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The genome and transcriptome of the pine saprophyte Ophiostoma piceae, and a comparison with the bark… Haridas, Sajeet; Wang, Ye; Lim, Lynette; Alamouti, Sepideh M; Jackman, Shaun; Docking, Rod; Robertson, Gordon; Birol, Inanc; Bohlmann, Jörg; Breuil, Colette Jun 2, 2013

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RESEARCH ARTICLE Open AccessThe genome and transcriptome of the pinesaprophyte Ophiostoma piceae, and a comparisonwith the bark beetle-associated pine pathogenGrosmannia clavigeraSajeet Haridas1, Ye Wang1, Lynette Lim1, Sepideh Massoumi Alamouti1, Shaun Jackman2, Rod Docking2,Gordon Robertson2, Inanc Birol2, Jörg Bohlmann3 and Colette Breuil1*AbstractBackground: Ophiostoma piceae is a wood-staining fungus that grows in the sapwood of conifer logs and lumber.We sequenced its genome and analyzed its transcriptomes under a range of growth conditions. A comparison withthe genome and transcriptomes of the mountain pine beetle-associated pathogen Grosmannia clavigera highlightsdifferences between a pathogen that colonizes and kills living pine trees and a saprophyte that colonizes woodand the inner bark of dead trees.Results: We assembled a 33 Mbp genome in 45 scaffolds, and predicted approximately 8,884 genes. The genomesize and gene content were similar to those of other ascomycetes. Despite having similar ecological niches, O. piceaeand G. clavigera showed no large-scale synteny. We identified O. piceae genes involved in the biosynthesis ofmelanin, which causes wood discoloration and reduces the commercial value of wood products. We also identifiedgenes and pathways involved in growth on simple carbon sources and in sapwood, O. piceae’s natural substrate. Likethe pathogen, the saprophyte is able to tolerate terpenes, which are a major class of pine tree defense compounds;unlike the pathogen, it cannot utilize monoterpenes as a carbon source.Conclusions: This work makes available the second annotated genome of a softwood ophiostomatoid fungus,and suggests that O. piceae’s tolerance to terpenes may be due in part to these chemicals being removed fromthe cells by an ABC transporter that is highly induced by terpenes. The data generated will provide the researchcommunity with resources for work on host-vector-fungus interactions for wood-inhabiting, beetle-associatedsaprophytes and pathogens.Keywords: Ophiostoma piceae, Genome, Transcriptome, Wood-staining fungus, SaprobeBackgroundPine trees and processed wood (lumber and logs) arecolonized by ascomycete ophiostomatoid fungi that in-clude pathogens and saprobes [1,2]. As they grow in thephloem and sapwood of the trees or in the sapwood oflogs or lumber, most of these fungi produce a dark mel-anin pigment that causes a wood discoloration known asblue stain or sap stain. Ophiostomatoid sap stain fungiwere first described more than 100 years ago [3,4] andhave been recognized as an economic problem for forestindustries worldwide. Currently, the group contains atleast five genera that include Ophiostoma and Grosmannia(Figure 1). Ophiostomatoid fungi produce sticky sexualand asexual spores that are readily vectored by specific orgeneralist bark beetles that colonize trees or processedwood [5]. Before 1995, in Canada, Ophiostoma specieswere reported as the major cause of pine discolor-ation [1,6]. However, since 1995, in western Canada, themountain pine beetle (MPB; Dendroctonus ponderosae)has expanded its range, and its fungal associates from the* Correspondence: colette.breuil@ubc.ca1Department of Wood Science, University of British Columbia, Vancouver, BCV6T1Z4, CanadaFull list of author information is available at the end of the article© 2013 Haridas et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Haridas et al. BMC Genomics 2013, 14:373http://www.biomedcentral.com/1471-2164/14/373genera Grosmannia (mainly G. clavigera and Lepto-graphium longiclavatum) and Ophiostoma (O. montium)have become the main cause of pine wood discoloration.As well, this beetle-fungal complex has killed large areas ofpine trees in western North American conifer forests [7,8].Wood discoloration is caused by melanin, a dark pigmentthat is synthesized inside the fungal cell and is released assmall black globules into the cell wall and outside of thecell (Figure 2).O. piceae is a saprobe that is dispersed by generalistbark beetles [10]. This fungal species has been reportedin North America, Europe and Asia [5,6,11]. In contrastto Grosmannia species, which penetrate deeply into thesapwood of pine logs and reach the heartwood boundary,O. piceae is a more superficial sap stain fungus that be-comes established in the outer two to three centimetresof sapwood [1,12,13]. Species in the O. piceae complexhave retained the attention of wood industry researchersbecause they cause stain in processed wood and were themost commonly isolated species of sap stain fungi inCanadian saw mills [6]. In contrast to G. clavigera, whichis specific to pine, O. piceae is able to grow not only onpine, but also on wood of other conifers in Canada, in-cluding black and white spruce, balsam fir and hemlock[6]. Because members of the O. piceae complex growpoorly on freshly cut pine logs and prefer the dryer en-vironment of lumber or dead trees [1,13], their stainingeffects can be minimized by keeping logs frozen orsaturated with water, or by prompt log processing.Green lumber can be protected by kiln drying to below20% moisture content, or by chemical and biologicaltreatments [14-16].Both the pathogen G. clavigera and the saprophyteO. piceae acquire nutrients from pine species by se-creting extracellular enzymes to break down largemolecules like polysaccharides (e.g. hemicellulose andstarch), proteins and lipids. They do not degradewood and do not affect wood structural properties[1,16,17], so likely have limited or incomplete cellulo-lytic and/or lignolytic activities. However, in order tocolonize conifers (e.g. lodgepole pine), fungi and theirbark beetle vectors have to cope with the host’spreformed and induced defense chemicals, which in-clude terpenoid and phenolic compounds [18-20].Pathogens like G. clavigera have evolved mechanismsto overcome these defences [8,21]. However, the role ofsuch host defence compounds in cut logs and lumber,where saprophytes like O. piceae are generally found,Figure 1 ITSs-rDNA maximum parsimony tree places O. piceae within the Pezizomycotina. O. piceae was originally described by Münch in1907 [4]; this species and closely related species (e.g. O. setosum, O. canum, O. novo-ulmi, O. ulmi and O. floccosum O. quercus) are reported as theO. piceae complex. The numbers on the branches of the tree are bootstrap values based on 1000 replicates and the heuristic option [9].Haridas et al. BMC Genomics 2013, 14:373 Page 2 of 15http://www.biomedcentral.com/1471-2164/14/373has not been reported. It is important to note that thecomposition of defence chemicals, especially