BACKGROUND
grapevine powdery mildew is one of the most widespread and devastating diseases of wine, table and raisin grapes, the vast majority of which are cultivars of vitis vinifera.
this disease is caused by the fungus erysiphe necator schw.
, an obligate biotroph that can infect all green tissues of a grapevine .
infected leaves exhibit reduced photosynthesis and often undergo premature senescence and abscission.
early berry infection causes berries to crack, and the overall impact on the crop includes decreased yields, increased acidity, and decreased anthocyanin and sugar content of mature fruit  <cit> .
even low levels of powdery mildew infection on the berries can lead to ruined table grapes and wines with negative sensory attributes and decreased varietal character  <cit> .figure  <dig>
powdery mildew disease symptoms and phylogenetic relation of
e. necator
with other grapevine fungal pathogens and other powdery mildew pathogens.
e. necator infections are initiated when conidia come in contact with a susceptible host and germinate, forming hyphae with multilobed appressoria and penetration pegs.
haustoria are formed within the epidermal cell membrane to maintain the parasitic relationship with the host.
young colonies are macroscopically visible and appear white on the surface of  leaves,  fruit, and other green tissue.
multiseptate conidophores form along the hyphae perpendicularly to the epidermis, and  conidia begin to develop within a few days of the initial infection .
phylogenetic relationship of e. necator with other powdery mildews  and grape fungal pathogens .
the neighbor-joining tree was constructed in mega <dig>  <cit>  using the complete rdna its .
multiple publicly available its sequences per species were used independently to confirm clustering.
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  is shown next to the branches  <cit> .
only bootstrap values greater than  <dig> are shown.
the evolutionary distances were computed using the maximum composite likelihood method  <cit>  and are in the units of the number of base substitutions per site.
the analysis involved  <dig> nucleotide sequences.
all positions containing gaps and missing data were eliminated.
there were a total of  <dig> positions in the final dataset.
most cultivated varieties are susceptible to powdery mildew, and as a consequence growers are forced to apply fungicides to control the disease, often as frequently as every 7– <dig> days when disease pressure is high.
it has been estimated that as much as 20% of the total costs associated with wine grape production in california goes to expenses related to powdery mildew control  <cit> .
elemental sulfur was the first effective fungicide recommended for vineyards in  <dig> to control powdery mildew, and it continues to be widely used, mainly due to its efficacy and low cost  <cit> .
although its multi-site mode of action remains effective at controlling powdery mildew, the limitations to sulfur use include phytotoxicity at higher temperatures, the need for application as a protectant at frequent intervals, potential off-characters in wine, and the risk of unintended environmental consequences  <cit> .
since the 1960s, new classes of fungicides have been developed and introduced, many with single-site modes of action and beneficial properties such as systemic effectiveness and longer times between applications  <cit> .
one important class of single-site fungicides is the sterol demethylase inhibitors , which includes the azole fungicides.
dmis inhibit fungal growth by targeting the cytochrome p <dig> lanosterol c-14α-demethylase .
cyp <dig> is a key enzyme in eukaryotic sterol biosynthesis, catalyzing the removal of the 14-α carbon of lanosterol during biosynthesis of ergosterol, the major fungal sterol and an important membrane component  <cit> .
ergosterol depletion and accumulation of deleterious methylated sterols caused by cyp <dig> inhibition affect membrane integrity and functionality, reducing fungal growth  <cit> .
dmis are prone to resistance issues due to their single metabolic target.
triadimefon was the first dmi fungicide to be approved for powdery mildew control in vineyards in  <dig>  and resistance was documented as soon as  <dig>  <cit> .
resistance to other dmis has since been reported, including cross-resistance between different compounds  <cit> .
dmi resistance in fungi has been associated with several molecular mechanisms, such as cyp <dig> mutations  <cit>  and increased expression of cyp51
<cit> .
a single point mutation in cyp <dig> causing a tyrosine for phenylalanine substitution at position  <dig>  is associated with dmi resistance in e. necator
<cit> , and similar mutations conferring dmi resistance have been identified in other fungi including blumeria graminis f. sp.
hordei , candida albicans
<cit> , and mycosphaerella graminicola
<cit> .
the amino acid  <dig> is within the highly conserved cr <dig> domain involved in substrate recognition  <cit> .
structural modeling has demonstrated that dmis bind cyp <dig> at the sixth ligand of the heme group, and it has been suggested that the y136f mutation confers resistance by obstructing the heme-binding site  <cit> .
pathogen populations, particularly species with mixed sexual and asexual reproduction systems like e. necator, rapidly evolve to circumvent host resistance genes or to counteract chemical control methods  <cit> .
though new fungicides are being developed and breeding for genetic resistance is ongoing, little is known regarding how e. necator evolves to become resistant to fungicides or how it overcomes host immune responses.
whole-genome sequencing, transcriptomics, and comparative genomic approaches have been successfully applied to other fungal plant pathogens  <cit> .
however, publicly available genomic information for e. necator is scarce and limited to ssr markers , likely reflecting the difficulty of working with an obligate parasite.
genomic analyses of related powdery mildew species , including the ascomycetes that cause powdery mildew in barley  and wheat  have provided insights into their evolution and mechanisms of pathogenicity.
in this study we applied a shotgun approach to sequence the genomes of five isolates of e. necator, and used rna-seq and comparative genomics to predict and annotate protein-coding genes.
our results not only show that the e. necator genome is exceptionally large and repetitive compared to other fungal plant pathogens , but also that frequent structural variations occur between field isolates.
structural variation resulted in a wide range of copy numbers in the dmi fungicide target gene cyp <dig> .
a panel of  <dig> additional e. necator isolates collected from both synthetic fungicide-treated and non-treated vineyard sites was screened for both copy number variation and presence/absence of the y136f mutation in the encyp <dig> gene.
the vast majority  of isolates with increased copy number were collected from fungicide-treated vineyards.
increased encyp <dig> copy numbers were almost always  detected with the encyp <dig> mutant allele , suggesting that an increase in copy number becomes advantageous only when the encyp <dig> gene is present in its fungicide-tolerant allelic form.
we also show that encyp <dig> genomic copy number is correlated with both its mrna transcript level and with fungal growth in the presence of dmi fungicide.
taken together, our results demonstrate that copy number variation can be adaptive in the development of resistance to dmi fungicides in e. necator.
RESULTS
genome sequencing and assembly of five e. necatorisolates
between  <dig> and  <dig>   <dig> e. necator isolates were collected from seven vineyard sites in california .
five isolates with diverse genetic and geographical backgrounds  were selected for genome sequencing based on microsatellite profiling results, collection site, and fungicide treatment history .
the branching and e1- <dig> isolates were collected from vineyard sites without fungicide treatment, while the c-strain, lodi, and ranch <dig> isolates were all collected from sites regularly treated with synthetic fungicides to control powdery mildew.
we used an illumina shotgun sequencing approach to sequence the haploid genomes of the five selected e. necator isolates.
we generated both medium reads  and short reads  using the illumina miseq and hiseq <dig> sequencers, respectively.
the longer reads were used to assemble the draft genome sequence for the c-strain isolate, while the shorter reads were used for resequencing and de novo assembly of the other isolates.
after quality trimming and filtering, most sequencing reads  were assembled into  <dig>  ±  <dig> scaffolds with a total length of  <dig>  ±  <dig>  mb .
most of the reads of each isolate  aligned as pair-ends to the assembled contigs of the other isolates suggesting a similar genome completeness and representation across all five isolates.
