's advantages in assembly quality and cost-effectiveness. Integration of transcriptomic and epigenomic data demonstrates that the genomic and methylation divergence across haplotypes drives substantial allelic expression differentiation. Time-course RNA-seq further reveals, for the first time, that 55% of genes exhibit divergent allelic expression, with dynamic shifts in dominant or suppressed alleles during tuber development. Additionally, our assembly resolves high-resolution haplotype-specific structures in centromeres and subtelomeres, as well as haplotype divergence of structural rearrangements. It also shows neocentromere formation via the expansion of megabase-scale satellite arrays. CONCLUSIONS: These findings provide insights into the architecture of autopolyploid genomes and establish a foundation for genomics-assisted breeding of polyploid potatoes.