terpe-noids, varies with different pine genotypes across thelandscape and can be affected by the environment [22].Further, wood processing and drying affect the concen-trations of chemicals in wood products, and so logs andlumber typically contain lower concentrations of thesubset of terpenoids that are volatile [23,24].In previous work we reported the annotated genomeof the pine pathogen G. clavigera, and showed that thisfungus is able to tolerate and utilize pine defence com-pounds, specifically terpenoids found in pine oleoresin[8,21]. Here, we report the annotated genome of thesaprophyte O. piceae, and its gene expression responsesin a range of growth conditions that include wood nutri-ents and host defence chemicals. We compare these re-sults to corresponding results from G. clavigera, andhighlight differences between a pathogen that colonizesand kills living pine trees and a saprophyte that lives ondead trees or processed wood. Neither fungus has alignocellulolytic enzyme system that would allow it todegrade wood. Both fungi overcome terpenoid defencechemicals in their pine niches; however, only the patho-gen, but not the saprophyte, can metabolize terpenoidsas a carbon source. Both fungi have a similar ABC effluxtransporter that is highly induced with monoterpenetreatments. The functionality of the O. piceae trans-porter remains to be fully characterized.ResultsGenome assemblyWe used ABySS [25] to assemble 100-nt reads from200- and 700-nt insert Illumina HiSeq 2000 libraries,and ~300-nt reads from an 8-kb insert 454 Titanium(Table 1, Methods). The Illumina libraries provided >100×coverage for assembly and initial scaffolding, while the454 reads supported long-range scaffolding. Refining thisassembly with two iterations of Anchor resulted in agenome that consisted of 244 scaffolds, each of whichwas at least 1,000 nt in length. The assembly contained335 false gaps represented by a single lowercase ‘n’ (seeMethods). Of these, 219 were resolved by mapping Trinity-assembled RNA-seq transcripts to the genome using exon-erate est2genome [26]. The remaining 116 gaps wereresolved by using exonerate to find small overlaps (<5 bp)at the ends of contigs that were joined by an ‘n’.We removed from the final assembly 187 scaffolds andcontigs smaller than 10,000 bp (including gaps) that rep-resented 1% of the assembly because they contained nogenes or t-RNAs. The corrected 32.8-Mbp genome as-sembly consisted of 45 scaffolds. One percent of thegenome consisted of 342 gaps (N’s). Half of the genomewas in nine scaffolds that had an N50 of approximately1.45 Mbp, while 90% was represented in 27 scaffolds thathad an N90 of approximately 0.38 Mbp. Using CEGMA[27], we identified complete copies of 233 of 248 conservedeukaryotic genes and partial copies of an additional five,which suggests that our assembly represents 94% - 96% ofthe O. piceae gene space [28]. The genome characteristicsof O. piceae, and three other ascomycetes [8,29,30] also inFigure 2 Micrographs of O. piceae mycelium, sexual and asexual structures. It grows as filamentous hyphae on solid media and as yeastlike form in liquid media. O. piceae mycelium, perithecium and synemata are highly melanized while both asexual and sexual spores are notpigmented. The pigment accumulates as small black granules in the cell wall and the external sheath surrounding the hyphae (a: electronmicrograph); Melanin is also present in fruiting body (perithecium) (b: light micrograph), as well as in the stem of the synemata (c: lightmicrograph; d: confocal micrograph), an aggregation of branched hyphae that produce abundant asexual spores. The fruiting bodies are easilyobtained in artificial media or wood but require the pairing of two individuals with different mating types.Table 1 Sequencing strategy for O. piceae genomeSequencingtechnologyRead length(nt)Insert length(nt)Read pairs(Millions)Illumina Hiseq 100 200 87.8Illumina Hiseq 100 700 32.2454 Titanium 318 (median) 8000 0.3Haridas et al. BMC Genomics 2013, 14:373 Page 3 of 15http://www.biomedcentral.com/1471-2164/14/373the class of the Sordariomycetes and found on dead treeand wood products are summarized in Table 2.Genome features and annotationWe used the Maker annotation pipeline [31] to predictgenes. Within the annotated genome of O. piceae, weidentified genes and gene families for secondary me-tabolite processing, cytochrome P450 as well as ABCtransporters. We also identified homologous O. piceae andG. clavigera proteins based on reciprocal best BLAST hits.We further characterized the MAT idiomorph that is re-sponsible for the mating type of the sequenced strain.Maker predicted 8,884 proteins within our acceptancecriteria (see Methods), of which 8,723 were at least 100amino acids long. Almost 65% (5,786) of the predictedproteins encoded by the gene models had a known Pfamdomain. Some of the major gene families in O. piceaeare shown in Table 3. About a third of the predictedgenes (3,026) had only one exon and only 1,283 tran-scripts were encoded by four or more exons. In this com-pact genome, genes, not including their upstream anddownstream untranslated regions (UTRs) represent 45%of the assembly. Almost a quarter (1,984) of the pre-dicted gene coding sequences (CDS) was within 500 bpof their respective neighbouring CDS, and almost half(4349) were within 1,000 bp of its neighbour. Our ana-lysis predicted that 778 CDSs encode secreted proteins.Although O. piceae and G. clavigera share hosts,cause sap-stain in pine, and are in sister clades in theOphiostomatales [32] (Figure 1), their genomes showedno large-scale synteny (Additional file 1). This isconsistent with synteny being lost with evolutionarytime between members of the class Dothideomycetes[33]. Dothideomycetes is a sister clade of the classSordariomycetes, which include O. piceae and G. clavigera,and we anticipate that similar synteny losses have occurredwithin this class. A BLAST comparison of the two pre-dicted proteomes showed that 5,450 proteins were recipro-cal best hits. These included most of the major metabolicfunctions. The O. piceae proteins with no significanthomolog in the G. clavigera genome were overrepresentedby protein kinases (GO:0004672), sequence-specific DNAbinding RNA polymerase II transcription factors (GO:0000981) and zinc ion binding proteins (GO:0008270)(Additional file 2). In addition, proteins involved in trans-membrane transport (GO:0055085) were also significantlyoverrepresented in this group of 3,469 proteins (Additionalfile 2). Over 40% (1,397) of the O. piceae proteinswith no evident homologs in G. clavigera were pro-teins of unknown function (predicted or hypotheticalproteins). None of the six carboxylic ester hydrolases(GO:0052689) in the O. piceae genome had a homologin the G. clavigera genome.We searched for genes that may be involved in produ-cing secondary metabolites (SMs). Such genes are typic-ally organized as contiguous genomic clusters and can beidentified by tools like SMURF [34], which uses hiddenMarkov models that consider genomic context and do-main content. The first step in fungal SM biosynthesis isusually catalyzed by ‘backbone’ genes like nonribosomalpeptide synthases (NRPSs), polyketide synthases (PKSs),hybrid NRPS-PKS enzymes, prenyltransferases, and ter-pene cyclases [34]. SMURF, which does not identifyclusters containing terpene cyclases, identified thirteenbackbone genes of which eleven are in SM clusters inO. piceae (Additional file 3), and nineteen genes infourteen clusters in G. clavigera [35]. All the O. piceaeSM genes have homologs in G. clavigera.Melanin is a secondary metabolite that is produced byO. piceae and related species, but, as in O. piceae, thegenes responsible for its production do not always occurin a cluster. Melanin is synthesized through the 1,8–dihydroxynaphthalene (DHN) pathway [36]. In O. piceaeTable 2 Characteristics of the O. piceae (Op) genomeassembly and annotation and a comparison with otherrelated genomesOp Gca Nc (10) b TrcGenome size (Mbp) 32.8 30 41 33.5Number of scaffolds 45 289 7 d 87N50 (Mbp) 1.45 2 1.56 1.12Number of ungapped contigs 388 478 956 231Genome GC content (%) 52.8 53.4 48.25 52.7Non-coding genome (%) 54 54.28 56Number of genes 8,884 8,312 9,733 9,129Median CDS length (bp) 1,401 1,350 1,673 1,299Exon GC content (%) 59.7 60.4 57.8a. G. clavigera (Gc); b. Neurospora crassa Sequencing Project, Broad Institute ofHarvard and MIT (NC10). c. Trichoderma reesei (Tr). d. Chromosome numbers.Table 3 Major gene families in O. piceae (Op) and in threeother ascomycetesGene family Op Gc* Nc* Tr*MFS transporters 289 227 161 236ABC transporters 34 40 36 48ATPases 308 349 356 352NAD binding proteins 258 254 211 301FAD binding proteins 130 146 122 144Cytochrome P450s 45 54 43 73Methyltransferases 112 159 126 125Transcription factors 115 133 106 218Glycoside hydrolases 140 126 168 170Glycosyl transferases 63 64 76 79*G. clavigera (Gc); Neurospora crassa (Nc) and Trichoderma reesei (Tr).Haridas et al. BMC Genomics 2013, 14:373 Page 4 of 15http://www.biomedcentral.com/1471-2164/14/373we identified a number of genes that were similar to genesthat have major roles in the DHN pathway in Ophiostoma,Grosmannia and Ceratocystis species [12,37,38]. Thesegenes included a PKS (OPP_00823), two reductases(OPP_02710, OPP_00820) and a scytalone dehydratase(OPP_07153). PKS catalyze both the elongation of fiveketide subunits and the cyclization of these units toform the base ring of naphthalene. The first reductase(OPP_02710) converts 1,3,6,8-hydroxynaphthalene toscytalone, while the second (OPP_00820) transformsscytalone to vermelone.O. piceae is a heterothallic species, so requires two indi-viduals with different mating types for sexual reproductionand production of fertile fruiting bodies. Our genomeannotation identified O. piceae’s MAT1-2 idiomorph(OPP_06680). A truncated MAT1-1 gene was next to theMAT1-2 gene, as in Grosmannia and related species[8,39,40]. We have produced perithecia for O. piceae bymating UAMH-11346 with UAMH- 11672 (Figure 2), andwe have successfully amplified and sequenced the fulllength of the MAT1-1 loci in this latter strain; the alphabox (~978 bp) was missing in the sequenced strain.Gene expression patternsTo identify genes that may be critical for the saprophyteO. piceae to grow in the presence of the nutrients anddefence chemicals that are characteristic of its naturalpine sapwood substrate, we profiled gene expression forthe fungus growing on solid agar media supplementedwith simple carbon sources (i.e. sugars and lipids), withpine sawdust, or with pine terpenes (see Methods). Map-ping the RNA-seq reads to the predicted gene modelsidentified 7,157 genes that had an abundance of at least10 FPKM (fragments per kilobase of exon per millionfragments mapped) in any of the conditions tested.To select genes that were highly differentially regu-lated under different growth conditions, we required agene to have an FPKM abundance that was at least tentimes higher in a specific condition, or a related set ofconditions, than in all other growth conditions. This ap-proach identified 677 genes whose transcripts were dif-ferentially abundant in at least one growth condition(Additional file 4), and 173 genes whose transcripts weredifferentially abundant in only one condition (Figure 3).By manually comparing the set of 173 genes to func-tional information in the Gene Ontology (http://www.geneontology.org/) and to reference metabolic pathwaysKEGG (http://www.genome.jp), we identified pathwaysthat were likely involved in the response of O. piceae tothe growth conditions tested in our experiments. Weadded support for these pathways by manually identify-ing genes from the 677-gene set (Additional file 4)whose transcripts, while up-regulated, did not pass thestringent 10-fold filter used to identify the set of 173 genes.We also assessed alternative transcript splicing acrossthe range of growth conditions used for this study(Additional file 5). Splicing appeared not to be an im-portant factor under these conditions.Growth on mannose, fatty acid (oleic acid) and triglyceridesMannose is a simple monomeric epimer of glucose andcan be readily utilized as a carbon source by O. piceae.We found five genes whose expression was at least tentimes higher with mannose than in any other conditionstested (Additional file 4 shown light blue). These includedtwo transporters, one oxidoreductase and two hypotheticalproteins. Our data suggests that mannose uptake involvestwo transporters (OPP_03031, OPP_05665), and a simpleisomerisation/ epimerization reaction by an oxidoreductase(OPP_00733) converts it into glucose. The function of tworemaining up-regulated genes (OPP_02416, OPP_07274) isunknown.We grew O. piceae on triglycerides and fatty acids,which are important lipid compounds in lodgepole pinesapwood, and are a major source of carbon for O. piceae[41]. Because most sources of triglycerides contain asmall proportion of fatty acids, it was not surprisingthat most of the 129 genes whose transcripts were dif-ferentially abundant between these conditions werehighly up-regulated in both of the conditions. Of the 25up-regulated genes that were significantly induced onlyin these two conditions (shown in gold in Additionalfile 4), 18 were predicted to produce secreted proteins.Unexpectedly, the differentially up-regulated genes in-cluded no fungal lipases, which are necessary for thehydrolysis of triglycerides (Additional file 4). Twenty-three of the 25 up-regulated genes were predicted to beinvolved in the breakdown of carbohydrates and sugars;these included eight genes coding for secreted proteinsin the glycoside hydrolase family and four genes for se-creted proteins involved in carbohydrate and starch bind-ing. We identified a transcription factor (OPP_02429)that showed significant up-regulation in the presence oftriglycerides and oleic acid.One of the genes differentially expressed between oliveoil and oleic acid was