unambiguously mapped reads were used to assess single nucleotide polymorphism  frequencies between the isolates following the gatk pipeline for snp calling.
the gatk analysis identified an average snp frequency of  <dig>  ±  <dig>  snps/kb .table  <dig>
e. necator
genome assembly statistics
1
1genome size estimated by k-mer frequency was 126 ± 18 mb.
based on the distribution of dna k-mer counts  <cit> , we estimated the genome size of e. necator at 126 ± 18 mb, which is similar to the 120 mb estimated genome size of bgh
<cit>  and significantly smaller than the 180 mb genome size estimated for bgt
<cit> .
the discrepancy between assembled scaffold size and estimated genome size suggests that a large proportion of the e. necator genome is highly repetitive and collapsed into common contigs during assembly.
this hypothesis is also supported by the high sequencing coverage observed in the highly fragmented and gene-poor fraction of the assemblies .
in contrast, the longest and most gene-rich contigs exhibit the expected sequencing coverage based on estimated genome size and sequencing depth .
depth of coverage was used to estimate the size of the interspersed repeats.
repeats, including transposable elements , microsatellites, and low-complexity regions, were estimated to represent  <dig>  ± 3% of the e. necator genome , a proportion similar to the repeat content estimated for the bgh genome .
tes were annotated using a combination of ab initio discovery and comparative analysis.
tes accounted for  <dig>  ±  <dig> % of the total repeats, with ltr retrotransposons and lines being the most abundant classes of tes in the genome .
tes were abundant among the sequenced transcripts, suggesting that in e. necator tes are transcriptionally active .
to assess the quality and completeness of the assembled gene space, we applied the cegma pipeline  <cit>  to map a set of  <dig> low copy core eukaryotic orthologous groups  which are conserved across higher eukaryotes.
on average  <dig>  ±  <dig> % of the kogs aligned as complete gene copies to the scaffolds, compared to  <dig>  ±  <dig> % that aligned as fragmented partial gene copies .
these results indicate that our shotgun sequencing approach generated a nearly complete assembly of the e. necator gene space, despite the obvious failure in resolving the structure of the repetitive fraction of the genome.
for the analyses described below we used the genome assembly of the c-strain isolate as a reference, because it appeared to be the most complete based on assembly metrics and cegma evaluation .
gene prediction and annotation
to enhance gene discovery and gene structure prediction, we sequenced and assembled the e. necator transcripts expressed during the interaction with grape leaves at  <dig> ,  <dig>   <dig>  and 6 days after conidia inoculation .
assembled transcripts were then used as evidence in a gene prediction pipeline that combined evidence-based, homology-based, and ab initio methods .
after removing  <dig>  genes associated with tes, we estimated that the e. necator genome contains  <dig>  protein-coding genes.
a similar total number of protein-coding genes was identified in bgh  and bgt .
nearly 92% of the predicted genes have detectable similarity to proteins known from other ascomycetes .
percent blastp matches and alignment coverage between the peptides of e. necator and other fungi, support the overall quality of the gene prediction pipeline results .
prediction accuracy was also supported by the identification of higher frequencies in nucleotide variants in the intergenic space  and intronic regions  than in the exons .
an average of  <dig>  introns per gene were predicted and, similarly to observations in other fungal genomes  <cit> , intron lengths had a narrow distribution with mean size of  <dig>  bp .
genes on longer scaffolds displayed shorter intergenic spaces than those on smaller scaffolds, suggestive of an uneven distribution of genes within the genome .
as reported in previous analyses of powdery mildews  <cit>  and other obligate biotrophs , the e. necator genome showed a loss of enzymes involved in secondary metabolism, nitrate and sulfate metabolism, further supporting the convergent adaptation of obligate biotrophy .
we did not find any of the genes known to be required for repeat induced point mutation  suggesting that the mechanisms that usually control tes in fungi are only partially functional in e. necator, as previously observed in other powdery mildew pathogens .
secretome and candidate effector proteins
microbial plant pathogens secrete effector proteins, some of which have been shown to subvert the plant innate immune response and enable infection  <cit> .
the predicted secretome of e. necator contained a total of  <dig> genes.
go term enrichment analysis of the secretome showed enrichment in hydrolase and peptidase molecular function  terms , although many of these enzymes possess β- <dig> -glucanase or chitinase activity and are likely involved in remodeling the pathogen’s own cell wall during early infection instead of breaking down the host tissue as in necrotrophy  <cit> .
the e. necator secretome also included several classes of proteins that were previously identified as pathogen candidate effectors, such as eka-like proteins  <cit>  and ribonucleases-like proteins  <cit> .
these putative effector proteins were detected in transcriptome data from early infection time points suggesting that some might be involved in the very early stages of infection .
following the same approach described in  <cit> , we identified  <dig> candidate secreted effector proteins , which displayed a significant enrichment in sequence motifs associated with secreted peptides of haustoria-forming pathogenic fungi .
these candidate effectors did not present any signature of positive selection unlike those described in other plant pathogens .
the absence of elevated levels of non-synonymous polymorphisms among the candidate effectors may hypothetically reflect the absence of antagonistic evolution in the host, since almost all cultivated grape varieties are fully susceptible to powdery mildew.
structural variation between e. necatorisolates
copy number variations  are unbalanced changes in the structure of the genome and include deletions, insertions, and duplications of >1 kb in size  <cit> .
although short sequencing read length and limited insert size do not allow reliable characterization of the type of structural variation underlying cnv, variation in sequencing coverage in genome assemblies can be used as an indicator of cnv between an assembled genome and sequencing reads from another genome .
to detect structural variants between the five sequenced isolates, we applied the cnv-seq pipeline to the mapped reads  <cit> .
cnv-seq calculates the read-depth signal across all genomic coordinates using a sliding window and was shown to provide more accurate relative copy number estimation than other cnv-detection tools  <cit> .
genome-wide cnv-seq results are summarized in table  <dig>  while the sizes of the detected cnvs were similar across the isolates, the total number of cnv sites had a broad range from  <dig> to  <dig> , corresponding to  <dig> % and  <dig> % of the assemblies, respectively.
the identified cnv loci not only spanned tes , but also  <dig> protein-coding genes .
a broad diversity of genes was observed within cnv loci with no detectable enrichment for any particular function.
cnv-seq estimations of copy number polymorphisms were validated by quantitative pcr .figure  <dig>
structural variation between
e. necator
isolates estimated by sequencing depth analysis and qpcr.
line plot showing examples of genomic loci  without copy number variation , and  with cnv across the five genomes.
loci with copy number polymorphisms were detected by analyzing sequence read depth with cnv-seq  <cit> .
gray boxes depict protein-coding sequences.
validation by qpcr of cnv-seq copy number calls for four sample cnv loci in the five sequenced isolates.
scatterplots show the correlation between estimated copy numbers for both methods.
a linear trend line is shown.
copy numbers by qpcr were estimated based on 2−Δct values normalized to the single copy e. necator elongation factor gene enef <dig> .
genome-wide estimates of copy number variation in the five sequenced
e. necator
isolates determined by cnv-seq analysis
e1-1011
ranch91
c-strain1
lodi1
branching2
1values represent cnv-seq results when branching scaffolds were used as reference.
