BACKGROUND
as the first plant to be adapted for tissue culture and among the first to be genetically engineered  <cit> , tobacco made two key contributions to molecular plant biology.
more recently, potential applications in biopharmaceutical  <cit>  and biofuel  <cit>  production have generated renewed interest in the species.
improved tobacco genomic resources are necessary to facilitate such applications, but genome size and complexity has slowed their development.
polyploidisation has occurred during the evolutionary history of the majority of flowering plants, suggesting a possible role in conferring selective advantages  <cit> .
such events are highly prevalent in the nicotiana genus  <cit> , including the relatively young allotetraploid n. tabacum , which arose less than  <dig>  ma ago through the hybridisation of the ancestral parents n. sylvestris  and n. tomentosiformis  .
efforts such as the tobacco genome initiative  provided sequence data for a low coverage of bacterial artificial chromosomes  and active parts of the n. tabacum genome , which allowed for genome-scale characterisation of gene families such as transcription factors  <cit> .
similarly, the tgi and other efforts generated expressed sequence tags , which provided insight into the gene content of n. tabacum and facilitated studies of gene expression in the species  <cit> .
however, development of more complete genomic resources was constrained by the relatively high cost and low output of traditional sequencing methods.
the emergence of next generation sequencing  technologies over the past-decade  has reduced these barriers and made sequencing efforts in species with complex genomes like n. tabacum more feasible.
the tobacco genome is estimated to be approximately  <dig> gb in size  <cit> , which is smaller than the combined estimated sizes of the n. sylvestris and n. tomentosiformis genomes .
a low coverage of ngs data was sufficient to demonstrate that this reduction in size was due to the preferential loss of repetitive sequence from the t-genome of tobacco  <cit> .
more recently draft genome sequences were made available for the species as well as its ancestral parents  <cit> , which represented a significant step forwards for research in the plant.
although the assemblies currently available for three different cultivars of n. tabacum  <cit>  provide a reasonable level of coverage, their utility is limited by less than 20% of the genome being anchored onto pseudomolecules.
as with many other crop species, tobacco can be categorized into multiple market classes that are differentiated by area of cultivation, agronomy, harvesting and curing methodologies, as well as plant genetics.
the burley market class is one of the major classes of tobacco, and is characterised by a high degree of chlorophyll deficiency that is most evident on the stems, stalks, and leaf mid-veins.
this trait is conferred by a double homozygous recessive genotype at the yellow burley  <dig>  and yellow burley  <dig>  loci , which were recently mapped to locations on tobacco linkage groups  <dig> and  <dig>  <cit> .
in addition to the chlorophyll deficiency, mutations at the yb loci have also been shown to confer changes to tobacco leaf chemistry  <cit> , including increased alkaloid levels and leaf nitrate nitrogen  that together likely contribute to the higher level of tobacco specific nitrosamine  class of toxicants associated with these plants  <cit> .
the yb loci also confer reduced nitrogen utilization efficiency  and nitrogen use efficiency   <cit> , which is consistent with recommended nitrogen fertilizer application rates being considerably higher for burley tobaccos  compared to other tobacco classes, such as the virginia market class .
improving uptake and utilisation of nitrogen by plants represents a significant challenge for sustainable agricultural production in future, given needs to increase agricultural production to feed a growing world population in the face of increasing fertilizer costs and a need to reduce environmental externalities associated with agrochemical use  <cit> .
here we present an improved de novo genome assembly for n. tabacum and use it to map-based clone the yb loci, identifying a pair of homeologous genes that may have implications for understanding and improving nute and nue in tobacco as well as other crop plants.
RESULTS
an improved genome assembly for tobacco
in order to improve the genomic resources available for n. tabacum we have generated a new next generation sequencing  assembly for the species .
the assembly covers over 4gb of non-n sequence , which is an increase from  <dig> gb  in the previously published version  <cit> .
analysis of gene content coverage  <cit>  showed lower levels of missing and fragmented sequences compared to the previously available tobacco genome assemblies , which together with the increased assembly size  suggests that it provides more complete coverage of the tobacco genome.
furthermore, the low level of missed or fragmented single-copy orthologs in the n. tabacum assembly is comparable to, or lower than the level shown by the tomato and potato genome assemblies respectively , supporting the quality of this current genome assembly for tobacco.
user-friendly access to the n. tabacum assembly is available via the solanaceae genomics network .fig.
<dig> the tobacco genome.
circos plot showing the  <dig> pseudomolecules  generated by the tobacco genome assembly.
with tracks for  gene density,  n. sylvestris sequence coverage,  n. tomentosiformis coverage,  regions of t-  or s-  putative genome origin and  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
table showing assembly statistics for the different levels of the tobacco genome assembly.
statistics for the s and t sub-genomes are based on calling ancestral origin of nitab <dig>  ngs assembly following mapping of sequence reads from n. sylvestris and n. tomentosiformis  <cit>
fig.
