BACKGROUND
rust fungi are an important group of plant pathogens that cause devastating diseases in agricultural, silvicultural and natural ecosystems.
throughout history rust epidemics have caused significant losses in cereal production and today they are considered a major threat to global food security due to the recent evolution and spread of the highly-virulent ug <dig> strain of the wheat stem rust pathogen  and persistent worldwide losses due to wheat stripe rust disease   <cit> .
in addition, many other cultivated plants, including soybean, coffee, poplar and pine, suffer rust diseases that drastically reduce productivity  and the recent incursion of myrtle rust  into australia threatens native ecosystems and forestry production  <cit> .
consequently, there is great interest in understanding how these fungal pathogens infect their hosts and how plants can be better protected from rust disease.
the infection of flax  by the flax rust fungus  has been an important model system for understanding rust biology since the pioneering studies of harold flor in the late 1930’s and early 1940’s.
flor determined that the success or failure of pathogen attack is dictated by a ‘gene-for-gene’ interaction between resistance genes in the plant and avirulence genes in the pathogen  <cit> .
when a pathogen expressing an avirulence gene infects a plant expressing a corresponding resistance gene, the plant is able to recognise the presence of the pathogen and trigger a multifaceted defence response, which includes localised cell death at the point of attempted infection  <cit> .
most disease resistance genes encode cytoplasmic receptor proteins with a nucleotide binding  site and leucine rich repeat  domain  <cit> , whereas avirulence genes encode small, secreted proteins that are thought to be part of a diverse array of secreted effectors with virulence functions that assist in the establishment and maintenance of pathogen infection .
five avirulence genes  have been identified at four different loci in m. lini .
they encode secreted proteins produced in a specialised fungal structure called the haustorium, which is a major site for signal exchange and nutrient acquisition in rust pathogens and other biotrophic fungi  <cit> .
genome sequencing of plant pathogenic fungi has become an important approach to identify effector genes that might be important during infection.
for species with relatively small genomes  and a low percentage of repetitive dna, high quality reference genome assemblies have been produced with large scaffolds that often represent complete chromosomes from telomere to telomere .
by contrast, species with larger genomes or a high percentage of repetitive dna have proved more difficult to assemble .
rust fungi have repetitive genomes  that are amongst the largest known for plant pathogens, with genome sizes estimated by flow cytometry that range from  <dig> mb in puccinia triticina to  <dig>  gb in uromyces bidentis .
in addition, they produce dikaryotic spores that are highly heterozygous  <cit> , which makes genome assembly particularly challenging.
the wheat stem rust  and poplar rust  pathogen genome assemblies were produced by sanger sequencing, yet these still contain  <dig> and  <dig> scaffolds, respectively  <cit> .
rust fungal genome assemblies produced using next-generation sequencing  technologies are considerably more fragmented  <cit> .
for example, the m. lini genome assembly was produced by illumina sequencing and contains  <dig>  scaffolds, which together span  <dig> mb of the predicted ~ <dig> mb genome.
the longest scaffold in the assembly is only  <dig>  kb and  <dig> % of the assembly comprises scaffolds of  <dig>  kb or less  <cit> .
one strategy to improve the utility of fragmented genome assemblies is to anchor the scaffolds onto a genetic linkage map.
in recent years, ngs technologies have been developed that allow simultaneous identification and scoring of single nucleotide polymorphism  markers, the two most commonly-used methods being the genotyping-by-sequencing  technique described by elshire et al.
<cit>  and restriction-site associated dna sequencing .
gbs and radseq are reduced representation sequencing technologies that rely on restriction digest of genomic dna to reduce sequence complexity, allowing the high depth of coverage required for accurate snp identification to be achieved economically.
using these technologies to sequence mapping families it is possible to produce high-density genetic maps that contain thousands of sequence-characterised markers, which can be used for quantitative trait loci  analysis, map-based cloning, anchoring genome assemblies and genome-wide analysis of recombination rate .
in fungi, qtls linked to temperature ensitivity and melanisation have been identified in zymoseptoria tritici using radseq markers  <cit>  and a gbs-based linkage map was used to anchor the pyrenophora teres f. sp.
teres genome assembly  <cit> .
however, these powerful technologies have yet to be applied widely in fungal genetics research.
we used radseq to identify and map genetic markers in an f <dig> family derived by self-fertilisation of m. lini strain ch <dig>  <cit> .
the resulting high-density genetic map comprises  <dig> linkage groups that contain  <dig>  radseq markers and nine of the ten avirulence genes known to segregate in this mapping family.
in addition the map includes the i- <dig> avirulence inhibitor locus, which prevents pathogen detection by flax plants containing the l <dig>  l <dig>  l <dig>  l <dig> and m <dig> resistance genes  <cit> .
the radseq marker sequences were used to anchor  <dig>  % of the ch <dig> m. lini genome assembly onto the genetic map.
the genetic map and anchored assembly were then used to examine recombination frequency and distribution across the m. lini genome, show that loss of avirulence in some ch5-derived mutants is associated with large-scale chromosomal duplications and deletions and identify two new avirulence genes  by map-based cloning.
RESULTS
ch <dig> genetic map
we used radseq to identify snp-based markers for construction of a m. lini genetic map.
genomic dnas extracted from strains c and h, their f <dig> offspring  and  <dig> f <dig> progeny obtained by self-fertilisation of ch <dig>  <cit>  were digested with psti or nsii and used to generate individually barcoded radseq libraries that were sequenced using  <dig> bp paired-end illumina sequencing.
after filtering to remove poor quality reads and pcr duplicates, on average  <dig>  million psti and  <dig>  million nsii read pairs per f <dig> individual were retained for further analysis .
the sequence data were used to identify  <dig>  genetic markers that were heterozygous in ch <dig>  scored in at least  <dig> of the  <dig> f <dig> individuals and segregated in a pattern not significantly different from the 1:2: <dig> ratio expected of a single-copy, codominant marker .
after filtering to remove potential genotyping errors and low-quality markers , a genetic map was constructed at a logarithm of odds  score of seven that contained  <dig>  genetic markers  in  <dig> linkage groups .
the map spanned a total of  <dig> cm and contained  <dig> recombination bins.
the individual linkage groups ranged in size from  <dig> to  <dig> cm and contained  <dig> to  <dig> genetic markers .table  <dig> linkage groups in the ch <dig> genetic map
ten avirulence genes segregate in the ch <dig> f <dig> family and confer  <dig> avirulence specificities , with all except avrn and avrp inherited from strain h. nine of these genes were placed on the genetic map along with the i-1avirulence inhibitor , which prevents pathogen detection by flax plants containing the l <dig>  l <dig>  l <dig>  l <dig> and m <dig> resistance genes  <cit> .
only avrl <dig> could not be accurately mapped in this family as it is inhibited by i- <dig>  <cit>  and there were too few f <dig> individuals for which the avrl <dig> genotype could be determined based on the infection phenotype alone .
the avirulence genes and i- <dig> are distributed throughout the m. lini genome, with no clustering observed.
although avrl <dig> and avrl <dig> are both located on lg <dig>  they are at least  <dig>  cm apart.
similarly, avrm and avrn are located at least  <dig>  cm apart on lg <dig>  significantly, co-segregating markers were identified for avrl <dig>  avrl <dig>  avrm <dig>  avrm <dig>  avrm <dig>  avrn and i- <dig> , none of which had been cloned.fig.
<dig> ch <dig> genetic map.
the positions of avirulence genes and the i- <dig> avirulence inhibitor are indicated; where an avirulence phenotype co-segregates with markers in more than one recombination bin, the map position is indicated using a bracket.
recombination coldspots are indicated by coloured triangles.
<dig> we show in this manuscript that the avrm <dig> and avrm <dig> avirulence specificities are conferred by a single gene, which we have named avrm14
anchoring the ch <dig> genome assembly
the radseq markers were used to anchor the m. lini ch <dig> genome assembly  <cit>  onto the genetic map.
in total,  <dig> of the  <dig>  scaffolds were tagged by radseq markers, including  <dig>  of the  <dig> scaffolds in the assembly that were > <dig> kb in length.
the anchored scaffolds had a cumulative length of  <dig>  mb  and contained  <dig>   of the  <dig>   <cit>  predicted m. lini protein coding genes.
physical locations of markers are provided in additional file  <dig>
to estimate the error rate in the genetic map, we compared map positions of  <dig> pairs of markers derived from either overlapping reads from adjacent restriction sites  or back-to-back reads surrounding a single restriction site .
disagreement in marker placement was observed in just  <dig> cases: four were caused by a single missing genotype score;  <dig> by potential single genotyping errors; and two by both a missing score and a potential genotyping error.
by extrapolation, we estimate that just  <dig>  % of markers are misplaced by a mean of  <dig>  cm .
there was also a high level of concordance between the genetic map and the ch <dig> genome assembly.
of the  <dig> scaffolds that contained one or more recombinant marker pairs,  <dig> showed colinearity in physical and genetic marker order allowing unambiguous assignment of scaffold orientation relative to the genetic map .
for another  <dig> scaffolds, disagreement between physical and genetic marker order may have resulted from single genotyping errors as described above.
however,  <dig> scaffolds showed substantial differences between genetic and physical maps, including  <dig> that contained markers in more than one linkage group and  <dig> with widely separated markers from the same linkage group .
none of these scaffolds contained markers positioned in the correct order to indicate a physical connection between the linkage groups.
therefore, it is likely that these are chimeric scaffolds resulting from errors in sequence assembly.table  <dig> scaffold anchoring summary
recombination frequency in the m. lini genome
a total of  <dig> recombination events were detected in the  <dig> members of our f <dig> mapping family.
of these,  <dig>  were single recombination events and only  <dig> locations were detected at which two recombination events had occurred between flanking markers .
this suggests that the region of the genome covered by the genetic map is close to marker saturation given the amount of recombination information available in our f <dig> family.
the number of recombination breakpoints detected per f <dig> individual ranged from  <dig> to  <dig> with a mean of  <dig>  .
amongst linkage groups, there was a strong positive correlation between physical length and the number of markers  and between physical length and the number of recombination events .
however, within linkage groups recombination events were not evenly distributed.
although  <dig> % of markers were located in bins containing nine markers or less, some bins contained up to  <dig> markers  suggesting that there may be recombination coldspots in the genome.
to estimate the physical distance between recombination events across the genome, the cumulative scaffold length per recombination bin was calculated .
whilst  <dig>  % of bins were associated with a cumulative scaffold length of  <dig> kb or less, there were  <dig> bins  associated with a physical distance of  <dig> kb or more .
comparing regions of the genome with differing recombination rates, we found an increase in repetitive dna content from ~ <dig> to ~ <dig> % as recombination rate decreased, along with a slight decrease in gene and effector gene content .
there were no differences in gc content between regions with different recombination rates .
of the mapped m. lini avirulence genes, most were located in recombination bins associated with a physical distance of  <dig> kb or less .
the exceptions were avrl <dig> and avrl <dig>  which were located in large recombination coldspots of  <dig> and  <dig> kb, respectively.fig.