2values represent cnv-seq results when e- <dig> scaffolds were used as reference.
copy number variation of the encyp51locus in the five sequenced isolates
the encyp <dig> gene, which encodes the enzyme targeted by dmi fungicides, was among the protein-coding genes with a wide range of copy number variation across the five sequenced genomes.
sequencing coverage, confirmed by qpcr, estimated the presence of  <dig>   <dig>  and  <dig> encyp <dig> copies in ranch  <dig>  c-strain, and lodi, respectively, and single copies in e1- <dig> and branching .
remarkably, the three isolates collected from fungicide-treated vineyards showed multiple copies of encyp <dig> with all copies being the y136f mutated allele, while the two isolates from vineyards not treated with fungicides both had single copies of the encyp <dig> gene in its fungicide-susceptible allelic form .
the y136f substitution was the only non-synonymous polymorphism detected between the isolates in the encyp <dig> coding region .
all encyp <dig> alleles were confirmed by sanger sequencing, which also confirmed that all encyp <dig> duplicated copies were in the same allelic form .figure  <dig>
copy number variation of the
encyp51
locus.
log <dig> ratio plot of sequencing depth in the encyp <dig> locus of e1- <dig>  c-strain, lodi, and ranch <dig> relative to the sequence depth in the encyp <dig> locus of branching.
cnv was confirmed in all pair-wise comparisons using cnv-seq.
depth of coverage of the encyp <dig> locus in the branching isolate is presented as it provided the longest assembled scaffold of the locus across all isolates.
arrows and boxes depict protein coding sequences and transposable elements, respectively.
duplication boundaries upstream and downstream of the encyp <dig> coding region were common in all isolates with multiple copies of encyp <dig>  only in lodi did an additional duplication event occur involving a shorter region of  <dig>  kb.
cnv-seq estimates of encyp <dig> copy numbers were validated using qpcr.
scatterplot in inset shows the correlation between cnv-seq and qpcr copy number estimates .
in all isolates the duplication events did not involve the two flanking genes, en-g <dig> and en-g <dig>  which were confirmed by qpcr to be single copy in all genomes .
duplication boundaries, approximately  <dig>  and  <dig>  kb upstream and downstream of the encyp <dig> coding region, respectively, were common in all isolates .
only in lodi did an additional duplication event occur involving a shorter region of  <dig>  kb .
in all isolates, the duplication events did not involve the two flanking genes, en-g <dig> and en-g <dig>  which were confirmed by qpcr to be single copy in all genomes.
in all isolates the downstream duplication boundary coincided with a fragment of a gypsy element .
although numerous other tes were found throughout the sequence flanking the duplicated region, we did not detect repeated homologous sequences that could provide the substrate for non-allelic homologous recombination and allow us to hypothesize the mechanism underlying the copy number polymorphisms in the locus  <cit> .
microsynteny of the encyp <dig> locus between e. necator, bgh, and bgt  both confirms the accuracy of the sequence assembly and also shows the structural conservation in this region of the genome across species despite the frequent structural rearrangements observed in the encyp <dig> locus.figure  <dig>
microsynteny of the
encyp51
locus between
e. necator
,
blumeria graminis
f.sp.
hordei
, and
blumeria graminis
f.sp.
tritici
.
scaffold alignment based on translated nucleotides  and similarity visualization was done using act-artemis comparison tool .
red and blue lines indicate similar regions between scaffolds .
blue lines indicate inversions.
arrows correspond to protein-coding genes.
cnvs can modify the expression of genes that localize within the cnv in a gene dose-dependent manner  <cit> .
to determine the impact of cnv on encyp <dig> expression, we measured mrna transcript expression levels at  <dig> and 5 days post conidia inoculation using qrt-pcr.
our results show that encyp <dig> was differentially expressed in the different isolates at both time points  with a strong correlation between gene expression levels and encyp <dig> copy number .figure  <dig>
impact of cnv on
cyp51
expression.
bar graph presenting qrt-pcr data for the relative expression of encyp <dig> in sequenced isolates at  <dig> and 5 days post inoculation.
expression values are presented as fold-enef <dig> , calculated using the Δct method as described in  <cit> .
data were analyzed using a two-way anova.
values are the mean ± standard error of three biological replicates.
different letters represent significant results .
scatterplot showing the correlation between relative expression of encyp <dig> by qrt-pcr and copy number predicted by sequencing coverage.
a linear trend is shown.
copy number variation of the encyp51locus occurs frequently in e. necatorand is correlated with fungal growth under fungicide treatment
to determine the extent of encyp <dig> cnv in e. necator beyond the five sequenced isolates, we screened an additional  <dig> isolates collected from multiple vineyard locations .
all isolates tested for cnv were also genotyped to determine the allelic forms of encyp <dig>  out of the  <dig> total isolates, 48% carried the wild-type allele, while 52% carried the y136f mutation .
the majority  of the isolates with the wild type allele were collected from non-sprayed vineyards, while almost all  of the isolates with the y136f mutation came from sprayed vineyards .
quantitative pcr analysis showed the presence of multiple copies of encyp <dig> in  <dig> isolates with a wide range of copy numbers, from as few as  <dig> copies and up to  <dig> copies .
the two genes that flank encyp <dig> were mostly single copy confirming the conservation of the duplication boundaries .
remarkably, 94% of the isolates with multiple copies of encyp <dig> also carried the mutant y136f allele, while 96% of the isolates with single copies of encyp <dig> were wild type, further supporting the hypothesis that the acquisition of multiple copies of the fungicide-tolerant allele contributes to the evolutionary fitness.
hypergeometric tests supported the significant association between fungicide treatment and both presence of the y136f allele  and multiple copies of encyp <dig> .
the encyp <dig> cnv and its allelic form did not correlate with ssr-based clustering  suggesting that the y136f mutation and the acquisition of multiple of copies of the encyp <dig> gene occurred multiple times in the e. necator populations screened.figure  <dig>
copy number variation of the
cyp51
locus occurs frequently in
e. necator
and correlates with fungal growth under fungicide treatment.
circular visualization of the allelic form  and copy number of encyp <dig> in the  <dig> california isolates, and the fungicide spray regime of each collection site.
at the center of the circular plot is a hierarchical clustering dendrogram determined by ssr marker-based fingerprinting .
bar plots showing the proportion of wild type and mutant encyp <dig> alleles collected from sites sprayed or not sprayed with fungicides.
box plots showing  the distribution of encyp <dig> copy number in isolates containing the mutant allele compared to those with the wild type allele, and  box plots showing the distribution of copy numbers of the two genes that flank the conserved duplication boundaries observed in the individuals with multiple encyp <dig> copies.
scatterplot shows the correlation between mean relative growth measured by qpcr and copy number estimates.
the capability of isolates with different copy numbers of encyp <dig> to grow in the presence a dmi fungicide  was measured.
to account for growth rate differences among isolates in absence of the fungicide, growth results in presence of the fungicide were calculated as a percentage of the fungal biomass accumulation in absence of the fungicide.
growth values are the mean of two separate assays, each run with three biological replicates.
to test the hypothesis that an increase in encyp <dig> copy number leads to stronger resistance to dmi fungicides, we tested the capability of isolates with different copy numbers of encyp <dig> to grow in the presence of rally® 40wsp, a dmi fungicide.
eight isolates with a range of cnvs in encyp <dig> were tested, including the five isolates we sequenced.
a qpcr analysis was performed to quantify the accumulation of e. necator biomass eight days after conidia inoculation.
as expected, under fungicide treatment we did not detect any growth of branching and e1- <dig>  which are the two isolates that carry the wild type and single copy encyp <dig>  while a positive correlation between fungal growth in presence of the dmi fungicide and the estimated number of encyp <dig> copies was observed in the isolates containing the y136f mutation .
