A nearly gapless, highly contiguous reference genome for a doubled haploid line of Populus ussuriensis, enabling advanced genomic studies.

Populus species, particularly P. trichocarpa, have long served as model trees for genomics research, owing to fully sequenced genomes. However, the high heterozygosity, and the presence of repetitive regions, including centromeres and ribosomal RNA gene clusters, have left 59 unresolved gaps, accounting for approximately 3.32% of the P. trichocarpa genome. In this study, the callus induction method was improved to derive a doubled haploid (DH) callus line from P. ussuriensis anthers. Leveraging long-read sequencing, we successfully assembled a nearly gap-free, telomere-to-telomere (T2T) P. ussuriensis genome spanning 412.13 Mb. This genome assembly contains only seven gaps and has a contig N50 length of 19.50 Mb. Annotation revealed 34,953 protein-coding genes in this genome, which is 465 more than that of P. trichocarpa. Notably, centromeric regions are characterized by higher-order repeats, we identified and annotated centromere regions in all DH genome chromosomes, a first for poplars. The derived DH genome exhibits high collinearity with P. trichocarpa and significantly fills gaps present in the latter's genome. This T2T P. ussuriensis reference genome will not only enhance our understanding of genome structure, and functions within the poplar genus but also provides valuable resources for poplar genomic and evolutionary studies.