A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency

Abstract

Background: Tobacco (Nicotiana tabacum) is an important plant model system that has played a key role in the early development of molecular plant biology. The tobacco genome is large and its characterisation challenging because it is an allotetraploid, likely arising from hybridisation between diploid N. sylvestris and N. tomentosiformis ancestors. A draft assembly was recently published for N. tabacum, but because of the aforementioned genome complexities it was of limited utility due to a high level of fragmentation.

Results: Here we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an N₅₀ size of 2.17 Mb and enables anchoring of 64% of the genome to pseudomolecules; a significant increase from the previous value of 19%. We use this assembly to identify two homeologous genes that explain the differentiation of the burley tobacco market class, with potential for greater understanding of Nitrogen Utilization Efficiency and Nitrogen Use Efficiency in plants; an important trait for future sustainability of agricultural production.

Conclusions: Development of an improved genome assembly for N. tabacum enables what we believe to be the first successful map-based gene discovery for the species, and demonstrates the value of an improved assembly for future research in this model and commercially-important species.

Keywords: EGY1; Map-based cloning; Nicotiana; Nicotiana tabacum; Nitrogen use efficiency; Nitrogen utilization efficiency; Polyploidy; Sequencing; Solanaceae; tobacco.

Fig. 1

The tobacco genome. Circos plot showing the 24 pseudomolecules (Nt1–Nt24) generated by the tobacco genome assembly. With tracks for (a) gene density, (b) N. sylvestris sequence coverage, (c) N. tomentosiformis coverage, (d) regions of T- (red bars; inner track) or S- (blue bars; outer track) putative genome origin and (e) Physical super-scaffolds generated by hybrid assembly of NGS and optical map data anchored to the genetic map. Note that track e is split over two levels due to the density of the super-scaffolds visible at the displayed scale. Synteny between pseudomolecules is represented by coloured linkers across the centre of the plot. Tracks a, b and c represent density over 50 kb bins

Fig. 2

Tobacco Gene space (a) Analysis of completeness of the tobacco genome assembly versus other plant genome assemblies based on mapping of a set of universal single-copy orthologs using BUSCO [27]. Bar charts showing missing- (red), fragmented- (amber), complete duplicated- (green) and complete single-copy genes (blue) shown for the presented assembly (N. tabacum K326), along with the previously published N. tabacum assemblies for cultivars K326 and TN90 [15], N. benthamiana [66] tomato (ITAG2.4), potato (v3.4) and Arabidopsis (TAIR10). b Venn diagram showing the cross-over of gene families between tobacco (N. tabacum; green), tomato (Solanum lycopersicum; red), potato (S. tuberosum; purple) and Arabidopsis (A. thaliana; blue). Number of gene families is show for each intersection, with number of individual genes contained within each set shown below in parentheses. Table summarising the number of genes and gene families within each species

Fig. 3

Ancestral origin of the tobacco genome (a) pie chart showing percentage of the tobacco genome assembly that is mapped by sequence reads from N. tomentosiformis (red) and N. sylvestris (blue), neither species (Not mapped; grey), or both species (Collapsed; purple). b, pie chart showing percentage of the Not mapped regions of the tobacco genome from (a) that are contained in Non-genic sequence (grey), exons (green), or introns (orange). c, pie chart showing percentage of the Collapsed regions of the tobacco genome from (a) that are contained in Non-genic sequence (purple), exons (green), or introns (orange). d, Number of genes (with percentage of total genes shown below in parentheses) that could be assigned to N. tomentosiformis (red) and N. sylvestris (blue) origin, or were not mapped (grey) or mapped by both species (Collapsed; purple) displayed. Genes in the collapsed set that could be putatively assigned to N. tomentosiformis (dark red), or N. sylvestris (dark blue) origin based on conserved sequence polymorphisms are also shown

Fig. 4

Map-based cloning of the yb mutant genes NtEGY1 and NtEGY2. a picture showing yellow, chlorotic phenotype of yb1 yb2 genotype NIL (left) versus wild type YB1 YB2 parent (right) in one of the lines used in mapping of yb loci (Cultivar SC58). b, High density genetic map for tobacco (N. tabacum 30 k Infinium HD consensus map 2015; https://solgenomics.net/cview/map.pl?map_version_id=178) showing location of SNP markers linked to yb1 (blue box) on Nt24 and yb2 (red box) on Nt5. Mapping of yb1 (c) and yb2 (d) loci showing position of SNP markers linked to the loci on (i) genetic and (ii) physical maps. Physical map shows position of super-scaffolds (alternating light and dark green bars) and underlying sequence scaffolds/contigs (blue bars), as well as genes (green triangles). Position of NtEGY1 and NtEGY2 in physical map shown (iii) with schematic representation of exons (wide dark blue boxes), introns (narrow light blue bar) and 5’ and 3’ UTRs (intermediate blue boxes), with direction of gene indicated by white arrow-head at 3’end. Sequence polymorphisms between wild type and mutant alleles indicated, showing single base insertion in exon 9 of NtEGY2 (c) and 8 bp deletion in exon 2 of NtEGY1 (d). e, protein alignment based on predicted sequence translated from cDNA of NtEGY1 and NtEGY2 from YB1 YB2 genotype K326 and yb1 yb2 genotype TN90 cultivars, showing truncated proteins produced from the TN90 alleles of the genes. Coloured regions of alignment indicate sequence identity between the four proteins (dark blue 100%, green 60–80%, and grey <60%)