<dig> recombination frequency in the m. lini genome.
a histogram of the number of recombination breakpoints per individual in the  <dig> individuals of the ch <dig> f <dig> mapping family.
b scatter plot showing the correlation between cumulative scaffold length and the number of markers or number of recombination breakpoints per linkage group.
best-fit linear regression lines are shown for each data series.
c histogram of the cumulative scaffold length associated with each recombination bin in the ch <dig> genetic map.
recombination coldspots were considered bins for which the cumulative scaffold length exceeded  <dig> kb.
the cumulative scaffold length associated with bins containing avirulence genes and the i- <dig> avirulence inhibitor are indicated; where an avirulence gene is associated with more than one bin, the bin with the largest cumulative scaffold length is shown.
avrp/p <dig> and avrp <dig> are not shown as they do not co-segregate with any markers in the ch <dig> genetic map.
see additional file  <dig> for a description of how cumulative scaffold length was calculated.
d dna sequence content in fractions of the genome with differing recombination rate.
the  <dig> recombination bins were ordered by increasing cumulative scaffold length and grouped to produce ten genome fractions of approximately equal physical size , with group  <dig> representing regions of the genome with the highest recombination rate and group  <dig> representing regions of the genome with the lowest recombination rate.
gene density, effector density and the percentage repeat-masked dna were then determined for each genome fraction.
best-fit linear regression lines are shown for each data series
analysis of loss-of-avirulence mutants
as one aim of our research was to identify m. lini avirulence genes, we examined five ch <dig> mutants that had spontaneously lost either the avrl <dig> , avrm <dig> and avrm <dig>  or avrn  avirulence genes during asexual propagation.
genomic dnas from these individuals were used for radseq analysis and the  <dig> nsii markers used in genetic map construction were scored for each line.
with the exception of missing data and a small number of potential genotyping errors, all nsii markers were heterozygous in ms <dig> and ms <dig>  including those located on lg <dig> that co-segregated with avrl <dig> in the f <dig> family.
therefore, loss of avrl <dig> in these mutants must have involved a small-scale mutation event that did not affect the adjacent markers.
by contrast, loss of heterozygosity  was observed for large numbers of markers in ms <dig>  ms <dig> and ms <dig>  suggesting that loss of avirulence in these mutants was caused by large-scale chromosomal rearrangements.fig.
<dig> loss of heterozygosity in ch <dig> mutants.
regions containing markers for which only the strain h allele is present are shown in green, those for which only the strain c allele is present are shown in blue and those that are heterozygous are shown in grey.
the number of markers and cumulative scaffold distance associated with each loh event are indicated .
a genotypes of lg <dig> markers in ms <dig> and ms <dig>  avrm <dig> and avrm <dig> were inherited from strain h and co-segregate in the ch <dig> f <dig> family  <cit> .
we show later in this paper that they are conferred by a single gene, which we have called avrm <dig>  b genotypes of lg <dig> and lg <dig> markers in ms <dig>  avrn was inherited from strain c  and avrm was inherited from strain h  <cit> .
therefore, ms <dig> has retained the avirulence allele of avrm but has lost the avirulence allele of avrn
loss of avrm <dig> and avrm <dig> in ms <dig> and ms <dig> was associated with loh for  <dig> consecutive nsii markers located between  <dig> and  <dig>  cm in lg <dig> .
these markers were located on  <dig> different scaffolds with a cumulative length of  <dig>  mb.
to determine the cause of loh, total read depth and percentage of reads from the strain h allele were examined for all nsii markers scored in ms <dig>  ms <dig> and ch <dig>  as shown in fig.
4a and b, total read depth was uniform across all markers in ch <dig>  but in ms <dig> the region suffering loh was characterised by a mean read depth of  <dig>  compared to  <dig>  for the rest of the genome, consistent with deletion of part of the strain h-derived chromosome that carried avrm <dig> and avrm <dig>  however, this analysis detected a further change in the genome of ms <dig> that appeared unrelated to loss of avrm <dig> and avrm4; a region of  <dig> consecutive nsii markers located between  <dig> and  <dig>  cm in lg <dig> had higher read depth than the rest of the genome .
these markers were located on  <dig> scaffolds with a cumulative length of  <dig> kb.
in this region the mean percentage of reads from the strain h allele was  <dig>  % compared to  <dig>  % for the rest of the genome, consistent with a duplication of this region of the strain h chromosome.
very similar results were obtained for ms <dig> in both abnormal regions .
as ms <dig> and ms <dig> were isolated in the same mutant screen, it is likely that they are clonal individuals.fig.
<dig> read depth and percentage of h allele reads in ch <dig> mutants.
a read depth and percentage of h allele reads per marker across the ch <dig> genetic map.
markers are ordered according to their position in the linkage group.
linkage groups are shown in numerical order, which was assigned according to linkage group size and may not reflect relative position in the genome.
identified regions showing chromosome duplication  or loss of heterozygosity  are marked by black arrows.
identified regions in ms <dig> are as follows: dupl.
lg10– <dig> nsii markers located between  <dig> and  <dig>  cm; loh lg19– <dig> nsii markers located between  <dig> and  <dig>  cm.
identified regions in ms <dig> are as follows: dupl.
lg2– <dig> nsii markers located between  <dig>  and  <dig>  cm; dupl.
lg5– <dig> nsii markers located between  <dig> and  <dig>  cm; loh lg5– <dig> nsii markers located between  <dig>  and  <dig>  cm; dupl.
lg14– <dig> nsii markers located between  <dig>  and  <dig>  cm; loh lg27– <dig> nsii markers located between  <dig> and  <dig>  cm.
b and  mean read depth and mean percentage of h allele reads per marker in identified regions compared to the rest of the genome.
significant differences between identified regions and the rest of the genome for a given individual are marked with an asterisk .
error bars represent ± one standard error
in ms <dig>  loh was observed for all  <dig> nsii markers in lg <dig> and for  <dig> consecutive nsii markers located between  <dig>  cm and the end of lg <dig> .
however, in this case avirulence gene loss was not caused by chromosomal deletion alone, as the mean read depth in these two regions was not significantly different to that seen for the rest of the ms <dig> genome .
instead, the  <dig> heterozygous nsii markers located from  <dig> to  <dig>  cm in lg <dig> had both increased read depth  and increased percentage of h allele reads , suggesting that loss of avrn was associated with duplication of the strain h chromosome corresponding to lg <dig> accompanied by deletion of the region containing avrn from the equivalent chromosome from strain c. a similar chromosome duplication and deletion event could also explain loh without loss of read depth as observed for lg <dig> and suggested that lg <dig> and lg <dig> might be adjacent regions of the same chromosome.
indeed, when re-analysed at lod <dig>  markers from these two linkage groups formed a single group separated by a gap of  <dig>  cm.
altogether, the combined lg5/lg <dig> deletion in ms <dig> affected markers on  <dig> scaffolds with a cumulative length of  <dig>  mb.
the duplication and deletion events in lg <dig> and lg <dig> were not the only large-scale chromosomal changes detected in ms <dig>  increased read depth was also observed for  <dig> nsii markers located between  <dig>  and  <dig>  cm in the middle of lg <dig> and for  <dig> nsii markers located between  <dig>  cm and the end of the linkage group in lg <dig>  in both cases the increased read depth and decreased percentage of h allele reads compared to the rest of the genome was consistent with partial duplication of the chromosomal regions inherited from strain c , in contrast to the lg5/lg <dig> duplication event.
the duplication in lg <dig> affected markers on  <dig> scaffolds with a cumulative scaffold length of  <dig>  mb, whereas the duplication in lg <dig> affected markers on  <dig> scaffolds with a cumulative length of  <dig>  mb.
given the duplication and deletions observed in ms45/ <dig> and ms <dig>  we were interested to know if any of the f <dig> individuals harboured similar chromosomal changes.
nsii marker read depth was examined in all  <dig> individuals and evidence of h chromosome duplication was observed in ch5f2- <dig> .
the duplicated region affected  <dig> consecutive nsii markers located from  <dig>  to  <dig>  cm in the middle of lg <dig>  this genetic interval was associated with  <dig> anchored scaffolds that had a cumulative length of  <dig>  mb.
interestingly the duplicated region contained the largest recombination coldspot in the m. lini genome  and spanned the map location of the avrl <dig> avirulence gene.
identification of avrm14
flor  <cit>  showed that avrm <dig> and avrm <dig> co-segregated in a mapping family that used strain h as the avirulence source and the phenotypic data for the ch <dig> f <dig> family were consistent with this observation, although the presence of the i- <dig> inhibitor gene precluded scoring of avrm <dig> directly in many f <dig> individuals  <cit> .
we identified five genetic markers located at  <dig>  cm in lg <dig> that co-segregated with avrm <dig> in this family .
four markers were located on scaffold  <dig>  in the ch <dig> genome assembly, which also contained three adjacent markers at  <dig>  cm, and the fifth marker was the only marker located on sc <dig> .
these scaffolds were examined for the presence of genes encoding small-secreted proteins, which are generally considered candidates for effectors.
however, none of the gene models annotated on the assembly by nemri et al.
<cit>  fitted these expectations.
therefore, the region was examined manually and a candidate gene was identified at 1588– <dig> bp on sc <dig> that had not been predicted by the annotation pipeline, but was supported by rnaseq transcript assembly data  <cit>  and an incomplete cdna sequence obtained from the haustorial cdna library made by catanzariti et al.
