Abstract
<jats:title>Abstract</jats:title> <jats:p> Conifers, which comprise nearly two-thirds of extant gymnosperm species, are ecologically and economically important but remain genomically understudied because of their exceptionally large, repeat-rich genomes. Here, we report a chromosome-level assembly of the haploid genome of <jats:italic>Cupressus sempervirens</jats:italic> generated using PacBio HiFi reads and scaffolded with optical and genetic maps. The 10 Gb assembly shows exceptional contiguity for a conifer genome (contig N50 = 29.8 Mb) and was organized into 11 pseudomolecules. Iso-Seq-supported annotation identified 42,980 protein-coding genes. Repetitive elements account for over 80% of the genome, with LTR retrotransposons alone representing 52.5%. Transposable elements (TE) are pervasive in both intergenic and genic regions and have a major impact on gene architecture: TE insertions within introns generate ultra-long introns, often exceeding 100 kb, and drive gene size expansion. Analyses of LTR retrotransposon dynamics indicate that genome enlargement in <jats:italic>C. sempervirens</jats:italic> was driven not by recent transpositional bursts, but by the long-term accumulation and incomplete removal of ancient LTR retrotransposons. Consistent with this pattern, paleogenomic reconstruction across representative gymnosperms found no evidence of whole-genome duplication in the Cupressus lineage. This reference genome provides a valuable resource for studying conifer genome evolution, gene structure, and traits of agronomic and ecological interest, including cypress pollinosis. </jats:p>