Long-read and chromosome-scale assembly of the hexaploid wheat genome achieves high resolution for research and breeding

Abstract

Background: The sequencing of the wheat (Triticum aestivum) genome has been a methodological challenge for many years owing to its large size (15.5 Gb), repeat content, and hexaploidy. Many initiatives aiming at obtaining a reference genome of cultivar Chinese Spring have been launched in the past years and it was achieved in 2018 as the result of a huge effort to combine short-read sequencing with many other resources. Reference-quality genome assemblies were then produced for other accessions, but the rapid evolution of sequencing technologies offers opportunities to reach high-quality standards at lower cost.

Results: Here, we report on an optimized procedure based on long reads produced on the Oxford Nanopore Technology PromethION device to assemble the genome of the French bread wheat cultivar Renan.

Conclusions: We provide the most contiguous chromosome-scale assembly of a bread wheat genome to date. Coupled with an annotation based on RNA-sequencing data, this resource will be valuable for the crop community and will facilitate the rapid selection of agronomically important traits. We also provide a framework to generate high-quality assemblies of complex genomes using ONT.

Keywords: genome assembly; haplotype characterization; hexaploid genome; introgressions; long-reads; nanopore sequencing; wheat.

Figure 1:

Genome overview of the 21 chromosomes of hexaploid T. aestivum Renan (the 7A chromosomes are in blue, the 7B chromosomes in orange, and the 7D chromosomes in green). From inner to outer track: (a) Coverage with short reads, (b) Coverage with long reads, (c) coverage with A. ventricosa short reads, (d) red dots represent large deletions (>500 kb), (e) gene density, (f) density of CACTA (DNA transposon) elements, (g) density of Copia elements, (h) density of Gypsy elements. All densities and coverage are calculated in 1-Mb windows; yellow and red colours in density plots indicate lower and higher values, respectively.

Figure 2:

Comparison of existing hexaploid genome assemblies. A. contig N50 values in Mb. B. Proportion of complete BUSCO genes found in each assembly (N = 4,896). C. Number of gaps in each chromosome. D. chromosome length in Mb.

Figure 3:

Dot plot comparisons of the 21 chromosomes of Renan (y axis) with the Chinese Spring RefSeq v2.1 assembly (x axis).

Figure 4:

Representation of haplotype blocks in chromosome 6A for the 11 chromosome-scale cultivars (based on 1-Mb blocks). Regions with the same colour represent common regions in wheat lines, except white regions, which are not contained in haplotype blocks. The grey and black regions represent haplotypes respectively shared by ≥10 cultivars or specific to a given cultivar.

Figure 5:

Haplotypic blocks in wheat chromosomes. Colours represent common regions in wheat cultivars. The grey and black regions represent haplotypes respectively shared by ≥10 cultivars or specific to a given cultivar. The orange curve, when present, represents coverage with A. ventricosa short reads. The red boxes frame the introgressions. A. Known introgressions in chromosomes 3D and 2B in LongReach Lancer. Regions in black represent genomic regions that are specific to LongReach Lancer and are respectively Triticum ponticum and Triticum timopheevii introgressions as described previously [8]. B. A. ventricosa introgression on chromosome 3D in CDC Stanley, Mace, SY Mattis, and Jagger. This known introgression is also present in Renan. The dark blue block represents the region shared across the 5 cultivars. C. Validation of the introgression in Renan (chromosome 2A from 1 to 34.2 Mb) using Bionano maps. D. Comparison of the contig composition of the first megabases from the introgression point in Jagger and Renan cultivars.

Figure 6:

Haplotypic blocks in wheat chromosomes. Colours represent common regions in wheat cultivars. The grey and black regions represent haplotypes respectively shared by ≥10 cultivars or specific to a given cultivar. The orange curve represents coverage with A. ventricosa short reads. Non-zero coverage of the D subgenome is expected because this subgenome is evolutionarily close to the Dv genome of A. ventricosa. The red boxes frame the introgressions. A. Candidate introgression (green block) on chromosomes 2D in Julius, ArinaLrFor, SY Mattis, Jagger, and Renan. B. Candidate introgressions (black blocks) on chromosome 3D in LongReach Lancer and SY Mattis. C. A. ventricosa introgression (black block) on chromosome 7D in Renan.

Figure 7:

Comparative view of an important locus on chromosome 1B containing prolamin and resistance genes, tandemly duplicated. A. Representation of the region with gaps and genes on the 2 assemblies of Renan and CS. B. Zoomed view on the ω-gliadin gene cluster. C. Proportion of the length of the proteins that were aligned in the genomic region of Renan and CS. Aligned protein sequences were annotated in CS by Huo et al. [35]. D. Alignment view of Bionano maps on the Renan cluster; coloured diamond shapes represent genes belonging to the ω-gliadin gene cluster. The optical maps are in blue and the chromosome sequence in green. Restriction sites are represented by vertical lines and are joined between the sequence and the map when properly aligned.

Figure 8:

Comparison of the Sm1 loci. A. Representation of haplotype blocks (5-kb bins) of the region surrounding the Sm1 gene on chromosome 2B. Colours represent common regions in wheat cultivars. The genomic region of CDC Landmark (15–16 Mb) was aligned against other cultivars to localize the Sm1 loci. The Sm1 gene in CDC Landmark and Renan, the 2 Sm1 carrier cultivars, is represented by a red star. B. Comparison of the contig composition in the Sm1 region of CDC Landmark and Renan, and validation of the assembly structure in Renan using Bionano optical maps. The optical map is in blue and the chromosome sequence in green. Restriction sites are represented by vertical lines and are joined between the sequence and the map when properly aligned.