One of China’s most important resources is flax (Linum usitatissimum L.), an ancient crop with significant nutritional and therapeutic benefits. Despite its importance, existing flax reference genomes remain incomplete, with many unassembled sequences. Here, we report a gapless 482.51 Mb telomere-to-telomere (T2T) flax genome assembly, predicting 46 634 genes, of which 42 805 were functionally annotated. Repetitive sequences constitute 60.05% of the genome, and we identified 30 telomeres and 15 centromeres across the chromosomes. Whole-genome duplication (WGD) events were detected at approximately 11.5, 53.5, and 114 million years ago (MYA) based on synonymous substitution rates (Ks). The T2T assembly enabled the reconstruction of the fatty acid metabolic pathway, identifying 49 related genes, including six newly annotated ones. Furthermore, genomic colocalization was observed between fatty acid metabolism pathway-related genes and transposable elements, suggesting that functional differentiation of these genes in flax evolution may have occurred through transposon-mediated duplication events. Phylogenetic analysis of SAD and FAD gene families revealed that FAD genes segregate into FAD2 and FAD3/7/8 subfamilies. Gene structure and motif analyses demonstrated conserved exon–intron architectures and motif organization within each phylogenetic clade of SAD and FAD genes. Promoter region characterization identified numerous cis-acting elements responsive to phytohormones (MeJA and abscisic acid) and abiotic stresses (low temperature and anaerobic induction) in both SAD and FAD genes. Our knowledge of the evolution of the flax genome is improved by this excellent genome assembly, which also offers a strong basis for enhancing agricultural attributes and speeding up molecular breeding.

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