<dig> tobacco gene space  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  <cit> .
bar charts showing missing- , fragmented- , complete duplicated-  and complete single-copy genes  shown for the presented assembly , along with the previously published n. tabacum assemblies for cultivars k <dig> and tn <dig>  <cit> , n. benthamiana  <cit>  tomato , potato  and arabidopsis .
b venn diagram showing the cross-over of gene families between tobacco , tomato , potato  and arabidopsis .
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
genome annotation identified repeat families covering 67% of the assembly , and predicted  <dig>  genes with structures comparable to genes from other solanaceae .
gene family and gene ontology analysis also showed good cross-over with the related solanaceae species tomato and potato, in addition to other flowering plants .table  <dig> comparison of gene numbers and structures across the solanaceae
n. tabacum
n. benthamiana
n. sylvestris
n. tomentosiformis
s. lycopersicum
n. tabacum/
s. lycopersicum
table showing statistics for predicted gene models from n. tabacum assembly as well as other nicotiana species and tomato .
data for other nicotiana species based early access to the n. benthamiana v <dig> .
<dig> assembly and re-annotations of the n. sylvestris and n. tomentosiformis genomes  <cit>  using maker and the publicly available transcriptomic data provided by the n. benthamiana genome sequencing consortium
evolution of the tobacco genome
mapping of sequence reads generated from n. sylvestris and n. tomentosiformis  <cit>  covered more than 80% of the tobacco genome assembly, which allowed the ancestral origin of much of the sequence to be established .
mapping of reads from n. otophora  <cit> , which has also been proposed as a potential paternal genome donor for n. tabacum , only covered  <dig> % of the genome assembly, and these reads showed a tendency to be located in the same regions as n. tomentosformis reads, which supports n. tomentosiformis as the paternal genome donor, given the higher level of genome coverage shown by this species.
the parts of the genome assembly that were unmapped by either n. sylvestris or n. tomentosiformis reads  may partially be explained by introgressions from other nicotiana species introduced during commercial breeding for disease resistance in modern cultivars.
for example, n. otophora reads mapped to just over one tenth of the of the  <dig> % of the genome that was not mapped by n. sylvestris or n. tomentosiformis reads, which supports the possibility that such parts of the genome may originate from other nicotiana species.
however, approximately 98% of the unmapped regions were located outside of gene space , which was significantly greater than might be expected relative to the whole assembly  and may be due to lower selective pressures, resulting in more rapid divergence from the ancestral sequence in these regions.fig.
<dig> ancestral origin of the tobacco genome  pie chart showing percentage of the tobacco genome assembly that is mapped by sequence reads from n. tomentosiformis  and n. sylvestris , neither species , or both species .
b, pie chart showing percentage of the not mapped regions of the tobacco genome from  that are contained in non-genic sequence , exons , or introns .
c, pie chart showing percentage of the collapsed regions of the tobacco genome from  that are contained in non-genic sequence , exons , or introns .
d, number of genes  that could be assigned to n. tomentosiformis  and n. sylvestris  origin, or were not mapped  or mapped by both species  displayed.
genes in the collapsed set that could be putatively assigned to n. tomentosiformis , or n. sylvestris  origin based on conserved sequence polymorphisms are also shown
our results demonstrate a reduced contribution of n. tomentosiformis to the tobacco genome ; consistent with the loss of repetitive sequence from the t-genome  <cit> .
interestingly, this reduction in repetitive sequence makes the t-genome of n. tabacum easier to assemble as demonstrated by assembly statistics for the two sub-genomes .
reads from both ancestral species mapped equally well to  <dig> % of the assembly, suggesting potential collapse of the assembly at these locations, or sequence loss from one parental genome during the evolution of tobacco .
approximately 24% of this sequence was present in gene space ; significantly higher than would be expected by chance .
polymorphisms between the n. sylvestris and n. tomentosiformis sequences allowed the majority of these genes to be putatively assigned to an ancestral genome, supporting the suggestion that the homeologous gene from the other ancestral parent has been lost in these  <dig> cases .
this low level of putative gene loss is consistent with the high number of predicted genes identified  compared to other solanaceae, and the combined total of predicted genes from the n. sylvestris and n. tomentosiformis genomes .
a low level of gene loss is also consistent with the relatively high number of duplicated copies of proposed single-copy orthologs identified in the tobacco genome assembly compared with other sequenced diploid plant species such as tomato, potato and arabidopsis .
unlike in the inter-genic sequence, our analysis showed no evidence for preferential loss of genes from either ancestral parent .
transcriptomic analysis of three different tissues showed evidence of expression for a majority of predicted genes , with  <dig>  expressed in all conditions .
this suggests that, in the majority of cases, both t- and s-genome copies of homeologous genes have not only been maintained, but are also expressed.
