Long-read assembly of the Brassica napus reference genome Darmor-bzh

Abstract

Background The combination of long reads and long-range information to produce genome assemblies is now accepted as a common standard. This strategy not only allows access to the gene catalogue of a given species but also reveals the architecture and organization of chromosomes, including complex regions such as telomeres and centromeres. The Brassica genus is not exempt, and many assemblies based on long reads are now available. The reference genome for Brassica napus, Darmor-bzh, which was published in 2014, was produced using short reads and its contiguity was extremely low compared with current assemblies of the Brassica genus. Findings Herein, we report the new long-read assembly of Darmor-bzh genome (Brassica napus) generated by combining long-read sequencing data and optical and genetic maps. Using the PromethION device and 6 flowcells, we generated ∼16 million long reads representing 93× coverage and, more importantly, 6× with reads longer than 100 kb. This ultralong-read dataset allows us to generate one of the most contiguous and complete assemblies of a Brassica genome to date (contig N50 > 10 Mb). In addition, we exploited all the advantages of the nanopore technology to detect modified bases and sequence transcriptomic data using direct RNA to annotate the genome and focus on resistance genes. Conclusion Using these cutting-edge technologies, and in particular by relying on all the advantages of the nanopore technology, we provide the most contiguous Brassica napus assembly, a resource that will be valuable to the Brassica community for crop improvement and will facilitate the rapid selection of agronomically important traits.

Local Duplication of TIR-NBS-LRR Gene Marks Clubroot Resistance in Brassica napus cv. Tosca

“…The Darmor-bzh 4.1 reference assembly was constructed prior to the advance of long-read sequencing technologies mainly based on Illumina short-read sequencing and is thus highly fragmented (Lee et al, 2020). We have to note that between the submission and publication of this article, an upgraded, Oxford Nanoporebased version of Darmor-bzh genome (v10) was published, in which the region in question is assembled in agreement with the results of our study and other long-read assemblies (Rousseau-Gueutin et al, 2020). Moreover, it has been shown that in B. napus more than 50% of known RGA copies do not occur in a single reference genotype assembly but require analysis of pangenome assemblies for detection (Dolatabadian et al, 2020).…”

A Modified Meiotic Recombination in Brassica napus Largely Improves Its Breeding Efficiency

“…The context sequences of each SNP marker were physically localized on the reference genome B. rapa Chiifu v1.5 [45] and B. napus cv. Darmor-bzh v10 [46] using BLASTn (ver. 2.9.0, min.…”

“…Darmor A01 chromosome was selected for its pericentromeric localization. The context sequences of the SNP markers (derived from Brassica 60 K Illumina infinium array) flanking this QTL were retrieved and physically localized on the B. napus reference genome Darmor-bzh v10 [46] using BLASTn [47].…”

“…The centromeric regions were defined by blasting several centromeric-specific repeats (CentBr1, CentBr2, TR238, TR805, PCRBr and CRB; [50,51]) against B. napus cv. Darmorbzh v10 [46] and by examining the plot density of the BLASTn results (e-value less than 1 × 10 −20 ). The pericentromeric borders for each chromosome were inferred by using the mean gene density along the chromosomes (as presented in [52]) and defined as the regions surrounding the centromere with a gene density below the chromosome average.…”

Sequencing and Chromosome-Scale Assembly of Plant Genomes, <em>Brassica rapa</em> as a Use Case

“…Gene prediction was performed using several proteomes: 8 from other genotypes of B. napus [51] (Westar, Zs11, QuintaA, Zheyou73, N02127, GanganF73, Tapidor3, and Shen-gli3), Arabidopsis thaliana (UP000006548) and the 2021 annotation of Darmor-bzh [30]. Regions of low complexity in genomic sequences were masked with the DustMasker algorithms [52] (version 1.0.0 from the blast 2.10.0 package).…”