<cit> .
this gene encoded a predicted protein of  <dig> amino acids with a  <dig> amino acid predicted signal peptide and showed homology to proteins of the nudix hydrolase superfamily .fig.
<dig> identification of avrm <dig>  a physical and genetic maps of the region surrounding avrm <dig>  the position of avrm <dig> is shown in red.
b alignment of the predicted avrm14-a and avrm14-b protein sequences.
the predicted signal peptide is shaded green and polymorphic amino acids are shaded blue.
the nudix box  is shown in red, where x represents any amino acid and u represents an aliphatic, hydrophobic amino acid.
c genetic mapping of avrm <dig> using a caps marker.
pcr products were amplified from genomic dnas and then digested with bspei.
the phenotype of each individual on m <dig> flax plants is indicated as ‘a’ for avirulent or ‘v’ for virulent.
approximate product sizes are shown in base-pairs.
d rt-pcr analysis of avrm <dig> gene expression.
products were amplified from genomic dna  extracted from strain c , strain h  and ch <dig>  products were also amplified from cdna made from rna isolated from flax leaves  <dig> days post-infection with ch <dig>  rt+ indicates a cdna reaction that contained reverse transcriptase whereas rt- indicates an identical reaction that lacked reverse transcriptase.
approximate product sizes are shown in base-pairs.
e expression of avirulence gene candidates in flax leaves using agrobacterium tumefaciens-mediated transient transformation.
expression constructs were generated using cdna sequences that lacked the region encoding the predicted signal peptide .
avrm14-a is the allele found in strain h, which is avirulent on m <dig> and m <dig> flax.
avrm14-b is the allele found in strain c, which is virulent on m <dig> and m <dig> flax.
constructs were expressed in flax lines that lacked all known resistance genes , or contained only the m <dig> resistance gene  or only the m <dig> resistance gene .
plants were photographed  <dig> days after infiltration
sequences corresponding to the avirulence gene candidate were amplified from strain h and strain c genomic dna.
sequencing revealed seven nucleotide polymorphisms within the coding region  that resulted in six amino acid polymorphisms between the predicted proteins, none of which were located in conserved residues of the nudix box motif .
one of the nucleotide polymorphisms created a bspei restriction site polymorphism and this was used as the basis of a cleaved amplified polymorphic sequence  marker.
the caps marker co-segregated with avrm <dig> in the ch <dig> f <dig> family and only the strain c  allele was found in ms <dig> and ms <dig> deletion mutants .
reverse transcriptase-polymerase chain reaction  showed that both alleles of the avirulence gene candidate were expressed in ch5-infected flax leaves  <dig> days post-infection and confirmed the presence of a  <dig> bp intron in the coding region .
to test for recognition of the candidate avirulence gene by the corresponding m <dig> and m <dig> resistance genes, cdna sequences of the two alleles were used to generate t-dna constructs for expression in planta.
constructs were produced that contained or lacked the region encoding the predicted signal peptide, as all flax resistance genes cloned to date  encode predicted cytoplasmic nb-lrr proteins  <cit>  and the interaction between m. lini avirulence proteins and their corresponding nb-lrr proteins occurs inside the plant cell  <cit> .
agrobacterium tumefaciens-mediated transient expression of the strain h-derived allele without the predicted signal peptide induced strong necrosis in flax plants containing the m <dig> resistance gene, slightly weaker necrosis in flax plants containing the m <dig> resistance gene and no symptoms in flax plants lacking these resistance genes, including near-isogenic lines .
therefore, both the avrm <dig> and avrm <dig> avirulence specificities are conferred by a single gene, which we have named avrm14-a  in keeping with the naming convention previously established for avrl <dig> and avrp <dig>  <cit> .
by contrast, no necrosis or chlorosis was observed on any of the plants infiltrated with the strain c-derived virulence allele, which we have named avrm14-b .
no response was observed when the full-length avrm14-a gene was expressed in m <dig> or m <dig> plants .
identification of avrl2
the ch <dig> genetic map showed that avrl <dig> co-segregated with  <dig> markers at map position  <dig>  cm in lg <dig> .
co-segregation was observed in  <dig> of the  <dig> f <dig> progeny.
however, one individual was heterozygous for all markers that co-segregated with avrl <dig> in the rest of the f <dig> family, yet was virulent on l <dig> flax .
this was analogous to the situation seen for the ms <dig> and ms <dig> mutants, suggesting that all three individuals might have lost avrl <dig> avirulence specificity by a similar process.
to identify additional markers that might help to locate avrl <dig>  we re-analysed the radseq data using different genotyping calling and filtering parameters that would allow inclusion of multi-copy sequences .
in addition, as approximately  <dig> % of the radseq reads did not align to the ch <dig> genome assembly , the data were also analysed by de novo assembly.
this led to the identification of  <dig> additional markers from reference-aligned reads and  <dig> markers from de novo assembly, five of which co-segregated with avrl <dig> in the  <dig> f <dig> progeny and were represented by only the strain c allele in ch5f2- <dig>  ms <dig> and ms <dig>  three of these markers were located on sc <dig> and two were derived from repetitive sequences that could not be placed accurately in the ch <dig> genome assembly .
scaffold  <dig> contained an annotated gene  predicted to encode a small, secreted protein.
two other scaffolds contained closely-related sequences  and both of these contained markers that co-segregated with avrl <dig>  suggesting the presence of a small gene family at this location.
because of the sequence duplications and assembly difficulties in this region  we used pcr to confirm the sequences of these candidate genes in each haplotype.
three genes  were found in homozygous avirulent f <dig> individuals and two genes  were found in homozygous virulent f <dig> individuals .
the coding regions of avrl2-a and avrl2-b are highly related, as are the coding regions of avrl2-c and avrl2-d .
between avrl2-a/b and avrl2-c/d there is much lower identity at the dna level , but all four genes share a conserved  <dig> bp intron within the coding region  and avrl2-a, -c and -d encode proteins with conserved n-terminal sequences .
the predicted proteins are 150– <dig> aa in length with relatively long predicted signal peptides  as predicted by signalp v  <dig>   <cit> .
the mature proteins are rich in proline and charged amino acids and contain predicted intrinsically disordered regions , but have no homology to other proteins of known function.fig.
<dig> identification of an avrl <dig> gene family.
a physical and genetic maps of the region surrounding avrl <dig>  the positions of avrl <dig> family members are shown in red.
genetic markers that were not used in ch <dig> genetic map construction are shown in italics.
b avrl <dig> family members found in avirulent  and virulent  ch <dig> f <dig> individuals.
the percentage dna identity between coding regions is shown along with the predicted protein size in amino acids .
c alignment of introns found in avrl <dig> family genes.
polymorphic nucleotides are shaded blue.
d alignment of predicted avrl <dig> family proteins.
predicted signal peptides are shaded green.
identical amino acids are marked by ‘*’, conservative amino acid substitutions are marked by ‘:’ and semi-conservative amino acid substitutions are marked by ‘.’.
predicted regions of intrinsic disorder are shown in red
the avrl2-b coding region was identical in virulent and avirulent individuals, indicating that this gene was not responsible for avirulence on l <dig> flax.
we could not detect expression of avrl2-b by rt-pcr in either genotype at  <dig> days post infection  and mapping of rnaseq reads from ch5-infected flax  did not detect any specific reads corresponding to this gene.
however, avrl2-a, avrl2-c and avrl2-d were expressed , so we developed sequence characterised amplified region  markers specific to each gene and mapped them in the ch <dig> f <dig> family .
avrl2-c and -d segregated as alleles and co-segregated with markers in the recombination bin spanning the avrl <dig> locus, but like the other linked markers in this bin they were heterozygous in ch5f2- <dig>  ms <dig> and ms <dig>  however, avrl2-a not only co-segregated with the avrl <dig> avirulence phenotype in the f <dig> family, but was also absent from ch5f2- <dig>  ms <dig> and ms <dig>  indicating that it was included in the small deletion event that gave rise to these virulent lines.
the full length cdna sequence of avrl2-a was confirmed by mapping of rnaseq reads from infected flax  <cit>  onto the genomic sequence and corresponded to a single clone sequenced from a haustorial-specific cdna library  <cit> .
to test for avirulence function, avrl2-a expression constructs were generated using cdna sequences that contained or lacked the region encoding the predicted signal peptide.
initially the signal peptide was predicted using signalp v <dig>   <cit> , which suggested a signal peptide cleavage site between amino acids  <dig> and  <dig> .
when expressed in flax leaves by agrobacterium tumefaciens-mediated transient transformation, no symptoms were observed for either construct when expressed in plants that lacked the l <dig> resistance gene .
however, when expressed in l <dig> flax plants, the avrl2-a full-length construct induced strong necrosis of infiltrated leaves and the construct lacking the predicted signal peptide-encoding region  induced chlorosis and weak necrosis.
the weak response to the dsp <dig> construct was unexpected given that avirulence protein recognition is thought to occur inside the plant cell  <cit> .
however, at  <dig> amino acids the signal peptide predicted by signalp v <dig>  is unusually long.
therefore, we tested three other constructs that lacked the region encoding the n-terminal  <dig> ,  <dig>  or  <dig>  amino acids, the latter corresponding to a signal peptide predicted by predisi  <cit> .
when co-expressed with l <dig> in nicotiana tabacum, the avrl2-a full-length, dsp <dig>  dsp <dig> and dsp <dig> constructs all elicited a strong necrotic response, whereas no response was observed upon co-expression of avrl2-a dsp <dig> and l <dig> .
we therefore conclude that avrl <dig> avirulence specificity is conferred by the avrl2-a gene.fig.
