BACKGROUND
the acquisition of a completed genomic reference sequence has numerous benefits over a near or partial completed sequence.
fully completed genomes have higher impact in terms of the comparative power with other species to evaluate the divergence of pathways and the expansion and contraction of gene families.
completed genomes can also be used to align syntenic regions between related species and thereby improve the accuracy and efficacy of other genome assembly projects  <cit> .
in addition, comparative gene finding tools can be used to annotate other related species  <cit>  and define species specific genes.
the fusaria are globally important ascomycete fungi of growing agricultural, horticultural, medical and ecological importance.
different species of fusaria can infect and cause disease on a wide diversity of host species including cereal and non-cereal crops, for example f. graminearum, only non-cereal crops , forestry trees , turf grass , animals, fish, shell fish and humans .
whereas other fusarium species are a major source of a high value edible protein consumed by humans  or are used to produce specific biomolecules, for example plant growth promoting gibberellins .
as a consequence over the past decade, multiple fusarium sequencing projects have taken place  <cit>  or are currently in progress  <cit> .
although the haploid fusarium genomes are small in size, typically 35- <dig> mbp, for no fusarium species is the complete genome sequence available.
this is counter-intuitive to providing high quality genomic resources for research however a full spectrum of genomic resources currently exists.
this is problematic for the international fusarium community.
the most complete genomic reference sequence of the fusaria clade is available for f. graminearum.
the assembled contigs/supercontigs for this species have been assigned to four large chromosomes by using information from a genetic map  <cit> .
but the assembled genome 10 years later still contains >  <dig>  unknown bases , and lacks telomeric and centromeric sequences.
although incomplete this genome has provided the basis to align and assemble genomes of four other plant pathogenic fusarium species, namely f. fujikuroi b <dig>  <cit> , f. verticillioides, f. oxysporum, f. sp lycopersici  <cit> , f. pseudograminearum cs <dig>  and isolates of f. pseudograminearum  <cit>  including the highly virulent f. pseudograminearum cs <dig> isolate  <cit> .
unfortunately, each of these other genomes also consists of multiple scaffolds which are heavily interspersed with unknown bases.
consequently their gene annotations may be incomplete.
the value of a complete genomic reference sequence is complemented by high-quality annotation of gene and non-coding rna annotation because all targets of interest can be identified and tested.
gene deletion studies to probe the function of proteins/metabolites potentially linked to virulence or other phenotypic traits of interest  <cit>  are dependent upon the quality of the reference genome and the annotation provided.
therefore an increased confidence in research findings based upon genome and annotation use is observed using a complete reference/annotation versus an incomplete.
for example, deletion or silencing of a class of enzymes that are implicated in virulence is not hindered by unknown genes of the same class which maintain the virulence effect.
some fusarium species, for example f. graminearum, are ideal targets for full genome completion given the predicted low repetitive sequence content and small genome size.
completion of the f. graminearum reference genome would be the first completed within the class sordariomycetes, which contains fungal species with very diverse lifestyles ranging from free living saprophytes, plant invading endophytes, to pathogens of microbes, plants and/or animals.
ongoing comparative analysis between and within fusarium species is important to food security due to their role in infecting hosts which include; wheat and barley , maize and sorghum , tomato, tobacco, legumes, cucurbits, sweet potatoes and banana , and rice .
fusarium graminearum is a fungus of world-wide economic importance because it causes fusarium head blight  disease on wheat and barley, also known as head scab disease, and fusarium stalk and ear rot disease on maize  <cit> .
the infection results in reduced yield affecting grain/kernel mass and quality, and mycotoxin contamination  <cit> , which is a threat to human health  <cit> .
consequently mycotoxin content in the harvested grain has to be expensively monitored due to maximum tolerated levels of toxins permitted in human foods and animal feeds  <cit> .
research to reduce fhb disease in cereal species has used the ph- <dig> strain as a model to identify and understand virulence mechanisms and pathways, with the ultimate aim of developing novel species specific fungicides and/or resistant crop plants  <cit> .
the f. graminearum ph- <dig> strain was originally sequenced using sanger technology, assembled into  <dig> contigs and  <dig> supercontigs, anchored to a genetic map, and annotated by the broad institute  <cit> .
later on the sequence was improved by broad, and the gene model annotations were refined by munich information services for protein sequences   <cit> , resulting in  <dig>  mrna transcripts in the mips annotation .
however there remain > <dig> gaps of unknown sequences within the current assembly represented either by sequential n bases or completely lacking sequence as indicated by the absence of the eight telomeres.
in addition, no sequence is available for the four centromeres and  <dig>  bp remained unassembled in small contigs.
for comparative purposes both the reference and annotation are both referred to in the text as mips v <dig> .
in this study, to complete the f. graminearum genome, whole shotgun re-sequencing of the strain ph- <dig> to 85-fold coverage was done using short paired end reads followed by de novo assembly using various k-mer values.
the accompanying annotation was produced by transferring the mips v <dig>  annotation, then refining the gene model annotation integrating multiple gene prediction algorithms within the maker <dig> software  <cit>  as well as by using available rna-seq from two wild type f. graminearum isolates  to provide additional gene model support.
finally, manual inspection was done on all the gene models.
in addition, the ph- <dig> mitochondrial draft  which was  <dig>  bp in length was re-evaluated, corrected and reduced to  <dig>  bp.
to complete this project, the rres v <dig>  genome sequence and the mitochondrial sequence were submitted to european nuclear archive  .
the completed f. graminearum genome is available within ensembl fungi  <cit>  and displayed on the plant pathogen specific phytopath database  <cit> .
RESULTS
genome closure
assembly
the de novo assembly using a sequencing depth of 85x coverage created a f. graminearum genome of  <dig> ,796 bp in length, assembled into four chromosomes and a mitochondrial genome of  <dig>  bp in length.
there was high accordance between the alignments of the rres v <dig>  genome to the previous mips v <dig>  reference genome.
the alignment of the de novo contigs identified a previous mis-assembly at the amino end of chromosome  <dig> with the 1- <dig>  bp sequence observed to be the reverse complement of the true sequence at this position.
this was confirmed by the extension at the amino end to a telomere sequence and the manual inspection of mapped reads throughout the genome to detect soft clipping of reads which are indicators of misassemblies.
the de novo assembly also provided extension of the amino and carboxyl sequence of chromosome 1- <dig> and identified a common telomere sequence  and reverse complement  respectively  <cit>  which is found in all vertebrates  <cit> .
this new data completes the four chromosomes at the amino and carboxyl end and confirms the new orientation of the amino terminus of chromosome  <dig>  in addition, a total of  <dig>  n bases have been removed to create the new assembly.
three previously constructed supercontigs namely  <dig> ,  <dig> , and  <dig>  had a similar depth of coverage  to the reads that mapped to the remainder of the genomic reference .
therefore each of these sequences became the targets for amalgamation into chromosomes  <dig> to  <dig> sequences.
after the alignment of contigs from the de novo assemblies and replacement of unknown bases these three supercontigs were successfully placed into the genome.
supercontig  <dig>  was placed at the carboxyl end of chromosome  <dig>  to provide the subtelomeric at-rich sequence and the missing telomere sequence.
supercontigs  <dig>  and  <dig>  were placed respectively in chromosomes  <dig> at position  <dig> ,775- <dig> , <dig> in which a new gene prediction is found , and chromosome  <dig> at position  <dig> ,855- <dig> , <dig> table  <dig> chromosome and supercontig sequence coverage of the f. graminearum ph- <dig> isolate and mips reference.
identification of underrepresented repeating sequences represented in the mips reference sequence could be calculated by dividing the total corrected average observed coverage by the original sequencing depth of  <dig>
4:  <dig> ,943+ bpa
achromosome  <dig> from  <dig> ,943 bp onwards, this is the start of the repeating rna annotated sequence
bthe read depth observed across the length of the sequence
cpercentage of bases that are a,c,t,g but not n
dthe calculated n bp corrected coverage
ethe number of copies of the sequence that should be represented in the reference sequence
fig.
<dig> visual representation of at rich and repeat rich regions in the rres and mips references.
all values are approximations to the nearest 1 kb and images have been scaled to equivalency.
the green line represents the at percentage and the blue the cg percentage to give a combined total of 100 %.
the labelling is in the format of a number which represents the chromosome i.e.
1 = chromosome  <dig>  a = the end of a reverse complement section, and b denotes regions in the mips version that were extended in the rres version.
the  <dig>  label represents where the supercontig  <dig>  was placed and “c” represents where fgrres_ <dig> is located.
the amino terminus set   reveals the at rich extensions of chromosomes 2- <dig> and the sequence that was reverse complemented in chromosome  <dig>  the centromere set  shows chromosome  <dig> at rich region extension from  <dig> kbp to  <dig> kbp mips to rres respectively within  <dig>  mbp region, chromosome  <dig> extension from  <dig> kbp to  <dig> kbp mips to rres respectively within  <dig>  mbp region, chromosome  <dig> extension from  <dig> kbp to  <dig> kbp mips to rres respectively within  <dig>  mbp region, and chromosome  <dig> extension from  <dig> kbp to  <dig> kbp mips to rres respectively within  <dig>  mbp region.
the carboxyl terminus set  shows chromosome  <dig> extension at the carboxyl end of  <dig> kbp, chromosome  <dig> extension at the carboxyl end of  <dig> kbp, chromosome  <dig> extension at the carboxyl end of  <dig> kbp, chromosome  <dig> extension at the carboxyl end of a repetitive sequence  <dig>  mbp
chromosome  <dig> was found to lack a telomere sequence at the carboxyl terminus but was found instead to possess a region of highly repetitive sequences solely annotated as rrna coding.
this large feature of the genome was found to extend for at least  <dig>  mbp and was identified using the following manual curation procedure.
mapping the reads of the re-sequenced ph- <dig> isolate to the mips reference sequence revealed a disproportionately higher read coverage for  <dig> of the  <dig> supercontigs not yet placed within the genome compared to the rest of the de novo assembled genome .
this suggested that some or all of these supercontig sequences were represented in multiple copies.
the high copy number of each supercontig may have contributed to the difficulty experienced in prior efforts to place these sequences within chromosomes 1- <dig>  <cit> .
following manual inspection, ten of these repetitive sequences containing supercontigs could be aligned to the carboxyl end of chromosome  <dig>  each of these supercontigs contained rrna encoding repetitive sequence.
this manual procedure permitted the chromosome  <dig> to be extended by  <dig>  mbp at the carboxyl terminus by further extrapolation of rrna sequence by division of the total mapped read coverage of this region by the expected  <dig> x coverage.
supercontig  <dig>  was identified as mitochondrial dna and was transferred into the mitochondrial assembly pipeline.
the unknown sequence within supercontig  <dig>   was corrected and the only supercontig remaining which had multiple copy coverage  but did not align partially or completely to chromosome 1- <dig> sequences or the mitochondria.
manual curation of the mapped reads suggests this supercontig to be either a repeating unit or a circular sequence of 5846 bp with no known genomic loci.
consequently this supercontig was reduced to one single repeating unit.
the identification of genomic loci of the remaining supercontigs from the original sanger sequencing and unknown sequence replacement completes chromosomes 1- <dig> sequences.
the remaining  <dig>  million re-sequenced un-mapped reads underwent de novo assembly of which the resulting  <dig>  remaining reads and  <dig>  contigs were analysed using blast with a  <dig>  significance value followed by megan  <cit> .
these reads and contigs were shown to represent contamination  with  <dig> sequences that had fungal hits.
the length of these  <dig> individual contigs did not exceed 400 bp and the concatenated length was  <dig>  bp.
the  <dig>  unplaced contigs from the original sanger sequencing used in the mips v <dig>  genome were mapped to the new reference and mitochondrial sequence, rres v <dig> .
the majority mapped to these sequences using bwa mem with only  <dig> sequences remaining to be placed of a combined length of  <dig>  bp.
these remaining sequences were assigned using blast with a  <dig>  significance value and megan to mouse  <dig>  bp , low complexity  <dig>  bp , no hits  <dig>  bp , and not assigned  <dig>  bp .
the blast and megan analysis of the remaining sequencing data from both sequencing platforms suggests that there are no additional chromosomes to the four represented in rres v <dig> .
the completed f. graminearum genome
the revised rres v <dig>  of the f. graminearum ph- <dig> strain genomic reference, deposited to ena, contains chromosomes  <dig> through  <dig> and comprises  <dig> ,796 bp with  <dig> ‘n’ bases.
this number excludes the significantly extended repetitive rrna encoding region at the carboxyl end of chromosome- <dig> .
in contrast in the mips v <dig>  genome, chromosomes 1- <dig>  comprises  <dig> ,856 bp with  <dig>  ‘n’ bases whilst the broad fg <dig>  version is in the form of multiple large supercontigs  <dig> - <dig>  and comprises  <dig> ,610 bp  with  <dig>  ‘n’ bases  <cit> .
the genomic length of rres v <dig>  chromosomes 1- <dig> is  <dig>  bp longer than that of mips v <dig> .
in order that this figure was not distorted, the calculations excluded the repetitive rrna annotated supercontigs within mips and complementary sequence at the carboxyl end of chromosome  <dig> in both the mips and rres v <dig>  reference because of significant extension in rres v <dig> .
