Allopolyploid oilseed rape (Brassica napus) is an important oil crop and vegetable. However, the latest version of its reference genome, with collapsed duplications, gaps, and other issues, prevents comprehensive genomic analysis. Herein, we report a gap-free assembly of the rapeseed cv. Xiang5A genome using a combination of ONT (Oxford Nanopore Technologies) ultra-long reads, PacBio high-fidelity reads, and Hi-C datasets. It includes gap-free assemblies of all 19 chromosomes and telomere-to-telomere assemblies of eight chromosomes. Compared with previously published genomes of B. napus, our gap-free genome, with a contig N50 length of 50.70 Mb, has complete assemblies of 9 of 19 chromosomes without manual intervention, and greatly improves contiguity and completeness, thereby representing the highest quality genome assembly to date. Our results revealed that B. napus Xiang5A underwent nearly complete triplication and allotetraploidy relative to Arabidopsis thaliana. Using the gap-free assembly, we found that 917 flowering-related genes were affected by structural variation, including BnaA03.VERNALIZATION INSENSITIVE 3 and BnaC04.HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1. These genes may play crucial roles in regulating flowering time and facilitating the adaptation of Xiang5A in the Yangtze River Basin of China. This reference genome provides a valuable genetic resource for rapeseed functional genomic studies and breeding.

Cold stress affects the seed germination and early growth of winter rapeseed, leading to yield losses. We employed transmission electron microscopy, physiological analyses, metabolome profiling, and transcriptome sequencing to understand the effect of cold stress (0 °C, LW) on the cotyledons of cold-tolerant (GX74) and -sensitive (XY15) rapeseeds. The mesophyll cells in cold-treated XY15 were severely damaged compared to slightly damaged cells in GX74. The fructose, glucose, malondialdehyde, and proline contents increased after cold stress in both genotypes; however, GX74 had significantly higher content than XY15. The pyruvic acid content increased after cold stress in GX74, but decreased in XY15. Metabolome analysis detected 590 compounds, of which 32 and 74 were differentially accumulated in GX74 (CK vs. cold stress) and XY15 (CK vs. cold stressed). Arachidonic acid and magnoflorine were the most up-accumulated metabolites in GX74 subjected to cold stress compared to CK. There were 461 and 1481 differentially expressed genes (DEGs) specific to XY15 and GX74 rapeseeds, respectively. Generally, the commonly expressed genes had higher expressions in GX74 compared to XY15 in CK and cold stress conditions. The expression changes in DEGs related to photosynthesis-antenna proteins, chlorophyll biosynthesis, and sugar biosynthesis-related pathways were consistent with the fructose and glucose levels in cotyledons. Compared to XY15, GX74 showed upregulation of a higher number of genes/transcripts related to arachidonic acid, pyruvic acid, arginine and proline biosynthesis, cell wall changes, reactive oxygen species scavenging, cold-responsive pathways, and phytohormone-related pathways. Taken together, our results provide a detailed overview of the cold stress responses in rapeseed cotyledons.