The genome and transcriptome of the pine saprophyte Ophiostoma piceae, and a comparison with the bark beetle-associated pine pathogen Grosmannia clavigera
© Haridas et al.; licensee BioMed Central Ltd. 2013
Received: 5 December 2012
Accepted: 10 May 2013
Published: 2 June 2013
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 with the genome and transcriptomes of the mountain pine beetle-associated pathogen Grosmannia clavigera highlights differences between a pathogen that colonizes and kills living pine trees and a saprophyte that colonizes wood and the inner bark of dead trees.
We assembled a 33 Mbp genome in 45 scaffolds, and predicted approximately 8,884 genes. The genome size and gene content were similar to those of other ascomycetes. Despite having similar ecological niches, O. piceae and G. clavigera showed no large-scale synteny. We identified O. piceae genes involved in the biosynthesis of melanin, which causes wood discoloration and reduces the commercial value of wood products. We also identified genes and pathways involved in growth on simple carbon sources and in sapwood, O. piceae’s natural substrate. Like the 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.
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 from the cells by an ABC transporter that is highly induced by terpenes. The data generated will provide the research community with resources for work on host-vector-fungus interactions for wood-inhabiting, beetle-associated saprophytes and pathogens.
KeywordsOphiostoma piceae Genome Transcriptome Wood-staining fungus Saprobe
O. piceae is a saprobe that is dispersed by generalist bark beetles . This fungal species has been reported in North America, Europe and Asia [5, 6, 11]. In contrast to Grosmannia species, which penetrate deeply into the sapwood of pine logs and reach the heartwood boundary, O. piceae is a more superficial sap stain fungus that becomes established in the outer two to three centimetres of sapwood [1, 12, 13]. Species in the O. piceae complex have retained the attention of wood industry researchers because they cause stain in processed wood and were the most commonly isolated species of sap stain fungi in Canadian saw mills . In contrast to G. clavigera, which is specific to pine, O. piceae is able to grow not only on pine, but also on wood of other conifers in Canada, including black and white spruce, balsam fir and hemlock . Because members of the O. piceae complex grow poorly on freshly cut pine logs and prefer the dryer environment of lumber or dead trees [1, 13], their staining effects can be minimized by keeping logs frozen or saturated with water, or by prompt log processing. Green lumber can be protected by kiln drying to below 20% moisture content, or by chemical and biological treatments [14–16].
Both the pathogen G. clavigera and the saprophyte O. piceae acquire nutrients from pine species by secreting extracellular enzymes to break down large molecules like polysaccharides (e.g. hemicellulose and starch), proteins and lipids. They do not degrade wood and do not affect wood structural properties [1, 16, 17], so likely have limited or incomplete cellulolytic and/or lignolytic activities. However, in order to colonize conifers (e.g. lodgepole pine), fungi and their bark beetle vectors have to cope with the host’s preformed and induced defense chemicals, which include terpenoid and phenolic compounds [18–20]. Pathogens like G. clavigera have evolved mechanisms to overcome these defences [8, 21]. However, the role of such host defence compounds in cut logs and lumber, where saprophytes like O. piceae are generally found, has not been reported. It is important to note that the composition of defence chemicals, especially terpenoids, varies with different pine genotypes across the landscape and can be affected by the environment . Further, wood processing and drying affect the concentrations of chemicals in wood products, and so logs and lumber typically contain lower concentrations of the subset of terpenoids that are volatile [23, 24].
In previous work we reported the annotated genome of the pine pathogen G. clavigera, and showed that this fungus is able to tolerate and utilize pine defence compounds, specifically terpenoids found in pine oleoresin [8, 21]. Here, we report the annotated genome of the saprophyte O. piceae, and its gene expression responses in a range of growth conditions that include wood nutrients and host defence chemicals. We compare these results to corresponding results from G. clavigera, and highlight differences between a pathogen that colonizes and kills living pine trees and a saprophyte that lives on dead trees or processed wood. Neither fungus has a lignocellulolytic enzyme system that would allow it to degrade wood. Both fungi overcome terpenoid defence chemicals in their pine niches; however, only the pathogen, but not the saprophyte, can metabolize terpenoids as a carbon source. Both fungi have a similar ABC efflux transporter that is highly induced with monoterpene treatments. The functionality of the O. piceae transporter remains to be fully characterized.