our previous research of transcriptomic data showed limited evidence for neo-functionalisation in n. tabacum  <cit> .
this indicates that a high level of redundancy is present between n. tabacum genes, and suggests that this species may be too young in evolutionary terms for mechanisms such as gene-loss or neo-functionalisation to have contributed broadly to the shape of the tobacco genome through natural- and domestication-based selective pressures.
consistent with this, the n. benthamiana genome, which has had a longer period of time since the polyploidization event that formed the species for mechanisms such as gene loss to occur  <cit> , shows a higher number of single-copy genes compared to n. tabacum .
organisation of the tobacco genome
whole genome physical maps have been shown to provide invaluable frameworks for scaffolding ngs assemblies  <cit> .
using a bionano genomics optical map for tobacco we were able to consolidate  <dig> gb of the assembly onto  <dig> scaffolds with an n <dig> size of  <dig>  mb; nearly an 8-fold increase from the  <dig>  mb n <dig> size of the ngs assembly alone .
this facilitated anchoring of approximately 64%  of the tobacco genome into pseudomolecules based on their locations on a high-density consensus genetic map .
the  <dig> pseudomolecules represented the diploid number of chromosomes of n. tabacum and, with the exception of chromosome nt <dig>  showed good separation based upon t- and s-origins .
the results of chromosome nt <dig> may be due to varying introgressions or lineage specific chromosomal rearrangements  <cit>  in this group.
based on synteny, seven pairs of homeologous chromosomes could be clearly identified .
chromosomes nt <dig>  nt <dig>  nt <dig> and nt <dig> exhibited more complex relationships, which indicated that further rearrangements may have occurred between chromosomes of the n. sylvestris and n. tomentosiformis genomes, either before or after the formation of n. tabacum .
additional intra-genome rearrangements appear to have occurred based upon synteny between nt <dig> and both nt <dig> and nt <dig>  which also exhibit synteny with regions of n. sylvestris origin on nt <dig> and nt <dig> .
however, both nt <dig> and nt <dig> appear to contain large blocks of sequence of n. tomentosiformis origin, which exhibit synteny with chromosome nt <dig> of n. sylvestris origin, suggesting that an inter-genome rearrangement has occurred between these set of chromosomes .
a further rearrangement is indicated at the end of nt <dig>  which exhibits a block of n. tomentosiformis sequence in a chromosome of predominantly n. sylvestris origin, and a small cross over in the opposite direction at the end of nt <dig>  which is supported by previous results  <cit> , suggesting a reciprocal cross-over between these chromosomes .
these observations are consistent with the proposed number of inter-genome recombinant chromosomes present in cultivated tobacco based on previous genomic in situ hybridisation  analysis  <cit> .
genetic differentiation of burley tobacco
tobacco can be categorized into multiple market classes.
the k <dig> cultivar sequenced in this study is of the virginia market class, the widest-grown class.
varieties of burley market class tobacco exhibit a strong chlorophyll deficient phenotype , known to be conferred by a double homozygous recessive genotype at the yb <dig> and yb <dig> loci , which have also been associated with other plant physiology and leaf chemistry traits  <cit> .
the yb <dig> yb <dig> genotype was recently shown to cause reduced nitrogen utilization efficiency, and increase levels of alkaloids and leaf nitrate   <cit> , likely contributing factors to higher levels of carcinogenic tsna compounds typically found in this market class  <cit> .fig.
<dig> map-based cloning of the yb mutant genes ntegy <dig> and ntegy <dig>  a picture showing yellow, chlorotic phenotype of yb <dig> yb <dig> genotype nil  versus wild type yb <dig> yb <dig> parent  in one of the lines used in mapping of yb loci .
b, high density genetic map for tobacco  showing location of snp markers linked to yb <dig>  on nt <dig> and yb <dig>  on nt <dig>  mapping of yb <dig>  and yb <dig>  loci showing position of snp markers linked to the loci on  genetic and  physical maps.
physical map shows position of super-scaffolds  and underlying sequence scaffolds/contigs , as well as genes .
position of ntegy <dig> and ntegy <dig> in physical map shown  with schematic representation of exons , introns  and 5’ and 3’ utrs , 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  <dig> of ntegy <dig>  and 8 bp deletion in exon  <dig> of ntegy <dig> .
e, protein alignment based on predicted sequence translated from cdna of ntegy <dig> and ntegy <dig> from yb <dig> yb <dig> genotype k <dig> and yb <dig> yb <dig> genotype tn <dig> cultivars, showing truncated proteins produced from the tn <dig> alleles of the genes.
coloured regions of alignment indicate sequence identity between the four proteins
a combination of high levels of redundancy between genes in such a large and complex genome, together with a historical absence of molecular markers and genomic resources, has made identification and subsequent mapping of interesting mutants a very difficult prospect in tobacco.