the  <dig> unknown bases in the rres v <dig>  are found at a single poly-a locus within the centromere of chromosome  <dig>  there were  <dig>  homozygous mutations observed when mapping the re-sequenced ph- <dig> to the mips v <dig>  genomic reference consisting of:  <dig>  snps with a transition/transversion ratio of  <dig> ,  <dig> insertions, and  <dig> deletions.
these mutations may have arisen due to strain passaging or a consequence of sequencing errors from the original sequencing but the observation of these mutations clustering at loci suggests due to passaging.
readers are reminded that all genomic reference sequences are snapshots of a individual or a subset of individuals of a population.
the size of the four chromosomes was found to be; chromosome 1:  <dig> ,938 bp , chromosome 2:  <dig> ,558 bp, chromosome 3:  <dig> ,947 bp, and chromosome 4:  <dig> ,501 bp.table  <dig> comparative statistics on the length and nucleotide composition of the four f. graminearum chromosomes.
comparison of the mips  and rres  versions of the reference genome
anumber of total bases minus ‘n’ bases  used in difference comparison
bapproximate total length of repeating sequence at the carboxyl end of chromosome 4
to complete the f. graminearum mitochondrial genome,  <dig> snps were corrected,  <dig> indels deleted and  <dig> indels were inserted.
as a result, the new mitochondrial reference genome deposited at ena has a length that is slightly lesser in size at  <dig>  bp, compared to the original size of  <dig>  bp.
both the original sanger sequence reads and the new mitochondrial genome obtained by paired end reads exhibited no polymorphisms.
this result not only confirms the reliability of the new version of the genome, but clearly demonstrates that there has been no change in the mitochondrial sequence of strain ph- <dig> in over a decade of repeated fungal growth in vitro, in planta and long term storage at -80c.
resolving two specific genome features: telomeres and centromeres
the placement and extension of at rich sequence is normally difficult to resolve during genome assembly due to repetitive dna content.
these sequences predominate at the amino and carboxyl ends of each chromosome and represent the subtelomeric regions.
in most eukaryotic species there are multiple such regions within the relative vicinity of the repeating telomere sequence .
this was found to be the case in the seven assembled subtelomeric regions.
in fig.
<dig>  the original and de novo extended at rich sequences at the end of each chromosome are depicted.
to achieve the subtelomeric assembly, a multi k-mer approach was used in which a range of values from  <dig> to  <dig> was sequentially aligned to extend the sequence.
however, this difficult part of the assembly was most likely achieved because of the previously noted intrinsically low abundance of repeat sequences in f. graminearum ph- <dig>  <cit> .
the extension of an at rich region within each chromosome permitted the four centromere regions to be defined and accurately positioned.
in chromosome  <dig>  an at region starting at  <dig>  mbp was extended from  <dig> kbp to  <dig> kbp, in chromosome  <dig> at  <dig>  mbp a  <dig> kbp region was extended to  <dig> kbp, in chromosome  <dig> at  <dig>  mbp a  <dig> kbp region was extended to  <dig> kbp, and in chromosome  <dig> at  <dig>  mbp a  <dig> kbp region was to  <dig> kbp.
the centromere position quoted is directly related to the at rich positions originally noted in the mips version, see fig.
<dig>  these results indicate that each centromere has an asymmetric position in each chromosome.
therefore each of the four f. graminearum chromosomes is submetacentric with respect to centromere positioning.
the evidence supporting these regions are centromere sequences was tested by the connolly et al.
study  <cit> .
where there was centromeric sequence present, which was only in chromosome  <dig> in the mips version of the reference, then h3k9me <dig> binding was observed but no h3k27me <dig>  see additional file  <dig>
annotation of the completed fusarium graminearum
the revised gene call
from a total of  <dig>  mrna transcripts presented in the mips v <dig>  genome most were transferred to the rres v <dig>  genome.
however, sixteen were deleted due to sequence changes, two were redundant isoforms and removed ,  <dig> gene models were removed due to merging and five gene models were split.
in addition, gene models that had alternative model predictions which were longer were explored using interproscan <dig> and manually corrected where applicable.
the gene models that either did not 100 % match after transfer or had been revised by extension, splitting or merging have been given the id extension “_m”, representing modification.
some of the most common types of changes made are described in the following three generic examples:  an extended or reduced gene, for example, fgsg_  <dig>  splitting of a gene into two orfs results in the new gene designations 17172_a_m and 17172_b_m, and  joining of a gene with other gene models i.e.
<dig> and  <dig>  was designated as 17172_3_m keeping only the unique digit for the second gene if sequential id’s or if not sequential then the full gene id for each was used i.e.
<dig> and  <dig> becomes 17172_16222_m.
these changes to the gene names were done to signify that these gene models had been changed from the previous mips v <dig>  annotation but this approach also ensured the retention of the original gene id.
in total,  <dig> modified gene models are presented in the rres v <dig>  genome annotation.
also,  <dig> new gene models have been predicted of which  <dig> have supporting rna-seq evidence from cultured mycelium or spore transcriptome assemblies.
these have arisen because of the replacement of the ‘n’ sequences or alternative gene prediction software.
the new gene models have been given sequential numbers from  <dig> , beginning at the amino end of chromosome- <dig> and ending at the carboxyl end of chromosome- <dig>  the maker annotation software was used to transfer gene annotation id’s, predict gene models to correct mips gene models v <dig>  and add additional new genes using as evidence, rna-seq for the mycelium and spores of ph- <dig> , z- <dig> mycelia harvested 24 h after inoculation of wild-type conidia in complete medium rna-seq , proteins from uniprot from fusarium with evidence, and cogeme est transcripts  <cit> .
all evidence and final gene model predictions are available through the ensembl fungi tracks as “fusarium graminearum”.
the total number of gene models identified in the completed f. graminearum genome is  <dig>  which is in contrast to  <dig>  and  <dig>  in the broad and mips v <dig>  annotations respectively .
a comparison of the length of proteins between the rres and mips v <dig>  annotations shows proportionally the total percentage of proteins with a length below  <dig> aa, and between  <dig> and  <dig> aa to increase and decrease respectively for the rres set .
this figure is a reflection of the greater proportion of genes that are predicted as a single exon in the rres v <dig>  vs the mips v <dig>  set .table  <dig> basic statistics of the different reference and annotation versions of f. graminearum.
a comparison of f. graminearum genome version statistics between broad, mips, and rres
genome size a
scaffoldsb
gc  contentc
aincluding all scaffolds, n bases, and the mitochondria
bincluding all scaffolds, n bases excluding the mitochondria
cexcluding n’s and mitochondria
dexcluding n’s, mitochondria and large repetitive sequence at the carboxyl end of chromosome
the gene annotation
an analysis of the rres v <dig>  annotation using blast2go with the ncbi nr database and interproscan <dig> revealed a total of  <dig>  genes that were blast2go annotated or had blast hits, whilst  <dig>  genes have no go annotation .
there are an additional  <dig> level- <dig> go biological process go annotations and  <dig> molecular function go annotations in the rres v <dig>  annotation compared to the mips v <dig>  .
the number of proteins with alpha/beta hydrolase fold- <dig> or zn-c <dig> fungal-type dna-binding domain have increased from  <dig> to  <dig> and  <dig> to  <dig>  respectively .
the greatest increases for biological processes were in the categories; biological regulation, single-organism process, cellular process, metabolic process, and for molecular functions; catalytic activity, binding, and nucleic acid binding transcription factor activity.table  <dig> go term  summary of the  <dig> new gene annotations.
go terms summaries for biological process and molecular function of the  <dig> new genes annotated in the rres v <dig>  gene set
13
81
12
358
250
3
aone of these proteins was only partial
the go annotation assigned to the new genes are detailed in table  <dig> .
the new genes given enzyme designation codes are listed in table  <dig>  some of the new genes potentially have roles in primary metabolism, secondary metabolism or cell signalling.
some newly predicted genes of particular interest due to coding for metabolic pathway enzymes or orthologous in virulence mechanisms are as follows: fgrres_ <dig> is predicted to have alpha-n-arabinofuranosidase activity , is located in an average % gc region of approximately 2 kb in length neighboured by two at rich regions of approximately 9 kb length at the carboxyl end of chromosome  <dig>  fgrres_ <dig> and fgrres_ <dig> are both serine threonine protein kinases .
fgrres_ <dig> is predicted to be a cutinase .
fgrres_ <dig> is predicted to be related to the enzyme galactoside o-acetyltransferase .
fgrres_ <dig> is predicted to be involved in the inositol phosphate metabolism pathway, .
fgrres_ <dig> is predicted to be involved in pyruvate metabolism acetyl-coa to homocitrate and lysine biosynthesis of 2-oxoglutarate to homocitrate .
finally, fgrres_ <dig> has a pathogenicity protein description and go transporter activity.table  <dig> new enzyme’s from the new rres gene set.
new enzyme codes designated to the  <dig> new rres v <dig>  gene set
an additional  <dig> new enzymes have been identified from the modified rres gene model set, see table  <dig>  in total,  <dig> new biological process level go terms have been identified and  <dig> new molecular function level  <dig> go terms were found .
of the modified genes, two genes were of particular interest in terms of virulence, fgrres_17235_m, because this gene originally had no functional annotation but was extended to be identified as coding for a cysteine rich secretory protein, allergen v5/tpx-1-related with cap and signal peptide domains, which were previously linked with plant pathogenesis proteins of the pr- <dig> family  <cit>  and identified in the highly virulent f. graminearum strain cs <dig> .
the second gene 15917_m was identified as a endo- <dig> -beta-xylanase enzyme acting on plant cell walls by endohydrolysis of -beta-d-xylosidic linkages in xylans, also identified as part of the secretome.table  <dig> new enzyme codes from the modified rres gene set.
enzymes codes identified in the modified rres gene subset  not found in the mips annotation
for nine genes from the mips v <dig>  annotation, which were in regions of unknown sequence an improved level of annotation was possible for one gene.
fgrres_ <dig> previously classified as a hypothetical protein of  <dig> amino acid  length but was extended to  <dig> aa and identified as an uaa transporter.
the remaining eight corrected gene models could not be assigned additional functionality.
a total of  <dig> rna annotations were found across the four fully assembled chromosomes including  <dig> trna,  <dig> rrna,  <dig> ncrna, and  <dig> tmrna see table  <dig>  in addition a further  <dig> rrna were identified in the extensive repetitive region present at the carboxyl of chromosome  <dig>  a non-coding rna  <dig> s rna that has sequence similarity to a plant_srp  and fungi_srp   <cit>  of  <dig>  e- <dig> and  <dig>  e- <dig>  which is the only one identified in the genome at position  <dig> ,584 bp on chromosome  <dig>  may be important to the effectiveness of the secretome and pathogenicity.
signal recognition particles  are universally conserved ribonucleoproteins that direct the traffic of proteins within the cell allowing them to be secreted  <cit>  and therefore are a potentially important mechanism for virulence.table  <dig> the number of coding and non-coding gene models predicted in the rres and mips versions.
summary of coding and non-coding annotations of both the rres and mips annotations across chromosomes 1- <dig> and supercontigs
4a
awithout carboxyl end repeating units with rna annotation
bcarboxyl end repeating units with rna annotation
to complete the genome annotation, the remaining rres v <dig>  supercontig  <dig>  was found to contain four genes including an additional new gene of  <dig> aa length .
the only gene to have interproscan or blastp  annotation was fgrres_ <dig> which contains an intron endonuclease, group  <dig> domain and is a member of the giy-yig nuclease superfamily  <cit> , typically found predominantly in phage and mitochondria.
this supercontig may represent phage dna rather than mitochondrial due to the lack of alignment with the mitochondrial dna.
in addition to annotation using four gene predictors, open reading frame annotation of this contig showed no additional proteins that matched within the ncbi database therefore there are unlikely to be any small genes in addition to these four predicted genes.
deoxynivalenol  produced by fusarium graminearum is a potent b-type trichothecene mycotoxin  <cit>  and virulence factor to disease formation in wheat floral tissue  <cit> .
commonly found in harvested cereal grains, don lowers grain quality and is a serious health concern to humans and animals  <cit> .
don synthesis has been assigned to  <dig> genes , which is structurally related to don.
the f. graminearum ph- <dig> strain lacks functional copies of the tri <dig> and tri <dig> genes resulting in the inability to produce niv.
zearalenone induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals, therefore it is of agricultural importance to limit levels in the harvested grain.
zearalenone synthesis requires four genes in a cluster on chromosome  <dig>  namely fgrres_ <dig>  fgrres_ <dig>  fgrres_ <dig>  and fgrres_ <dig>  <cit> .
in the zearalenone gene cluster, the ps-dh domain of the protein 15980_m has increased from  <dig> aa to  <dig> aa and the ketoacy-synt-c domain has increase from  <dig> aa to  <dig> aa.
although the don production pathway is well studied, the regulatory mechanisms are less well characterised  <cit>  and unknowns such as the oxygenation step involving converting the c <dig> hydroxyl to a carbonyl group and the deacetylation of c <dig> have not been assigned an enzyme  <cit> .
a greater understanding of genetic factors regulating toxin biosynthesis could lead to the development of new strategies aimed at reducing toxin accumulation in the host tissue.
in the mips annotation there are eight gene predictions with aflatoxin biosynthesis regulatory protein annotations with two that have transcription factor domains.
although aflatoxin is not known to be produced by f. graminearum there role may be associated with another mycotoxin pathway such as don.
two modified gene predictions from the rres v <dig>  set that are annotated as aflatoxin biosynthesis regulatory proteins and one that also has a transcription factor domain  are additions to this small clade of functionally unknown regulatory genes.
analysis of the predicted secretome
the secretome of a fungus is the set of secreted proteins that frequently defines and facilitates the host infection process  <cit> .
during a fungal pathogens interaction with a host, the invading fungal cells secrete various proteins that can block host responses, i.e.
the fungus ustilagomaydis secretes cmu <dig> which can move through plant cells and redirect plant metabolic pathways to favour fungal infection  <cit> , or kill host cells i.e.
for f. graminearum,  <dig> genes coding for secreted cell wall degrading enzymes have previously been identified  <cit> .
characterisation of the secretome is fundamental to the understanding of the mechanisms of host infection and pathogen virulence.
using the mips v <dig>  assembly and annotation in  <dig>  brown et al.