while we know the number of encyp <dig> copies and the encyp <dig> allelic form in each isolate, we were unable to control for genetic differences in other genes in the genome that may mask or confound the role of encyp <dig>  for example it has been suggested that abc transporters contribute to dmi resistance  <cit> , and although we did not see sequence polymorphisms and cnvs that may suggest a contribution of abc transporters to the differential dmi susceptibility of the isolates we tested, we could not exclude the possibility that other loci in the genome are responsible for the differential growth in response to the fungicide treatment.
discussion
this study describes for the first time the genome of one of the most widespread and devastating diseases of grapevines.
with an estimated size of ~125 mb, the e. necator genome is among the largest sequenced ascomycete genomes.
the e. necator genome also possessed a characteristic reduction in protein-coding genes, a feature also seen in the genomes of other biotrophic fungi  <cit> .
the expansion of genome size in powdery mildews , including e. necator, is associated with the proliferation of repetitive dna, particularly tes, which account for about 63% of the e. necator genome.
tes were also detected in the e. necator transcriptome, indicating that they are transcriptionally active at least during host infection.
active tes are a major source of mutations in the genome.
transposition of tes not only contributes to the expansion of genome size  <cit> , but can also cause chromosome breaks and rearrangements  <cit> , gene deletions  <cit> , gene duplications  <cit> , illegitimate recombination  <cit> , and changes in gene expression  <cit> .
te activity has also been associated with the evolution of pathogen virulence factors  <cit> .
frequent structural variations in the genome also point to a highly unstable genomic architecture in e. necator, although a direct link between te proliferation and cnv cannot yet be established based on our results.
in this study, the analysis of read depth showed that up to 5% of the genome assemblies, corresponding to approximately  <dig>  mb, are cnv, which indicates that at least among the five isolates we sequenced cnv involves larger genomic regions than dna sequence polymorphisms .
studies with model organisms, including fungi, plants, and animals, have already shown that the extent of genetic differences between individual organisms due to alterations in the number of gene copies can often be greater than differences in the nucleotide sequences  <cit> .
cnvs can result from different types of structural variations, such as deletions, translocations, inversions, tandem duplications, and novel insertions.
because read depth analysis cannot identify with certainty the type of rearrangement that underlies the cnv, characterization of the type of medium- and large-scale structural genomic variations would require longer reads, larger inserts between paired-end reads, and longer scaffolds than those used in this study.
nonetheless, qpcr validation of cnvs  confirmed that read depth analysis can be used to reliably detect cnv regions in the genome.
cnvs can influence phenotypic variation and adaptation by modification of gene dosage, gene expression, or disruption of genes that span boundaries of structural rearrangements.
cnvs have been associated with phenotypic differences and complex diseases in humans  <cit> , adaptation to different soil types in arabidopsis lyrata
<cit> , higher aluminum tolerance in maize  <cit> , nematode resistance in soybean  <cit> , drug resistance in plasmodium falciparum
<cit> , and ddt resistance in drosophila melanogaster
<cit>
.
a role of cnv in contributing to effector protein evolution was described for the oomycete plant pathogen, phytophtora sojae
<cit> .
here we show that  the dmi fungicide target encyp <dig> is cnv in 95% of isolates collected from vineyards treated with fungicides,  increasing encyp <dig> copy number leads to higher expression of encyp <dig>  and  increasing encyp <dig> copy number correlates with increased ability to withstand fungicide treatment.
furthermore, sanger sequencing confirmed that the vast majority  of the isolates with multiple cyp <dig> copies carried the mutant y136f allele in every gene copy.
our results also show that isolates with identical or very similar ssr haplotypes can have contrasting encyp <dig> alleles and copy numbers, which suggests that structural rearrangement in the locus and subsequent selection under fungicide pressure happened frequently and on multiple occasions in the populations we screened.
this observation is compatible with findings of high cnv rates over family generational scales  <cit> .
while the y136f mutation reduces the affinity between cyp <dig> and the inhibitor  <cit> , sensitivity to dmis has also been shown to depend on the expression levels of cyp <dig>  in other plant pathogens the overexpression of cyp <dig> has resulted in decreased susceptibility to dmis .
segmental duplication of a 126-bp fraction of the cyp <dig> promoter led to increased expression of cyp <dig> in a fungicide resistant strain of penicillium digitatum
<cit> , while duplication of the entire chromosome bearing the cyp <dig> gene resulted in increased fungicide resistance in the yeast candida glabrata
<cit> .
although polymorphisms have been found in resistant cyp <dig> genes  <cit> , no clear association between a specific mutation and an increase in cyp <dig> expression has been reported in fungi.
based on the strong association between cnv, the y136f allele, and fungicide treatment, we hypothesize that in e. necator cnv can provide an additional layer of genetic diversity that contributes to pathogen fitness once a favorable allele is acquired.
the development of increasing quantitative fungicide resistance could happen in two steps.
first, occasional e. necator individuals with the y136f mutation in encyp <dig> survive dmi fungicide treatments and, as a consequence, y136f allelic frequency increases in vineyards treated with dmis.
second, structural rearrangements that increase the number of encyp <dig> copies occur in individuals carrying the y136f, which are then favored by further dmi treatments.
we do not rule out the possibility that structural rearrangements in the encyp <dig> locus may occur with the same frequency in the presence of the wild type allele, but based on the low frequency of cnv in the wild type allele we observed, the acquisition of multiple copies of the wild type alleles does not seem to provide evolutionary fitness in fungicide treated vineyards.
alternatively, the strong connection between the mutation and multiple copies in e. necator could also be a result of a fitness cost due to the y136f mutation.
the mutation occurs in a highly conserved substrate recognition site  <cit> , and perhaps the substitution leads to a less efficient sterol biosynthesis in e. necator and the increased copy number is needed to compensate the loss of affinity to the substrate.
the compensatory effect provided by the acquisition of multiple cnv copies may explain why out of the  <dig> isolates we studied none carried the y136f allele as a single copy gene.
however, we did not observe any obvious impact of the y136f mutation on fungal growth when growth rates of wild type and mutant strains in the absence of fungicide were compared .
taken together, our results show that cnv of the encyp <dig> gene contributes to e. necator fitness by providing increasing quantitative protection against dmi fungicide treatments in a gene-dosage dependent manner.
monitoring powdery mildew evolution for the development of fungicide resistance should include measuring the number of the encyp <dig> gene copies.
this knowledge will help in managing fungicide programs and maintaining effective control of powdery mildew while minimizing the development of populations increasingly resistant to dmi treatments.
CONCLUSIONS
as part of this study we generated valuable genomic information for the grape powdery mildew pathogen, including:  whole-shotgun sequence assemblies of five isolates of e. necator,  an estimate of the distribution of genetic diversity as both sequence polymorphisms and structural variants among e. necator isolates,  characterization of the expression of e. necator genes in infected tissues using rnaseq analysis,  gene models of  <dig>  protein-coding genes identified with an a hybrid approach that involved both ab initio and reference based predictions using genome-guided de novo assembled transcripts.
in addition to providing the first description of the large and highly repetitive genome, in this work we show that frequent structural variations occur between field isolates, including copy number variation in encyp <dig>  the target of sterol demethylase inhibitor fungicides.
the encyp <dig> gene in isolates collected from fungicide-treated vineyards contained both a mutation known to result in higher fungicide tolerance and an increased copy number that correlated with gene expression and fungal growth in the presence of fungicides.
our results also suggest that cnv can provide an additional layer of genetic diversity that contributes to pathogen fitness once a favorable allele is acquired.
the results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures.

