A high-contiguity Brassica nigra genome localizes active centromeres and defines the ancestral Brassica genome

Abstract

It is only recently, with the advent of long-read sequencing technologies, that we are beginning to uncover previously uncharted regions of complex and inherently recursive plant genomes. To comprehensively study and exploit the genome of the neglected oilseed Brassica nigra, we generated two high-quality nanopore de novo genome assemblies. The N50 contig lengths for the two assemblies were 17.1 Mb (12 contigs), one of the best among 324 sequenced plant genomes, and 0.29 Mb (424 contigs), respectively, reflecting recent improvements in the technology. Comparison with a de novo short-read assembly corroborated genome integrity and quantified sequence-related error rates (0.2%). The contiguity and coverage allowed unprecedented access to low-complexity regions of the genome. Pericentromeric regions and coincidence of hypomethylation enabled localization of active centromeres and identified centromere-associated ALE family retro-elements that appear to have proliferated through relatively recent nested transposition events (<1 Ma). Genomic distances calculated based on synteny relationships were used to define a post-triplication Brassica-specific ancestral genome, and to calculate the extensive rearrangements that define the evolutionary distance separating B. nigra from its diploid relatives.

Fig. 1. Genomic features of the B. nigra Ni100-LR assembly.

Bands: (1) chromosomes with centromere positions (black band); (2) class I retrotransposons (nucleotides per 100-kb bins); (3) class II DNA repeats (nucleotides per 100-kb bins); (4) gene density (genes per 100-kb bins); (5) gene expression in leaf tissue (log₁₀[average TPM] in 100-kb bins); (6) ONT CG methylation profile (ratio per 100 kb); (7) whole-genome bisulfite methylation profile (nucleotides per 100-kb bins). CG, blue; CHG, yellow; CHH, red.

Fig. 2. Comparison of B. nigra assemblies.

a, Chromosome-level genome alignment of the Ni100-SR (NS) assembly (centre) against the LR assemblies, C2-LR (bottom) and Ni100-LR (top). The plot was created using Synvisio (https://github.com/kiranbandi/synvisio). b, Circular map generated using Circos showing the alignment of the SR and LR assemblies for chromosome B5 of Ni100.

Fig. 3. Annotation of FL-LTR-RTs in B. nigra genomes.

a, Copy number of FL-LTR-RTs from 14 different families. b, Age distribution of FL-LTR-RTs in three B. nigra assemblies. c, Comparison of insertion sites of two FL-LTR-RTs (ALE and OTA) in the ONT assemblies. Source data

Fig. 4. Comparison of methylation data from WGBS and ONT sequencing in Ni100.

a, Genome-wide WGBS and ONT methylation profile of syntenic genes: CpG (purple), CHG (green), CHH (grey) and CpG by ONT (red). b, Genome features of the B2 chromosome of the Ni100-LR assembly, from outer to inner circle: gene density, class II DNA transposons, class I retrotransposons, chromosome cartoon, methylation profile from ONT data, methylation profile based on WGBS, ALE copia, CRB, B. nigra-specific centromeric tandem repeat, putative centromere region. This plot was developed using the AccuSyn tool (https://accusyn.usask.ca/). c–e, Comparison of 5-mC frequency detected by WGBS and ONT; frequency distribution plot without filtering (c) and with filtering based on either calls P ≤ 0.05 (d) or minimum (min.) ONT read depth of 10 (e).

Fig. 5. Characterization of centromeric region of chromosome B5 of Ni100-LR genome.

a, Distribution of various genomic features on the 5-Mb centromere region, including genes, methylome (ONT and WGBS) and full-length LTRs (ALE-LTR and 13 other family LTRs); distribution of young (<1 Ma) and old LTRs (>1 Ma); and distribution of centromeric repeat sequences of B. nigra based on chromatin immunoprecipitation (ChIP) analysis of CENH3 (ref. ). b, Nested insertion of full-length LTRs in the centromeric region. Age (in Ma) is shown above each element.

Fig. 6. Genome rearrangements and evolution of Brassica species.

a, Development of B. rapa, B. nigra and B. oleracea genomes based on ancestral genome. Blocks are ‘painted’ with colours corresponding to ancestral chromosomes. b, Divergence time estimation based on Ks distributions. Gaussian mixture models fitted to frequency distributions of Ks values obtained by comparing pairs of syntelogs between different Brassica species or the subgenomes of each species are shown. c, Phylogenetic relationship between the subgenomes of different Brassica species. A maximum-likelihood tree constructed based on concatenated sequences of 1,150 syntelogs between A. thaliana and each of the subgenomes (LF, MF1 and MF2) of B. rapa, B. oleracea and B. nigra is presented. Clade support values near nodes represent bootstrap proportions in percentages. All unmarked nodes have absolute support.