<cit>  predicted the secretome to contain  <dig> proteins .
using the new rres v <dig>  predicted protein set and updated versions of the software we obtain  <dig> proteins using the brown et al.
pipeline to represent the refined secretome .
<dig> of the rres predictions were confirmed using proteomics from the yang et al.
<cit>  and paper et al.
<cit>  studies.
<dig> of the  <dig> proteins identified in the yang et al.
study from proteins identified in f. graminearum culture supernatants after growth in wheat or barley flour-containing medium, and  <dig> of the  <dig> from the extracellular proteins in vitro and in planta from the pathogenic fungus f. graminearum identified in the paper et al.
study were confirmed in the in silico predictions.
of the  <dig> proteins predicted,  <dig> are from modified genes and  <dig> are from newly predicted genes.
in total,  <dig> protein id’s of the newly refined secretome set match the original predictions.
whereas  <dig> secreted proteins are unique to brown et al.
and  <dig> unique to rres v <dig>  .
of the complete predicted secretome,  <dig> proteins have some level of annotation whilst  <dig> proteins lack annotation and  <dig> proteins  of the newly refined secretome have a cysteine content of > 5 %.
although genomic distribution of the secretome is not altered, an overview diagram of the secretome genes and trna annotated genes shows an overlap of clustering, in particular on chromosome  <dig> and  <dig>  see fig.
<dig> fig.
<dig> gene and rna distribution across the four chromosome .
each vertical bar represents a single gene or rna annotation, aligned next to a heat map for genetic recombination .
rows  <dig> through to rows  <dig> denote the following: all gene annotations, recombinant frequency, at rich regions  greater than 2 kb , predicted secretome , trna annotations, all rna annotations, and new gene annotations only.
the location of each centromere is highlighted in row  <dig> with a red arrow
analysis of genes predicted to possess nucleic acid binding transcription factor activity
proteins that bind to specific dna sequences and regulate transcription of genes are termed transcription factors.
they represent an important family of proteins controlling activities of the organism that regulate traits important for growth, development, stress response, pathogenesis, and toxin production, therefore are of importance to the development of fungicides for intervention.
within the rres v <dig>  gene set we have modified  <dig> gene annotations of the mips set and predicted  <dig> new genes .
the modified set of gene annotations shows  <dig> gene models that lacked a zn-c <dig> fungal-type dna-binding domain in the mips annotation to have been extended to include it in the rres v <dig>  set, and  <dig> of these have been identified as having gal <dig> protein domain and therefore are potential positive regulators for the gene expression of galactose-induced genes.
of the new set of gene annotations predicted with transcription factor activity: fgrres_ <dig> is annotated as having oxidoreductase activity, fgrres_ <dig> is annotated as a component of a protein kinase ck <dig> complex, and fgrres_ <dig> is annotated as having lipid glycosylation activity which is part of a family of transferases involved in the final stages of the biosynthesis of antibiotics vancomycin and related chloroeremomycin.
genes located in close proximity to the telomere and centromere sequences
the centromeres are the region of the chromosomes that determine kinetochore formations.
each centromere enables pairing of the sister chromatids and interacts with spindle microtubules during mitotic chromatid segregation and homologous chromosome segregation during meiosis.
centromere structure in f. graminearum and more generally in eukaryotes is poorly understood  <cit> .
for eukaryotes it was suggested that centromeres may be derived from subtelomeric regions  <cit> .
the four centromeres identified in f. graminearum chromosome 1- <dig> respectively are  <dig> ,  <dig> ,  <dig> ,  <dig>  bp long and are marked by their similar high at content,  <dig> ,  <dig> ,  <dig> , and  <dig>  % respectively.
chromosome  <dig> is the only chromosome to have an additional ~ ¼ sized at rich region not located within the proximity of the subtelomeres, at position  <dig> ,437- <dig> ,387 bp , of  <dig>  bp length and an at content of  <dig>  %.
here we surveyed the functional annotation of neighbouring genes of the newly integrated centromere, neocentromere and subtelomeric sequences in f. graminearum .
although no overall pattern was found within 40 kb regions in proximity to the centromeres or the amino and carboxyl telomere sequences, two genes in common between these regions were found on the chromosome  <dig>  these two genes  possess a nb-arc signalling domain  implicated in programmed cell death  <cit> .
genes and other features associated with the proposed ancient chromosome fusion sites
in previous studies, f. graminearum was shown to contain only four chromosomes  <cit> , whilst in related fusarium species a considerably higher numbers of chromosomes exist.
for example, for f. verticilloides the presence of  <dig> chromosomes, and for f. oxysporum,  <dig> chromosomes were demonstrated using a whole-genome sequencing and comparative genomics approach  <cit> .
ma et al.
<cit>  used the whole-genome alignment of f. graminearum and f. verticilloides to show that the  <dig> sequenced f. verticilloides chromosomes could be mapped to the four f. graminearum chromosomes and proposed that the smaller number of chromosomes in f. graminearum was the result of a series of ancient chromosome fusion events.
the identified potential fusion points matched regions of high genomic diversity .
closer inspection of the centromeres in the rres v <dig>  reference sequence revealed repeat sequences of either marcry-1_fo or ltr-gypsy to embody a large proportion of the sequence content and also the at rich regions within the subtelomeric loci.
the fusion event proposed by ma et al that led to f. graminearum centromeres were likely a result of fusion at these at rich regions, which are found at various loci within each chromosome of f. verticilloides.
this hypothesis is supported by repeating the alignment by ma et al.
but using the rres v <dig>  genomic reference sequence and focusing upon the centromere regions of f. graminearum that were not identified in the mips reference.
regions that aligned contained a number of annotated and non-annotated genes sequentially homologous in sequence and context.
different flanking sequences of at rich regions of f. verticilloides from the same chromosomes align to flanks of the centromeres found in f. graminearum .
the observation of different flanking sequences adjacent to at rich regions from the same chromosome aligning to f. graminearum chromosome centromeres flanking sequences, suggests that fusing of multiple at rich sequences to make the centromeres in f. graminearum did not include exclusively ancient centromere sequences either in part or full but incorporated other at rich regions into the recombination event.
this analysis provides further evidence in support of the existence of ancient chromosome fusion events in the f. graminearum genome which were not a direct result of the maintenance of ancient centromeres.
genes previously tested for a role in the disease causing ability of f. graminearum
the pathogen-host interactions database  stores molecular and biological information on pathogen genes for which mutant phenotypes in pathogen-host interactions have been tested experimentally  <cit> .
phi-base currently provides information on >  <dig>  pathogen genes, involving ~ <dig> pathogenic species.
there were f. graminearum  <dig> entries, including  <dig> putative transcription factors  <cit>  and  <dig> kinases  <cit> , an atp citrate lyase acl <dig>  <cit>  and a syntaxin-like snare gene gzsyn <dig>  <cit>  from phi-base that had modified gene sequences in the rres v <dig>  gene set versus mips v <dig>  .
the kinases and transcription factor genes were analysed in high-throughput gene deletion studies for their virulence  <cit> .
five of these genes were essential genes in f. graminearum, four genes when mutated caused reduced virulence, and one gene loss of pathogenicity.
the remaining  <dig> genes were neither implicated in virulence nor essential for life.
due to changes in the gene model a re-evaluation of the function of each gene is recommended.
recombinant regions
telomeres are established in many species as sites of high single nucleotide polymorphism  frequency and structural rearrangement .
f. graminearum was shown to exhibit this trait but was also found to show interstitial regions of high diversity  <cit> .
this was suggested to represent ancient telomeres as a consequence of chromosomal fusion of progenitor chromosomes.
the mapping of markers used to establish these recombinant regions to the rres v <dig>  genome confirms these results with no observable changes in marker locations.
the identification of the centromeres in the rres genome and mapping of previously established markers shows that the high recombinant loci at the telomeres is on the long arm of the chromosomes, see fig.
<dig>  however, it is worth noting that markers are lacking for the carboxyl ends of the short arms of chromosome  <dig> and  <dig> due to new sequence inserted for the rres v <dig>  genome and may still show these regions to be highly recombinant in future studies.
a comparison of total genes, secretome predicted genes, unique genes, and at content by chromosomal arm reveals no pattern differences between areas of high or low recombination, .
however it is clear that the secretome genes are predominantly found at areas of high recombination and within the subtelomeric regions, see fig.
<dig>
repeat motifs and transposons
in the original broad analysis, the f. graminearum genome was found to contain very few transposon sequences annotated with gene id’s and none were considered to be biologically active  <cit> .
active elements such as transposons are of interest because these are important contributors to genome evolution, including the creation of novel host genes and disruption of existing genes.
the gene annotations identified with transposon protein domains are shown in additional file  <dig>  of particular note was the new gene annotation, fgrres_ <dig> is a gypsy-type long-terminal-repeat  retrotransposon, not previously included in the mips v <dig>  assembly and the only retroviral transposon gene annotation found.
rna-seq supporting expression data in both the mycelium and spore was found suggesting this transposon is actively transcribed.
however it was not found to have been included within any gene annotation leading to a truncation of the protein coding sequence.
the sequence has been previously reported  as a hypothetical protein partial mrna in another ph- <dig> sequencing and annotation project but appears to have been an automated process with no reference in print.
a comparison of centromere and subtelomeric at rich regions repeat content shows a ltr gypsy sequence or marcry-1_fo represented in each of these regions .
this study confirms previous findings in other species demonstrating the presence of transposon sequence in the centromeres  <cit> .
the potential neocentromere identified in chromosome  <dig> shows a higher proportion of transposon content to the chromosome  <dig> centromere of  <dig>  and 53 % respectively.
in contrast to the subtelomeric regions there is a general trend towards a lower transposon content which further supports the hypothesis of a neocentromere at this location.
blast comparisons
the original blastp analysis of the broad f. graminearum against f. asiaticum, f. boothii, f. culmorum, and f. pseudograminearum identified  <dig> species specific genes  <cit> .
a similar blastp analysis  using high quality fusarium genome sequences found on ensembl fungi including: f. culmorum, f. fujikuroi, f. oxysporum, f. verticilloides, and nectria haematococca using the rres v <dig>  genome annotations identified  <dig> species specific genes .
visualisation of these species specific genes loci shows no observable clustering .
blastp against the fusarium cereal infecting subset, f. culmorum, f. verticillioides, and f. fujikuroi identified  <dig> unique genes.
a conservative evalue of  <dig>  for excluding proteins as part of the clade was used as little difference was observed as this value decreased.
comparison of the fusarium and cereal clade species specific genes within the predicted rres v <dig>  secretome identified six genes that were f. graminearum unique and contain signal peptide sequences.
translated blastn of these six secreted f. graminearum unique sequences were explored against all fusarium taxon entries in ncbi 12/12/ <dig> to test if the identifications were a result of missing or incorrect gene annotations.
this analysis revealed one, fgrres_ <dig>  to have a hit below evalue  <dig>  in both nectria haematococca and f. fujikuroi and therefore this sequence is not unique to f. graminearum.
the remaining five secreted sequences identified as unique to f. graminearum are; fgrres_ <dig>  fgrres_ <dig>  fgrres_ <dig>  fgrres_ <dig>  and fgrres_ <dig>
the f. graminearum isolate genomes cs <dig> and cs <dig> are incomplete.
this somewhat hinders inter-strain comparative analysis.
however, our analysis of f. graminearum isolates cs <dig> and cs <dig> has revealed a total of  <dig> and  <dig> proteins, respectively identified as unique compared to f. graminearum ph- <dig>  however a combinational analysis using complete genome sequences to identify snp/indel polymorphisms, expression analysis differences and unique proteins between isolates and the rres v <dig>  reference is required to identify the underlying reasoning behind these isolates genomic and phenotypic differences.
the original broad analysis of the f. graminearum genome had identified very little evidence of gene duplication and had discovered an active rip mechanism that had eliminated duplicated sequences.