however, having anchored 64% of the genome assembly to chromosomal locations, a possibility now exists to apply map-based gene discovery approaches in the species.
map-based cloning of the yb loci
genotypic analysis of three previously described pairs of nearly isogenic lines , differing from their respective parents at genomic regions carrying yb <dig> and yb <dig> loci,  <cit>  resulted in the identification of two genomic regions that consistently distinguished the nils from their isogenic parents .
in agreement with previous mapping efforts  <cit> , these loci were located on chromosomes nt <dig> and nt <dig>  which were donated by n. sylvestris and n. tomentosiformis, respectively.
given the proposed ancestral origin of the yb loci  <cit> , this suggested that yb <dig> and yb <dig> are represented by genes present on nt <dig> and nt <dig>  respectively.
to confirm linkage and support fine mapping of yb <dig> and yb <dig>  several markers across the two loci were assayed in two mapping populations designed to be homozygous recessive for one locus and segregating for the other.
this located yb <dig> to within a  <dig>  centi-morgan  interval between snp markers yb24- <dig> and yb24- <dig>  and yb <dig> within  <dig>  cm of snp marker yb5- <dig> .
earlier characterisation of the yb loci suggested that their genetic effects were manifested in the leaves rather than the root  <cit> , and it has been speculated that the difference may be related to chlorophyll synthesis; with lower levels of precursors and higher chlorophyllase activity shown in burley cultivars  <cit> .
it could also be speculated that genes involved in nitrogen assimilation or transport might underlie the observed chlorophyll deficiency of burley tobacco plants, although previous analysis indicated few differences in gene expression in major nitrogen assimilation genes in burley tobacco  <cit> .
linking the genetic map to the genome assembly allowed the identification of genes in the vicinity of the two loci .
genes predicted to be associated with nitrogen assimilation, nitrogen use physiology, or chloroplast activities were prioritised for analysis and a comparison was made of their sequence from this study  to those from the previously sequenced yb <dig> yb <dig> burley cultivar tn <dig>  <cit> .
homologs of arabidopsis thaliana ethylene-dependent gravitropism-deficient and yellow-green  <dig>  were identified as strong candidate genes , as they contained sequence polymorphisms predicted to result in truncated proteins in the tn <dig> alleles .
manual annotation of the gene models indicated a gap in the sequence of ntegy <dig>  which affected the end of exon  <dig> and start of exon  <dig>  to fill in this gap and validate the predicted gene models and sequence polymorphisms, cdna sequences were generated from rna of the k <dig> and tn <dig> cultivars .
predicted ntegy <dig> and ntegy <dig> proteins shared  <dig> % identity, and both proteins showed strong alignment to ategy <dig>  as well as with related proteins from other plant species, suggesting that these genes are members of the chloroplast-targeted merops m <dig> family  <cit>  .
this analysis also confirmed the ancestral origin of ntegy <dig>  and ntegy <dig> .
together with synteny between chromosomes nt <dig> and nt <dig>  this strongly suggested ntegy <dig> and ntegy <dig> to be homeologs.
consistent with ntegy <dig> and ntegy <dig> being homologs of the arabidopsis gene ategy <dig>  their expression patterns were similar to ategy <dig>  with high expression levels in the shoot apex and leaves, and low levels in the root .
this expression pattern is also consistent with the yb phenotype being manifested from the leaf  <cit> .
ategy <dig> encodes a membrane-bound, plastid-targeted, and atp-independent metalloprotease site- <dig> protease that is required for development of thylakoid grana, a well-organized lamellae system, and accumulation of chlorophyll and chlorophyll a/b binding proteins in chloroplast membranes  <cit> .
arabidopsis egy <dig> mutants exhibit a yellow-green plant phenotype  <cit>  similar to the observed chlorotic phenotype in yb <dig> yb <dig> genotype tobacco plants .
however, whereas in arabidopsis this phenotype is visible throughout plant development, yb <dig> yb <dig> double mutants are generally not distinguishable from wild-type individuals until about 40 days after germination.
this is more similar to the phenotype shown in mutants for the tomato egy1-like gene, lutescent <dig> , which results in an enhanced rate of chlorophyll loss in leaves and fruits as plants mature  <cit> .
genotypic markers specific to ntegy <dig> and ntegy <dig> alleles exhibited complete linkage with the chlorophyll-deficient phenotype in more than  <dig> individuals from the mapping populations described above.
twelve additional white-stemmed burley cultivars tested also carried these alleles, while other market classes showed genotypes consistent with these genes underlying the yb phenotype and the differentiation of the burley market class .