 grapevine powdery mildew is one of the most widespread and devastating diseases of wine, table and raisin grapes, the vast majority of which are cultivars of vitis vinifera. this disease is caused by the fungus erysiphe necator schw., an obligate biotroph that can infect all green tissues of a grapevine. infected leaves exhibit reduced photosynthesis and often undergo premature senescence and abscission. early berry infection causes berries to crack, and the overall impact on the crop includes decreased yields, increased acidity, and decreased anthocyanin and sugar content of mature fruit. even low levels of powdery mildew infection on the berries can lead to ruined table grapes and wines with negative sensory attributes and decreased varietal character.figure powdery mildew disease symptoms and phylogenetic relation of e. necator with other grapevine fungal pathogens and other powdery mildew pathogens. e. necator infections are initiated when conidia come in contact with a susceptible host and germinate, forming hyphae with multilobed appressoria and penetration pegs. haustoria are formed within the epidermal cell membrane to maintain the parasitic relationship with the host. young colonies are macroscopically visible and appear white on the surface of leaves, fruit, and other green tissue. multiseptate conidophores form along the hyphae perpendicularly to the epidermis, and conidia begin to develop within a few days of the initial infection. phylogenetic relationship of e. necator with other powdery mildews and grape fungal pathogens. the neighbor-joining tree was constructed in mega using the complete rdna its. multiple publicly available its sequences per species were used independently to confirm clustering. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. only bootstrap values greater than are shown. the evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. the analysis involved nucleotide sequences. all positions containing gaps and missing data were eliminated. there were a total of positions in the final dataset. most cultivated varieties are susceptible to powdery mildew, and as a consequence growers are forced to apply fungicides to control the disease, often as frequently as every 7– days when disease pressure is high. it has been estimated that as much as 20% of the total costs associated with wine grape production in california goes to expenses related to powdery mildew control. elemental sulfur was the first effective fungicide recommended for vineyards in to control powdery mildew, and it continues to be widely used, mainly due to its efficacy and low cost. although its multi-site mode of action remains effective at controlling powdery mildew, the limitations to sulfur use include phytotoxicity at higher temperatures, the need for application as a protectant at frequent intervals, potential off-characters in wine, and the risk of unintended environmental consequences. since the 1960s, new classes of fungicides have been developed and introduced, many with single-site modes of action and beneficial properties such as systemic effectiveness and longer times between applications. one important class of single-site fungicides is the sterol demethylase inhibitors, which includes the azole fungicides. dmis inhibit fungal growth by targeting the cytochrome p lanosterol c-14α-demethylase. cyp is a key enzyme in eukaryotic sterol biosynthesis, catalyzing the removal of the 14-α carbon of lanosterol during biosynthesis of ergosterol, the major fungal sterol and an important membrane component. ergosterol depletion and accumulation of deleterious methylated sterols caused by cyp inhibition affect membrane integrity and functionality, reducing fungal growth. dmis are prone to resistance issues due to their single metabolic target. triadimefon was the first dmi fungicide to be approved for powdery mildew control in vineyards in and resistance was documented as soon as. resistance to other dmis has since been reported, including cross-resistance between different compounds. dmi resistance in fungi has been associated with several molecular mechanisms, such as cyp mutations and increased expression of cyp51. a single point mutation in cyp causing a tyrosine for phenylalanine substitution at position is associated with dmi resistance in e. necator, and similar mutations conferring dmi resistance have been identified in other fungi including blumeria graminis f. sp. hordei, candida albicans, and mycosphaerella graminicola. the amino acid is within the highly conserved cr domain involved in substrate recognition. structural modeling has demonstrated that dmis bind cyp at the sixth ligand of the heme group, and it has been suggested that the y136f mutation confers resistance by obstructing the heme-binding site. pathogen populations, particularly species with mixed sexual and asexual reproduction systems like e. necator, rapidly evolve to circumvent host resistance genes or to counteract chemical control methods. though new fungicides are being developed and breeding for genetic resistance is ongoing, little is known regarding how e. necator evolves to become resistant to fungicides or how it overcomes host immune responses. whole-genome sequencing, transcriptomics, and comparative genomic approaches have been successfully applied to other fungal plant pathogens. however, publicly available genomic information for e. necator is scarce and limited to ssr markers, likely reflecting the difficulty of working with an obligate parasite. genomic analyses of related powdery mildew species, including the ascomycetes that cause powdery mildew in barley and wheat have provided insights into their evolution and mechanisms of pathogenicity. in this study we applied a shotgun approach to sequence the genomes of five isolates of e. necator, and used rna-seq and comparative genomics to predict and annotate protein-coding genes. our results not only show that the e. necator genome is exceptionally large and repetitive compared to other fungal plant pathogens, but also that frequent structural variations occur between field isolates. structural variation resulted in a wide range of copy numbers in the dmi fungicide target gene cyp. a panel of additional e. necator isolates collected from both synthetic fungicide-treated and non-treated vineyard sites was screened for both copy number variation and presence/absence of the y136f mutation in the encyp gene. the vast majority of isolates with increased copy number were collected from fungicide-treated vineyards. increased encyp copy numbers were almost always detected with the encyp mutant allele, suggesting that an increase in copy number becomes advantageous only when the encyp gene is present in its fungicide-tolerant allelic form. we also show that encyp genomic copy number is correlated with both its mrna transcript level and with fungal growth in the presence of dmi fungicide. taken together, our results demonstrate that copy number variation can be adaptive in the development of resistance to dmi fungicides in e. necator.  genome sequencing and assembly of five e. necatorisolates between and e. necator isolates were collected from seven vineyard sites in california. five isolates with diverse genetic and geographical backgrounds were selected for genome sequencing based on microsatellite profiling results, collection site, and fungicide treatment history. the branching and e1- isolates were collected from vineyard sites without fungicide treatment, while the c-strain, lodi, and ranch isolates were all collected from sites regularly treated with synthetic fungicides to control powdery mildew. we used an illumina shotgun sequencing approach to sequence the haploid genomes of the five selected e. necator isolates. we generated both medium reads and short reads using the illumina miseq and hiseq sequencers, respectively. the longer reads were used to assemble the draft genome sequence for the c-strain isolate, while the shorter reads were used for resequencing and de novo assembly of the other isolates. after quality trimming and filtering, most sequencing reads were assembled into  ±  scaffolds with a total length of  ±   mb. most of the reads of each isolate aligned as pair-ends to the assembled contigs of the other isolates suggesting a similar genome completeness and representation across all five isolates. unambiguously mapped reads were used to assess single nucleotide polymorphism frequencies between the isolates following the gatk pipeline for snp calling. the gatk analysis identified an average snp frequency of  ±  snps/kb.table e. necator genome assembly statistics 1 1genome size estimated by k-mer frequency was 126 ± 18 mb. based on the distribution of dna k-mer counts, we estimated the genome size of e. necator at 126 ± 18 mb, which is similar to the 120 mb estimated genome size of bgh and significantly smaller than the 180 mb genome size estimated for bgt. the discrepancy between assembled scaffold size and estimated genome size suggests that a large proportion of the e. necator genome is highly repetitive and collapsed into common contigs during assembly. this hypothesis is also supported by the high sequencing coverage observed in the highly fragmented and gene-poor fraction of the assemblies. in contrast, the longest and most gene-rich contigs exhibit the expected sequencing coverage based on estimated genome size and sequencing depth. depth of coverage was used to estimate the size of the interspersed repeats. repeats, including transposable elements, microsatellites, and