an integral protein of the rip mechanism is the presence of a dna methyltransferase rip defective gene  , reported in neurospora crassa  <cit> .
blastp of the proteome of f. graminearum versus the rid gene from n. crassa reveals one protein, fgrres_ <dig>  of similar identity  but with an additional dna methylase c- <dig> cytosine-specific active site.
this protein is hypothesised to represent the rid gene in f. graminearum and further support the activity of rip.
the transposon class tcmar-fot <dig>  fgrres_ <dig>  fgrres_ <dig>  fgrres_ <dig>  fgrres_13431_m are all single exon predictions but for fgrres_13431_m which is multi-exon.
a previous analysis by cuomo et al.
has shown rip induced snps in this class of transposons however an alignment was not reported.
a blastn alignment of the region of fgrres_13431_m versus the other tcmar-fot <dig> class of transposons reveals that the transposon has had two stop codons introduced which explains the multi-exon prediction from the gene software.
fgrres_ <dig> has also a stop codon introduced causing a truncation.
in addition to the induced snps between these transposons we can report that this has led to stop codon introduction, likely terminating the functionality of the transposons discussed and additional evidence to the process of rip in f. graminearum.
a reciprocal blastp of the f. graminearum proteome identified two duplicated protein sequences, one of which had 100 % nucleotide identity.
the first is a tigger transposon  at position  <dig> , <dig> chromosome  <dig> and position  <dig> , <dig> chromosome  <dig> respectively which had identical nucleotides.
the second is the histone h <dig> protein fgrres_ <dig>  and fgrres_05491_m with  <dig> snps: c:g =  <dig>  c:a =  <dig>  t:g =  <dig>  g:a =  <dig> .
the sequence of fgrres_05491_m is longer than fgrres_ <dig> with an additional  <dig> aa at the amino end of the protein which includes a signal peptide of  <dig> aa which is predicted to target the protein to the mitochondria.
within the rres v <dig>  annotation there are two h <dig> proteins, one h <dig>  one h1/h <dig>  three h <dig> , but three h <dig> proteins of which two have the aforementioned 100 % matching sequences present.
in a further analysis, we explored gene families based upon protein sequence similarity which grouped sequences into no more than four using a similar methodology to klenk et al.
and heidelberg et al.
<cit> .
the average family size is  <dig>  and the largest  <dig> families identified included  <dig> families of amino acid transporters, three major facilitator families, three transposon families, three tyrosinases  families, one aldehyde/histidinol dehydrogenase family, one unknown function family and a family of nucleoside triphosphase hydrolases.
the remaining families were no greater than  <dig>  this suggests that few genes have formed via multi-duplications arising from a single progenitor gene to form these small multigene families.
discussion
the de novo genome assembly using de bruijn assembler software, such as soapdenovo <dig>  <cit> , requires the use of a k-mer value which requires a set sequence length in order to align and join sequenced reads but this is limited to the length of the reads i.e.
100 bp illumina reads.
as the k-mer value rises, this increases the specificity but reduces sensitivity.
therefore contigs of a repetitive nature such as at rich regions within centromeres, have a higher chance of assembling into a single sequence as the k-mer value increases.
there is a limitation to this approach, because as the k-mer value is increased greater sequencing coverage is needed to compensate for the reduced sensitivity.
using 100 bp illumina reads and a ~ <dig> x genome coverage, a k-mer value of approximately  <dig> resulted in the highest n <dig> assembly.
the presence of at rich regions or repetitive transposons loci are likely to cause breaks in the resulting contigs  <cit> .
using a greater k-mer value can in some cases create longer sequence contigs for these loci and potentially result in contigs that span these repetitive regions along with the non-repetitive flanks.
however combining such assemblies can be manually time consuming for genomes with many repetitive loci and genomes larger than  <dig> mbp.
the multi k-mer approaches like those that are available to soapdenovo <dig> can combine these two scenarios to produce a superior assembly with little manual curation.
we were successful in using this approach to fully assemble the f. graminearum genome.
for genomes of a similar size but contain more repetitive sequences, this strategy might be less successful but is worthwhile investigating.
for f. graminearum the multi-kmer approach resulted in the insertion of previously unknown sequences, identification of a significant mis-assembly in chromosome  <dig> at the amino end of the mips v <dig>  genome, extending several internal at rich regions, placing  <dig> supercontigs within chromosomes, and for the first time identifying for each of the four chromosomes the telomeric sequence  and centromeric dna.
the analysis of centromeric dna in filamentous fungi is difficult due to the fact that centromeric regions are comprised of heterogenous, repetitive, at-rich sequences  <cit> .
identification of the size and position of the centromeres on each of the four f. graminearum chromosomes permitted an examination of the annotations within the immediate vicinity versus annotation within the rest of the chromosome arms.
however comparison of total gene content, the secretome gene subset, f. graminearum unique genes, and gc content revealed no obvious patterns .
in saccharomyces cerevisiae short point centromeres of 120 bp exist  <cit> .
in contrast in many other fungi including schizosaccharomyces pombe, the human pathogenic fungus candida albicans and in the filamentous fungus n. crassa regional centromeres of considerable length are prevalent  <cit> .
in c. albicans, regional centromeres are composed of unique 3- <dig>  kb sequences  <cit> .
in the case of n. crassa, centromeres are composed of degenerate transposons, retrotransposons and simple sequence repeats, with no gene sequences.
our blast analysis  confirmed the absence of genes or distinctive dna elements for the four centromeric regions in f. graminearum.
the centromeric composition, despite its at rich >90 %, was sufficiently varied to assemble using the four re-sequencing data sets we had available with an insert distance of between  <dig> and  <dig> and the use of k-mer values > <dig>  see additional file  <dig>  the average size of the four centromeres was found to be  <dig>  bp, and for chromosome  <dig> our analysis revealed the existence of an additional smaller at-rich region of 15 kb.
this region is approximately ¼ the size of the designated centromeric region and may comprise a neocentromeric region or the remainder of centromere repositioning event.
the genes localised immediately adjacent to the four centromeres did not include genes implicated in virulence.
several sequences with predicted functions in programmed cell death and chromosome segregation, replication and chromatin remodelling were found to be located in close proximity to the centromeres.
we propose that the availability of centromeric sequence information for f. graminearum may be exploited in several ways.
although no evidence exists, they may help construct artificial chromosomes.
currently none are available for filamentous fungi.
assist in cancer research.
in several types of human cancers, centromeres were found to be deleted and kinetochores instead assemble on neocentromeres.
experiments in both s. pombe and c. albicans were successful in deleting centromeric regions and identifying neocentromeres.
to identify fungal specific drug targets.
kinetochore proteins binding to the centromeric regions in filamentous fungi during mitosis were proposed to be highly fungal-specific and provide pertinent specific drug targets  <cit> .
binding studies between fungal specific proteins and centromeric dna regions may reveal suitable small-molecule drugs.
the annotation of the genome was comprehensive.
first we took the broad  genome and the gene models provided by mips .
then we included additional annotation from a range of different sources to provide evidence for new gene models and to modify existing gene models.
this additional annotation evidence was obtained by using rna-seq data from the mycelium and spores of strain ph- <dig>  as well as the mycelium of strain z- <dig> harvested 24 h after inoculation of wild-type conidia in complete medium rna-seq .
in addition fusarium protein data was taken from uniprot and we also used the extensive est libraries generated by sanger sequencing available from the cogeme est transcripts database  <cit> .
as a consequence, a total  <dig> new gene models were predicted and  <dig> gene models have been modified.
the new  <dig> gene predictions and curated gene models are a significant step towards a fully annotated genome.
however further manual curation is required by the community using rna-seq supporting evidence from multiple biological situations to achieve the high level of novel transcriptome information needed to complete this important activity.
currently, this process is organised using the public ensembl fungi websites  <cit>  and the phytopathogenic specific website, phytopathdb  <cit> .
custom tracks can be displayed against the completed f. graminearum alongside any new gene model annotations and all changes are shared via a mailing list.
group members can contribute, view and discuss updated gene models prior to being incorporated into future ensembl fungi versions .
the study of the f. graminearum gene repertoire predicted to code for secreted proteins is highly significant to research aimed at understanding the pathogenic process due to the their hypothesised roles in penetration, host tissue necrosis and host immune subversion  <cit> .
the rapid rearrangement in these regions may promote rapid evolution of secretory genes in response to host responses leading to species specific genes and virulence hot spots.
for example, in the aspergillus species, species unique secondary metabolite genes are enriched in subtelomeric regions  <cit> .
the only similarly secreted and f. graminearum unique protein that has a cysteine content of above 5 %  is fgrres_ <dig> and is located in the subtelomeric region of chromosome  <dig>  at  <dig> ,286 bp.
these characteristics are a hallmark of an effector protein in other filamentous pathogenic species and of potential interest to gene deletion studies.
the reconfirmation of the identification of secretory genes and the identification of new secretory genes within these subtelomeric and the high recombinant regions previously demonstrated by ma et al.
and brown et al.
supports this hypothesis.
the identification of a predominance of the trna annotations within the genes predicted to code for secreted proteins, not previously noted, may be hypothesised to represent an evolutionary bias for improved response to stimulus such as host detection resulting in a rapid response to infection and an overall switch to the pathogenic process.
the detailed analysis of the newly predicted f. graminearum secretome, revealed two new secretory and one non-secretory proteins found in the rres v <dig>  predictions of potential importance to virulence and also serves to illustrate the significance of the complete genome and high quality annotations to the conclusions of research findings going forward.
fgrres_ <dig> is annotated as secreted and possessing alpha-n-arabinofuranosidase activity and is located in a new coding sequence island approximately 2 kb within two at rich regions of approximately 9 kb length at the carboxyl end of chromosome  <dig>  whilst fgrres_ <dig> is annotated as a secreted pathogenicity protein with transporter activity.
both genes were previously only identified in the ngs genome study of the high virulence f. graminearum australia isolate cs <dig> .
a third example, is the new cysteine-rich potential virulence gene  previously only reported in the highly virulent strain cs <dig> of f. graminearum which contains several features characteristic of many apoplastic fungal effectors shown to inhibit plant chitinases used to prevent pathogen associated molecular pattern triggered immunity thereby preventing induction of host defences.
the previous identification of these proteins in cs <dig> but not ph- <dig> was therefore the result of the incompleteness of the ph- <dig> genome.
the reanalysis of the well-studied tri and zearalenone gene clusters proved to be interesting.
a previous study had observed the co-expression of three flanking genes, namely  <dig> ,  <dig>  and  <dig> with the core tri genes and extended the list of genes involved in trichothecene don production and maintenance  <cit> .
in the current study, one gene was modified in the tri gene cluster and in the zearalenone gene clusters.
however with the publically available rna-seq data and manual curation of other newly identified genes, then it may be possible to identify and associate other sequences to these two key pathways and iteratively improve their curation and annotation.
these results highlight the need for re-appraisal of existing functional data to inform the current protein function studies on these two important toxin pathways.
the absence of genes that arose from duplications in f. graminearum reflects the activity of a process found in many fungi  <cit>  namely, repeat-induced point mutation  in which mutations are caused in duplicated sequences  <cit> .
this process is hypothesised to be a defence mechanism against transposable elements which are common to viral/bacterial infection.
a previous study has shown the introduction of c:g to t:a transition mutations during the sexual cycle in f. graminearum transposons  <cit> .
the identification of two histone h <dig> proteins of identical protein content but different dna sequences suggests these two sequences may be the consequence of a recent duplication event which is in the process of rip.
however the introns of the proteins are substantially different and snp c:g or t:a transitions are not predominant in the shared coding regions.
this suggests that these h <dig> proteins containing identical coding sequences but for an additional sequence including a signal peptide that is predicted to target the mitochondria for fgrres_05491_m, may not be in the process of rip but is present due to important functional reasons currently unknown.
the large repeating rrna containing sequence at the carboxyl end of chromosome  <dig> is similarly found in the human genome on chromosome  <dig> with adjacent unknown bases.
it is highly likely to exist in other genome reference sequences at the end of a chromosome interspersed with n bases and/or as an unplaced contigs.
by manual investigation such features where found in the genome references sequences  of f. fujikuroi and f. oxysporum f. sp.
lycopersici at the respective amino and carboxyl ends of chromosome  <dig>  in f. solani such sequences were found as unscaffolded contigs which are likely to originate from the amino or carboxyl end of a chromosome sequence yet unplaced.
this feature may be common among the fusarium clade and beyond but requires a greater number of genomic reference sequences to be completed before this feature can be thoroughly explored.
the genome and annotation sequences have been deposited into ena and displayed at ensembl fungi and with the dedicated plant pathogen phytopathdb database to permit public access to the data and tracking of versions.
information can be linked to the genome from other databases, such as the pathogen-host interaction entries  <cit>  to efficiently access phenotypic information from pathogenicity testing involving single and multiple gene deletion isolates generated mostly in the reference strain ph- <dig>  the presentation in ensembl fungi/phytopathdb will aid comparative genomics studies by the identification of homologous and orthologous genes with other representative fungal genomes which are continually increasing and being updated.