CONCLUSIONS
improving the tobacco genome assembly, and in particular increasing the anchorage of the tobacco genome to chromosomal locations from 19%  <cit>  to 64%, has provided a genomic roadmap that will help serve acceleration of many aspects of tobacco and plant science research.
although further improvements to the assembly would be desirable, the current research represents a substantial step forward, with the benefits of optical mapping for improving the coherency of assemblies in such complex genomes being readily apparent.
using the assembly, we were able to map-based clone ntegy <dig> and ntegy <dig>   mutations in these genes have been selected for during the domestication and breeding of an entire market class of tobacco.
we believe that this represents the first reported instance of successful map-based cloning in tobacco, and indeed one of the few successful examples alongside wheat  <cit>  of this approach being used in species with large polyploid genomes.
the added complexity of mapping mutants in polyploid species - particularly highly redundant ones such as tobacco - is well demonstrated by the requirement for mutations in both homeologous genes ntegy <dig> and ntegy <dig> to uncover the recessive yb mutant phenotype.
modification of these genes could have implications for management of tsna toxicants in tobacco, as well as for improving nitrogen utilization efficiency in plants and contributing towards more sustainable agricultural production.

 as the first plant to be adapted for tissue culture and among the first to be genetically engineered, tobacco made two key contributions to molecular plant biology. more recently, potential applications in biopharmaceutical and biofuel production have generated renewed interest in the species. improved tobacco genomic resources are necessary to facilitate such applications, but genome size and complexity has slowed their development. polyploidisation has occurred during the evolutionary history of the majority of flowering plants, suggesting a possible role in conferring selective advantages. such events are highly prevalent in the nicotiana genus, including the relatively young allotetraploid n. tabacum, which arose less than  ma ago through the hybridisation of the ancestral parents n. sylvestris and n. tomentosiformis. efforts such as the tobacco genome initiative provided sequence data for a low coverage of bacterial artificial chromosomes and active parts of the n. tabacum genome, which allowed for genome-scale characterisation of gene families such as transcription factors. similarly, the tgi and other efforts generated expressed sequence tags, which provided insight into the gene content of n. tabacum and facilitated studies of gene expression in the species. however, development of more complete genomic resources was constrained by the relatively high cost and low output of traditional sequencing methods. the emergence of next generation sequencing technologies over the past-decade has reduced these barriers and made sequencing efforts in species with complex genomes like n. tabacum more feasible. the tobacco genome is estimated to be approximately gb in size, which is smaller than the combined estimated sizes of the n. sylvestris and n. tomentosiformis genomes. a low coverage of ngs data was sufficient to demonstrate that this reduction in size was due to the preferential loss of repetitive sequence from the t-genome of tobacco. more recently draft genome sequences were made available for the species as well as its ancestral parents, which represented a significant step forwards for research in the plant. although the assemblies currently available for three different cultivars of n. tabacum provide a reasonable level of coverage, their utility is limited by less than 20% of the genome being anchored onto pseudomolecules. as with many other crop species, tobacco can be categorized into multiple market classes that are differentiated by area of cultivation, agronomy, harvesting and curing methodologies, as well as plant genetics. the burley market class is one of the major classes of tobacco, and is characterised by a high degree of chlorophyll deficiency that is most evident on the stems, stalks, and leaf mid-veins. this trait is conferred by a double homozygous recessive genotype at the yellow burley and yellow burley loci, which were recently mapped to locations on tobacco linkage groups and. in addition to the chlorophyll deficiency, mutations at the yb loci have also been shown to confer changes to tobacco leaf chemistry, including increased alkaloid levels and leaf nitrate nitrogen that together likely contribute to the higher level of tobacco specific nitrosamine class of toxicants associated with these plants. the yb loci also confer reduced nitrogen utilization efficiency and nitrogen use efficiency, which is consistent with recommended nitrogen fertilizer application rates being considerably higher for burley tobaccos compared to other tobacco classes, such as the virginia market class. improving uptake and utilisation of nitrogen by plants represents a significant challenge for sustainable agricultural production in future, given needs to increase agricultural production to feed a growing world population in the face of increasing fertilizer costs and a need to reduce environmental externalities associated with agrochemical use. here we present an improved de novo genome assembly for n. tabacum and use it to map-based clone the yb loci, identifying a pair of homeologous genes that may have implications for understanding and improving nute and nue in tobacco as well as other crop plants.  