low-complexity regions, were estimated to represent  ± 3% of the e. necator genome, a proportion similar to the repeat content estimated for the bgh genome. tes were annotated using a combination of ab initio discovery and comparative analysis. tes accounted for  ±  % of the total repeats, with ltr retrotransposons and lines being the most abundant classes of tes in the genome. tes were abundant among the sequenced transcripts, suggesting that in e. necator tes are transcriptionally active. to assess the quality and completeness of the assembled gene space, we applied the cegma pipeline to map a set of low copy core eukaryotic orthologous groups which are conserved across higher eukaryotes. on average  ±  % of the kogs aligned as complete gene copies to the scaffolds, compared to  ±  % that aligned as fragmented partial gene copies. these results indicate that our shotgun sequencing approach generated a nearly complete assembly of the e. necator gene space, despite the obvious failure in resolving the structure of the repetitive fraction of the genome. for the analyses described below we used the genome assembly of the c-strain isolate as a reference, because it appeared to be the most complete based on assembly metrics and cegma evaluation. gene prediction and annotation to enhance gene discovery and gene structure prediction, we sequenced and assembled the e. necator transcripts expressed during the interaction with grape leaves at, and 6 days after conidia inoculation. assembled transcripts were then used as evidence in a gene prediction pipeline that combined evidence-based, homology-based, and ab initio methods. after removing genes associated with tes, we estimated that the e. necator genome contains protein-coding genes. a similar total number of protein-coding genes was identified in bgh and bgt. nearly 92% of the predicted genes have detectable similarity to proteins known from other ascomycetes. percent blastp matches and alignment coverage between the peptides of e. necator and other fungi, support the overall quality of the gene prediction pipeline results. prediction accuracy was also supported by the identification of higher frequencies in nucleotide variants in the intergenic space and intronic regions than in the exons. an average of introns per gene were predicted and, similarly to observations in other fungal genomes, intron lengths had a narrow distribution with mean size of  bp. genes on longer scaffolds displayed shorter intergenic spaces than those on smaller scaffolds, suggestive of an uneven distribution of genes within the genome. as reported in previous analyses of powdery mildews and other obligate biotrophs, the e. necator genome showed a loss of enzymes involved in secondary metabolism, nitrate and sulfate metabolism, further supporting the convergent adaptation of obligate biotrophy. we did not find any of the genes known to be required for repeat induced point mutation suggesting that the mechanisms that usually control tes in fungi are only partially functional in e. necator, as previously observed in other powdery mildew pathogens. secretome and candidate effector proteins microbial plant pathogens secrete effector proteins, some of which have been shown to subvert the plant innate immune response and enable infection. the predicted secretome of e. necator contained a total of genes. go term enrichment analysis of the secretome showed enrichment in hydrolase and peptidase molecular function terms, although many of these enzymes possess β- -glucanase or chitinase activity and are likely involved in remodeling the pathogen’s own cell wall during early infection instead of breaking down the host tissue as in necrotrophy. the e. necator secretome also included several classes of proteins that were previously identified as pathogen candidate effectors, such as eka-like proteins and ribonucleases-like proteins. these putative effector proteins were detected in transcriptome data from early infection time points suggesting that some might be involved in the very early stages of infection. following the same approach described in, we identified candidate secreted effector proteins, which displayed a significant enrichment in sequence motifs associated with secreted peptides of haustoria-forming pathogenic fungi. these candidate effectors did not present any signature of positive selection unlike those described in other plant pathogens. the absence of elevated levels of non-synonymous polymorphisms among the candidate effectors may hypothetically reflect the absence of antagonistic evolution in the host, since almost all cultivated grape varieties are fully susceptible to powdery mildew. structural variation between e. necatorisolates copy number variations are unbalanced changes in the structure of the genome and include deletions, insertions, and duplications of >1 kb in size. although short sequencing read length and limited insert size do not allow reliable characterization of the type of structural variation underlying cnv, variation in sequencing coverage in genome assemblies can be used as an indicator of cnv between an assembled genome and sequencing reads from another genome. to detect structural variants between the five sequenced isolates, we applied the cnv-seq pipeline to the mapped reads. cnv-seq calculates the read-depth signal across all genomic coordinates using a sliding window and was shown to provide more accurate relative copy number estimation than other cnv-detection tools. genome-wide cnv-seq results are summarized in table  while the sizes of the detected cnvs were similar across the isolates, the total number of cnv sites had a broad range from to, corresponding to % and % of the assemblies, respectively. the identified cnv loci not only spanned tes, but also protein-coding genes. a broad diversity of genes was observed within cnv loci with no detectable enrichment for any particular function. cnv-seq estimations of copy number polymorphisms were validated by quantitative pcr.figure structural variation between e. necator isolates estimated by sequencing depth analysis and qpcr. line plot showing examples of genomic loci without copy number variation, and with cnv across the five genomes. loci with copy number polymorphisms were detected by analyzing sequence read depth with cnv-seq. gray boxes depict protein-coding sequences. validation by qpcr of cnv-seq copy number calls for four sample cnv loci in the five sequenced isolates. scatterplots show the correlation between estimated copy numbers for both methods. a linear trend line is shown. copy numbers by qpcr were estimated based on 2−Δct values normalized to the single copy e. necator elongation factor gene enef. genome-wide estimates of copy number variation in the five sequenced e. necator isolates determined by cnv-seq analysis e1-1011 ranch91 c-strain1 lodi1 branching2 1values represent cnv-seq results when branching scaffolds were used as reference. 2values represent cnv-seq results when e- scaffolds were used as reference. copy number variation of the encyp51locus in the five sequenced isolates the encyp gene, which encodes the enzyme targeted by dmi fungicides, was among the protein-coding genes with a wide range of copy number variation across the five sequenced genomes. sequencing coverage, confirmed by qpcr, estimated the presence of and encyp copies in ranch c-strain, and lodi, respectively, and single copies in e1- and branching. remarkably, the three isolates collected from fungicide-treated vineyards showed multiple copies of encyp with all copies being the y136f mutated allele, while the two isolates from vineyards not treated with fungicides both had single copies of the encyp gene in its fungicide-susceptible allelic form. the y136f substitution was the only non-synonymous polymorphism detected between the isolates in the encyp coding region. all encyp alleles were confirmed by sanger sequencing, which also confirmed that all encyp duplicated copies were in the same allelic form.figure copy number variation of the encyp51 locus. log ratio plot of sequencing depth in the encyp locus of e1- c-strain, lodi, and ranch relative to the sequence depth in the encyp locus of branching. cnv was confirmed in all pair-wise comparisons using cnv-seq. depth of coverage of the encyp locus in the branching isolate is presented as it provided the longest assembled scaffold of the locus across all isolates. arrows and boxes depict protein coding sequences and transposable elements, respectively. duplication boundaries upstream and downstream of the encyp coding region were common in all isolates with multiple copies of encyp only in lodi did an additional duplication event occur involving a shorter region of  kb. cnv-seq estimates of encyp copy numbers were validated using qpcr. scatterplot in inset shows the correlation between cnv-seq and qpcr copy number estimates. in all isolates the duplication events did not involve the two flanking genes, en-g and en-g which were confirmed by qpcr to be single copy in all genomes. duplication boundaries, approximately and  kb upstream and downstream of the encyp coding region, respectively, were common in all isolates. only in lodi did an additional duplication event occur involving a shorter region of  kb. in all isolates, the duplication events did not involve the two flanking genes, en-g and en-g which were confirmed by qpcr to be single copy in all genomes. in all isolates the downstream duplication boundary coincided with a fragment of a gypsy element. although numerous other