<dig> identification of avrl <dig>  a rt-pcr analysis of avrl <dig> family gene expression.
gene-specific primers were used to amplify products by pcr.
products were amplified from genomic dna  extracted from a ch <dig> f <dig> individual virulent on l <dig> flax , a ch <dig> f <dig> individual avirulent on l <dig> flax  and ch <dig>  products were also amplified from cdna made from rna isolated from flax leaves  <dig> days post-infection with ch <dig>  rt+ indicates a cdna reaction that contained reverse transcriptase whereas rt- indicates an identical reaction that lacked reverse transcriptase.
approximate product sizes are shown in base-pairs.
b genetic mapping of individual avrl <dig> family members using gene-specific scar markers.
multiplex pcr was performed using primers that amplify an internal control fragment of  <dig> bp.
avrl <dig> family members were amplified using gene-specific primers and product sizes are as follows: avrl2-a,  <dig> bp; avrl2-c,  <dig> bp; and avrl2-d,  <dig> bp.
the phenotype of each individual on l <dig> flax plants is indicated as ‘a’ for avirulent or ‘v’ for virulent.
approximate product sizes are shown in base-pairs.
c expression of avirulence gene candidates in flax leaves using agrobacterium tumefaciens-mediated transient transformation.
expression constructs were generated using the full-length avrl2-a cdna sequence  or a sequence that lacked the region encoding the first  <dig> amino acids .
constructs were expressed in near-isogenic flax lines that contained or lacked the l <dig> resistance gene .
plants were photographed  <dig> days after infiltration.
d expression of avirulence gene candidates in nicotiana tabacum leaves using agrobacterium tumefaciens-mediated transient transformation.
expression constructs were generated using the full-length avrl2-a cdna sequence  or sequences that lacked the region encoding the first  <dig>  ,  <dig> ,  <dig>  or  <dig>  amino acids.
for co-infiltrations, cultures containing avrl <dig> and l <dig> constructs were mixed together in equal ratio.
leaves were photographed  <dig> days after infiltration
discussion
we have developed a high-density genetic map for the flax rust fungus  that contains  <dig>  radseq markers in  <dig> linkage groups.
as  <dig>  % of the  <dig> recombination events detected were single recombination events, it is likely that the region of the genome covered by the map is close to marker saturation given the amount of recombination information available in the ch <dig> f <dig> family.
despite the high marker density, the map contains  <dig> linkage groups whereas the m. lini haploid genome contains  <dig> chromosomes  <cit> .
in part, this may be due to the fact we selected markers for map construction that are codominant, predicted single-copy sequences with no strong segregation distortion.
therefore, linkage groups might be broken in regions where markers could not be developed  or in regions composed entirely of repetitive dna, provided that sufficient recombination occurred in the region to produce a genetic gap that exceeded the  <dig> cm threshold used in map construction.
gaps exceeding  <dig> cm are also expected in regions of strong segregation distortion such as fungal mating type loci, two of which are expected to be heterozygous in every member of the ch <dig> f <dig> family  <cit> .
nevertheless, despite m. lini having a repetitive genome  <cit> , at least nine chromosomes are represented by a single linkage group and the high marker density, low level of missing data  and low estimated marker misplacement rate  makes this genetic map a valuable tool for genomics research.
genetic maps have been used to anchor a number of fungal genome assemblies, including fusarium graminearum and pyrenophora teres f. sp.
teres  <cit> .
f. graminearum has a high quality genome assembly  <cit> ,  <dig>  % of which could be anchored using a genetic map of just  <dig> markers  <cit> .
by contrast, we were only able to anchor  <dig>  % of the m. lini genome assembly onto the genetic map and provide unambiguous orientation to  <dig> scaffolds.
this is due to the fact that the number of genetic markers required to anchor a genome assembly is inversely proportional to the size of sequence scaffolds.
although the radseq markers are an average of  <dig>  kb apart ,  <dig> % of the assembly is comprised of scaffolds of  <dig>  kb or less , which limits improvement of the anchored assembly.
during the anchoring process we identified  <dig> scaffolds that contained markers from different linkage groups, or different parts of the same linkage group.
the strict alignment parameters used make it unlikely that this is due to incorrect marker placement on the reference genome and none of these markers showed aberrant segregation or high read depth that would indicate they were derived from multi-copy sequences.
instead, the most likely explanation is that the scaffold sequences are chimeric, a common error in assemblies of complex genomes resulting from the inability of sequence assemblers to resolve repetitive dna stretches from short read sequence data  <cit> .
scaffolding errors are very difficult to detect by analysis of the assembly alone, prompting some authors to suggest that genetic maps are an essential component of any genome sequencing project .
recombination is an essential part of the meiotic cell division process and at least one recombination event  is required per chromosome pair to ensure proper segregation of homologous chromosomes during the first meiotic division.
however, as aberrant segregation can lead to severe genetic disorders , in most species recombination is tightly controlled to produce a low number of widely spaced recombination events on each chromosome  <cit> .
in caenorhabditis elegans, arabidopsis thaliana and many mammalian species, only 1– <dig> crossover events occur per chromosome pair .
by contrast, we saw an average of  <dig>  recombination breakpoints per f <dig> individual and for the largest linkage group  up to  <dig> breakpoints were detected in some f <dig> individuals.
based on the former observation, we infer an average of  <dig>  recombination events per chromosome pair during meiosis in m. lini, which is in excess of the number of recombination events thought to be required for correct chromosome disjunction alone.
high recombination rates may be typical for fungi as an average of  <dig>  recombination events per meiosis have been detected across the  <dig> chromosome pairs of a diploid saccharomyces cerevisiae strain  <cit>  and many fungi have relatively large genetic maps given the physical size of their genome.
for example, a genetic map of  <dig> cm has been constructed for the  <dig> mb genome of the pine fusiform rust fungus, cronartium quercuum f. sp.
fusiforme  <cit> .
this is the only other rust fungus for which a comprehensive genetic map currently exists and the size of the map suggests a comparable rate of recombination to that seen here in m. lini.
understanding what controls recombination is an active area of research in many species , but we know very little about what controls this process in filamentous fungi.
the high rate of fungal recombination is of great benefit in experimental studies as it means that high-resolution mapping can be conducted using relatively small population sizes.
in a biological sense, the rapid shuffling of alleles and potential for intragenic recombination  might produce new genetic combinations that are better adapted to changing environmental conditions.
despite an overall high rate of recombination in the m. lini genome, some regions were devoid of recombination and  <dig> coldspots were identified in which a recombination bin was associated with a physical region of  <dig> kb or more .
in many species strong suppression of recombination is observed at centromeres, which are sites of sister chromatid adhesion during cell division and play a major role in ensuring that chromosomes segregate correctly during mitosis and meiosis  <cit> .
therefore, some of the coldspots observed in the m. lini genome might correspond to centromeric regions.
however, as some linkage groups contained more than one coldspot , other explanations are possible.
low recombination rates have been associated with non-centromeric heterochromatin and other repeat-rich regions  <cit>  and we observed an increase in mean repetitive dna content as recombination rate decreased .
alternatively, lack of recombination could be caused by chromosomal inversions, translocations or regions of low sequence homology that are specific to the parental combination used to construct the ch <dig> f <dig> mapping family.
we used genetic markers to examine five mutants that arose spontaneously during asexual propagation of strain ch <dig>  and, in three cases, found that conversion to virulence was associated with large-scale chromosomal alterations.
in ms <dig> and ms <dig>  over  <dig> mb of sequence that contained the avrm <dig> gene was deleted from the strain h chromosome corresponding to the end of lg <dig>  and a small duplication of the strain h chromosome corresponding to the end of lg <dig> was also observed in these mutants.
the relationship between these two events is currently unknown, but perhaps deletion of lg <dig> can only be sustained if there is an associated duplication of a function located on lg <dig>  alternatively, the tip of the lg <dig> chromosome could have replaced the region deleted from the lg <dig> chromosome during a translocation event.
ms <dig> sustained the most substantial and complex chromosomal rearrangements of the mutants examined.
in this mutant, loss of avrn was associated with deletion of over  <dig> mb from the strain c chromosome corresponding to lg <dig> and half of lg <dig> .
the deletion in lg <dig> began in a large recombination coldspot at  <dig>  cm that might represent a centromere, suggesting that the deletion may involve loss of a complete chromosome arm.
in addition we observed duplication of the entire strain h chromosome corresponding to lg5/lg <dig> and duplication of strain c sequences corresponding to the central region of lg <dig> and the end of lg <dig> .
again, the relationship between these changes is unclear.
however, the fact that deletion of lg5/ <dig> from the strain c-derived nucleus was accompanied by duplication of lg5/ <dig> from the strain h-derived nucleus suggests that there may be physical association or communication between the two haploid nuclei, which have traditionally been considered separate entities in this dikaryotic life stage.
chromosome length polymorphisms and dispensable chromosomes have been observed in many fungal species  <cit>  and loss of heterozygosity has been associated with changes in pathogenicity in the oomycete pathogen, phytophthora capsici  <cit> , but to our knowledge this is the first demonstration of gross chromosomal changes affecting virulence in a rust fungus.
large-scale chromosomal changes may impose a fitness cost in the field.
however, it will be interesting to determine whether such changes contribute to genome evolution in natural populations of rust fungi, particularly for heteroecious species that require two host species to complete their life cycle in regions where reproduction is necessarily asexual due to absence of the sexual stage host.
a major aim of our research was to identify genes controlling avirulence in m. lini and to this end we identified co-segregating markers for six avirulence genes and the i- <dig> inhibitor gene, none of which had been cloned.
in addition, we successfully identified the avrm <dig> and avrl <dig> genes by map-based cloning.
notably, although recognised by allelic host resistance genes that encode highly homologous proteins , avrl <dig> is unrelated to avrl <dig> and similarly avrm <dig> is unrelated to avrm.
avrm <dig> is a dual specificity avirulence protein that is recognised by both the flax m <dig> and m <dig> resistance proteins.
significantly, avrm <dig> has homology to proteins of the nudix hydrolase superfamily and is the first rust avirulence protein for which a prediction of biochemical function can be made from the protein sequence alone.
nudix hydrolases are commonly found in both prokaryotes and eukaryotes and are responsible for hydrolysing a wide range of organic pyrophosphates  <cit> .
several nudix hydrolases have been implicated in plant-pathogen interactions.
the phytophthora sojae avr3b avirulence protein has proven nudix hydrolase activity  <cit>  and effectors from colletotrichum truncatum  and ralstonia solanacearum  are predicted nudix hydrolase proteins.
an arabidopsis thaliana nudix hydrolase has also been identified that negatively regulates plant defence signalling .
therefore, secreted pathogen nudix hydrolases might play an important role in manipulating defence activation and cell death responses by the plant, thereby facilitating infection.