an improved genome assembly for tobacco in order to improve the genomic resources available for n. tabacum we have generated a new next generation sequencing assembly for the species. the assembly covers over 4gb of non-n sequence, which is an increase from gb in the previously published version. analysis of gene content coverage showed lower levels of missing and fragmented sequences compared to the previously available tobacco genome assemblies, which together with the increased assembly size suggests that it provides more complete coverage of the tobacco genome. furthermore, the low level of missed or fragmented single-copy orthologs in the n. tabacum assembly is comparable to, or lower than the level shown by the tomato and potato genome assemblies respectively, supporting the quality of this current genome assembly for tobacco. user-friendly access to the n. tabacum assembly is available via the solanaceae genomics network.fig. the tobacco genome. circos plot showing the pseudomolecules generated by the tobacco genome assembly. with tracks for gene density, n. sylvestris sequence coverage, n. tomentosiformis coverage, regions of t- or s- putative genome origin and 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 table showing assembly statistics for the different levels of the tobacco genome assembly. statistics for the s and t sub-genomes are based on calling ancestral origin of nitab ngs assembly following mapping of sequence reads from n. sylvestris and n. tomentosiformis fig. tobacco gene space 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. bar charts showing missing-, fragmented-, complete duplicated- and complete single-copy genes shown for the presented assembly, along with the previously published n. tabacum assemblies for cultivars k and tn, n. benthamiana tomato, potato and arabidopsis. b venn diagram showing the cross-over of gene families between tobacco, tomato, potato and arabidopsis. 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 genome annotation identified repeat families covering 67% of the assembly, and predicted genes with structures comparable to genes from other solanaceae. gene family and gene ontology analysis also showed good cross-over with the related solanaceae species tomato and potato, in addition to other flowering plants.table comparison of gene numbers and structures across the solanaceae n. tabacum n. benthamiana n. sylvestris n. tomentosiformis s. lycopersicum n. tabacum/ s. lycopersicum table showing statistics for predicted gene models from n. tabacum assembly as well as other nicotiana species and tomato. data for other nicotiana species based early access to the n. benthamiana v. assembly and re-annotations of the n. sylvestris and n. tomentosiformis genomes using maker and the publicly available transcriptomic data provided by the n. benthamiana genome sequencing consortium evolution of the tobacco genome mapping of sequence reads generated from n. sylvestris and n. tomentosiformis covered more than 80% of the tobacco genome assembly, which allowed the ancestral origin of much of the sequence to be established. mapping of reads from n. otophora, which has also been proposed as a potential paternal genome donor for n. tabacum, only covered % of the genome assembly, and these reads showed a tendency to be located in the same regions as n. tomentosformis reads, which supports n. tomentosiformis as the paternal genome donor, given the higher level of genome coverage shown by this species. the parts of the genome assembly that were unmapped by either n. sylvestris or n. tomentosiformis reads may partially be explained by introgressions from other nicotiana species introduced during commercial breeding for disease resistance in modern cultivars. for example, n. otophora reads mapped to just over one tenth of the of the % of the genome that was not mapped by n. sylvestris or n. tomentosiformis reads, which supports the possibility that such parts of the genome may originate from other nicotiana species. however, approximately 98% of the unmapped regions were located outside of gene space, which was significantly greater than might be expected relative to the whole assembly and may be due to lower selective pressures, resulting in more rapid divergence from the ancestral sequence in these regions.fig. ancestral origin of the tobacco genome pie chart showing percentage of the tobacco genome assembly that is mapped by sequence reads from n. tomentosiformis and n. sylvestris, neither species, or both species. b, pie chart showing percentage of the not mapped regions of the tobacco genome from that are contained in non-genic sequence, exons, or introns. c, pie chart showing percentage of the collapsed regions of the tobacco genome from that are contained in non-genic sequence, exons, or introns. d, number of genes that could be assigned to n. tomentosiformis and n. sylvestris origin, or were not mapped or mapped by both species displayed. genes in the collapsed set that could be putatively assigned to n. tomentosiformis, or n. sylvestris origin based on conserved sequence polymorphisms are also shown our results demonstrate a reduced contribution of n. tomentosiformis to the tobacco genome; consistent with the loss of repetitive sequence from the t-genome. interestingly, this reduction in repetitive sequence makes the t-genome of n. tabacum easier to assemble as demonstrated by assembly statistics for the two sub-genomes. reads from both ancestral species mapped equally well to % of the assembly, suggesting potential collapse of the assembly at these locations, or sequence loss from one parental genome during the evolution of tobacco. approximately 24% of this sequence was present in gene space; significantly higher than would be expected by chance. polymorphisms between the n. sylvestris and n. tomentosiformis sequences allowed the majority of these genes to be putatively assigned to an ancestral genome, supporting the suggestion that the homeologous gene from the other ancestral parent has been lost in these cases. this low level of putative gene loss is consistent with the high number of predicted genes identified compared to other solanaceae, and the combined total of predicted genes from the n. sylvestris and n. tomentosiformis genomes. a low level of gene loss is also consistent with the relatively high number of duplicated copies of proposed single-copy orthologs identified in the tobacco genome assembly compared with other sequenced diploid plant species such as tomato, potato and arabidopsis. unlike in the inter-genic sequence, our analysis showed no evidence for preferential loss of genes from either