tes were found throughout the sequence flanking the duplicated region, we did not detect repeated homologous sequences that could provide the substrate for non-allelic homologous recombination and allow us to hypothesize the mechanism underlying the copy number polymorphisms in the locus. microsynteny of the encyp locus between e. necator, bgh, and bgt both confirms the accuracy of the sequence assembly and also shows the structural conservation in this region of the genome across species despite the frequent structural rearrangements observed in the encyp locus.figure microsynteny of the encyp51 locus between e. necator, blumeria graminis f.sp. hordei, and blumeria graminis f.sp. tritici. scaffold alignment based on translated nucleotides and similarity visualization was done using act-artemis comparison tool. red and blue lines indicate similar regions between scaffolds. blue lines indicate inversions. arrows correspond to protein-coding genes. cnvs can modify the expression of genes that localize within the cnv in a gene dose-dependent manner. to determine the impact of cnv on encyp expression, we measured mrna transcript expression levels at and 5 days post conidia inoculation using qrt-pcr. our results show that encyp was differentially expressed in the different isolates at both time points with a strong correlation between gene expression levels and encyp copy number.figure impact of cnv on cyp51 expression. bar graph presenting qrt-pcr data for the relative expression of encyp in sequenced isolates at and 5 days post inoculation. expression values are presented as fold-enef, calculated using the Δct method as described in. data were analyzed using a two-way anova. values are the mean ± standard error of three biological replicates. different letters represent significant results. scatterplot showing the correlation between relative expression of encyp by qrt-pcr and copy number predicted by sequencing coverage. a linear trend is shown. copy number variation of the encyp51locus occurs frequently in e. necatorand is correlated with fungal growth under fungicide treatment to determine the extent of encyp cnv in e. necator beyond the five sequenced isolates, we screened an additional isolates collected from multiple vineyard locations. all isolates tested for cnv were also genotyped to determine the allelic forms of encyp out of the total isolates, 48% carried the wild-type allele, while 52% carried the y136f mutation. the majority of the isolates with the wild type allele were collected from non-sprayed vineyards, while almost all of the isolates with the y136f mutation came from sprayed vineyards. quantitative pcr analysis showed the presence of multiple copies of encyp in isolates with a wide range of copy numbers, from as few as copies and up to copies. the two genes that flank encyp were mostly single copy confirming the conservation of the duplication boundaries. remarkably, 94% of the isolates with multiple copies of encyp also carried the mutant y136f allele, while 96% of the isolates with single copies of encyp were wild type, further supporting the hypothesis that the acquisition of multiple copies of the fungicide-tolerant allele contributes to the evolutionary fitness. hypergeometric tests supported the significant association between fungicide treatment and both presence of the y136f allele and multiple copies of encyp. the encyp cnv and its allelic form did not correlate with ssr-based clustering suggesting that the y136f mutation and the acquisition of multiple of copies of the encyp gene occurred multiple times in the e. necator populations screened.figure copy number variation of the cyp51 locus occurs frequently in e. necator and correlates with fungal growth under fungicide treatment. circular visualization of the allelic form and copy number of encyp in the california isolates, and the fungicide spray regime of each collection site. at the center of the circular plot is a hierarchical clustering dendrogram determined by ssr marker-based fingerprinting. bar plots showing the proportion of wild type and mutant encyp alleles collected from sites sprayed or not sprayed with fungicides. box plots showing the distribution of encyp copy number in isolates containing the mutant allele compared to those with the wild type allele, and box plots showing the distribution of copy numbers of the two genes that flank the conserved duplication boundaries observed in the individuals with multiple encyp copies. scatterplot shows the correlation between mean relative growth measured by qpcr and copy number estimates. the capability of isolates with different copy numbers of encyp to grow in the presence a dmi fungicide was measured. to account for growth rate differences among isolates in absence of the fungicide, growth results in presence of the fungicide were calculated as a percentage of the fungal biomass accumulation in absence of the fungicide. growth values are the mean of two separate assays, each run with three biological replicates. to test the hypothesis that an increase in encyp copy number leads to stronger resistance to dmi fungicides, we tested the capability of isolates with different copy numbers of encyp to grow in the presence of rally® 40wsp, a dmi fungicide. eight isolates with a range of cnvs in encyp were tested, including the five isolates we sequenced. a qpcr analysis was performed to quantify the accumulation of e. necator biomass eight days after conidia inoculation. as expected, under fungicide treatment we did not detect any growth of branching and e1- which are the two isolates that carry the wild type and single copy encyp while a positive correlation between fungal growth in presence of the dmi fungicide and the estimated number of encyp copies was observed in the isolates containing the y136f mutation. while we know the number of encyp copies and the encyp allelic form in each isolate, we were unable to control for genetic differences in other genes in the genome that may mask or confound the role of encyp for example it has been suggested that abc transporters contribute to dmi resistance, and although we did not see sequence polymorphisms and cnvs that may suggest a contribution of abc transporters to the differential dmi susceptibility of the isolates we tested, we could not exclude the possibility that other loci in the genome are responsible for the differential growth in response to the fungicide treatment. discussion this study describes for the first time the genome of one of the most widespread and devastating diseases of grapevines. with an estimated size of ~125 mb, the e. necator genome is among the largest sequenced ascomycete genomes. the e. necator genome also possessed a characteristic reduction in protein-coding genes, a feature also seen in the genomes of other biotrophic fungi. the expansion of genome size in powdery mildews, including e. necator, is associated with the proliferation of repetitive dna, particularly tes, which account for about 63% of the e. necator genome. tes were also detected in the e. necator transcriptome, indicating that they are transcriptionally active at least during host infection. active tes are a major source of mutations in the genome. transposition of tes not only contributes to the expansion of genome size, but can also cause chromosome breaks and rearrangements, gene deletions, gene duplications, illegitimate recombination, and changes in gene expression. te activity has also been associated with the evolution of pathogen virulence factors. frequent structural variations in the genome also point to a highly unstable genomic architecture in e. necator, although a direct link between te proliferation and cnv cannot yet be established based on our results. in this study, the analysis of read depth showed that up to 5% of the genome assemblies, corresponding to approximately  mb, are cnv, which indicates that at least among the five isolates we sequenced cnv involves larger genomic regions than dna sequence polymorphisms. studies with model organisms, including fungi, plants, and animals, have already shown that the extent of genetic differences between individual organisms due to alterations in the number of gene copies can often be greater than differences in the nucleotide sequences. cnvs can result from different types of structural variations, such as deletions, translocations, inversions, tandem duplications, and novel insertions. because read depth analysis cannot identify with certainty the type of rearrangement that underlies the cnv, characterization of the type of medium- and large-scale structural genomic variations would require longer reads, larger inserts between paired-end reads, and longer scaffolds than those used in this study. nonetheless, qpcr validation of cnvs confirmed that read depth analysis can be used to reliably detect cnv regions in the genome. cnvs can influence phenotypic variation and adaptation by modification of gene dosage, gene expression, or disruption of genes that span boundaries of structural rearrangements. cnvs have been associated with phenotypic differences and complex diseases in humans, adaptation to different soil types in arabidopsis lyrata, higher aluminum tolerance in maize, nematode resistance in soybean, drug resistance in plasmodium falciparum, and ddt resistance in drosophila melanogaster. a role of cnv in contributing to effector protein evolution was described for the oomycete plant pathogen, phytophtora sojae. here we show that the dmi fungicide target encyp is cnv in 95% of isolates