Sequencing strategy for O. piceae genome
Read length (nt)
Insert length (nt)
Read pairs (Millions)
Characteristics of the O. piceae (Op) genome assembly and annotation and a comparison with other related genomes
Nc (10) b
Genome size (Mbp)
Number of scaffolds
Number of ungapped contigs
Genome GC content (%)
Non-coding genome (%)
Number of genes
Median CDS length (bp)
Exon GC content (%)
Genome features and annotation
We used the Maker annotation pipeline  to predict genes. Within the annotated genome of O. piceae, we identified genes and gene families for secondary metabolite processing, cytochrome P450 as well as ABC transporters. We also identified homologous O. piceae and G. clavigera proteins based on reciprocal best BLAST hits. We further characterized the MAT idiomorph that is responsible for the mating type of the sequenced strain.
Major gene families in O. piceae (Op) and in three other ascomycetes
NAD binding proteins
FAD binding proteins
Although O. piceae and G. clavigera share hosts, cause sap-stain in pine, and are in sister clades in the Ophiostomatales  (Figure 1), their genomes showed no large-scale synteny (Additional file 1). This is consistent with synteny being lost with evolutionary time between members of the class Dothideomycetes . Dothideomycetes is a sister clade of the class Sordariomycetes, which include O. piceae and G. clavigera, and we anticipate that similar synteny losses have occurred within this class. A BLAST comparison of the two predicted proteomes showed that 5,450 proteins were reciprocal best hits. These included most of the major metabolic functions. The O. piceae proteins with no significant homolog in the G. clavigera genome were overrepresented by protein kinases (GO:0004672), sequence-specific DNA binding RNA polymerase II transcription factors (GO: 0000981) and zinc ion binding proteins (GO:0008270) (Additional file 2). In addition, proteins involved in transmembrane transport (GO:0055085) were also significantly overrepresented in this group of 3,469 proteins (Additional file 2). Over 40% (1,397) of the O. piceae proteins with no evident homologs in G. clavigera were proteins of unknown function (predicted or hypothetical proteins). None of the six carboxylic ester hydrolases (GO:0052689) in the O. piceae genome had a homolog in the G. clavigera genome.
We searched for genes that may be involved in producing secondary metabolites (SMs). Such genes are typically organized as contiguous genomic clusters and can be identified by tools like SMURF , which uses hidden Markov models that consider genomic context and domain content. The first step in fungal SM biosynthesis is usually catalyzed by ‘backbone’ genes like nonribosomal peptide synthases (NRPSs), polyketide synthases (PKSs), hybrid NRPS-PKS enzymes, prenyltransferases, and terpene cyclases . SMURF, which does not identify clusters containing terpene cyclases, identified thirteen backbone genes of which eleven are in SM clusters in O. piceae (Additional file 3), and nineteen genes in fourteen clusters in G. clavigera. All the O. piceae SM genes have homologs in G. clavigera.
Melanin is a secondary metabolite that is produced by O. piceae and related species, but, as in O. piceae, the genes responsible for its production do not always occur in a cluster. Melanin is synthesized through the 1,8–dihydroxynaphthalene (DHN) pathway . In O. piceae we identified a number of genes that were similar to genes that have major roles in the DHN pathway in Ophiostoma, Grosmannia and Ceratocystis species [12, 37, 38]. These genes included a PKS (OPP_00823), two reductases (OPP_02710, OPP_00820) and a scytalone dehydratase (OPP_07153). PKS catalyze both the elongation of five ketide subunits and the cyclization of these units to form the base ring of naphthalene. The first reductase (OPP_02710) converts 1,3,6,8-hydroxynaphthalene to scytalone, while the second (OPP_00820) transforms scytalone to vermelone.
O. piceae is a heterothallic species, so requires two individuals with different mating types for sexual reproduction and production of fertile fruiting bodies. Our genome annotation identified O. piceae’s MAT1-2 idiomorph (OPP_06680). A truncated MAT1-1 gene was next to the MAT1-2 gene, as in Grosmannia and related species [8, 39, 40]. We have produced perithecia for O. piceae by mating UAMH-11346 with UAMH- 11672 (Figure 2), and we have successfully amplified and sequenced the full length of the MAT1-1 loci in this latter strain; the alpha box (~978 bp) was missing in the sequenced strain.