avrl <dig> is located in the region of the m. lini genome with the lowest recombination rate, which may be a centromere or other heterochromatic or repeat rich region.
a number of avirulence genes have been identified in repetitive regions with unique characteristics, such as leptosphaeria maculans effectors in at-rich isochores  <cit>  and phytophthora infestans effectors in gene-sparse genomic regions  <cit> .
the region surrounding avrl <dig> shares neither of these characteristics .
however, we found at least two independent mutants in which conversion to virulence was associated with deletion of the avrl2-a gene and another individual that contained duplication of the avrl <dig> locus and surrounding region.
this suggests that the avrl <dig> locus is prone to rearrangement, reminiscent of l. maculans avrlm <dig> and several magnaporthe oryzae avirulence genes that are located in unstable genomic regions .
by contrast, virulence alleles at the avrl <dig>  avrm, avrp <dig> and avrp <dig> loci all result from sequence diversification leading to the production of avirulence protein variants that are not recognised by flax plants containing the corresponding resistance genes  <cit> .
of these cloned avirulence genes, only avrl <dig> is located in a recombination coldspot, therefore the genomic context that influences recombination rate may also shape evolution at different m. lini avirulence loci.
the avrl <dig> family proteins have no homology to proteins of known function and this is a common feature of pathogen avirulence proteins.
however, they do contain several small predicted intrinsically disordered regions, which are known to provide structural flexibility in many proteins involved in cell signalling pathways by allowing them to adopt different conformations in response to post-translational modification and nucleic acid or protein binding  <cit> .
the predicted  <dig> amino acid, mature avrl2-a protein contains three small intrinsically disordered regions that together comprise  <dig> % of the protein sequence .
disordered regions have been identified in a number of pathogen effectors.
structural studies have shown that the oomycete avr3a and avh <dig> effectors contain n-terminal disordered regions  <cit>  and the bean rust rtp1p effector contains a predicted n-terminal disordered domain  <cit> .
moreover, intrinsically disordered regions are enriched in predicted bacterial effectors, leading marin et al.
<cit>  to propose these may be commonly occurring regions in pathogen effectors that provide the protein flexibility required to enter plant cells, mimic host functions or evade detection by the plant immune system.
CONCLUSIONS
the generation of a high-density genetic map has greatly improved the utility of the fragmented m. lini genome assembly and we have used these genetic resources together to identify two new avirulence genes by map-based cloning.
we have also identified radseq markers that co-segregate with six other avirulence genes and the i- <dig> inhibitor, which will greatly facilitate future identification of these important genes.
the genetic map and anchored assembly have been used to examine recombination rate and frequency across the m. lini genome and the ability to use marker read depth and allele frequency information to calculate dna copy number has provided novel insights into chromosome rearrangements that occur during asexual reproduction.
taken together, our data suggest that variability in the m. lini genome can be achieved through a high overall recombination rate, consistent with reports of high genetic diversity in natural, sexually reproducing m. lini populations  <cit> , and an ability of the asexual stage to withstand small- and large-scale chromosomal duplications and deletions, which can both impact upon the virulence phenotype.
several mapping families have been produced for other rust fungi, including the wheat stem rust, poplar rust and pine fusiform rust fungi  <cit> .
approaches like those described here may facilitate genome analysis and avirulence gene cloning in these rust fungi and other species.

 rust fungi are an important group of plant pathogens that cause devastating diseases in agricultural, silvicultural and natural ecosystems. throughout history rust epidemics have caused significant losses in cereal production and today they are considered a major threat to global food security due to the recent evolution and spread of the highly-virulent ug strain of the wheat stem rust pathogen and persistent worldwide losses due to wheat stripe rust disease. in addition, many other cultivated plants, including soybean, coffee, poplar and pine, suffer rust diseases that drastically reduce productivity and the recent incursion of myrtle rust into australia threatens native ecosystems and forestry production. consequently, there is great interest in understanding how these fungal pathogens infect their hosts and how plants can be better protected from rust disease. the infection of flax by the flax rust fungus has been an important model system for understanding rust biology since the pioneering studies of harold flor in the late 1930’s and early 1940’s. flor determined that the success or failure of pathogen attack is dictated by a ‘gene-for-gene’ interaction between resistance genes in the plant and avirulence genes in the pathogen. when a pathogen expressing an avirulence gene infects a plant expressing a corresponding resistance gene, the plant is able to recognise the presence of the pathogen and trigger a multifaceted defence response, which includes localised cell death at the point of attempted infection. most disease resistance genes encode cytoplasmic receptor proteins with a nucleotide binding site and leucine rich repeat domain, whereas avirulence genes encode small, secreted proteins that are thought to be part of a diverse array of secreted effectors with virulence functions that assist in the establishment and maintenance of pathogen infection. five avirulence genes have been identified at four different loci in m. lini. they encode secreted proteins produced in a specialised fungal structure called the haustorium, which is a major site for signal exchange and nutrient acquisition in rust pathogens and other biotrophic fungi. genome sequencing of plant pathogenic fungi has become an important approach to identify effector genes that might be important during infection. for species with relatively small genomes and a low percentage of repetitive dna, high quality reference genome assemblies have been produced with large scaffolds that often represent complete chromosomes from telomere to telomere. by contrast, species with larger genomes or a high percentage of repetitive dna have proved more difficult to assemble. rust fungi have repetitive genomes that are amongst the largest known for plant pathogens, with genome sizes estimated by flow cytometry that range from mb in puccinia triticina to gb in uromyces bidentis. in addition, they produce dikaryotic spores that are highly heterozygous, which makes genome assembly particularly challenging. the wheat stem rust and poplar rust pathogen genome assemblies were produced by sanger sequencing, yet these still contain and scaffolds, respectively. rust fungal genome assemblies produced using next-generation sequencing technologies are considerably more fragmented. for example, the m. lini genome assembly was produced by illumina sequencing and contains scaffolds, which together span mb of the predicted ~ mb genome. the longest scaffold in the assembly is only kb and % of the assembly comprises scaffolds of kb or less. one strategy to improve the utility of fragmented genome assemblies is to anchor the scaffolds onto a genetic linkage map. in recent years, ngs technologies have been developed that allow simultaneous identification and scoring of single nucleotide polymorphism markers, the two most commonly-used methods being the genotyping-by-sequencing technique described by elshire et al. and restriction-site associated dna sequencing. gbs and radseq are reduced representation sequencing technologies that rely on restriction digest of genomic dna to reduce sequence complexity, allowing the high depth of coverage required for accurate snp identification to be achieved economically. using these technologies to sequence mapping families it is possible to produce high-density genetic maps that contain thousands of sequence-characterised markers, which can be used for quantitative trait loci analysis, map-based cloning, anchoring genome assemblies and genome-wide analysis of recombination rate. in fungi, qtls linked to temperature ensitivity and melanisation have been identified in zymoseptoria tritici using radseq markers and a gbs-based linkage map was used to anchor the pyrenophora teres f. sp. teres genome assembly. however, these powerful technologies have yet to be applied widely in fungal genetics research. we used radseq to identify and map genetic markers in an f family derived by self-fertilisation of m. lini strain ch. the resulting high-density genetic map comprises linkage groups that contain radseq markers and nine of the ten avirulence genes known to segregate in this mapping family. in addition the map includes the i- avirulence inhibitor locus, which prevents pathogen detection by flax plants containing the l l l l and m resistance genes. the radseq marker sequences were used to anchor % of the ch m. lini genome assembly onto the genetic map. the genetic map and anchored assembly were then used to examine recombination frequency and distribution across the m. lini genome, show that loss of avirulence in some ch5-derived mutants is associated with large-scale chromosomal duplications and deletions and identify two new avirulence genes by map-based cloning.  ch genetic map we used radseq to identify snp-based markers for construction of a m. lini genetic map. genomic dnas extracted from strains c and h, their f offspring and f progeny obtained by self-fertilisation of ch were digested with psti or nsii and used to generate individually barcoded radseq libraries that were sequenced using bp paired-end illumina sequencing. after filtering to remove poor quality reads and pcr duplicates, on average million psti and million nsii read pairs per f individual were retained for further analysis. the sequence data were used to identify genetic markers that were heterozygous in ch scored in at least of the f individuals and segregated in a pattern not significantly different from the 1:2: ratio expected of a single-copy, codominant marker. after filtering to remove potential genotyping errors and low-quality markers, a genetic map was constructed at a logarithm of odds score of seven that contained genetic markers in linkage groups. the map spanned a total of cm and contained recombination bins. the individual linkage groups ranged in size from to cm and contained to genetic markers.table linkage groups in the ch genetic map ten avirulence genes segregate in the ch f family and confer avirulence specificities, with all except avrn and avrp inherited from strain h. nine of these genes were placed on the genetic map along with the i-1avirulence inhibitor, which prevents pathogen detection by flax plants containing the l l l l and m resistance genes. only avrl could not be accurately mapped in this family as it is inhibited by i- and there were too few f individuals for which the avrl genotype could be determined based on the infection phenotype alone. the avirulence genes and i- are distributed throughout the m. lini genome, with no clustering observed. although avrl and avrl are both located on lg they are at least cm apart. similarly, avrm and avrn are located at least cm apart on lg significantly, co-segregating markers were identified for avrl avrl avrm avrm avrm avrn and i-, none of which had been cloned.fig. ch genetic map. the positions of avirulence genes and the i- avirulence inhibitor are indicated; where an avirulence phenotype co-segregates with markers in more than one recombination bin, the map position is indicated using a bracket. recombination coldspots are indicated by coloured triangles. we show in this manuscript that the avrm and avrm avirulence specificities are conferred by a single gene, which we have named avrm14 anchoring the ch genome assembly the radseq markers were used to anchor the m. lini ch genome assembly onto the genetic map. in total, of the scaffolds were tagged by radseq markers, including of the scaffolds in the assembly that were > kb in length. the anchored scaffolds had a cumulative length of mb and contained of the predicted m. lini protein coding genes. physical locations of markers are provided in additional file to estimate the error rate in the genetic map, we compared map positions of pairs of markers derived from either overlapping reads from adjacent restriction sites or back-to-back reads surrounding a single restriction site. disagreement in marker placement was observed in just cases: four were caused by a single missing genotype score; by potential single genotyping errors; and two by both a missing score and a potential genotyping error. by extrapolation, we estimate that just % of markers are misplaced by a mean of cm. there was also a high level of concordance between the genetic map and the ch genome