Rapeseed (Brassica napus) is the second most important oilseed crop in the world but the genetic diversity underlying its massive phenotypic variations remains largely unexplored. Here, we report the sequencing, de novo assembly and annotation of eightB. napusaccessions. Using pan-genome comparative analysis, millions of small variations and 77.2–149.6 megabase presence and absence variations (PAVs) were identified. More than 9.4% of the genes contained large-effect mutations or structural variations. PAV-based genome-wide association study (PAV-GWAS) directly identified causal structural variations for silique length, seed weight and flowering time in a nested association mapping population with ZS11 (reference line) as the donor, which were not detected by single-nucleotide polymorphisms-based GWAS (SNP-GWAS), demonstrating that PAV-GWAS was complementary to SNP-GWAS in identifying associations to traits. Further analysis showed that PAVs in threeFLOWERING LOCUS Cgenes were closely related to flowering time and ecotype differentiation. This study provides resources to support a better understanding of the genome architecture and acceleration of the genetic improvement ofB. napus.

CRISPR/Cas9 is a valuable tool for both basic and applied research that has been widely applied to different plant species. Nonetheless, a systematical assessment of the efficiency of this method is not available for the allotetraploid Brassica napus—an important oilseed crop. In this study, we examined the mutation efficiency of the CRISPR/Cas9 method for 12 genes and also determined the pattern, specificity and heritability of these gene modifications in B. napus. The average mutation frequency for a single-gene targeted sgRNA in the T0 generation is 65.3%. For paralogous genes located in conserved regions that were targeted by sgRNAs, we observed mutation frequencies that ranged from 27.6% to 96.6%. Homozygotes were readily found in T0 plants. A total of 48.2% of the gene mutations, including homozygotes, bi-alleles, and heterozygotes were stably inherited as classic Mendelian alleles in the next generation (T1) without any new mutations or reversions. Moreover, no mutation was found in the putative off-target sites among the examined T0 plants. Collectively, our results demonstrate that CRISPR/Cas9 is an efficient tool for creating targeted genome modifications at multiple loci that are stable and inheritable in B. napus. These findings open many doors for biotechnological applications in oilseed crops.