Gene expression patterns
To identify genes that may be critical for the saprophyte O. piceae to grow in the presence of the nutrients and defence chemicals that are characteristic of its natural pine sapwood substrate, we profiled gene expression for the fungus growing on solid agar media supplemented with simple carbon sources (i.e. sugars and lipids), with pine sawdust, or with pine terpenes (see Methods). Mapping the RNA-seq reads to the predicted gene models identified 7,157 genes that had an abundance of at least 10 FPKM (fragments per kilobase of exon per million fragments mapped) in any of the conditions tested.
We also assessed alternative transcript splicing across the range of growth conditions used for this study (Additional file 5). Splicing appeared not to be an important factor under these conditions.
Growth on mannose, fatty acid (oleic acid) and triglycerides
Mannose is a simple monomeric epimer of glucose and can be readily utilized as a carbon source by O. piceae. We found five genes whose expression was at least ten times higher with mannose than in any other conditions tested (Additional file 4 shown light blue). These included two transporters, one oxidoreductase and two hypothetical proteins. Our data suggests that mannose uptake involves two transporters (OPP_03031, OPP_05665), and a simple isomerisation/ epimerization reaction by an oxidoreductase (OPP_00733) converts it into glucose. The function of two remaining up-regulated genes (OPP_02416, OPP_07274) is unknown.
We grew O. piceae on triglycerides and fatty acids, which are important lipid compounds in lodgepole pine sapwood, and are a major source of carbon for O. piceae. Because most sources of triglycerides contain a small proportion of fatty acids, it was not surprising that most of the 129 genes whose transcripts were differentially abundant between these conditions were highly up-regulated in both of the conditions. Of the 25 up-regulated genes that were significantly induced only in these two conditions (shown in gold in Additional file 4), 18 were predicted to produce secreted proteins. Unexpectedly, the differentially up-regulated genes included no fungal lipases, which are necessary for the hydrolysis of triglycerides (Additional file 4). Twenty-three of the 25 up-regulated genes were predicted to be involved in the breakdown of carbohydrates and sugars; these included eight genes coding for secreted proteins in the glycoside hydrolase family and four genes for secreted proteins involved in carbohydrate and starch binding. We identified a transcription factor (OPP_02429) that showed significant up-regulation in the presence of triglycerides and oleic acid.
One of the genes differentially expressed between olive oil and oleic acid was a cytochrome P450 (OPP_02426) with a significantly higher expression with triglyceride than with fatty acid. Like its G. clavigera homolog (CMQ_5365; CYP630B18) and homologs in several other species including Fusarium graminearum, Aspergillus niger, A. fumigates and others, this gene is in close proximity to genes encoding a myo-inositol transporter, ARCA-like protein and a cytochrome P450 reductase .
Growth on pine sapwood, a natural substrate for O. piceae
Gene clusters up-regulated in sawdust
Fungal-specific transcription factor domain protein
Salicylate hydroxylase (salicylate 1-monooxygenase)
NAD dependent epimerase
Amidohydrolase family protein
FAD binding domain protein
Retinol dehydrogenase 13
General alpha-glucoside permease
Major facilitator superfamily transporter
N-carbamoyl-l-amino acid hydrolase
Gal4-like transcription factor
Class ii aldolase adducin domain-containing protein
Isoflavone reductase family protein
Ethanolamine utilization protein
C6 zinc finger domain containing protein
Benzoate 4-monooxygenase cytochrome p450
NADPH-cytochrome p450 reductase
NAD binding rossmann fold
One of the above eight up-regulated genomic clusters contained seven genes that were involved in metabolizing quinic acid (OPP_8732 to OPP_8738). These include a quinate permease, two regulatory genes (an activator and a repressor), and the four genes of the quinate/shikamate catabolic pathway [43, 44]. The latter four catabolic genes (OPP_08735 to OPP_08738) suggest that O. 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, while G. clavigera cannot, we showed that the former, but not the latter, grows on YNB media with quinic acid as the sole carbon source (Additional file 6). Finally, we identified a secreted lipase (OPP_00605) with predicted triglyceride degradation activity, whose transcript abundance was at least 50-fold higher in the mycelium grown in pine sapwood than in the control mannose.