assembly. of the scaffolds that contained one or more recombinant marker pairs, showed colinearity in physical and genetic marker order allowing unambiguous assignment of scaffold orientation relative to the genetic map. for another scaffolds, disagreement between physical and genetic marker order may have resulted from single genotyping errors as described above. however, scaffolds showed substantial differences between genetic and physical maps, including that contained markers in more than one linkage group and with widely separated markers from the same linkage group. none of these scaffolds contained markers positioned in the correct order to indicate a physical connection between the linkage groups. therefore, it is likely that these are chimeric scaffolds resulting from errors in sequence assembly.table scaffold anchoring summary recombination frequency in the m. lini genome a total of recombination events were detected in the members of our f mapping family. of these, were single recombination events and only locations were detected at which two recombination events had occurred between flanking markers. this suggests that the region of the genome covered by the genetic map is close to marker saturation given the amount of recombination information available in our f family. the number of recombination breakpoints detected per f individual ranged from to with a mean of. amongst linkage groups, there was a strong positive correlation between physical length and the number of markers and between physical length and the number of recombination events. however, within linkage groups recombination events were not evenly distributed. although % of markers were located in bins containing nine markers or less, some bins contained up to markers suggesting that there may be recombination coldspots in the genome. to estimate the physical distance between recombination events across the genome, the cumulative scaffold length per recombination bin was calculated. whilst % of bins were associated with a cumulative scaffold length of kb or less, there were bins associated with a physical distance of kb or more. comparing regions of the genome with differing recombination rates, we found an increase in repetitive dna content from ~ to ~ % as recombination rate decreased, along with a slight decrease in gene and effector gene content. there were no differences in gc content between regions with different recombination rates. of the mapped m. lini avirulence genes, most were located in recombination bins associated with a physical distance of kb or less. the exceptions were avrl and avrl which were located in large recombination coldspots of and kb, respectively.fig. recombination frequency in the m. lini genome. a histogram of the number of recombination breakpoints per individual in the individuals of the ch f mapping family. b scatter plot showing the correlation between cumulative scaffold length and the number of markers or number of recombination breakpoints per linkage group. best-fit linear regression lines are shown for each data series. c histogram of the cumulative scaffold length associated with each recombination bin in the ch genetic map. recombination coldspots were considered bins for which the cumulative scaffold length exceeded kb. the cumulative scaffold length associated with bins containing avirulence genes and the i- avirulence inhibitor are indicated; where an avirulence gene is associated with more than one bin, the bin with the largest cumulative scaffold length is shown. avrp/p and avrp are not shown as they do not co-segregate with any markers in the ch genetic map. see additional file for a description of how cumulative scaffold length was calculated. d dna sequence content in fractions of the genome with differing recombination rate. the recombination bins were ordered by increasing cumulative scaffold length and grouped to produce ten genome fractions of approximately equal physical size, with group representing regions of the genome with the highest recombination rate and group representing regions of the genome with the lowest recombination rate. gene density, effector density and the percentage repeat-masked dna were then determined for each genome fraction. best-fit linear regression lines are shown for each data series analysis of loss-of-avirulence mutants as one aim of our research was to identify m. lini avirulence genes, we examined five ch mutants that had spontaneously lost either the avrl, avrm and avrm or avrn avirulence genes during asexual propagation. genomic dnas from these individuals were used for radseq analysis and the nsii markers used in genetic map construction were scored for each line. with the exception of missing data and a small number of potential genotyping errors, all nsii markers were heterozygous in ms and ms including those located on lg that co-segregated with avrl in the f family. therefore, loss of avrl in these mutants must have involved a small-scale mutation event that did not affect the adjacent markers. by contrast, loss of heterozygosity was observed for large numbers of markers in ms ms and ms suggesting that loss of avirulence in these mutants was caused by large-scale chromosomal rearrangements.fig. loss of heterozygosity in ch mutants. regions containing markers for which only the strain h allele is present are shown in green, those for which only the strain c allele is present are shown in blue and those that are heterozygous are shown in grey. the number of markers and cumulative scaffold distance associated with each loh event are indicated. a genotypes of lg markers in ms and ms avrm and avrm were inherited from strain h and co-segregate in the ch f family. we show later in this paper that they are conferred by a single gene, which we have called avrm b genotypes of lg and lg markers in ms avrn was inherited from strain c and avrm was inherited from strain h. therefore, ms has retained the avirulence allele of avrm but has lost the avirulence allele of avrn loss of avrm and avrm in ms and ms was associated with loh for consecutive nsii markers located between and cm in lg. these markers were located on different scaffolds with a cumulative length of mb. to determine the cause of loh, total read depth and percentage of reads from the strain h allele were examined for all nsii markers scored in ms ms and ch as shown in fig. 4a and b, total read depth was uniform across all markers in ch but in ms the region suffering loh was characterised by a mean read depth of compared to for the rest of the genome, consistent with deletion of part of the strain h-derived chromosome that carried avrm and avrm however, this analysis detected a further change in the genome of ms that appeared unrelated to loss of avrm and avrm4; a region of consecutive nsii markers located between and cm in lg had higher read depth than the rest of the genome. these markers were located on scaffolds with a cumulative length of kb. in this region the mean percentage of reads from the strain h allele was % compared to % for the rest of the genome, consistent with a duplication of this region of the strain h chromosome. very similar results were obtained for ms in both abnormal regions. as ms and ms were isolated in the same mutant screen, it is likely that they are clonal individuals.fig. read depth and percentage of h allele reads in ch mutants. a read depth and percentage of h allele reads per marker across the ch genetic map. markers are ordered according to their position in the linkage group. linkage groups are shown in numerical order, which was assigned according to linkage group size and may not reflect relative position in the genome. identified regions showing chromosome duplication or loss of heterozygosity are marked by black arrows. identified regions in ms are as follows: dupl. lg10– nsii markers located between and cm; loh lg19– nsii markers located between and cm. identified regions in ms are as follows: dupl. lg2– nsii markers located between and cm; dupl. lg5– nsii markers located between and cm; loh lg5– nsii markers located between and cm; dupl. lg14– nsii markers located between and cm; loh lg27– nsii markers located between and cm. b and mean read depth and mean percentage of h allele reads per marker in identified regions compared to the rest of the genome. significant differences between identified regions and the rest of the genome for a given individual are marked with an asterisk. error bars represent ± one standard error in ms loh was observed for all nsii markers in lg and for consecutive nsii markers located between cm and the end of lg. however, in this case avirulence gene loss was not caused by chromosomal deletion alone, as the mean read depth in these two regions was not significantly different to that seen for the rest of the ms genome. instead, the heterozygous nsii markers located from to cm in lg had both increased read depth and increased percentage of h allele reads, suggesting that loss of avrn was associated with duplication of the strain h chromosome corresponding to lg accompanied by deletion of the region containing avrn from the equivalent chromosome from strain c. a similar chromosome duplication and deletion event could also explain loh without loss of read depth as observed for lg and suggested that lg and lg might be adjacent regions of the same chromosome. indeed, when re-analysed at lod markers from these two linkage groups formed a single group separated by a gap of cm. altogether, the combined lg5/lg deletion in ms affected markers on scaffolds with a cumulative length of mb. the duplication and deletion events in lg and lg were not the only large-scale chromosomal changes detected in ms increased read depth was also observed for nsii markers located between and cm in the middle of lg and for nsii markers located between cm and the end of the linkage group in lg in both cases the increased read depth and decreased percentage of h allele reads compared to the rest of the genome was consistent with partial duplication of the chromosomal regions inherited from strain c, in contrast to the lg5/lg duplication event. the duplication in lg affected markers on scaffolds with a cumulative scaffold length of mb, whereas the duplication in lg affected markers on scaffolds with a cumulative length of mb. given the duplication and deletions observed in ms45/ and ms we were interested to know if any of the f individuals harboured similar chromosomal changes. nsii marker read depth was examined in all individuals and evidence of h chromosome duplication was observed in ch5f2-. the duplicated region affected consecutive nsii markers located from to cm in the middle of lg this genetic interval was associated with anchored scaffolds that had a cumulative length of mb. interestingly the duplicated region contained the largest recombination coldspot in the m. lini genome and spanned the map location of the avrl avirulence gene. identification of avrm14 flor showed that avrm and avrm co-segregated in a mapping family that used strain h as the avirulence source and the phenotypic data for the ch f family were consistent with this observation, although the presence of the i- inhibitor gene precluded scoring of avrm directly in many f individuals. we identified five genetic markers located at cm in lg that co-segregated with avrm in this family. four markers were located on scaffold in the ch genome assembly, which also contained three adjacent markers at cm, and the fifth marker was the only marker located on sc. these scaffolds were examined for the presence of genes encoding small-secreted proteins, which are generally considered candidates for effectors. however, none of the gene models annotated on the assembly by nemri et al. fitted these expectations. therefore, the region was examined manually and a candidate gene was identified at 1588– bp on sc that had not been predicted by the annotation pipeline, but was supported by rnaseq transcript assembly data and an incomplete cdna sequence obtained from the haustorial cdna library made by catanzariti et al.. this gene encoded a predicted protein of amino acids with a amino acid predicted signal peptide and showed homology to proteins of the nudix hydrolase superfamily.fig. identification of avrm a physical and genetic maps of the region surrounding avrm the position of avrm is shown in red. b alignment of the predicted avrm14-a and avrm14-b protein sequences. the predicted signal peptide is shaded green and polymorphic amino acids are shaded blue. the nudix box is shown in red, where x represents any amino acid and u represents an aliphatic, hydrophobic amino acid. c genetic mapping of avrm using a caps marker. pcr products were amplified from genomic dnas and then digested with bspei. the phenotype of each individual on m flax plants is indicated as ‘a’ for avirulent or ‘v’ for virulent. approximate product sizes are shown in base-pairs. d rt-pcr analysis of avrm gene expression. products were amplified from genomic dna extracted from strain c, strain h and ch products were also amplified from cdna made from rna isolated from flax leaves days post-infection with ch rt+ indicates a cdna reaction that contained reverse transcriptase whereas rt- indicates an identical reaction that lacked reverse transcriptase. approximate product sizes are shown