Haploid Mutation Mapping Identifies a Homoeologous Non‐Reciprocal Translocation Linked to Reduced Fibre and Enhanced Protein in <scp> <i>Brassica napus</i> </scp>

A key challenge for the genetic improvement of canola (Brassica napus), one of the world's most important oilseeds, is the limited natural variation for commercially important traits. The creation of new variation is hindered by the lack of functional knowledge about genes controlling these traits. Ploidy and genomic duplications in canola complicate the effective transfer of functional insights from Arabidopsis. Here, we report a novel functional genomics platform for rapid gene/trait discovery and optimisation. We established a double haploid population of 1240 lines from EMS-mutagenised microspores of the spring-type canola line, NRCDH4079. A platinum-quality reference genome, gene annotations and a gene expression atlas from developing seeds were generated for NRCDH4079. Exome sequencing of the mutagenised population resulted in the development of a 'TILLED' database, revealing 1243 premature stop codons across 1222 genes, along with 140 522 moderate-effect or modifier variants impacting 70 626 genes. Phenotypic analysis revealed significant variation in key seed traits, including oil, protein and acid detergent fibre (ADF). Notably, the mutant variant DP125410314 exhibited increased protein and reduced ADF, two important traits for improving the meal composition of canola. Genetic mapping of this variant identified a homoeologous non-reciprocal translocation between A1 and C1 chromosomes associated with reduced ADF content, highlighting the role of structural variations in trait development. This work establishes haploid mutagenesis as a powerful tool for crop improvement, with broader implications for other Brassica species. By enhancing our understanding of seed protein traits, it lays the foundation for canola varieties that meet future nutritional and market demands.