Tolerance of pine tree defence chemicals
In order to identify genes involved in terpene tolerance, we grew O. piceae on CM and treated it with a mixture of terpenes as previously described in our studies with G. clavigera[8, 21]. While the experiments for the two species were done at different times, we used the same conditions for both growth experiments and the same protocols for RNA extractions. We compared gene expression profiles of O. piceae after 14 h and 40 h treatments to profiles for untreated CM plates at the same time points. At 14 h, 295 genes were differentially abundant, 261 of which were down-regulated. While carbohydrate metabolism (GO:0005975) was associated with the down-regulated genes (p < 0.001), we were unable to identify any GO terms that were associated with the 34 up-regulated genes. After 40 h in the presence of terpenes, 264 genes were differentially abundant, 126 of which were up-regulated. While carbohydrate metabolism 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 carbon source, more than 250 genes were up-regulated by at least 2-fold at 12 h and 36 h in the presence of terpenes . Of the 34 O. piceae genes that were up-regulated at 14 h, 26 had homologs in G. clavigera, and nine of these G. clavigera genes were up-regulated at 12 h. Similarly, of the 126 O. piceae genes that were up-regulated at 40 h, 75 had G. clavigera homologs, twenty of which were up-regulated at 36 h.
The ecological niches of saprophytic and pathogenic wood-inhabiting filamentous fungi differ in moisture content, nutrients and defence chemicals. For such fungi to survive in and colonize their substrates and hosts, they need active transport systems that can excrete enzymes that break down complex external substrates and then import nutrients into the cells. As well, they need to modify or remove toxic host defense chemicals that have entered their cells. The wood of trees, logs and lumber has a wide range of moisture contents and a high carbon-to-nitrogen ratio . O. piceae grows more efficiently in drier pine wood than in freshly cut logs; G. clavigera, which is vectored by MPB, colonizes healthy or stressed living pine trees, which have high moisture and low oxygen contents. Neither organism degrades lignocellulosic wood fibers [1, 45]. O. piceae has to retrieve nutrients from a nutrient-poor substrate that typically contains very little nitrogen and relatively low levels of host defence chemicals. In contrast, G. clavigera has first to cope with high concentrations of defense chemicals produced by its pine host.
Recently, we reported the annotated genome sequence and transcriptomes of the pine pathogen G. clavigera. Here, we report the annotated genome and transcriptomes of the saprophyte O. piceae, the second pine wood-inhabiting ophiostomatoid fungus for which a complete genome has been sequenced. O. piceae’s genome size and the number of predicted genes and proteins were similar to those for G. clavigera and other sequenced saprophytic ascomycetes in the class Sordariomycetes (e.g. N. crassa, T. reesei). O. piceae’s predicted secretome is 10% larger than that of the pine-specific pathogen. Given its more diverse range of host trees (e.g. pines, hemlock, spruces), it is likely that the saprophyte requires more extracellular enzymes to degrade the different chemicals encountered in these substrates.
In both genomes we identified genes that are potentially involved in the biosynthesis or processing of SMs. In fungi, SMs are diverse and play a range of roles; some SMs are protective, while others are virulence factors . Both O. piceae and G. clavigera produce the SM melanin in artificial media and in their natural substrates. Fungal melanin may protect cells in harsh environments (e.g. UV radiation, extreme temperatures and toxic compounds), and may be involved in cellular development, differentiation and pathogenicity . In all conditions tested here, except with terpene treatments, O. piceae mycelia and asexual structures (i.e. synemata) were highly melanized. Scytalone dehydratase, which is a marker gene for the DHN pathway , was up-regulated in all conditions tested except with terpene treatments, and was most highly expressed in sawdust. Similarly, in G. clavigera, which is densely melanized in its pine host, scytalone dehydratase was down-regulated on CM with terpenes, but was up-regulated on other media and when monoterpenes were the only carbon source. In contrast to O. piceae, G. clavigera does not produce large numbers of asexual spores when it is actively growing on these media. It is likely that melanin protects O. piceae from the unfavourable environmental conditions that it encounters in lumber (e.g. dessication, UV), as well as being involved in cellular development. In contrast, for G. clavigera, melanin may be more important in protecting the fungus from host defense chemicals.
O. piceae and G. clavigera can grow on a variety of simple sugars that are present in phloem or in sapwood parenchyma cells , and can acquire additional sugars by degrading wood hemicelluloses [13, 14, 45]. Both fungi grow well with mannose and maltose, and can also use starch, a stored tree nutrient . For O. piceae, our data suggest that mannose uptake and the initial steps in its utilization are controlled by at least six genes that include two transporters. That none of the six were up-regulated with maltose suggests that maltose utilization involves an alternate pathway.
