Chromosome-scale assembly of the Cupressus sempervirens genome unravels new insights into the evolutionary history of conifers

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 Cupressus sempervirens 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 C. sempervirens 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.