in base-pairs. e expression of avirulence gene candidates in flax leaves using agrobacterium tumefaciens-mediated transient transformation. expression constructs were generated using cdna sequences that lacked the region encoding the predicted signal peptide. avrm14-a is the allele found in strain h, which is avirulent on m and m flax. avrm14-b is the allele found in strain c, which is virulent on m and m flax. constructs were expressed in flax lines that lacked all known resistance genes, or contained only the m resistance gene or only the m resistance gene. plants were photographed days after infiltration sequences corresponding to the avirulence gene candidate were amplified from strain h and strain c genomic dna. sequencing revealed seven nucleotide polymorphisms within the coding region that resulted in six amino acid polymorphisms between the predicted proteins, none of which were located in conserved residues of the nudix box motif. one of the nucleotide polymorphisms created a bspei restriction site polymorphism and this was used as the basis of a cleaved amplified polymorphic sequence marker. the caps marker co-segregated with avrm in the ch f family and only the strain c allele was found in ms and ms deletion mutants. reverse transcriptase-polymerase chain reaction showed that both alleles of the avirulence gene candidate were expressed in ch5-infected flax leaves days post-infection and confirmed the presence of a bp intron in the coding region. to test for recognition of the candidate avirulence gene by the corresponding m and m resistance genes, cdna sequences of the two alleles were used to generate t-dna constructs for expression in planta. constructs were produced that contained or lacked the region encoding the predicted signal peptide, as all flax resistance genes cloned to date encode predicted cytoplasmic nb-lrr proteins and the interaction between m. lini avirulence proteins and their corresponding nb-lrr proteins occurs inside the plant cell. agrobacterium tumefaciens-mediated transient expression of the strain h-derived allele without the predicted signal peptide induced strong necrosis in flax plants containing the m resistance gene, slightly weaker necrosis in flax plants containing the m resistance gene and no symptoms in flax plants lacking these resistance genes, including near-isogenic lines. therefore, both the avrm and avrm avirulence specificities are conferred by a single gene, which we have named avrm14-a in keeping with the naming convention previously established for avrl and avrp. by contrast, no necrosis or chlorosis was observed on any of the plants infiltrated with the strain c-derived virulence allele, which we have named avrm14-b. no response was observed when the full-length avrm14-a gene was expressed in m or m plants. identification of avrl2 the ch genetic map showed that avrl co-segregated with markers at map position cm in lg. co-segregation was observed in of the f progeny. however, one individual was heterozygous for all markers that co-segregated with avrl in the rest of the f family, yet was virulent on l flax. this was analogous to the situation seen for the ms and ms mutants, suggesting that all three individuals might have lost avrl avirulence specificity by a similar process. to identify additional markers that might help to locate avrl we re-analysed the radseq data using different genotyping calling and filtering parameters that would allow inclusion of multi-copy sequences. in addition, as approximately % of the radseq reads did not align to the ch genome assembly, the data were also analysed by de novo assembly. this led to the identification of additional markers from reference-aligned reads and markers from de novo assembly, five of which co-segregated with avrl in the f progeny and were represented by only the strain c allele in ch5f2- ms and ms three of these markers were located on sc and two were derived from repetitive sequences that could not be placed accurately in the ch genome assembly. scaffold contained an annotated gene predicted to encode a small, secreted protein. two other scaffolds contained closely-related sequences and both of these contained markers that co-segregated with avrl suggesting the presence of a small gene family at this location. because of the sequence duplications and assembly difficulties in this region we used pcr to confirm the sequences of these candidate genes in each haplotype. three genes were found in homozygous avirulent f individuals and two genes were found in homozygous virulent f individuals. the coding regions of avrl2-a and avrl2-b are highly related, as are the coding regions of avrl2-c and avrl2-d. between avrl2-a/b and avrl2-c/d there is much lower identity at the dna level, but all four genes share a conserved bp intron within the coding region and avrl2-a, -c and -d encode proteins with conserved n-terminal sequences. the predicted proteins are 150– aa in length with relatively long predicted signal peptides as predicted by signalp v. the mature proteins are rich in proline and charged amino acids and contain predicted intrinsically disordered regions, but have no homology to other proteins of known function.fig. identification of an avrl gene family. a physical and genetic maps of the region surrounding avrl the positions of avrl family members are shown in red. genetic markers that were not used in ch genetic map construction are shown in italics. b avrl family members found in avirulent and virulent ch f individuals. the percentage dna identity between coding regions is shown along with the predicted protein size in amino acids. c alignment of introns found in avrl family genes. polymorphic nucleotides are shaded blue. d alignment of predicted avrl family proteins. predicted signal peptides are shaded green. identical amino acids are marked by ‘*’, conservative amino acid substitutions are marked by ‘:’ and semi-conservative amino acid substitutions are marked by ‘.’. predicted regions of intrinsic disorder are shown in red the avrl2-b coding region was identical in virulent and avirulent individuals, indicating that this gene was not responsible for avirulence on l flax. we could not detect expression of avrl2-b by rt-pcr in either genotype at days post infection and mapping of rnaseq reads from ch5-infected flax did not detect any specific reads corresponding to this gene. however, avrl2-a, avrl2-c and avrl2-d were expressed, so we developed sequence characterised amplified region markers specific to each gene and mapped them in the ch f family. avrl2-c and -d segregated as alleles and co-segregated with markers in the recombination bin spanning the avrl locus, but like the other linked markers in this bin they were heterozygous in ch5f2- ms and ms however, avrl2-a not only co-segregated with the avrl avirulence phenotype in the f family, but was also absent from ch5f2- ms and ms indicating that it was included in the small deletion event that gave rise to these virulent lines. the full length cdna sequence of avrl2-a was confirmed by mapping of rnaseq reads from infected flax onto the genomic sequence and corresponded to a single clone sequenced from a haustorial-specific cdna library. to test for avirulence function, avrl2-a expression constructs were generated using cdna sequences that contained or lacked the region encoding the predicted signal peptide. initially the signal peptide was predicted using signalp v, which suggested a signal peptide cleavage site between amino acids and. when expressed in flax leaves by agrobacterium tumefaciens-mediated transient transformation, no symptoms were observed for either construct when expressed in plants that lacked the l resistance gene. however, when expressed in l flax plants, the avrl2-a full-length construct induced strong necrosis of infiltrated leaves and the construct lacking the predicted signal peptide-encoding region induced chlorosis and weak necrosis. the weak response to the dsp construct was unexpected given that avirulence protein recognition is thought to occur inside the plant cell. however, at amino acids the signal peptide predicted by signalp v is unusually long. therefore, we tested three other constructs that lacked the region encoding the n-terminal, or amino acids, the latter corresponding to a signal peptide predicted by predisi. when co-expressed with l in nicotiana tabacum, the avrl2-a full-length, dsp dsp and dsp constructs all elicited a strong necrotic response, whereas no response was observed upon co-expression of avrl2-a dsp and l. we therefore conclude that avrl avirulence specificity is conferred by the avrl2-a gene.fig. identification of avrl a rt-pcr analysis of avrl family gene expression. gene-specific primers were used to amplify products by pcr. products were amplified from genomic dna extracted from a ch f individual virulent on l flax, a ch f individual avirulent on l flax and ch products were also amplified from cdna made from rna isolated from flax leaves days post-infection with ch rt+ indicates a cdna reaction that contained reverse transcriptase whereas rt- indicates an identical reaction that lacked reverse transcriptase. approximate product sizes are shown in base-pairs. b genetic mapping of individual avrl family members using gene-specific scar markers. multiplex pcr was performed using primers that amplify an internal control fragment of bp. avrl family members were amplified using gene-specific primers and product sizes are as follows: avrl2-a, bp; avrl2-c, bp; and avrl2-d, bp. the phenotype of each individual on l flax plants is indicated as ‘a’ for avirulent or ‘v’ for virulent. approximate product sizes are shown in base-pairs. c expression of avirulence gene candidates in flax leaves using agrobacterium tumefaciens-mediated transient transformation. expression constructs were generated using the full-length avrl2-a cdna sequence or a sequence that lacked the region encoding the first amino acids. constructs were expressed in near-isogenic flax lines that contained or lacked the l resistance gene. plants were photographed days after infiltration. d expression of avirulence gene candidates in nicotiana tabacum leaves using agrobacterium tumefaciens-mediated transient transformation. expression constructs were generated using the full-length avrl2-a cdna sequence or sequences that lacked the region encoding the first,, or amino acids. for co-infiltrations, cultures containing avrl and l constructs were mixed together in equal ratio. leaves were photographed days after infiltration discussion we have developed a high-density genetic map for the flax rust fungus that contains radseq markers in linkage groups. as % of the recombination events detected were single recombination events, it is likely that the region of the genome covered by the map is close to marker saturation given the amount of recombination information available in the ch f family. despite the high marker density, the map contains linkage groups whereas the m. lini haploid genome contains chromosomes. in part, this may be due to the fact we selected markers for map construction that are codominant, predicted single-copy sequences with no strong segregation distortion. therefore, linkage groups might be broken in regions where markers could not be developed or in regions composed entirely of repetitive dna, provided that sufficient recombination occurred in the region to produce a genetic gap that exceeded the cm threshold used in map construction. gaps exceeding cm are also expected in regions of strong segregation distortion such as fungal mating type loci, two of which are expected to be heterozygous in every member of the ch f family. nevertheless, despite m. lini having a repetitive genome, at least nine chromosomes are represented by a single linkage group and the high marker density, low level of missing data and low estimated marker misplacement rate makes this genetic map a valuable tool for genomics research. genetic maps have been used to anchor a number of fungal genome assemblies, including fusarium graminearum and pyrenophora teres f. sp. teres. f. graminearum has a high quality genome assembly, % of which could be anchored using a genetic map of just markers. by contrast, we were only able to anchor % of the m. lini genome assembly onto the genetic map and provide unambiguous orientation to scaffolds. this is due to the fact that the number of genetic markers required to anchor a genome assembly is inversely proportional to the size of sequence scaffolds. although the radseq markers are an average of kb apart, % of the assembly is comprised of scaffolds of kb or less, which limits improvement of the anchored assembly. during the anchoring process we identified scaffolds that contained markers from different linkage groups, or different parts of the same linkage group. the strict alignment parameters used make it unlikely that this is due to incorrect marker placement on the reference genome and none of these markers showed aberrant segregation or high read depth that would indicate they were derived from multi-copy sequences. instead, the most likely explanation is that the scaffold sequences are chimeric, a common error in assemblies of complex genomes resulting from the inability of sequence assemblers to resolve repetitive dna stretches from short read sequence