a cytochrome P450 (OPP_02426)with a significantly higher expression with triglyceride thanwith fatty acid. Like its G. clavigera homolog (CMQ_5365;CYP630B18) and homologs in several other species includ-ing Fusarium graminearum, Aspergillus niger, A. fumigatesand others, this gene is in close proximity to genes encod-ing a myo-inositol transporter, ARCA-like protein and acytochrome P450 reductase [35].Growth on pine sapwood, a natural substrate for O. piceaeO. piceae has slower growth rates than G. clavigera on richmedia and on wood; for example, here, on MEA, O. piceaegrows 5.8 mm/day, while in Wang et al. [21], G. clavigeraHaridas et al. BMC Genomics 2013, 14:373 Page 5 of 15http://www.biomedcentral.com/1471-2164/14/373grew 14 mm/day. Of the treatments used in this study,sawdust obtained by grinding pine sapwood was the clos-est to the natural substrate. It contains a variety of car-bon sources including mannose, triglycerides and fattyacids. In this growth condition, 366 genes were up-regulated, 91 of which were up-regulated only in thepresence of sawdust (Additional file 4). The subset of 91genes was overrepresented in GO terms for transport(GO:0005215, GO:0006810; p < 0.0001) (Figure 4), whichmay reflect the complexity of the nutrient sources usedby O. piceae. The up-regulated transporters included sev-eral allantoate, urea, hexose, iron and sugar transporters,and other major facilitator superfamily (MFS) transporters.As well, oxidoreductase genes that code for putative pro-teins involved in the modification of aromatic compounds,including phenolics, were highly up-regulated (e.g. P450s,dehydrogenases).Among the 91 genes that were up-regulated on saw-dust, 32 were found in 8 genomic clusters, each of whichcontained four to seven genes that may be co-regulated(Table 4). Three of the clusters contain a fungal-specifictranscription factor, Zn2cys, which may be involved inprimary and secondary metabolism and drug resistance[42]. Four of the clusters contain at least one of theFigure 3 Expression pattern of 173 selected genes of O. piceae and a dendrogram based on Jensen-Shannon distances between theconditions. The expression of each gene (row) across the various growth conditions (column) is presented as log transformed values of fragmentsper kilobase of exon per million fragments mapped (FPKM). Red shows low expression and green shows high expression. Sawdust (SD); Triglyceride(TG/Olive oil, OO); Oleic acid (OA); CM at 14 hr (C14); CM at 40 hr (C40); Mannose (MA); CM + Terpene at 14 hr (T14); CM + Terpene at 40 hr (T40).Haridas et al. BMC Genomics 2013, 14:373 Page 6 of 15http://www.biomedcentral.com/1471-2164/14/373following: a putative secreted salicylate dehydroxylase, anNAD-dependant epimerase, an alpha-mannosyltransferanseand an FAD–binding protein. An additional 22 genes thatwere up-regulated with sawdust were also up-regulatedwith triglycerides and oleic acid (dark blue in Additionalfile 4). The overall set of 113 genes (i.e. the 91 and the 22)was overrepresented in GO terms for secreted proteins(GO:0005576; p < 0.001) and carbohydrate metabolism(GO:0005975, GO:0030246; p < 0.001).One of the above eight up-regulated genomic clusterscontained seven genes that were involved in metaboliz-ing quinic acid (OPP_8732 to OPP_8738). These includea quinate permease, two regulatory genes (an activatorand a repressor), and the four genes of the quinate/shikamate catabolic pathway [43,44]. The latter fourcatabolic genes (OPP_08735 to OPP_08738) suggest thatO. piceae uses quinic acid in wood as a carbon source.While this gene cluster has been reported in many fungi,we were unable to find the cluster in G. clavigera.To confirm that O. piceae can use quinic acid, whileG. clavigera cannot, we showed that the former, butnot the latter, grows on YNB media with quinic acidas the sole carbon source (Additional file 6). Finally,we identified a secreted lipase (OPP_00605) with pre-dicted triglyceride degradation activity, whose transcriptabundance was at least 50-fold higher in the myceliumgrown in pine sapwood than in the control mannose.Tolerance of pine tree defence chemicalsO. piceae did not grow when a mixture of monoterpenes(MT) (i.e. (+)-limonene, (+)-3-carene, racemic a-pineneand (−)-ß-pinene at a ratio of 5:3:1:1) was the only car-bon source in YNB. However, after a month of incuba-tion in the presence of MT, the inocula resumed normalgrowth when they were transferred from YNB +MT toMEA. This suggests that O. piceae is able to survive inthe presence of very high levels of monoterpenes. Whenthe fungus was inoculated on MEA and treated with dif-ferent amounts of MT, the growth rate was only signifi-cantly affected when at least 100 μl /plate (~ 0.7 g/l) ofMT was added (Figure 5). For all MT treatments, themycelia were more aerial and fluffy, while the asexualreproduction structures (i.e. formation of synemata)were highly inhibited (Additional file 7).In order to identify genes involved in terpene toler-ance, we grew O. piceae on CM and treated it with amixture of terpenes as previously described in our stud-ies with G. clavigera [8,21]. While the experiments forthe two species were done at different times, we usedthe same conditions for both growth experiments andthe same protocols for RNA extractions. We comparedgene expression profiles of O. piceae after 14 h and 40 htreatments to profiles for untreated CM plates at thesame time points. At 14 h, 295 genes were differentiallyabundant, 261 of which were down-regulated. Whilecarbohydrate metabolism (GO:0005975) was associatedwith the down-regulated genes (p < 0.001), we were un-able to identify any GO terms that were associated withthe 34 up-regulated genes. After 40 h in the presenceof terpenes, 264 genes were differentially abundant,126 of which were up-regulated. While carbohydrate me-tabolism was still associated with down-regulated genes(p < 0.001), several transporters (OPP_06758, OPP_05515,OPP_03974, OPP_02103) were significantly up-regulated.In G. clavigera, which is able to utilize terpenes as a carbonsource, more than 250 genes were up-regulated by at least2-fold at 12 h and 36 h in the presence of terpenes[8]. Of the 34 O. piceae genes that were up-regulatedFigure 4 Functional classification of up-regulated genes of O. piceae grown on sawdust using Blast2go. Potential biological roles of transcribedproteins during growth experiments on different media were inferred by first identifying genes that were exclusively up-regulated (e.g. in sawdust), thenassociating the encoded protein sequences with biological processes using Blast2go.Haridas et al. BMC Genomics 2013, 14:373 Page 7 of 15http://www.biomedcentral.com/1471-2164/14/373Table 4 Gene clusters up-regulated in sawdustGene IDs Putative function Secreted Log2(FC)*Cluster 1OPP_08738 Inositol monophosphatase No 3.98OPP_08737 Catabolic 3-dehydroquinase No 5.78OPP_08736 3-dehydroshikimate dehydratase No 7.20OPP_08735 Quinate dehydrogenase No 5.65Cluster 