CONCLUSIONS
using whole shotgun re-sequencing of f. graminearum we have comprehensively completed the genomic sequence by replacing unknown bases, placing supercontigs within their correct loci, correcting assembly errors, and inserting new sequences which include for the first time complete at rich sequences such as centromere sequences and subtelomeric regions.
the identification of a retroviral transposon on chromosome  <dig> shows a potential route that f. graminearum is continuing to evolve in particular to environmental stresses, such as fungicides, and diverge from related fungal species.
an analysis of  <dig> new gene models and  <dig> curated gene models reveals  <dig> new and  <dig> modified transcription regulator protein predictions, three new targets for virulence mechanisms, and includes a range of proteins that may require further re-testing as demonstrated by the  <dig> entries with modified sequences reported in phi-base which are reported as not being virulently associated.
all these changes will greatly improve the analysis of gene function and the reinterpretation of the considerable number of transcriptomics data sets deposited by the international fusarium community which are available at plexdb  <cit> .
the redefined subset of genes predicted to comprise the refined secretome, verifies the previously recognised secretome predominantly within previously identified hotspot location for this gene type in the genome, namely the subtelomeric and highly recombinatorial regions.
the f. graminearum genome can now be comprehensively studied with the knowledge that a full set of gene models and surrounding genomic sequence can be probed in combination with non-coding rna annotation to identify pathogenicity mechanisms.
in summary, we present a fully completed f. graminearum isolate ph- <dig> genome and manually curated annotation using rna-seq evidence which provides the optimum resource to perform interspecies comparative analyses and gene function studies using various reverse genetics approaches.
the availability of the rres v <dig>  genome should also permit in the near future a detailed inter-comparison of historic and modern f. graminearum isolates collected from diverse geographical locations, plant species as well as fungicide application and crop rotation regimes.
availability of data and materials
sequence read archive  accession numbers for the four cells of illumina sequencing are .
the rres genomic and mitochondrial sequence for the f. graminearum strain ph- <dig> is available at the european nucleotide archive .
an omnimap instance of the rres v <dig>  fg genome which includes files to reproduce images contained herein is available for download  <cit> .

 the acquisition of a completed genomic reference sequence has numerous benefits over a near or partial completed sequence. fully completed genomes have higher impact in terms of the comparative power with other species to evaluate the divergence of pathways and the expansion and contraction of gene families. completed genomes can also be used to align syntenic regions between related species and thereby improve the accuracy and efficacy of other genome assembly projects. in addition, comparative gene finding tools can be used to annotate other related species and define species specific genes. the fusaria are globally important ascomycete fungi of growing agricultural, horticultural, medical and ecological importance. different species of fusaria can infect and cause disease on a wide diversity of host species including cereal and non-cereal crops, for example f. graminearum, only non-cereal crops, forestry trees, turf grass, animals, fish, shell fish and humans. whereas other fusarium species are a major source of a high value edible protein consumed by humans or are used to produce specific biomolecules, for example plant growth promoting gibberellins. as a consequence over the past decade, multiple fusarium sequencing projects have taken place or are currently in progress. although the haploid fusarium genomes are small in size, typically 35- mbp, for no fusarium species is the complete genome sequence available. this is counter-intuitive to providing high quality genomic resources for research however a full spectrum of genomic resources currently exists. this is problematic for the international fusarium community. the most complete genomic reference sequence of the fusaria clade is available for f. graminearum. the assembled contigs/supercontigs for this species have been assigned to four large chromosomes by using information from a genetic map. but the assembled genome 10 years later still contains >  unknown bases, and lacks telomeric and centromeric sequences. although incomplete this genome has provided the basis to align and assemble genomes of four other plant pathogenic fusarium species, namely f. fujikuroi b, f. verticillioides, f. oxysporum, f. sp lycopersici, f. pseudograminearum cs and isolates of f. pseudograminearum including the highly virulent f. pseudograminearum cs isolate. unfortunately, each of these other genomes also consists of multiple scaffolds which are heavily interspersed with unknown bases. consequently their gene annotations may be incomplete. the value of a complete genomic reference sequence is complemented by high-quality annotation of gene and non-coding rna annotation because all targets of interest can be identified and tested. gene deletion studies to probe the function of proteins/metabolites potentially linked to virulence or other phenotypic traits of interest are dependent upon the quality of the reference genome and the annotation provided. therefore an increased confidence in research findings based upon genome and annotation use is observed using a complete reference/annotation versus an incomplete. for example, deletion or silencing of a class of enzymes that are implicated in virulence is not hindered by unknown genes of the same class which maintain the virulence effect. some fusarium species, for example f. graminearum, are ideal targets for full genome completion given the predicted low repetitive sequence content and small genome size. completion of the f. graminearum reference genome would be the first completed within the class sordariomycetes, which contains fungal species with very diverse lifestyles ranging from free living saprophytes, plant invading endophytes, to pathogens of microbes, plants and/or animals. ongoing comparative analysis between and within fusarium species is important to food security due to their role in infecting hosts which include; wheat and barley, maize and sorghum, tomato, tobacco, legumes, cucurbits, sweet potatoes and banana, and rice. fusarium graminearum is a fungus of world-wide economic importance because it causes fusarium head blight disease on wheat and barley, also known as head scab disease, and fusarium stalk and ear rot disease on maize. the infection results in reduced yield affecting grain/kernel mass and quality, and mycotoxin contamination, which is a threat to human health. consequently mycotoxin content in the harvested grain has to be expensively monitored due to maximum tolerated levels of toxins permitted in human foods and animal feeds. research to reduce fhb disease in cereal species has used the ph- strain as a model to identify and understand virulence mechanisms and pathways, with the ultimate aim of developing novel species specific fungicides and/or resistant crop plants. the f. graminearum ph- strain was originally sequenced using sanger technology, assembled into contigs and supercontigs, anchored to a genetic map, and annotated by the broad institute. later on the sequence was improved by broad, and the gene model annotations were refined by munich information services for protein sequences, resulting in mrna transcripts in the mips annotation. however there remain > gaps of unknown sequences within the current assembly represented either by sequential n bases or completely lacking sequence as indicated by the absence of the eight telomeres. in addition, no sequence is available for the four centromeres and  bp remained unassembled in small contigs. for comparative purposes both the reference and annotation are both referred to in the text as mips v. in this study, to complete the f. graminearum genome, whole shotgun re-sequencing of the strain ph- to 85-fold coverage was done using short paired end reads followed by de novo assembly using various k-mer values. the accompanying annotation was produced by transferring the mips v annotation, then refining the gene model annotation integrating multiple gene prediction algorithms within the maker software as well as by using available rna-seq from two wild type f. graminearum isolates to provide additional gene model support. finally, manual inspection was done on all the gene models. in addition, the ph- mitochondrial draft which was  bp in length was re-evaluated, corrected and reduced to  bp. to complete this project, the rres v genome sequence and the mitochondrial sequence were submitted to european nuclear archive. the completed f. graminearum genome is available within ensembl fungi and displayed on the plant pathogen specific phytopath database.  genome closure assembly the de novo assembly using a sequencing depth of 85x coverage created a f. graminearum genome of,796 bp in length, assembled into four chromosomes and a mitochondrial genome of  bp in length. there was high accordance between the alignments of the rres v genome to the previous mips v reference genome. the alignment of the de novo contigs identified a previous mis-assembly at the amino end of chromosome with the 1-  bp sequence observed to be the reverse complement of the true sequence at this position. this was confirmed by the extension at the amino end to a telomere sequence and the manual inspection of mapped reads throughout the genome to detect soft clipping of reads which are indicators of misassemblies. the de novo assembly also provided extension of the amino and carboxyl sequence of chromosome 1- and identified a common telomere sequence and reverse complement respectively which is found in all vertebrates. this new data completes the four chromosomes at the amino and carboxyl end and confirms the new orientation of the amino terminus of chromosome in addition, a total of  n bases have been removed to create the new assembly. three previously constructed supercontigs namely,, and had a similar depth of coverage to the reads that mapped to the remainder of the genomic reference. therefore each of these sequences became the targets for amalgamation into chromosomes to sequences. after the alignment of contigs from the de novo assemblies and replacement of unknown bases these three supercontigs were successfully placed into the genome. supercontig was placed at the carboxyl end of chromosome to provide the subtelomeric at-rich sequence and the missing telomere sequence. supercontigs and were placed respectively in chromosomes at position,775-, in which a new gene prediction is found, and chromosome at position,855-, table chromosome and supercontig sequence coverage of the f. graminearum ph- isolate and mips reference. identification of underrepresented repeating sequences represented in the mips reference sequence could be calculated by dividing the total corrected average observed coverage by the original sequencing depth of 4:,943+ bpa achromosome from,943 bp onwards, this is the start of the repeating rna annotated sequence bthe read depth observed across the length of the sequence cpercentage of bases that are a,c,t,g but not n dthe calculated n bp corrected coverage ethe number of copies of the sequence that should be represented in the reference sequence fig. visual representation of at rich and repeat rich regions in the rres and mips references. all values are approximations to the nearest 1 kb and images have been scaled to equivalency. the green line represents the at percentage and the blue the cg percentage to give a combined total of 100 %. the labelling is in the format of a number which represents the chromosome i.e. 1 = chromosome a = the end of a reverse complement section, and b denotes regions in the mips version that were extended in the rres version. the label represents where the supercontig was placed and “c” represents where fgrres_ is located. the amino terminus set reveals the at rich extensions of chromosomes 2- and the sequence that was reverse complemented in chromosome the centromere set shows chromosome at rich region extension from kbp to kbp mips to rres respectively within mbp region, chromosome extension from kbp to kbp mips to rres respectively within mbp region, chromosome extension from kbp to kbp mips to rres respectively within mbp region, and chromosome extension from kbp to kbp mips to rres respectively within mbp region. the carboxyl terminus set shows chromosome extension at the carboxyl end of kbp, chromosome extension at the carboxyl end of kbp, chromosome extension at the carboxyl end of kbp, chromosome extension at the carboxyl end of a repetitive sequence mbp chromosome was found to lack a telomere sequence at the carboxyl terminus but was found instead to possess a region of highly repetitive sequences solely annotated as rrna coding. this large feature of the genome was found to extend for at least mbp and was identified using the following manual curation procedure. mapping the reads of the re-sequenced ph- isolate to the mips reference sequence revealed a disproportionately higher read coverage for of the supercontigs not yet placed within the genome compared to the rest of the de novo assembled genome. this suggested that some or all of these supercontig sequences were represented in multiple copies. the high copy number of each supercontig may have contributed to the difficulty experienced in prior efforts to place these sequences within chromosomes 1-. following manual inspection, ten of these repetitive sequences containing supercontigs could be aligned to the carboxyl end of chromosome each of these supercontigs contained rrna encoding repetitive sequence. this manual procedure permitted the chromosome to be extended by mbp at the carboxyl terminus by further extrapolation of rrna sequence by division of the total mapped read coverage of this region by the expected x coverage. supercontig was identified as mitochondrial dna and was transferred into the mitochondrial assembly pipeline. the unknown sequence within supercontig was corrected and the only supercontig remaining which had multiple copy coverage but did not align partially or completely to chromosome 1- sequences or the mitochondria. manual curation of the mapped reads suggests this supercontig to be either a repeating unit or a circular sequence of 5846 bp with no known genomic loci. consequently this supercontig was reduced to one single repeating unit. the identification of genomic loci of the remaining supercontigs from the original sanger sequencing and unknown sequence replacement completes chromosomes 1- sequences. the remaining million re-sequenced un-mapped reads underwent de novo assembly of which the resulting remaining reads and contigs were analysed using blast with a significance value followed by megan. these reads and contigs were shown to represent contamination with sequences that had fungal hits. the length of these individual contigs did not exceed 400 bp and the concatenated length was  bp. the unplaced contigs from the original sanger sequencing used in the mips v genome were mapped to the new reference and mitochondrial sequence, rres v. the majority mapped to these sequences using bwa mem with only sequences remaining to be placed of a combined length of  bp. these remaining sequences were assigned using blast with a significance value and megan to mouse  bp, low complexity  bp, no hits  bp, and not assigned  bp. the blast and megan analysis of the remaining sequencing data from both sequencing platforms suggests that there are no additional chromosomes to the four represented in rres v. the completed f. graminearum genome the revised rres v of the f. graminearum ph- strain genomic reference, deposited to ena, contains chromosomes through and comprises,796 bp with ‘n’ bases. this number excludes the significantly extended repetitive rrna encoding region at the carboxyl end of chromosome-. in contrast in the mips v genome, chromosomes 1- comprises,856 bp with ‘n’ bases whilst the broad fg version is in the form of multiple large supercontigs - and comprises,610 bp with ‘n’ bases. the genomic length of rres v chromosomes 1- is  bp longer than that of mips v. in order that this figure was not distorted, the calculations excluded the repetitive rrna annotated supercontigs within mips and complementary sequence at the carboxyl end of chromosome in both the mips and rres v reference because of significant extension in rres v. the unknown bases in the rres v are found at a single poly-a locus within the centromere of chromosome there were homozygous mutations observed when mapping the re-sequenced ph- to the mips v genomic reference consisting of: snps with a transition/transversion ratio of, insertions, and deletions. these