data. scaffolding errors are very difficult to detect by analysis of the assembly alone, prompting some authors to suggest that genetic maps are an essential component of any genome sequencing project. recombination is an essential part of the meiotic cell division process and at least one recombination event is required per chromosome pair to ensure proper segregation of homologous chromosomes during the first meiotic division. however, as aberrant segregation can lead to severe genetic disorders, in most species recombination is tightly controlled to produce a low number of widely spaced recombination events on each chromosome. in caenorhabditis elegans, arabidopsis thaliana and many mammalian species, only 1– crossover events occur per chromosome pair. by contrast, we saw an average of recombination breakpoints per f individual and for the largest linkage group up to breakpoints were detected in some f individuals. based on the former observation, we infer an average of recombination events per chromosome pair during meiosis in m. lini, which is in excess of the number of recombination events thought to be required for correct chromosome disjunction alone. high recombination rates may be typical for fungi as an average of recombination events per meiosis have been detected across the chromosome pairs of a diploid saccharomyces cerevisiae strain and many fungi have relatively large genetic maps given the physical size of their genome. for example, a genetic map of cm has been constructed for the mb genome of the pine fusiform rust fungus, cronartium quercuum f. sp. fusiforme. this is the only other rust fungus for which a comprehensive genetic map currently exists and the size of the map suggests a comparable rate of recombination to that seen here in m. lini. understanding what controls recombination is an active area of research in many species, but we know very little about what controls this process in filamentous fungi. the high rate of fungal recombination is of great benefit in experimental studies as it means that high-resolution mapping can be conducted using relatively small population sizes. in a biological sense, the rapid shuffling of alleles and potential for intragenic recombination might produce new genetic combinations that are better adapted to changing environmental conditions. despite an overall high rate of recombination in the m. lini genome, some regions were devoid of recombination and coldspots were identified in which a recombination bin was associated with a physical region of kb or more. in many species strong suppression of recombination is observed at centromeres, which are sites of sister chromatid adhesion during cell division and play a major role in ensuring that chromosomes segregate correctly during mitosis and meiosis. therefore, some of the coldspots observed in the m. lini genome might correspond to centromeric regions. however, as some linkage groups contained more than one coldspot, other explanations are possible. low recombination rates have been associated with non-centromeric heterochromatin and other repeat-rich regions and we observed an increase in mean repetitive dna content as recombination rate decreased. alternatively, lack of recombination could be caused by chromosomal inversions, translocations or regions of low sequence homology that are specific to the parental combination used to construct the ch f mapping family. we used genetic markers to examine five mutants that arose spontaneously during asexual propagation of strain ch and, in three cases, found that conversion to virulence was associated with large-scale chromosomal alterations. in ms and ms over mb of sequence that contained the avrm gene was deleted from the strain h chromosome corresponding to the end of lg and a small duplication of the strain h chromosome corresponding to the end of lg was also observed in these mutants. the relationship between these two events is currently unknown, but perhaps deletion of lg can only be sustained if there is an associated duplication of a function located on lg alternatively, the tip of the lg chromosome could have replaced the region deleted from the lg chromosome during a translocation event. ms sustained the most substantial and complex chromosomal rearrangements of the mutants examined. in this mutant, loss of avrn was associated with deletion of over mb from the strain c chromosome corresponding to lg and half of lg. the deletion in lg began in a large recombination coldspot at cm that might represent a centromere, suggesting that the deletion may involve loss of a complete chromosome arm. in addition we observed duplication of the entire strain h chromosome corresponding to lg5/lg and duplication of strain c sequences corresponding to the central region of lg and the end of lg. again, the relationship between these changes is unclear. however, the fact that deletion of lg5/ from the strain c-derived nucleus was accompanied by duplication of lg5/ from the strain h-derived nucleus suggests that there may be physical association or communication between the two haploid nuclei, which have traditionally been considered separate entities in this dikaryotic life stage. chromosome length polymorphisms and dispensable chromosomes have been observed in many fungal species and loss of heterozygosity has been associated with changes in pathogenicity in the oomycete pathogen, phytophthora capsici, but to our knowledge this is the first demonstration of gross chromosomal changes affecting virulence in a rust fungus. large-scale chromosomal changes may impose a fitness cost in the field. however, it will be interesting to determine whether such changes contribute to genome evolution in natural populations of rust fungi, particularly for heteroecious species that require two host species to complete their life cycle in regions where reproduction is necessarily asexual due to absence of the sexual stage host. a major aim of our research was to identify genes controlling avirulence in m. lini and to this end we identified co-segregating markers for six avirulence genes and the i- inhibitor gene, none of which had been cloned. in addition, we successfully identified the avrm and avrl genes by map-based cloning. notably, although recognised by allelic host resistance genes that encode highly homologous proteins, avrl is unrelated to avrl and similarly avrm is unrelated to avrm. avrm is a dual specificity avirulence protein that is recognised by both the flax m and m resistance proteins. significantly, avrm has homology to proteins of the nudix hydrolase superfamily and is the first rust avirulence protein for which a prediction of biochemical function can be made from the protein sequence alone. nudix hydrolases are commonly found in both prokaryotes and eukaryotes and are responsible for hydrolysing a wide range of organic pyrophosphates. several nudix hydrolases have been implicated in plant-pathogen interactions. the phytophthora sojae avr3b avirulence protein has proven nudix hydrolase activity and effectors from colletotrichum truncatum and ralstonia solanacearum are predicted nudix hydrolase proteins. an arabidopsis thaliana nudix hydrolase has also been identified that negatively regulates plant defence signalling. therefore, secreted pathogen nudix hydrolases might play an important role in manipulating defence activation and cell death responses by the plant, thereby facilitating infection. avrl is located in the region of the m. lini genome with the lowest recombination rate, which may be a centromere or other heterochromatic or repeat rich region. a number of avirulence genes have been identified in repetitive regions with unique characteristics, such as leptosphaeria maculans effectors in at-rich isochores and phytophthora infestans effectors in gene-sparse genomic regions. the region surrounding avrl shares neither of these characteristics. however, we found at least two independent mutants in which conversion to virulence was associated with deletion of the avrl2-a gene and another individual that contained duplication of the avrl locus and surrounding region. this suggests that the avrl locus is prone to rearrangement, reminiscent of l. maculans avrlm and several magnaporthe oryzae avirulence genes that are located in unstable genomic regions. by contrast, virulence alleles at the avrl avrm, avrp and avrp loci all result from sequence diversification leading to the production of avirulence protein variants that are not recognised by flax plants containing the corresponding resistance genes. of these cloned avirulence genes, only avrl is located in a recombination coldspot, therefore the genomic context that influences recombination rate may also shape evolution at different m. lini avirulence loci. the avrl family proteins have no homology to proteins of known function and this is a common feature of pathogen avirulence proteins. however, they do contain several small predicted intrinsically disordered regions, which are known to provide structural flexibility in many proteins involved in cell signalling pathways by allowing them to adopt different conformations in response to post-translational modification and nucleic acid or protein binding. the predicted amino acid, mature avrl2-a protein contains three small intrinsically disordered regions that together comprise % of the protein sequence. disordered regions have been identified in a number of pathogen effectors. structural studies have shown that the oomycete avr3a and avh effectors contain n-terminal disordered regions and the bean rust rtp1p effector contains a predicted n-terminal disordered domain. moreover, intrinsically disordered regions are enriched in predicted bacterial effectors, leading marin et al. to propose these may be commonly occurring regions in pathogen effectors that provide the protein flexibility required to enter plant cells, mimic host functions or evade detection by the plant immune system.  the generation of a high-density genetic map has greatly improved the utility of the fragmented m. lini genome assembly and we have used these genetic resources together to identify two new avirulence genes by map-based cloning. we have also identified radseq markers that co-segregate with six other avirulence genes and the i- inhibitor, which will greatly facilitate future identification of these important genes. the genetic map and anchored assembly have been used to examine recombination rate and frequency across the m. lini genome and the ability to use marker read depth and allele frequency information to calculate dna copy number has provided novel insights into chromosome rearrangements that occur during asexual reproduction. taken together, our data suggest that variability in the m. lini genome can be achieved through a high overall recombination rate, consistent with reports of high genetic diversity in natural, sexually reproducing m. lini populations, and an ability of the asexual stage to withstand small- and large-scale chromosomal duplications and deletions, which can both impact upon the virulence phenotype. several mapping families have been produced for other rust fungi, including the wheat stem rust, poplar rust and pine fusiform rust fungi. approaches like those described here may facilitate genome analysis and avirulence gene cloning in these rust fungi and other species. 
rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. the marker sequences were used to anchor % of the m. lini genome assembly onto the genetic map. identifying avirulence genes is crucial for understanding how virulence evolves in the field. the map and anchored assembly were then used to: 1) show that m. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the avrl and avrm avirulence genes by map-based cloning. the m. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes. avrl is located in the region of the m. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. avrm is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. to facilitate avirulence gene cloning in the flax rust fungus, melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated dna sequencing data.
 rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. identifying avirulence genes is crucial for understanding how virulence evolves in the field.  to facilitate avirulence gene cloning in the flax rust fungus, melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated dna sequencing data. the map comprises radseq markers in linkage groups that together span cm and contain recombination bins. the marker sequences were used to anchor % of the m. lini genome assembly onto the genetic map. the map and anchored assembly were then used to: 1) show that m. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the avrl and avrm avirulence genes by map-based cloning. avrm is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. avrl is located in the region of the m. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein.  the m. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords avirulence genegenetic linkage maploss of heterozygosity map-based cloningmelampsora linirecombinationrestriction-site associated dna sequencing rust fungusscaffold anchoringhttp://dx.doi.org/ /501100000923australian research councildp130104098dp130104098issue-copyright-statement© the author 2016 