2OPP_06948 Allantoate permease No 8.05OPP_06946 Sarcosine oxidase No 5.97OPP_06944 Fungal-specific transcription factor domain protein No 4.50OPP_06943 Oxoglutarate 3-dioxygenase No 8.75Cluster 3OPP_07708 Sugar transporter No 6.06OPP_07707 Salicylate hydroxylase (salicylate 1-monooxygenase) Yes 7.75OPP_07706 NAD dependent epimerase Yes 3.62OPP_07705 Arylacetamide deacetylase No 4.69Cluster 4OPP_08830 Amidohydrolase family protein No 8.98OPP_08829 Aldehyde dehydrogenase No 7.04OPP_08827 FAD binding domain protein Yes 6.25OPP_08826 Retinol dehydrogenase 13 No 7.78OPP_08825 Cytochrome p450 No 9.25OPP_08824 General alpha-glucoside permease No 7.98Cluster 5OPP_07998 Xaa-pro dipeptidase No 3.34OPP_07997 Major facilitator superfamily transporter No 4.21OPP_07996 Hexose transporter No 5.56OPP_07995 Thymine dioxygenase No 4.95Cluster 6OPP_01495 N-carbamoyl-l-amino acid hydrolase No 9.85OPP_01494 Gal4-like transcription factor No 6.74OPP_01493 Class ii aldolase adducin domain-containing protein No 9.97OPP_01491 Isoflavone reductase family protein Yes 6.49Cluster 7OPP_05544 Hypothetical protein No 10.82OPP_05543 Alpha-mannosyltransferase Yes 8.083OPP_05542 Ethanolamine utilization protein No 7.16OPP_05541 C6 zinc finger domain containing protein No 4.99OPP_05540 Alpha-mannosyltransferase No 8.78Cluster 8OPP_02428 Myo-inositol transporter No 7.42OPP_02427 Arca-like protein No 3.81OPP_02426 Benzoate 4-monooxygenase cytochrome p450 No 4.45OPP_02425 NADPH-cytochrome p450 reductase No 5.12OPP_02424 NAD binding rossmann fold No 4.02* FC = fold change.Haridas et al. BMC Genomics 2013, 14:373 Page 8 of 15http://www.biomedcentral.com/1471-2164/14/373at 14 h, 26 had homologs in G. clavigera, and nine of theseG. clavigera genes were up-regulated at 12 h. Similarly, ofthe 126 O. piceae genes that were up-regulated at 40 h, 75had G. clavigera homologs, twenty of which were up-regulated at 36 h.We found 26 O. piceae genes that were up-regulatedonly in the presence of terpenes, at one or both timepoints. Eighteen of these had G. clavigera homologs. Themost highly up-regulated O. piceae gene (OPP_06758)encoded an ABC transporter that was homologous to theG. clavigera transporter (CMQ_4184; GcABC-G1) thatconfers terpene tolerance to the pathogen [21] (Figure 6).Approximately 1,500 bp upstream of the O. piceae ABCtransporter is a gene (OPP_06759) encoding a tran-scription factor whose expression, like that of thetransporter, was up-regulated only in the presence of ter-penes (Figure 6). We found that the O. piceae OPP_06758and the G. clavigera CMQ_4184 (GcABC-G1) ABCtransporters were placed in the same clade when weconstructed a phylogenetic tree for a subset of the fungalspecies recently analyzed by Wang et al. [21]. To ourknowledge, from currently available sequence data, thisclade is unique to these two fungal species (Figure 7); nosimilar fungal ABC transporter has been reported.DiscussionThe ecological niches of saprophytic and pathogenicwood-inhabiting filamentous fungi differ in moisturecontent, nutrients and defence chemicals. For such fungito survive in and colonize their substrates and hosts,they need active transport systems that can excrete en-zymes that break down complex external substrates andthen import nutrients into the cells. As well, they needto modify or remove toxic host defense chemicals thathave entered their cells. The wood of trees, logs andlumber has a wide range of moisture contents and ahigh carbon-to-nitrogen ratio [14]. O. piceae grows moreefficiently in drier pine wood than in freshly cut logs;G. clavigera, which is vectored by MPB, colonizes healthyor stressed living pine trees, which have high moistureand low oxygen contents. Neither organism degrades lig-nocellulosic wood fibers [1,45]. O. piceae has to retrievenutrients from a nutrient-poor substrate that typicallycontains very little nitrogen and relatively low levels ofhost defence chemicals. In contrast, G. clavigera has firstto cope with high concentrations of defense chemicalsproduced by its pine host.Recently, we reported the annotated genome sequenceand transcriptomes of the pine pathogen G. clavigera [8].Figure 5 Growth of O. piceae on malt extract agar (MEA) treated with various volumes of mixed monoterpenes (MT). Fresh fungalmycelia were used as starting material and treated with 50, 100, 200 μl MT (equivalent to 0.35, 0.7, 1.4 g/l respectively). Colony diameters weremeasured daily. The growth rates were calculated as mm/day at linear stage. Results are average of 3 replicates; error bars are standarddeviations. MT: (+)-limonene, (+)-3-carene, racemic α-pinene and (−)-β-pinene at a ratio of 5:3:1:1. Student t-test indicated that MT applied at 100and 200 μl/plate inhibited fungal growth significantly, but not at 50 μl/plate ( P value cutoff 0.05).Haridas et al. BMC Genomics 2013, 14:373 Page 9 of 15http://www.biomedcentral.com/1471-2164/14/373Here, we report the annotated genome and transcriptomesof the saprophyte O. piceae, the second pine wood-inhabiting ophiostomatoid fungus for which a completegenome has been sequenced. O. piceae’s genome sizeand the number of predicted genes and proteins weresimilar to those for G. clavigera and other sequencedsaprophytic ascomycetes in the class Sordariomycetes(e.g. N. crassa, T. reesei). O. piceae’s predicted secretome is10% larger than that of the pine-specific pathogen. Givenits more diverse range of host trees (e.g. pines, hemlock,spruces), it is likely that the saprophyte requires moreextracellular enzymes to degrade the different chemicalsencountered in these substrates.In both genomes we identified genes that are poten-tially involved in the biosynthesis or processing of SMs.In fungi, SMs are diverse and play a range of roles; someSMs are protective, while others are virulence factors[46]. Both O. piceae and G. clavigera produce the SMmelanin in artificial media and in their natural sub-strates. Fungal melanin may protect cells in harsh envi-ronments (e.g. UV radiation, extreme temperatures andtoxic compounds), and may be involved in cellular de-velopment, differentiation and pathogenicity [36]. In allconditions tested here, except with terpene treatments,O. piceae mycelia and asexual structures (i.e. synemata)were highly melanized. Scytalone dehydratase, which isa marker gene for the DHN pathway [47], was up-regulated in all conditions tested except with terpenetreatments, and was most highly expressed in sawdust.Similarly, in G. clavigera, which is densely melanized inits pine host, scytalone dehydratase was down-regulatedon CM with terpenes, but was up-regulated on otherFigure 6 Expression pattern of the most highly up-regulated ABC-G transporter (OPP_06758) and of a transcription factor (OPP_06759).Relative transcript abundance of the gene OPP_06758 encoding an ABC transporter at 40 hr after terpene treatment. Sawdust (SD); Triglyceride(TG/Olive oil, OO); Oleic acid (OA); CM at 14 hr (C14); CM at 40 hr (C40); Mannose (MA); CM + Terpene at 14 hr (T14); CM + Terpene at 40 hr (T40).Figure 7 Phylogenetic tree of ABC-G group I transporters inO. piceae (OPP_06758, orange) and G. clavigera (CMQ_4184, blue).We included two other ABC transporters from O. piceae (OPP_06275,OPP_07323 ) and from G. clavigera (CMQ_3147 and CMQ_7257) and asubset of ABC transporters from ascomycete species. These speciesare: Saccharomyces cerevisiae (YOR328W, YOR153W, YDR406W);Pyrenomycetes like Gibberella zea (FGSG04580, FGSG08312), NectriaHaematococca (NECHADRAFT_63187, NECHADRAFT_35467,NECHADRAFT_82055), Neurospora crassa (NCU05591), Magnaporthegrisea (MGG_13624).Haridas et al. BMC Genomics 2013, 14:373 Page 10 of 15http://www.biomedcentral.com/1471-2164/14/373media and when monoterpenes were the only carbonsource. In contrast to O. piceae, G. clavigera does not pro-duce large numbers of asexual spores when it is activelygrowing on these media. It is likely that melanin protectsO. piceae from the unfavourable environmental conditionsthat it encounters in lumber (e.g. dessication, UV), as wellas being involved in cellular development. In contrast, forG. clavigera, melanin may be more important in protectingthe fungus from host defense chemicals.O. piceae and G. clavigera can grow on a variety ofsimple sugars that are present in phloem or in sapwoodparenchyma cells [13], and can acquire additional sugarsby degrading wood hemicelluloses [13,14,45]. Both fungigrow well with mannose and maltose, and can also usestarch, a stored tree nutrient [48]. For O. piceae, ourdata suggest that mannose uptake and the initial steps inits utilization are controlled by at least six genes that in-clude two transporters. That none of the six were up-regulated with maltose suggests that maltose utilizationinvolves an alternate pathway.O. piceae and related species can use triglycerides andfatty acids in artificial media or wood; these lipids canaccount for up to 3% of the dry weight of sapwood [41].Triglycerides are hydrolyzed by extracellular lipases intofatty acids and glycerol, which are ultimately processedthrough ß-oxidation and glycolysis pathways [47]. Whilelipase and esterase genes were present in the O. piceaegenome and we noted that a lipase was expressed onsawdust, we were unable to detect up-regulated lipaseson triglycerides. It is possible that on triglycerides thelipase was produced very early in growth, as shown byGao and Breuil [49], who reported an optimum produc-tion of the enzyme at day 3, before the pH of themedium decreases due to the accumulation of fattyacids. Here, we collected the mycelium after seven daysof growth on a solid media with triglycerides. We identi-fied a glycerol kinase that was up-regulated for triglycer-ides and sawdust, which suggests that glycerol may bemetabolized by the fungus. Further, we noticed that tri-glycerides induced a genomic cluster that contained aP450 and a reductase (described in Results). Lah et al.[35] reported a similar genomic cluster organization andexpression pattern in G. clavigera, and it is likely thatthe clusters have similar roles. Lah et al. suggested thatthe cytochrome P450 and the reductase may be specificredox partners and may play a role in the conversion ofexogenous phenolics or fatty acids.O. piceae retrieves and metabolizes diverse nutrientsthat are present in low concentrations in sawdust, par-ticularly nitrogen sources, while removing or modifyingdiverse toxic compounds like terpenes, and aromaticcompounds that include simple phenolics. While thefungus grows more slowly on sawdust, diverse trans-porters were up-regulated. Some of these are involvedin acquiring nutrients like sugars and nitrogen, whileothers, like ABC or MFS transporters, are known tocontribute to drug resistance or chemical modificationor detoxification [50].While small amounts of simple sugars are available insapwood, O. piceae can retrieve additional sugars by de-grading pine hemicellulose [13,45]. Fleet et al. [13]reported that mannose was the most depleted sugar inlogs and lumber inoculated with Ophiostoma species. Inour data, the genes up-regulated on sawdust also in-cluded glycoside hydrolases (e.g. two xylanases and onepectinase), which are involved in degrading hemicellu-lose and pectin. As well, the fungus can retrieve quinicacid through a quinate permease, and can utilize thiscarbon source by processing it through the quinate/shikamate pathway, which was up-regulated on sawdust.Further, in artificial media O. piceae can readily use inor-ganic or organic nitrogen. However, in pine sapwood ni-trogen is found mainly as amino acids and proteins, andat very low concentrations (~0.05% of the wood dryweight) [51]. We have shown that O. piceae and relatedspecies have to produce proteases in order to retrieveorganic nitrogen from wood [52]. In the current work,an amino acid permease, and urea and ammoniumtransporters were up-regulated on sawdust. Urea can beused as a source of nitrogen by many fungi, and it canbe efficiently converted into ammonium by a urease en-zyme [53]. However, while ammonium is present in traceamounts in pine lumber [53,54], urea has not beenreported in wood.Mono- and diterpenes are well known biocides for mi-croorganisms, including fungi, and for beetle vectors[21,55]. Our data show that on artificial media O. piceaetolerates monoterpenes but does not use them as a car-bon source. It is not found in living trees, which havethe highest terpene concentrations. However, it is ableto remain viable for extended periods in the presence ofmonoterpenes, and likely in the presence of diterpenes,which can account for ~0.4% of pine sapwood dryweight [41]. Here, we show that monoterpenes affectedthe macroscopic morphology of O. piceae’s mycelia, andinhibited its production of synemata and asexual spores.Further, in the saprophyte, monoterpene/diterpene treat-ments rapidly up-regulated expression of genes involved inoxidative and reductive processes, as well as transmem-brane transport, suggesting that the fungus’ primary re-sponse involves protecting itself from these chemicals.During these processes, an ABC transporter (OPP_06758),which is homologous to the G. clavigera efflux transportercharacterized by Wang et al. [21], was highly expressed.We have shown that this G. clavigera ABC-G trans-porter is expressed in young trees and that the trans-porter excretes monoterpenes [21]. As we have not yetdemonstrated this function for the homologous gene inHaridas et al. BMC Genomics 2013, 14:373 Page 11 of 15http://www.biomedcentral.com/1471-2164/14/373O. piceae, at this time we can only suggest that thisunique transporter may play a similar role in the sapro-phyte by allowing it to survive in toxic mixtures of ter-penes. When O. piceae is treated with terpenes on richmedia, there is an initial growth delay, after which thefungus resumes its growth. In this growth phase, whilegenes providing most of the primary protective bio-logical functions were active, genes involved in degrad-ing hydrophobic