mutations may have arisen due to strain passaging or a consequence of sequencing errors from the original sequencing but the observation of these mutations clustering at loci suggests due to passaging. readers are reminded that all genomic reference sequences are snapshots of a individual or a subset of individuals of a population. the size of the four chromosomes was found to be; chromosome 1:,938 bp, chromosome 2:,558 bp, chromosome 3:,947 bp, and chromosome 4:,501 bp.table comparative statistics on the length and nucleotide composition of the four f. graminearum chromosomes. comparison of the mips and rres versions of the reference genome anumber of total bases minus ‘n’ bases used in difference comparison bapproximate total length of repeating sequence at the carboxyl end of chromosome 4 to complete the f. graminearum mitochondrial genome, snps were corrected, indels deleted and indels were inserted. as a result, the new mitochondrial reference genome deposited at ena has a length that is slightly lesser in size at  bp, compared to the original size of  bp. both the original sanger sequence reads and the new mitochondrial genome obtained by paired end reads exhibited no polymorphisms. this result not only confirms the reliability of the new version of the genome, but clearly demonstrates that there has been no change in the mitochondrial sequence of strain ph- in over a decade of repeated fungal growth in vitro, in planta and long term storage at -80c. resolving two specific genome features: telomeres and centromeres the placement and extension of at rich sequence is normally difficult to resolve during genome assembly due to repetitive dna content. these sequences predominate at the amino and carboxyl ends of each chromosome and represent the subtelomeric regions. in most eukaryotic species there are multiple such regions within the relative vicinity of the repeating telomere sequence. this was found to be the case in the seven assembled subtelomeric regions. in fig.  the original and de novo extended at rich sequences at the end of each chromosome are depicted. to achieve the subtelomeric assembly, a multi k-mer approach was used in which a range of values from to was sequentially aligned to extend the sequence. however, this difficult part of the assembly was most likely achieved because of the previously noted intrinsically low abundance of repeat sequences in f. graminearum ph-. the extension of an at rich region within each chromosome permitted the four centromere regions to be defined and accurately positioned. in chromosome an at region starting at mbp was extended from kbp to kbp, in chromosome at mbp a kbp region was extended to kbp, in chromosome at mbp a kbp region was extended to kbp, and in chromosome at mbp a kbp region was to kbp. the centromere position quoted is directly related to the at rich positions originally noted in the mips version, see fig.  these results indicate that each centromere has an asymmetric position in each chromosome. therefore each of the four f. graminearum chromosomes is submetacentric with respect to centromere positioning. the evidence supporting these regions are centromere sequences was tested by the connolly et al. study. where there was centromeric sequence present, which was only in chromosome in the mips version of the reference, then h3k9me binding was observed but no h3k27me see additional file annotation of the completed fusarium graminearum the revised gene call from a total of mrna transcripts presented in the mips v genome most were transferred to the rres v genome. however, sixteen were deleted due to sequence changes, two were redundant isoforms and removed, gene models were removed due to merging and five gene models were split. in addition, gene models that had alternative model predictions which were longer were explored using interproscan and manually corrected where applicable. the gene models that either did not 100 % match after transfer or had been revised by extension, splitting or merging have been given the id extension “_m”, representing modification. some of the most common types of changes made are described in the following three generic examples: an extended or reduced gene, for example, fgsg_ splitting of a gene into two orfs results in the new gene designations 17172_a_m and 17172_b_m, and joining of a gene with other gene models i.e. and was designated as 17172_3_m keeping only the unique digit for the second gene if sequential id’s or if not sequential then the full gene id for each was used i.e. and becomes 17172_16222_m. these changes to the gene names were done to signify that these gene models had been changed from the previous mips v annotation but this approach also ensured the retention of the original gene id. in total, modified gene models are presented in the rres v genome annotation. also, new gene models have been predicted of which have supporting rna-seq evidence from cultured mycelium or spore transcriptome assemblies. these have arisen because of the replacement of the ‘n’ sequences or alternative gene prediction software. the new gene models have been given sequential numbers from, beginning at the amino end of chromosome- and ending at the carboxyl end of chromosome- the maker annotation software was used to transfer gene annotation id’s, predict gene models to correct mips gene models v and add additional new genes using as evidence, rna-seq for the mycelium and spores of ph-, z- mycelia harvested 24 h after inoculation of wild-type conidia in complete medium rna-seq, proteins from uniprot from fusarium with evidence, and cogeme est transcripts. all evidence and final gene model predictions are available through the ensembl fungi tracks as “fusarium graminearum”. the total number of gene models identified in the completed f. graminearum genome is which is in contrast to and in the broad and mips v annotations respectively. a comparison of the length of proteins between the rres and mips v annotations shows proportionally the total percentage of proteins with a length below aa, and between and aa to increase and decrease respectively for the rres set. this figure is a reflection of the greater proportion of genes that are predicted as a single exon in the rres v vs the mips v set.table basic statistics of the different reference and annotation versions of f. graminearum. a comparison of f. graminearum genome version statistics between broad, mips, and rres genome size a scaffoldsb gc contentc aincluding all scaffolds, n bases, and the mitochondria bincluding all scaffolds, n bases excluding the mitochondria cexcluding n’s and mitochondria dexcluding n’s, mitochondria and large repetitive sequence at the carboxyl end of chromosome the gene annotation an analysis of the rres v annotation using blast2go with the ncbi nr database and interproscan revealed a total of genes that were blast2go annotated or had blast hits, whilst genes have no go annotation. there are an additional level- go biological process go annotations and molecular function go annotations in the rres v annotation compared to the mips v. the number of proteins with alpha/beta hydrolase fold- or zn-c fungal-type dna-binding domain have increased from to and to respectively. the greatest increases for biological processes were in the categories; biological regulation, single-organism process, cellular process, metabolic process, and for molecular functions; catalytic activity, binding, and nucleic acid binding transcription factor activity.table go term summary of the new gene annotations. go terms summaries for biological process and molecular function of the new genes annotated in the rres v gene set 13 81 12 358 250 3 aone of these proteins was only partial the go annotation assigned to the new genes are detailed in table . the new genes given enzyme designation codes are listed in table  some of the new genes potentially have roles in primary metabolism, secondary metabolism or cell signalling. some newly predicted genes of particular interest due to coding for metabolic pathway enzymes or orthologous in virulence mechanisms are as follows: fgrres_ is predicted to have alpha-n-arabinofuranosidase activity, is located in an average % gc region of approximately 2 kb in length neighboured by two at rich regions of approximately 9 kb length at the carboxyl end of chromosome fgrres_ and fgrres_ are both serine threonine protein kinases. fgrres_ is predicted to be a cutinase. fgrres_ is predicted to be related to the enzyme galactoside o-acetyltransferase. fgrres_ is predicted to be involved in the inositol phosphate metabolism pathway,. fgrres_ is predicted to be involved in pyruvate metabolism acetyl-coa to homocitrate and lysine biosynthesis of 2-oxoglutarate to homocitrate. finally, fgrres_ has a pathogenicity protein description and go transporter activity.table new enzyme’s from the new rres gene set. new enzyme codes designated to the new rres v gene set an additional new enzymes have been identified from the modified rres gene model set, see table  in total, new biological process level go terms have been identified and new molecular function level go terms were found. of the modified genes, two genes were of particular interest in terms of virulence, fgrres_17235_m, because this gene originally had no functional annotation but was extended to be identified as coding for a cysteine rich secretory protein, allergen v5/tpx-1-related with cap and signal peptide domains, which were previously linked with plant pathogenesis proteins of the pr- family and identified in the highly virulent f. graminearum strain cs. the second gene 15917_m was identified as a endo- -beta-xylanase enzyme acting on plant cell walls by endohydrolysis of -beta-d-xylosidic linkages in xylans, also identified as part of the secretome.table new enzyme codes from the modified rres gene set. enzymes codes identified in the modified rres gene subset not found in the mips annotation for nine genes from the mips v annotation, which were in regions of unknown sequence an improved level of annotation was possible for one gene. fgrres_ previously classified as a hypothetical protein of amino acid length but was extended to aa and identified as an uaa transporter. the remaining eight corrected gene models could not be assigned additional functionality. a total of rna annotations were found across the four fully assembled chromosomes including trna, rrna,  ncrna, and tmrna see table  in addition a further rrna were identified in the extensive repetitive region present at the carboxyl of chromosome a non-coding rna s rna that has sequence similarity to a plant_srp and fungi_srp of e- and e- which is the only one identified in the genome at position,584 bp on chromosome may be important to the effectiveness of the secretome and pathogenicity. signal recognition particles are universally conserved ribonucleoproteins that direct the traffic of proteins within the cell allowing them to be secreted and therefore are a potentially important mechanism for virulence.table the number of coding and non-coding gene models predicted in the rres and mips versions. summary of coding and non-coding annotations of both the rres and mips annotations across chromosomes 1- and supercontigs 4a awithout carboxyl end repeating units with rna annotation bcarboxyl end repeating units with rna annotation to complete the genome annotation, the remaining rres v supercontig was found to contain four genes including an additional new gene of aa length. the only gene to have interproscan or blastp annotation was fgrres_ which contains an intron endonuclease, group domain and is a member of the giy-yig nuclease superfamily, typically found predominantly in phage and mitochondria. this supercontig may represent phage dna rather than mitochondrial due to the lack of alignment with the mitochondrial dna. in addition to annotation using four gene predictors, open reading frame annotation of this contig showed no additional proteins that matched within the ncbi database therefore there are unlikely to be any small genes in addition to these four predicted genes. deoxynivalenol produced by fusarium graminearum is a potent b-type trichothecene mycotoxin and virulence factor to disease formation in wheat floral tissue. commonly found in harvested cereal grains, don lowers grain quality and is a serious health concern to humans and animals. don synthesis has been assigned to genes, which is structurally related to don. the f. graminearum ph- strain lacks functional copies of the tri and tri genes resulting in the inability to produce niv. zearalenone induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals, therefore it is of agricultural importance to limit levels in the harvested grain. zearalenone synthesis requires four genes in a cluster on chromosome namely fgrres_ fgrres_ fgrres_ and fgrres_. in the zearalenone gene cluster, the ps-dh domain of the protein 15980_m has increased from aa to aa and the ketoacy-synt-c domain has increase from aa to aa. although the don production pathway is well studied, the regulatory mechanisms are less well characterised and unknowns such as the oxygenation step involving converting the c hydroxyl to a carbonyl group and the deacetylation of c have not been assigned an enzyme. a greater understanding of genetic factors regulating toxin biosynthesis could lead to the development of new strategies aimed at reducing toxin accumulation in the host tissue. in the mips annotation there are eight gene predictions with aflatoxin biosynthesis regulatory protein annotations with two that have transcription factor domains. although aflatoxin is not known to be produced by f. graminearum there role may be associated with another mycotoxin pathway such as don. two modified gene predictions from the rres v set that are annotated as aflatoxin biosynthesis regulatory proteins and one that also has a transcription factor domain are additions to this small clade of functionally unknown regulatory genes. analysis of the predicted secretome the secretome of a fungus is the set of secreted proteins that frequently defines and facilitates the host infection process. during a fungal pathogens interaction with a host, the invading fungal cells secrete various proteins that can block host responses, i.e. the fungus ustilagomaydis secretes cmu which can move through plant cells and redirect plant metabolic pathways to favour fungal infection, or kill host cells i.e. for f. graminearum, genes coding for secreted cell wall degrading enzymes have previously been identified. characterisation of the secretome is fundamental to the understanding of the mechanisms of host infection and pathogen virulence. using the mips v assembly and annotation in brown et al. predicted the secretome to contain proteins. using the new rres v predicted protein set and updated versions of the software we obtain proteins using the brown et al. pipeline to represent the refined secretome. of the rres predictions were confirmed using proteomics from the yang et al. and paper et al. studies. of the proteins identified in the yang et al. study from proteins identified in f. graminearum culture supernatants after growth in wheat or barley flour-containing medium, and of the from the extracellular proteins in vitro and in planta from the pathogenic fungus f. graminearum identified in the paper et al. study were confirmed in the in silico predictions. of the proteins predicted, are from modified genes and are from newly predicted genes. in total, protein id’s of the newly refined secretome set match the original predictions. whereas secreted proteins are unique to brown et al. and unique to rres v. of the complete predicted secretome, proteins have some level of annotation whilst proteins lack annotation and proteins of the newly refined secretome have a cysteine content of > 5 %. although genomic distribution of the secretome is not altered, an overview diagram of the secretome genes and trna annotated genes shows an overlap of clustering, in particular on chromosome and see fig.  fig. gene and rna distribution across the four chromosome. each vertical bar represents a single gene or rna annotation, aligned next to a heat map for genetic recombination. rows through to rows denote the following: all gene annotations, recombinant frequency, at rich regions greater than 2 kb, predicted secretome, trna annotations, all rna annotations, and new gene annotations only. the location of each centromere is highlighted in row with a red arrow analysis of genes predicted to possess nucleic acid binding transcription factor activity proteins that bind to specific dna sequences and regulate transcription of genes are termed transcription factors. they represent an important family of proteins controlling activities of the organism that regulate traits important for growth, development, stress response, pathogenesis, and toxin production, therefore are of importance to the development of fungicides for intervention. within the rres v gene set we have modified gene annotations of the mips set and predicted new genes. the modified set of gene annotations shows gene models that lacked a zn-c fungal-type