collected from vineyards treated with fungicides, increasing encyp copy number leads to higher expression of encyp and increasing encyp copy number correlates with increased ability to withstand fungicide treatment. furthermore, sanger sequencing confirmed that the vast majority of the isolates with multiple cyp copies carried the mutant y136f allele in every gene copy. our results also show that isolates with identical or very similar ssr haplotypes can have contrasting encyp alleles and copy numbers, which suggests that structural rearrangement in the locus and subsequent selection under fungicide pressure happened frequently and on multiple occasions in the populations we screened. this observation is compatible with findings of high cnv rates over family generational scales. while the y136f mutation reduces the affinity between cyp and the inhibitor, sensitivity to dmis has also been shown to depend on the expression levels of cyp in other plant pathogens the overexpression of cyp has resulted in decreased susceptibility to dmis. segmental duplication of a 126-bp fraction of the cyp promoter led to increased expression of cyp in a fungicide resistant strain of penicillium digitatum, while duplication of the entire chromosome bearing the cyp gene resulted in increased fungicide resistance in the yeast candida glabrata. although polymorphisms have been found in resistant cyp genes, no clear association between a specific mutation and an increase in cyp expression has been reported in fungi. based on the strong association between cnv, the y136f allele, and fungicide treatment, we hypothesize that in e. necator cnv can provide an additional layer of genetic diversity that contributes to pathogen fitness once a favorable allele is acquired. the development of increasing quantitative fungicide resistance could happen in two steps. first, occasional e. necator individuals with the y136f mutation in encyp survive dmi fungicide treatments and, as a consequence, y136f allelic frequency increases in vineyards treated with dmis. second, structural rearrangements that increase the number of encyp copies occur in individuals carrying the y136f, which are then favored by further dmi treatments. we do not rule out the possibility that structural rearrangements in the encyp locus may occur with the same frequency in the presence of the wild type allele, but based on the low frequency of cnv in the wild type allele we observed, the acquisition of multiple copies of the wild type alleles does not seem to provide evolutionary fitness in fungicide treated vineyards. alternatively, the strong connection between the mutation and multiple copies in e. necator could also be a result of a fitness cost due to the y136f mutation. the mutation occurs in a highly conserved substrate recognition site, and perhaps the substitution leads to a less efficient sterol biosynthesis in e. necator and the increased copy number is needed to compensate the loss of affinity to the substrate. the compensatory effect provided by the acquisition of multiple cnv copies may explain why out of the isolates we studied none carried the y136f allele as a single copy gene. however, we did not observe any obvious impact of the y136f mutation on fungal growth when growth rates of wild type and mutant strains in the absence of fungicide were compared. taken together, our results show that cnv of the encyp gene contributes to e. necator fitness by providing increasing quantitative protection against dmi fungicide treatments in a gene-dosage dependent manner. monitoring powdery mildew evolution for the development of fungicide resistance should include measuring the number of the encyp gene copies. this knowledge will help in managing fungicide programs and maintaining effective control of powdery mildew while minimizing the development of populations increasingly resistant to dmi treatments.  as part of this study we generated valuable genomic information for the grape powdery mildew pathogen, including: whole-shotgun sequence assemblies of five isolates of e. necator, an estimate of the distribution of genetic diversity as both sequence polymorphisms and structural variants among e. necator isolates, characterization of the expression of e. necator genes in infected tissues using rnaseq analysis, gene models of protein-coding genes identified with an a hybrid approach that involved both ab initio and reference based predictions using genome-guided de novo assembled transcripts. in addition to providing the first description of the large and highly repetitive genome, in this work we show that frequent structural variations occur between field isolates, including copy number variation in encyp the target of sterol demethylase inhibitor fungicides. the encyp gene in isolates collected from fungicide-treated vineyards contained both a mutation known to result in higher fungicide tolerance and an increased copy number that correlated with gene expression and fungal growth in the presence of fungicides. our results also suggest that cnv can provide an additional layer of genetic diversity that contributes to pathogen fitness once a favorable allele is acquired. the results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures. 
the results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures. copy number variations are unbalanced changes in the structure of the genome that have been associated with complex traits. frequent structural variations were found between isolates and included copy number variation in encyp the target of the commonly used sterol demethylase inhibitor fungicides. a panel of additional e. necator isolates collected from diverse vineyard sites was screened for copy number variation in the encyp gene and for presence/absence of a point mutation known to result in higher fungicide tolerance. we show that an increase in encyp copy number is significantly more likely to be detected in isolates collected from fungicide-treated vineyards. increased encyp copy numbers were detected with the y136f allele, suggesting that an increase in copy number becomes advantageous only after the fungicide-tolerant allele is acquired. we also show that encyp copy number influences expression in a gene-dose dependent manner and correlates with fungal growth in the presence of a dmi fungicide. fungal genomicscopy number variationgenetic adaptationfungicide resistancecytochrome p450cyp51issue-copyright-statement© the author 2014 powdery mildew, caused by the obligate biotrophic fungus erysiphe necator, is an economically important disease of grapevines worldwide. our results show that the e. necator genome is exceptionally large and repetitive and suggest that transposable elements are responsible for genome expansion. taken together our results show that cnv can be adaptive in the development of resistance to fungicides by providing increasing quantitative protection in a gene-dosage dependent manner. a shotgun approach was applied to sequence and assemble the genome of five e. necator isolates, and rna-seq and comparative genomics were used to predict and annotate protein-coding genes. in addition to providing the first description of the large and highly repetitive genome of e. necator, this study describes the impact of genomic structural variation on fungicide resistance in erysiphe necator.
 powdery mildew, caused by the obligate biotrophic fungus erysiphe necator, is an economically important disease of grapevines worldwide. large quantities of fungicides are used for its control, accelerating the incidence of fungicide-resistance. copy number variations are unbalanced changes in the structure of the genome that have been associated with complex traits. in addition to providing the first description of the large and highly repetitive genome of e. necator, this study describes the impact of genomic structural variation on fungicide resistance in erysiphe necator.  a shotgun approach was applied to sequence and assemble the genome of five e. necator isolates, and rna-seq and comparative genomics were used to predict and annotate protein-coding genes. our results show that the e. necator genome is exceptionally large and repetitive and suggest that transposable elements are responsible for genome expansion. frequent structural variations were found between isolates and included copy number variation in encyp the target of the commonly used sterol demethylase inhibitor fungicides. a panel of additional e. necator isolates collected from diverse vineyard sites was screened for copy number variation in the encyp gene and for presence/absence of a point mutation known to result in higher fungicide tolerance. we show that an increase in encyp copy number is significantly more likely to be detected in isolates collected from fungicide-treated vineyards. increased encyp copy numbers were detected with the y136f allele, suggesting that an increase in copy number becomes advantageous only after the fungicide-tolerant allele is acquired. we also show that encyp copy number influences expression in a gene-dose dependent manner and correlates with fungal growth in the presence of a dmi fungicide.  taken together our results show that cnv can be adaptive in the development of resistance to fungicides by providing increasing quantitative protection in a gene-dosage dependent manner. the results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords fungal genomicscopy number variationgenetic adaptationfungicide resistancecytochrome p450cyp51issue-copyright-statement© the author 2014 