ancestral parent. transcriptomic analysis of three different tissues showed evidence of expression for a majority of predicted genes, with expressed in all conditions. this suggests that, in the majority of cases, both t- and s-genome copies of homeologous genes have not only been maintained, but are also expressed. our previous research of transcriptomic data showed limited evidence for neo-functionalisation in n. tabacum. this indicates that a high level of redundancy is present between n. tabacum genes, and suggests that this species may be too young in evolutionary terms for mechanisms such as gene-loss or neo-functionalisation to have contributed broadly to the shape of the tobacco genome through natural- and domestication-based selective pressures. consistent with this, the n. benthamiana genome, which has had a longer period of time since the polyploidization event that formed the species for mechanisms such as gene loss to occur, shows a higher number of single-copy genes compared to n. tabacum. organisation of the tobacco genome whole genome physical maps have been shown to provide invaluable frameworks for scaffolding ngs assemblies. using a bionano genomics optical map for tobacco we were able to consolidate gb of the assembly onto scaffolds with an n size of  mb; nearly an 8-fold increase from the  mb n size of the ngs assembly alone. this facilitated anchoring of approximately 64% of the tobacco genome into pseudomolecules based on their locations on a high-density consensus genetic map. the pseudomolecules represented the diploid number of chromosomes of n. tabacum and, with the exception of chromosome nt showed good separation based upon t- and s-origins. the results of chromosome nt may be due to varying introgressions or lineage specific chromosomal rearrangements in this group. based on synteny, seven pairs of homeologous chromosomes could be clearly identified. chromosomes nt nt nt and nt exhibited more complex relationships, which indicated that further rearrangements may have occurred between chromosomes of the n. sylvestris and n. tomentosiformis genomes, either before or after the formation of n. tabacum. additional intra-genome rearrangements appear to have occurred based upon synteny between nt and both nt and nt which also exhibit synteny with regions of n. sylvestris origin on nt and nt. however, both nt and nt appear to contain large blocks of sequence of n. tomentosiformis origin, which exhibit synteny with chromosome nt of n. sylvestris origin, suggesting that an inter-genome rearrangement has occurred between these set of chromosomes. a further rearrangement is indicated at the end of nt which exhibits a block of n. tomentosiformis sequence in a chromosome of predominantly n. sylvestris origin, and a small cross over in the opposite direction at the end of nt which is supported by previous results, suggesting a reciprocal cross-over between these chromosomes. these observations are consistent with the proposed number of inter-genome recombinant chromosomes present in cultivated tobacco based on previous genomic in situ hybridisation analysis. genetic differentiation of burley tobacco tobacco can be categorized into multiple market classes. the k cultivar sequenced in this study is of the virginia market class, the widest-grown class. varieties of burley market class tobacco exhibit a strong chlorophyll deficient phenotype, known to be conferred by a double homozygous recessive genotype at the yb and yb loci, which have also been associated with other plant physiology and leaf chemistry traits. the yb yb genotype was recently shown to cause reduced nitrogen utilization efficiency, and increase levels of alkaloids and leaf nitrate, likely contributing factors to higher levels of carcinogenic tsna compounds typically found in this market class.fig. map-based cloning of the yb mutant genes ntegy and ntegy a picture showing yellow, chlorotic phenotype of yb yb genotype nil versus wild type yb yb parent in one of the lines used in mapping of yb loci. b, high density genetic map for tobacco showing location of snp markers linked to yb on nt and yb on nt mapping of yb and yb loci showing position of snp markers linked to the loci on genetic and physical maps. physical map shows position of super-scaffolds and underlying sequence scaffolds/contigs, as well as genes. position of ntegy and ntegy in physical map shown with schematic representation of exons, introns and 5’ and 3’ utrs, 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 of ntegy and 8 bp deletion in exon of ntegy. e, protein alignment based on predicted sequence translated from cdna of ntegy and ntegy from yb yb genotype k and yb yb genotype tn cultivars, showing truncated proteins produced from the tn alleles of the genes. coloured regions of alignment indicate sequence identity between the four proteins a combination of high levels of redundancy between genes in such a large and complex genome, together with a historical absence of molecular markers and genomic resources, has made identification and subsequent mapping of interesting mutants a very difficult prospect in tobacco. however, having anchored 64% of the genome assembly to chromosomal locations, a possibility now exists to apply map-based gene discovery approaches in the species. map-based cloning of the yb loci genotypic analysis of three previously described pairs of nearly isogenic lines, differing from their respective parents at genomic regions carrying yb and yb loci, resulted in the identification of two genomic regions that consistently distinguished the nils from their isogenic parents. in agreement with previous mapping efforts, these loci were located on chromosomes nt and nt which were donated by n. sylvestris and n. tomentosiformis, respectively. given the proposed ancestral origin of the yb loci, this suggested that yb and yb are represented by genes present on nt and nt respectively. to confirm linkage and support fine mapping of yb and yb several markers across the two loci were assayed in two mapping populations designed to be homozygous recessive for one locus and segregating for the other. this located yb