dna-binding domain in the mips annotation to have been extended to include it in the rres v set, and of these have been identified as having gal protein domain and therefore are potential positive regulators for the gene expression of galactose-induced genes. of the new set of gene annotations predicted with transcription factor activity: fgrres_ is annotated as having oxidoreductase activity, fgrres_ is annotated as a component of a protein kinase ck complex, and fgrres_ is annotated as having lipid glycosylation activity which is part of a family of transferases involved in the final stages of the biosynthesis of antibiotics vancomycin and related chloroeremomycin. genes located in close proximity to the telomere and centromere sequences the centromeres are the region of the chromosomes that determine kinetochore formations. each centromere enables pairing of the sister chromatids and interacts with spindle microtubules during mitotic chromatid segregation and homologous chromosome segregation during meiosis. centromere structure in f. graminearum and more generally in eukaryotes is poorly understood. for eukaryotes it was suggested that centromeres may be derived from subtelomeric regions. the four centromeres identified in f. graminearum chromosome 1- respectively are,,,  bp long and are marked by their similar high at content,,,, and  % respectively. chromosome is the only chromosome to have an additional ~ ¼ sized at rich region not located within the proximity of the subtelomeres, at position,437-,387 bp, of  bp length and an at content of  %. here we surveyed the functional annotation of neighbouring genes of the newly integrated centromere, neocentromere and subtelomeric sequences in f. graminearum. although no overall pattern was found within 40 kb regions in proximity to the centromeres or the amino and carboxyl telomere sequences, two genes in common between these regions were found on the chromosome these two genes possess a nb-arc signalling domain implicated in programmed cell death. genes and other features associated with the proposed ancient chromosome fusion sites in previous studies, f. graminearum was shown to contain only four chromosomes, whilst in related fusarium species a considerably higher numbers of chromosomes exist. for example, for f. verticilloides the presence of chromosomes, and for f. oxysporum, chromosomes were demonstrated using a whole-genome sequencing and comparative genomics approach. ma et al. used the whole-genome alignment of f. graminearum and f. verticilloides to show that the sequenced f. verticilloides chromosomes could be mapped to the four f. graminearum chromosomes and proposed that the smaller number of chromosomes in f. graminearum was the result of a series of ancient chromosome fusion events. the identified potential fusion points matched regions of high genomic diversity. closer inspection of the centromeres in the rres v reference sequence revealed repeat sequences of either marcry-1_fo or ltr-gypsy to embody a large proportion of the sequence content and also the at rich regions within the subtelomeric loci. the fusion event proposed by ma et al that led to f. graminearum centromeres were likely a result of fusion at these at rich regions, which are found at various loci within each chromosome of f. verticilloides. this hypothesis is supported by repeating the alignment by ma et al. but using the rres v genomic reference sequence and focusing upon the centromere regions of f. graminearum that were not identified in the mips reference. regions that aligned contained a number of annotated and non-annotated genes sequentially homologous in sequence and context. different flanking sequences of at rich regions of f. verticilloides from the same chromosomes align to flanks of the centromeres found in f. graminearum. the observation of different flanking sequences adjacent to at rich regions from the same chromosome aligning to f. graminearum chromosome centromeres flanking sequences, suggests that fusing of multiple at rich sequences to make the centromeres in f. graminearum did not include exclusively ancient centromere sequences either in part or full but incorporated other at rich regions into the recombination event. this analysis provides further evidence in support of the existence of ancient chromosome fusion events in the f. graminearum genome which were not a direct result of the maintenance of ancient centromeres. genes previously tested for a role in the disease causing ability of f. graminearum the pathogen-host interactions database stores molecular and biological information on pathogen genes for which mutant phenotypes in pathogen-host interactions have been tested experimentally. phi-base currently provides information on >  pathogen genes, involving ~ pathogenic species. there were f. graminearum entries, including putative transcription factors and kinases, an atp citrate lyase acl and a syntaxin-like snare gene gzsyn from phi-base that had modified gene sequences in the rres v gene set versus mips v. the kinases and transcription factor genes were analysed in high-throughput gene deletion studies for their virulence. five of these genes were essential genes in f. graminearum, four genes when mutated caused reduced virulence, and one gene loss of pathogenicity. the remaining genes were neither implicated in virulence nor essential for life. due to changes in the gene model a re-evaluation of the function of each gene is recommended. recombinant regions telomeres are established in many species as sites of high single nucleotide polymorphism frequency and structural rearrangement. f. graminearum was shown to exhibit this trait but was also found to show interstitial regions of high diversity. this was suggested to represent ancient telomeres as a consequence of chromosomal fusion of progenitor chromosomes. the mapping of markers used to establish these recombinant regions to the rres v genome confirms these results with no observable changes in marker locations. the identification of the centromeres in the rres genome and mapping of previously established markers shows that the high recombinant loci at the telomeres is on the long arm of the chromosomes, see fig.  however, it is worth noting that markers are lacking for the carboxyl ends of the short arms of chromosome and due to new sequence inserted for the rres v genome and may still show these regions to be highly recombinant in future studies. a comparison of total genes, secretome predicted genes, unique genes, and at content by chromosomal arm reveals no pattern differences between areas of high or low recombination,. however it is clear that the secretome genes are predominantly found at areas of high recombination and within the subtelomeric regions, see fig.  repeat motifs and transposons in the original broad analysis, the f. graminearum genome was found to contain very few transposon sequences annotated with gene id’s and none were considered to be biologically active. active elements such as transposons are of interest because these are important contributors to genome evolution, including the creation of novel host genes and disruption of existing genes. the gene annotations identified with transposon protein domains are shown in additional file of particular note was the new gene annotation, fgrres_ is a gypsy-type long-terminal-repeat retrotransposon, not previously included in the mips v assembly and the only retroviral transposon gene annotation found. rna-seq supporting expression data in both the mycelium and spore was found suggesting this transposon is actively transcribed. however it was not found to have been included within any gene annotation leading to a truncation of the protein coding sequence. the sequence has been previously reported as a hypothetical protein partial mrna in another ph- sequencing and annotation project but appears to have been an automated process with no reference in print. a comparison of centromere and subtelomeric at rich regions repeat content shows a ltr gypsy sequence or marcry-1_fo represented in each of these regions. this study confirms previous findings in other species demonstrating the presence of transposon sequence in the centromeres. the potential neocentromere identified in chromosome shows a higher proportion of transposon content to the chromosome centromere of and 53 % respectively. in contrast to the subtelomeric regions there is a general trend towards a lower transposon content which further supports the hypothesis of a neocentromere at this location. blast comparisons the original blastp analysis of the broad f. graminearum against f. asiaticum, f. boothii, f. culmorum, and f. pseudograminearum identified species specific genes. a similar blastp analysis using high quality fusarium genome sequences found on ensembl fungi including: f. culmorum, f. fujikuroi, f. oxysporum, f. verticilloides, and nectria haematococca using the rres v genome annotations identified species specific genes. visualisation of these species specific genes loci shows no observable clustering. blastp against the fusarium cereal infecting subset, f. culmorum, f. verticillioides, and f. fujikuroi identified unique genes. a conservative evalue of for excluding proteins as part of the clade was used as little difference was observed as this value decreased. comparison of the fusarium and cereal clade species specific genes within the predicted rres v secretome identified six genes that were f. graminearum unique and contain signal peptide sequences. translated blastn of these six secreted f. graminearum unique sequences were explored against all fusarium taxon entries in ncbi 12/12/ to test if the identifications were a result of missing or incorrect gene annotations. this analysis revealed one, fgrres_ to have a hit below evalue in both nectria haematococca and f. fujikuroi and therefore this sequence is not unique to f. graminearum. the remaining five secreted sequences identified as unique to f. graminearum are; fgrres_ fgrres_ fgrres_ fgrres_ and fgrres_ the f. graminearum isolate genomes cs and cs are incomplete. this somewhat hinders inter-strain comparative analysis. however, our analysis of f. graminearum isolates cs and cs has revealed a total of and proteins, respectively identified as unique compared to f. graminearum ph- however a combinational analysis using complete genome sequences to identify snp/indel polymorphisms, expression analysis differences and unique proteins between isolates and the rres v reference is required to identify the underlying reasoning behind these isolates genomic and phenotypic differences. the original broad analysis of the f. graminearum genome had identified very little evidence of gene duplication and had discovered an active rip mechanism that had eliminated duplicated sequences. an integral protein of the rip mechanism is the presence of a dna methyltransferase rip defective gene, reported in neurospora crassa. blastp of the proteome of f. graminearum versus the rid gene from n. crassa reveals one protein, fgrres_ of similar identity but with an additional dna methylase c- cytosine-specific active site. this protein is hypothesised to represent the rid gene in f. graminearum and further support the activity of rip. the transposon class tcmar-fot fgrres_ fgrres_ fgrres_ fgrres_13431_m are all single exon predictions but for fgrres_13431_m which is multi-exon. a previous analysis by cuomo et al. has shown rip induced snps in this class of transposons however an alignment was not reported. a blastn alignment of the region of fgrres_13431_m versus the other tcmar-fot class of transposons reveals that the transposon has had two stop codons introduced which explains the multi-exon prediction from the gene software. fgrres_ has also a stop codon introduced causing a truncation. in addition to the induced snps between these transposons we can report that this has led to stop codon introduction, likely terminating the functionality of the transposons discussed and additional evidence to the process of rip in f. graminearum. a reciprocal blastp of the f. graminearum proteome identified two duplicated protein sequences, one of which had 100 % nucleotide identity. the first is a tigger transposon at position, chromosome and position, chromosome respectively which had identical nucleotides. the second is the histone h protein fgrres_ and fgrres_05491_m with snps: c:g =  c:a =  t:g =  g:a = . the sequence of fgrres_05491_m is longer than fgrres_ with an additional aa at the amino end of the protein which includes a signal peptide of aa which is predicted to target the protein to the mitochondria. within the rres v annotation there are two h proteins, one h one h1/h three h, but three h proteins of which two have the aforementioned 100 % matching sequences present. in a further analysis, we explored gene families based upon protein sequence similarity which grouped sequences into no more than four using a similar methodology to klenk et al. and heidelberg et al.. the average family size is and the largest families identified included families of amino acid transporters, three major facilitator families, three transposon families, three tyrosinases families, one aldehyde/histidinol dehydrogenase family, one unknown function family and a family of nucleoside triphosphase hydrolases. the remaining families were no greater than this suggests that few genes have formed via multi-duplications arising from a single progenitor gene to form these small multigene families. discussion the de novo genome assembly using de bruijn assembler software, such as soapdenovo, requires the use of a k-mer value which requires a set sequence length in order to align and join sequenced reads but this is limited to the length of the reads i.e. 100 bp illumina reads. as the k-mer value rises, this increases the specificity but reduces sensitivity. therefore contigs of a repetitive nature such as at rich regions within centromeres, have a higher chance of assembling into a single sequence as the k-mer value increases. there is a limitation to this approach, because as the k-mer value is increased greater sequencing coverage is needed to compensate for the reduced sensitivity. using 100 bp illumina reads and a ~ x genome coverage, a k-mer value of approximately resulted in the highest n assembly. the presence of at rich regions or repetitive transposons loci are likely to cause breaks in the resulting contigs. using a greater k-mer value can in some cases create longer sequence contigs for these loci and potentially result in contigs that span these repetitive regions along with the non-repetitive flanks. however combining such assemblies can be manually time consuming for genomes with many repetitive loci and genomes larger than mbp. the multi k-mer approaches like those that are available to soapdenovo can combine these two scenarios to produce a superior assembly with little manual curation. we were successful in using this approach to fully assemble the f. graminearum genome. for genomes of a similar size but contain more repetitive sequences, this strategy might be less successful but is worthwhile investigating. for f. graminearum the multi-kmer approach resulted in the insertion of previously unknown sequences, identification of a significant mis-assembly in chromosome at the amino end of the mips v genome, extending several internal at rich regions, placing supercontigs within chromosomes, and for the first time identifying for each of the four chromosomes the telomeric sequence and centromeric dna. the analysis of centromeric dna in filamentous fungi is difficult due to the fact that centromeric regions are comprised of heterogenous, repetitive, at-rich sequences. identification of the size and position of the centromeres on each of the four f. graminearum chromosomes permitted an examination of the annotations within the immediate vicinity versus annotation within the rest of the chromosome arms. however comparison of total gene content, the secretome gene subset, f. graminearum unique genes, and gc content revealed no obvious patterns. in saccharomyces cerevisiae short point centromeres of 120 bp exist. in contrast in many other fungi including schizosaccharomyces pombe, the human pathogenic fungus candida albicans and in the filamentous fungus n. crassa regional centromeres of considerable length are prevalent. in c. albicans, regional centromeres are composed of unique 3-  kb sequences. in the case of n. crassa, centromeres are composed of degenerate transposons, retrotransposons and simple sequence repeats, with no gene sequences. our blast analysis confirmed the absence of genes or distinctive dna elements for the four centromeric regions in f. graminearum. the centromeric composition, despite its at rich >90 %, was sufficiently varied to assemble using the four re-sequencing data sets we had available with an insert distance of between and and the use of k-mer values > see additional file the average size of the four centromeres was found to be  bp, and for chromosome our analysis revealed the existence of an additional smaller at-rich region of 15 kb. this region is approximately ¼ the size of the