Brassica napus NRCDH4079 Genome Assembly and Annotation

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 further 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 optimization. We established a double haploid population of 1,236 lines from EMS-mutagenized 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 mutagenized population resulted in the development of a 'TILLED' variant database, revealing 1,243 knockout mutations across 1,222 unique 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 meal acid detergent fiber (ADF) content. 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 in B. napus, 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.作为全球最重要的油料作物之一，油菜（Brassica napus）的遗传改良面临一项核心挑战：其商业重要性状的自然变异资源极为有限。而由于对控制这些性状的基因缺乏功能认知，新变异的创制进一步受阻。油菜的多倍性与基因组重复特性，使得从拟南芥（Arabidopsis）转移功能研究成果的工作变得复杂低效。本研究构建了一套全新的功能基因组学平台，用于快速开展基因与性状发掘及优化工作。我们以春性油菜品系NRCDH4079为材料，从其乙基甲磺酸酯（Ethyl Methanesulfonate, EMS）诱变的小孢子中，培育得到包含1236个株系的双单倍体（double haploid, DH）群体。为该品系NRCDH4079组装了白金级参考基因组、基因注释文件，并构建了发育种子的基因表达图谱。对诱变群体开展外显子组测序后，我们开发了'TILLED'变异数据库，共鉴定得到1222个独特基因上的1243个敲除突变，以及影响70626个基因的140522个中等效应或修饰性变异。表型分析显示，关键种子性状存在显著变异，包括含油量、蛋白质含量及粕类酸性洗涤纤维（acid detergent fiber, ADF）含量。值得注意的是，突变体DP125410314表现出蛋白质含量提升与ADF含量降低的特性，这两项性状均是优化油菜粕品质的关键指标。对该变异位点的遗传定位发现，A1与C1染色体间存在同源非相互易位，该结构变异与ADF含量降低相关，凸显了结构变异在性状形成中的重要作用。本研究确立了单倍体诱变技术作为甘蓝型油菜（B. napus）作物改良的高效工具，其研究成果对其他芸苔属（Brassica）物种亦具有广泛借鉴意义。通过加深我们对种子蛋白质性状的认知，本研究为培育满足未来营养需求与市场要求的油菜新品种奠定了坚实基础。