to within a centi-morgan interval between snp markers yb24- and yb24- and yb within  cm of snp marker yb5-. earlier characterisation of the yb loci suggested that their genetic effects were manifested in the leaves rather than the root, and it has been speculated that the difference may be related to chlorophyll synthesis; with lower levels of precursors and higher chlorophyllase activity shown in burley cultivars. it could also be speculated that genes involved in nitrogen assimilation or transport might underlie the observed chlorophyll deficiency of burley tobacco plants, although previous analysis indicated few differences in gene expression in major nitrogen assimilation genes in burley tobacco. linking the genetic map to the genome assembly allowed the identification of genes in the vicinity of the two loci. genes predicted to be associated with nitrogen assimilation, nitrogen use physiology, or chloroplast activities were prioritised for analysis and a comparison was made of their sequence from this study to those from the previously sequenced yb yb burley cultivar tn. homologs of arabidopsis thaliana ethylene-dependent gravitropism-deficient and yellow-green were identified as strong candidate genes, as they contained sequence polymorphisms predicted to result in truncated proteins in the tn alleles. manual annotation of the gene models indicated a gap in the sequence of ntegy which affected the end of exon and start of exon to fill in this gap and validate the predicted gene models and sequence polymorphisms, cdna sequences were generated from rna of the k and tn cultivars. predicted ntegy and ntegy proteins shared % identity, and both proteins showed strong alignment to ategy as well as with related proteins from other plant species, suggesting that these genes are members of the chloroplast-targeted merops m family. this analysis also confirmed the ancestral origin of ntegy and ntegy. together with synteny between chromosomes nt and nt this strongly suggested ntegy and ntegy to be homeologs. consistent with ntegy and ntegy being homologs of the arabidopsis gene ategy their expression patterns were similar to ategy with high expression levels in the shoot apex and leaves, and low levels in the root. this expression pattern is also consistent with the yb phenotype being manifested from the leaf. ategy encodes a membrane-bound, plastid-targeted, and atp-independent metalloprotease site- protease that is required for development of thylakoid grana, a well-organized lamellae system, and accumulation of chlorophyll and chlorophyll a/b binding proteins in chloroplast membranes. arabidopsis egy mutants exhibit a yellow-green plant phenotype similar to the observed chlorotic phenotype in yb yb genotype tobacco plants. however, whereas in arabidopsis this phenotype is visible throughout plant development, yb yb double mutants are generally not distinguishable from wild-type individuals until about 40 days after germination. this is more similar to the phenotype shown in mutants for the tomato egy1-like gene, lutescent, which results in an enhanced rate of chlorophyll loss in leaves and fruits as plants mature. genotypic markers specific to ntegy and ntegy alleles exhibited complete linkage with the chlorophyll-deficient phenotype in more than individuals from the mapping populations described above. twelve additional white-stemmed burley cultivars tested also carried these alleles, while other market classes showed genotypes consistent with these genes underlying the yb phenotype and the differentiation of the burley market class.  improving the tobacco genome assembly, and in particular increasing the anchorage of the tobacco genome to chromosomal locations from 19% to 64%, has provided a genomic roadmap that will help serve acceleration of many aspects of tobacco and plant science research. although further improvements to the assembly would be desirable, the current research represents a substantial step forward, with the benefits of optical mapping for improving the coherency of assemblies in such complex genomes being readily apparent. using the assembly, we were able to map-based clone ntegy and ntegy mutations in these genes have been selected for during the domestication and breeding of an entire market class of tobacco. we believe that this represents the first reported instance of successful map-based cloning in tobacco, and indeed one of the few successful examples alongside wheat of this approach being used in species with large polyploid genomes. the added complexity of mapping mutants in polyploid species - particularly highly redundant ones such as tobacco - is well demonstrated by the requirement for mutations in both homeologous genes ntegy and ntegy to uncover the recessive yb mutant phenotype. modification of these genes could have implications for management of tsna toxicants in tobacco, as well as for improving nitrogen utilization efficiency in plants and contributing towards more sustainable agricultural production. 
here we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an n size of  mb and enables anchoring of 64% of the genome to pseudomolecules; a significant increase from the previous value of 19%. 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. tobacco is an important plant model system that has played a key role in the early development of molecular plant biology. 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. 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. sequencingnicotiananicotiana tabacumtobaccosolanaceaenitrogen use efficiencynitrogen utilization efficiencyegy1map-based cloningpolyploidyissue-copyright-statement© the author 2017 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.
 tobacco 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.  here we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an n size of  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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords sequencingnicotiananicotiana tabacumtobaccosolanaceaenitrogen use efficiencynitrogen utilization efficiencyegy1map-based cloningpolyploidyissue-copyright-statement© the author 2017 