compounds were up-regulated. Thissuggests that, like G. clavigera, O. piceae may be able tomodify terpenes into less toxic compounds. However,while G. clavigera has a gene cluster that specifically re-sponds to terpenes and is potentially involved in metab-olizing terpenes [8], in O. piceae we found no such genecluster. Only five of the 30 genes in this G. clavigeracluster had homologs in O. piceae, and these five geneswere dispersed through the O. piceae genome. In on-going work we are characterizing O. piceae genes thatare involved in modifying terpenes.ConclusionsWe compared the genomes of O. piceae and G. clavigera.While we found no large-scale synteny, the ecologicalniches of both fungi involve growing in pine wood, andboth produce similar sets of diverse enzymes. Neither fun-gus produces a complete battery of cell-wall degrading en-zymes, and neither affects the structural properties ofwood. We began to clarify differences between the sapro-phyte and the pathogen, focusing on ABC transporters,CYP450s, genes that produce secondary metabolites likemelanin, genes involved in lipid metabolism, and genesthat detoxify terpenes and phenolics. G. clavigera, but notO. piceae, can use monoterpenes as a carbon source. How-ever, both O. piceae and G. clavigera have a similar ABC-Gtransporter that, for both fungi, may play an important rolein reducing the intracellular concentration of toxic com-pounds like monoterpenes. Similar specialized transportersmay have evolved in other ophiostomatoid fungi that arevectored by insects and inhabit the phloem and sapwoodof living or processed conifers.MethodsStrain and growth conditionsThe O. piceae strains used in this work, either for thegenome sequencing or for the mating experiments, hadbeen isolated from freshly sawn timber of Pinus contortaat Prince George in British Columbia (Canada) [6]. Thestrains have been deposited at the University of AlbertaMicrofungus Collection and Herbarium; ID: UAMH-11346 for the genome sequenced and ID: UMAH-11672for the mating experiments or for checking the growthcharacteristics. For growth and maintenance, spores orplugs of fungal mycelium were inoculated and grown atroom temperature on plates of MEA (1.6% Oxoid maltextract agar and 1.5% technical agar, pH 5–6). Growthand utilization of terpenes were performed as previouslydescribed by Wang et al. [21]. The only exception wasfor growth on sawdust where spores were inoculatedand germinated on 1% MEA (Difco) for 2 days, and thentransferred on sawdust plates (15% lodgepole pine saw-dust, mixed with 2% granulated agar) overlaid with cello-phane for one week. Growth experiments with orwithout terpenes or with different carbon sources wereat least repeated or carried out three times. For RNA-seq analysis, fungal hyphae grown on MEA for threedays were transferred to the respective treatment condi-tions as shown in Table 5.Genome sequencingDNA was extracted from fungal hyphae grown on MEAusing methods described by Haridas and Gantt [56]. Weused two sequencing technologies: Illumina HiSeq 2000,which generated 100 nt reads, and 454 Titanium, whichgenerated reads with a mean length of 318 nt. The li-braries for Illumina HiSeq 2000 had two different in-sert sizes, 200 and 700 nt, while the library for 454had an insert size of 8000 nt. Illumina HiSeq sequen-cing was done at the BC Genome Sciences Centre inVancouver, Canada and 454 sequencing was done at thePlate-forme d’Analyses Génomiques at Laval Universityin Québec, Canada.Genome assemblyReads generated by the two platforms were used with nofurther processing for genome assembly using ABySSv1.3.0 [25] with a kmer size of 60. This assembler filtersthe FASTQ sequences based on quality scores. In orderto efficiently use the 454 reads for scaffolding, we used aminimum contig size (1000) and read pairs for buildingTable 5 Growth conditions for RNA-seqMedium Carbon sources or treatment DurationCM No treatment 14 hCM No treatment 40 hCM 200 μl Terpene blend 14 hCM 200 μl Terpene blend 40 hYNB Mannose (1% w/v) 5 daysYNB *TG: Olive Oil (1% v/v) 5 daysYNB Oleic acid (0.5% v/v) 5 daysSawdust No treatment 1 weeks* Triglyceride: Olive oil 80% + Fatty acids 20%.All treatment times were calculated from the initial transfer of activelygrowing hyphae onto the appropriate medium. CM = 0.17% yeast nitrogenbase without amino acids, 1.5% granulated agar, 1% maltose, 0.1% potassiumhydrogen phthalate, 0.3% asparagine. YNB = 0.67% yeast nitrogen basewithout amino acid and 1.5% agar with carbon sources indicated as treatment.The terpene blend was a mixture of monoterpenes and diterpenes asdescribed by Lah et al. [35].Haridas et al. BMC Genomics 2013, 14:373 Page 12 of 15http://www.biomedcentral.com/1471-2164/14/373scaffolds (2) (SCAFFOLD_OPTIONS= ‘-s1000 -n2’). Theassembly was scrubbed and gaps closed with Anchor(v0.3.0; www.bcgsc.ca/platform/bioinfo/software/anchor).When Abyss is unable to find overlaps between contigswhere paired end data suggests that the contigs shouldoverlap, it joins the contigs with a single lowercase ‘n’.Such overlaps were resolved using transcriptome as-sembly (described below) or by finding small overlaps atthe ends of the contigs using exonerate v2.2.0 [26]. Gen-ome synteny was assessed by MUMmer [57].Transcriptome assemblyRNA-seq was performed on eight RNA samples extractedfrom the mycelia of O. piceae hyphae grown under variousconditions as shown in Table 5. For each RNA-seq library,we collected samples from three biological replicates,extracted RNA separately from each replicate, and pooledthe samples for paired-end sequencing on an IlluminaHiSeq (Canada’s Michael Smith Genome Science Center,Vancouver). Multiplexed sequencing with three librariesper lane was done using the Illumina HiSeq platform toobtain 100 bp paired end reads from 250 bp fragments.Reads were analysed using fastqc (www.bioinformatics.babraham.ac.uk/projects/fastqc/) and showed read biasand in the first few bases of the reads and poor quality inthe last few. Reads with minimum (Phred) quality scoresless than 20 were removed and the first six and last fourbases of all reads were trimmed using prinseq [58].Processed RNA-seq reads were assembled using Trinity[28] using the jaccard_clip option to minimize fusion tran-scripts. The best protein coding transcripts were identifiedusing the included scripts and aligned back to the assem-bled genome using exonerate v2.2.0 est2genome [26].Genome annotationWe used the Maker annotation pipeline (v2.26) for gen-ome annotation [31]. In addition to the trinity assem-bled best candidates, we also used two additionalsources of evidence in Maker. The first was transcriptspredicted by the Core Eukaryotic Genes Mapping Ap-proach [27], (CEGMA) and the second was coding se-quences of transcripts assembled by cufflinks [59] fromRNA-seq reads mapped to the assembled genome.Within the Maker framework, we trained SNAP v2006-07-28 [60] using the Trinity assembled transcripts, genemodels of Magnaporthe