designated centromeric region and may comprise a neocentromeric region or the remainder of centromere repositioning event. the genes localised immediately adjacent to the four centromeres did not include genes implicated in virulence. several sequences with predicted functions in programmed cell death and chromosome segregation, replication and chromatin remodelling were found to be located in close proximity to the centromeres. we propose that the availability of centromeric sequence information for f. graminearum may be exploited in several ways. although no evidence exists, they may help construct artificial chromosomes. currently none are available for filamentous fungi. assist in cancer research. in several types of human cancers, centromeres were found to be deleted and kinetochores instead assemble on neocentromeres. experiments in both s. pombe and c. albicans were successful in deleting centromeric regions and identifying neocentromeres. to identify fungal specific drug targets. kinetochore proteins binding to the centromeric regions in filamentous fungi during mitosis were proposed to be highly fungal-specific and provide pertinent specific drug targets. binding studies between fungal specific proteins and centromeric dna regions may reveal suitable small-molecule drugs. the annotation of the genome was comprehensive. first we took the broad genome and the gene models provided by mips. then we included additional annotation from a range of different sources to provide evidence for new gene models and to modify existing gene models. this additional annotation evidence was obtained by using rna-seq data from the mycelium and spores of strain ph- as well as the mycelium of strain z- harvested 24 h after inoculation of wild-type conidia in complete medium rna-seq. in addition fusarium protein data was taken from uniprot and we also used the extensive est libraries generated by sanger sequencing available from the cogeme est transcripts database. as a consequence, a total new gene models were predicted and gene models have been modified. the new gene predictions and curated gene models are a significant step towards a fully annotated genome. however further manual curation is required by the community using rna-seq supporting evidence from multiple biological situations to achieve the high level of novel transcriptome information needed to complete this important activity. currently, this process is organised using the public ensembl fungi websites and the phytopathogenic specific website, phytopathdb. custom tracks can be displayed against the completed f. graminearum alongside any new gene model annotations and all changes are shared via a mailing list. group members can contribute, view and discuss updated gene models prior to being incorporated into future ensembl fungi versions. the study of the f. graminearum gene repertoire predicted to code for secreted proteins is highly significant to research aimed at understanding the pathogenic process due to the their hypothesised roles in penetration, host tissue necrosis and host immune subversion. the rapid rearrangement in these regions may promote rapid evolution of secretory genes in response to host responses leading to species specific genes and virulence hot spots. for example, in the aspergillus species, species unique secondary metabolite genes are enriched in subtelomeric regions. the only similarly secreted and f. graminearum unique protein that has a cysteine content of above 5 % is fgrres_ and is located in the subtelomeric region of chromosome at,286 bp. these characteristics are a hallmark of an effector protein in other filamentous pathogenic species and of potential interest to gene deletion studies. the reconfirmation of the identification of secretory genes and the identification of new secretory genes within these subtelomeric and the high recombinant regions previously demonstrated by ma et al. and brown et al. supports this hypothesis. the identification of a predominance of the trna annotations within the genes predicted to code for secreted proteins, not previously noted, may be hypothesised to represent an evolutionary bias for improved response to stimulus such as host detection resulting in a rapid response to infection and an overall switch to the pathogenic process. the detailed analysis of the newly predicted f. graminearum secretome, revealed two new secretory and one non-secretory proteins found in the rres v predictions of potential importance to virulence and also serves to illustrate the significance of the complete genome and high quality annotations to the conclusions of research findings going forward. fgrres_ is annotated as secreted and possessing alpha-n-arabinofuranosidase activity and is located in a new coding sequence island approximately 2 kb within two at rich regions of approximately 9 kb length at the carboxyl end of chromosome whilst fgrres_ is annotated as a secreted pathogenicity protein with transporter activity. both genes were previously only identified in the ngs genome study of the high virulence f. graminearum australia isolate cs. a third example, is the new cysteine-rich potential virulence gene previously only reported in the highly virulent strain cs of f. graminearum which contains several features characteristic of many apoplastic fungal effectors shown to inhibit plant chitinases used to prevent pathogen associated molecular pattern triggered immunity thereby preventing induction of host defences. the previous identification of these proteins in cs but not ph- was therefore the result of the incompleteness of the ph- genome. the reanalysis of the well-studied tri and zearalenone gene clusters proved to be interesting. a previous study had observed the co-expression of three flanking genes, namely, and with the core tri genes and extended the list of genes involved in trichothecene don production and maintenance. in the current study, one gene was modified in the tri gene cluster and in the zearalenone gene clusters. however with the publically available rna-seq data and manual curation of other newly identified genes, then it may be possible to identify and associate other sequences to these two key pathways and iteratively improve their curation and annotation. these results highlight the need for re-appraisal of existing functional data to inform the current protein function studies on these two important toxin pathways. the absence of genes that arose from duplications in f. graminearum reflects the activity of a process found in many fungi namely, repeat-induced point mutation in which mutations are caused in duplicated sequences. this process is hypothesised to be a defence mechanism against transposable elements which are common to viral/bacterial infection. a previous study has shown the introduction of c:g to t:a transition mutations during the sexual cycle in f. graminearum transposons. the identification of two histone h proteins of identical protein content but different dna sequences suggests these two sequences may be the consequence of a recent duplication event which is in the process of rip. however the introns of the proteins are substantially different and snp c:g or t:a transitions are not predominant in the shared coding regions. this suggests that these h proteins containing identical coding sequences but for an additional sequence including a signal peptide that is predicted to target the mitochondria for fgrres_05491_m, may not be in the process of rip but is present due to important functional reasons currently unknown. the large repeating rrna containing sequence at the carboxyl end of chromosome is similarly found in the human genome on chromosome with adjacent unknown bases. it is highly likely to exist in other genome reference sequences at the end of a chromosome interspersed with n bases and/or as an unplaced contigs. by manual investigation such features where found in the genome references sequences of f. fujikuroi and f. oxysporum f. sp. lycopersici at the respective amino and carboxyl ends of chromosome in f. solani such sequences were found as unscaffolded contigs which are likely to originate from the amino or carboxyl end of a chromosome sequence yet unplaced. this feature may be common among the fusarium clade and beyond but requires a greater number of genomic reference sequences to be completed before this feature can be thoroughly explored. the genome and annotation sequences have been deposited into ena and displayed at ensembl fungi and with the dedicated plant pathogen phytopathdb database to permit public access to the data and tracking of versions. information can be linked to the genome from other databases, such as the pathogen-host interaction entries to efficiently access phenotypic information from pathogenicity testing involving single and multiple gene deletion isolates generated mostly in the reference strain ph- the presentation in ensembl fungi/phytopathdb will aid comparative genomics studies by the identification of homologous and orthologous genes with other representative fungal genomes which are continually increasing and being updated.  using whole shotgun re-sequencing of f. graminearum we have comprehensively completed the genomic sequence by replacing unknown bases, placing supercontigs within their correct loci, correcting assembly errors, and inserting new sequences which include for the first time complete at rich sequences such as centromere sequences and subtelomeric regions. the identification of a retroviral transposon on chromosome shows a potential route that f. graminearum is continuing to evolve in particular to environmental stresses, such as fungicides, and diverge from related fungal species. an analysis of new gene models and curated gene models reveals new and modified transcription regulator protein predictions, three new targets for virulence mechanisms, and includes a range of proteins that may require further re-testing as demonstrated by the entries with modified sequences reported in phi-base which are reported as not being virulently associated. all these changes will greatly improve the analysis of gene function and the reinterpretation of the considerable number of transcriptomics data sets deposited by the international fusarium community which are available at plexdb. the redefined subset of genes predicted to comprise the refined secretome, verifies the previously recognised secretome predominantly within previously identified hotspot location for this gene type in the genome, namely the subtelomeric and highly recombinatorial regions. the f. graminearum genome can now be comprehensively studied with the knowledge that a full set of gene models and surrounding genomic sequence can be probed in combination with non-coding rna annotation to identify pathogenicity mechanisms. in summary, we present a fully completed f. graminearum isolate ph- genome and manually curated annotation using rna-seq evidence which provides the optimum resource to perform interspecies comparative analyses and gene function studies using various reverse genetics approaches. the availability of the rres v genome should also permit in the near future a detailed inter-comparison of historic and modern f. graminearum isolates collected from diverse geographical locations, plant species as well as fungicide application and crop rotation regimes. availability of data and materials sequence read archive accession numbers for the four cells of illumina sequencing are. the rres genomic and mitochondrial sequence for the f. graminearum strain ph- is available at the european nucleotide archive. an omnimap instance of the rres v  fg genome which includes files to reproduce images contained herein is available for download. 
the position of a potential neocentromere was also defined. we have comprehensively completed the genomic,796 bp sequence by replacing unknown bases, placing supercontigs within their correct loci, correcting assembly errors, and inserting new sequences which include for the first time complete at rich sequences such as centromere sequences, subtelomeric regions and the telomeres. completion was achieved by combining the broad sanger sequenced draft, with the gene predictions from munich information services for protein sequences v, with de novo whole-genome shotgun re-sequencing, re-annotation of the gene models using rna-seq evidence and fgenesh, snap, genemark and augustus prediction algorithms, followed by manual curation. this fully completed f. graminearum ph- genome and manually curated annotation, available at ensembl fungi, provides the optimum resource to perform interspecies comparative analyses and gene function studies. accurate genome assembly and gene model annotation are critical for comparative species and gene functional analyses. within the genome gene models have been modified and new gene models predicted, with a total gene call of. regions with high recombination previously noted to be rich in secretome encoding genes were also found to be rich in trna sequences. the re-annotation impacts upon entries held within the pathogen-host interactions database which stores information on genes for which mutant phenotypes in pathogen-host interactions have been experimentally tested, of which are putative transcription factors, kinases, atp citrate lyase, and syntaxin-like snare gene. the full content of the proposed ancient chromosome fusion sites has also been revealed and investigated. a preferentially higher frequency of genetic recombination was observed at the end of the longer arm of each chromosome. although the completed f. graminearum contains very few transposon sequences, a previously unrecognised and potentially active gypsy-type long-terminal-repeat retrotransposon was identified. here we present the completed genome sequence and annotation of the reference strain ph- of fusarium graminearum, the causal agent of head scab disease of small grain cereals which threatens global food security. gibberella zeaefusarium head blight diseasefungal centromere sequencesfungal telomere sequencesfungal virulence gene hotspotscompleted sordariomycetes genomeissue-copyright-statement© the author 2015 this study has identified 741 f. graminearum species specific genes and provides the first complete genome assembly for a sordariomycetes species.
 accurate genome assembly and gene model annotation are critical for comparative species and gene functional analyses. here we present the completed genome sequence and annotation of the reference strain ph- of fusarium graminearum, the causal agent of head scab disease of small grain cereals which threatens global food security. completion was achieved by combining the broad sanger sequenced draft, with the gene predictions from munich information services for protein sequences v, with de novo whole-genome shotgun re-sequencing, re-annotation of the gene models using rna-seq evidence and fgenesh, snap, genemark and augustus prediction algorithms, followed by manual curation.  we have comprehensively completed the genomic,796 bp sequence by replacing unknown bases, placing supercontigs within their correct loci, correcting assembly errors, and inserting new sequences which include for the first time complete at rich sequences such as centromere sequences, subtelomeric regions and the telomeres. each of the four f. graminearium chromosomes was found to be submetacentric with respect to centromere positioning. the position of a potential neocentromere was also defined. a preferentially higher frequency of genetic recombination was observed at the end of the longer arm of each chromosome. within the genome gene models have been modified and new gene models predicted, with a total gene call of. the re-annotation impacts upon entries held within the pathogen-host interactions database which stores information on genes for which mutant phenotypes in pathogen-host interactions have been experimentally tested, of which are putative transcription factors, kinases, atp citrate lyase, and syntaxin-like snare gene. although the completed f. graminearum contains very few transposon sequences, a previously unrecognised and potentially active gypsy-type long-terminal-repeat retrotransposon was identified. in addition, each of the sub-telomeres and centromeres contained either a ltr or marcry-1_fo element. the full content of the proposed ancient chromosome fusion sites has also been revealed and investigated. regions with high recombination previously noted to be rich in secretome encoding genes were also found to be rich in trna sequences. this study has identified 741 f. graminearum species specific genes and provides the first complete genome assembly for a sordariomycetes species.  this fully completed f. graminearum ph- genome and manually curated annotation, available at ensembl fungi, provides the optimum resource to perform interspecies comparative analyses and gene function studies. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords gibberella zeaefusarium head blight diseasefungal centromere sequencesfungal telomere sequencesfungal virulence gene hotspotscompleted sordariomycetes genomeissue-copyright-statement© the author 2015 