grisea for Augustus (v2.5.5) [61]and an hmm file for Genemark-ES (v2.3) [62] using anindependent run. The UniProtKB/Swiss-Prot (release2012_01) fasta file was provided as protein homology evi-dence and pred_flank was set to 50 to minimize fusiontranscripts. Predicted genes smaller than 100 amino acidswere removed unless they were at least 80 amino acidslong and had transcript, protein or CEGMA evidence.Selected gene models were manually curated. Functionalidentification of predicted genes was done using Blast2go(v2.5.1) [63]. tRNA’s were identified using tRNAscan-SE(v1.3.1) [64]. Relative synonymous codon usage (RSCU)was calculated using a local installation of the graphicalcodon usage analyser [65]. Secretome predictions weremade with TargetP [66] and Phobius [67]. A protein wasconsidered to be secreted if either TargetP or Phobiussuggested that it was secreted and this result was not inconflict with the other. Identification of secondary me-tabolism genes and clusters was done using the Second-ary Metabolite Unique Regions Finder (SMURF) [34].RNA-Seq analysisQuality trimmed RNA-seq reads were aligned to theO. piceae genome using Bowtie (v0.12.7), Tophat (v2.0.4),Cufflinks (v2.0.2) as described by Trapnell et al. [59].Because mapping the RNA-seq reads to the genomewithout providing fixed gene models resulted in anunacceptable number of predicted fusion transcripts,reads were mapped using the curated gene models pre-dicted by the Maker pipeline.Data availabilityThe sequences reported in this paper are being depos-ited in NCBI gene bank as assembly and annotations,Project NO. PRJNA182071.Additional filesAdditional file 1: Comparison of the genomes of O. piceae andG. clavigera, using MUMmer [57]. A dot plot genomic comparisonshowed no large scale synteny between the assembled genomes ofO. piceae and G. clavigera. The genome of O. piceae is representedon the X-axis and that of G. clavigera on the Y-axis.Additional file 2: GO term enrichment in a set of 3,469 genes ofO. piceae which were not reciprocal best blast hits in the G. clavigeragenome. #Test represents the number of genes annotated with therespective GO term in the set of 3,469 genes and #Ref represents thenumber of genes with the GO classification in the entire O. piceae genome.P-value is calculated for Fisher’s exact test and FDR is the false discovery rate.Additional file 3: The eleven secondary metabolism clusters inO. piceae predicted by SMURF [34]. The backbone gene in each clusteris highlighted in yellow.Additional file 4: Expression patterns of the 677 significantlydifferentially regulated genes. Expression values measured as FPKM(fragments per kilobase of exon per million fragments mapped). Genesthat showed a 10 times or greater up-regulation in one condition or arelated set of conditions are colour coded as described.Additional file 5: Alternative splicing. To assess how importantalternative splicing and transcripts were, we used Tophat and Cufflinks tomap the RNA-seq reads to the genome assembly using the techniquesdescribed by Trapnell et al. The results suggested that approximately 150alternative transcripts were expressed; however, all of these appeared tobe false positives. The dominant cause of these false positive predictionswas that closely spaced genes with overlapping UTRs weremisassembled as single contigs, and differential regulation of such genesunder different growth conditions appeared as alterative isoforms. Inother cases, mapping errors produced false gene calls and alternativeisoforms. These results indicated that alter415native splicing is notimportant under the growth conditions used for this study.Haridas et al. BMC Genomics 2013, 14:373 Page 13 of 15http://www.biomedcentral.com/1471-2164/14/373Additional file 6: Growth of O.piceae and G. clavigera on mannose,oleic acid and quinic acid. Fungal plugs were inoculated onto YNBplates (pH ~ 7, adjusted by KH2PO3-K2HPO3 buffer) containing a singlecarbon source; the plates with the fungus were incubated for 2 weeks.The growth of O. piceae is slower than G. clavigera, as shown withmannose. Control: YNB with no carbon.Additional file 7: Growth of O. piceae on MEA with or without theaddition of monoterpenes for a week. A) Growth on MEA alone(arrow a: synemata and spores, arrow b: mycelium), B) Growth on MEAwith monoterpenes, the mycelium was more aerial and fluffy (arrow c)while the production of asexual structures was highly inhibited.AbbreviationsMbp: Mega base pair; MPB: Mountain pine beetle; nt: Nucleotide; bp: Basepair; UTR: Untranslated region; CDS: Coding sequence; SM: Secondarymetabolite; NRPS: Nonribosomal peptide synthase; PKS: Polyketide synthase;DHN: 1,8-dihydroxynaphthalene; FPKM: Fragments per kilobase of exon permillion fragments mapped; MFS: Major facilitator superfamily; YNB: Yeastnitrogen base; MT: Monoterpene; MEA: Malt extract agar; CM: Completemedium; UV: Ultra violet; CEGMA: Core Eukaryotic Gene Mapping Approach;RSCU: Relative synonymous codon usage; SMURF: Secondary MetaboliteUnique Regions Finder.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsSJ and RD performed the initial genome assembly, and SH the finalassembly. SH annotated gene models, analyzed transcriptomes, and draftedthe results for the manuscript. YW participated in and finalized thetranscriptome analyses. LL prepared DNA, mRNA, performed growthexperiments and participated in the analyses. SMA did the phylogenetictrees and micrographs. IB supervised the sequencing of the genome andtranscriptomes. GR, JB and CB wrote and completed the manuscript. CB andJB conceived and directed the project. All co-authors critically reviewed,edited and approved the manuscript.AcknowledgementsThe authors would like to thank Anthony Raymond and Mack Yuen forstimulating discussions on bioinformatics issues. Simon Chan for uploadingthe sequences to NCBI. The work was funded by grants from the NaturalSciences and Engineering Research Council of Canada (NSERC; grant to JBand CB), and funds from Genome Canada, Genome BC and Genome Alberta(grant to JB, CB) that supported the Tria project (www.thetriaproject.ca).Author details1Department of Wood Science, University of British Columbia, Vancouver, BCV6T1Z4, Canada. 2Canada’s Michael Smith Genome Sciences Centre,Vancouver, BC V5Z 4E6, Canada. 3Michael Smith Laboratories, University ofBritish Columbia, Vancouver, BC V6T 1Z4, Canada.Received: 5 December 2012 Accepted: 10 May 2013Published: 2 June 2013References1. Seifert K: Sapstain of commercial lumber by species of Ophiostoma andCeratocystis. In Ceratocystis and Ophiostoma: taxonomy, ecology, andpathogenicity. Edited by Wingfield M, Seifert K, Webber J. St Paul, Minnesota:APS Press; 1993:141–151.2. Harrington TC: Diseases of conifers caused by species of Ophiostoma andLeptographium. 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BMC Genomics 2013, 14:373 Page 15 of 15http://www.biomedcentral.com/1471-2164/14/373

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