O. piceae and related species can use triglycerides and fatty acids in artificial media or wood; these lipids can account for up to 3% of the dry weight of sapwood . Triglycerides are hydrolyzed by extracellular lipases into fatty acids and glycerol, which are ultimately processed through ß-oxidation and glycolysis pathways . While lipase and esterase genes were present in the O. piceae genome and we noted that a lipase was expressed on sawdust, we were unable to detect up-regulated lipases on triglycerides. It is possible that on triglycerides the lipase was produced very early in growth, as shown by Gao and Breuil , who reported an optimum production of the enzyme at day 3, before the pH of the medium decreases due to the accumulation of fatty acids. Here, we collected the mycelium after seven days of growth on a solid media with triglycerides. We identified a glycerol kinase that was up-regulated for triglycerides and sawdust, which suggests that glycerol may be metabolized by the fungus. Further, we noticed that triglycerides induced a genomic cluster that contained a P450 and a reductase (described in Results). Lah et al. reported a similar genomic cluster organization and expression pattern in G. clavigera, and it is likely that the clusters have similar roles. Lah et al. suggested that the cytochrome P450 and the reductase may be specific redox partners and may play a role in the conversion of exogenous phenolics or fatty acids.
O. piceae retrieves and metabolizes diverse nutrients that are present in low concentrations in sawdust, particularly nitrogen sources, while removing or modifying diverse toxic compounds like terpenes, and aromatic compounds that include simple phenolics. While the fungus grows more slowly on sawdust, diverse transporters were up-regulated. Some of these are involved in acquiring nutrients like sugars and nitrogen, while others, like ABC or MFS transporters, are known to contribute to drug resistance or chemical modification or detoxification .
While small amounts of simple sugars are available in sapwood, O. piceae can retrieve additional sugars by degrading pine hemicellulose [13, 45]. Fleet et al.  reported that mannose was the most depleted sugar in logs and lumber inoculated with Ophiostoma species. In our data, the genes up-regulated on sawdust also included glycoside hydrolases (e.g. two xylanases and one pectinase), which are involved in degrading hemicellulose and pectin. As well, the fungus can retrieve quinic acid through a quinate permease, and can utilize this carbon source by processing it through the quinate/shikamate pathway, which was up-regulated on sawdust. Further, in artificial media O. piceae can readily use inorganic or organic nitrogen. However, in pine sapwood nitrogen is found mainly as amino acids and proteins, and at very low concentrations (~0.05% of the wood dry weight) . We have shown that O. piceae and related species have to produce proteases in order to retrieve organic nitrogen from wood . In the current work, an amino acid permease, and urea and ammonium transporters were up-regulated on sawdust. Urea can be used as a source of nitrogen by many fungi, and it can be efficiently converted into ammonium by a urease enzyme . However, while ammonium is present in trace amounts in pine lumber [53, 54], urea has not been reported in wood.
Mono- and diterpenes are well known biocides for microorganisms, including fungi, and for beetle vectors [21, 55]. Our data show that on artificial media O. piceae tolerates monoterpenes but does not use them as a carbon source. It is not found in living trees, which have the highest terpene concentrations. However, it is able to remain viable for extended periods in the presence of monoterpenes, and likely in the presence of diterpenes, which can account for ~0.4% of pine sapwood dry weight . Here, we show that monoterpenes affected the macroscopic morphology of O. piceae’s mycelia, and inhibited its production of synemata and asexual spores. Further, in the saprophyte, monoterpene/diterpene treatments rapidly up-regulated expression of genes involved in oxidative and reductive processes, as well as transmembrane transport, suggesting that the fungus’ primary response involves protecting itself from these chemicals. During these processes, an ABC transporter (OPP_06758), which is homologous to the G. clavigera efflux transporter characterized by Wang et al. , was highly expressed.
We have shown that this G. clavigera ABC-G transporter is expressed in young trees and that the transporter excretes monoterpenes . As we have not yet demonstrated this function for the homologous gene in O. piceae, at this time we can only suggest that this unique transporter may play a similar role in the saprophyte by allowing it to survive in toxic mixtures of terpenes. When O. piceae is treated with terpenes on rich media, there is an initial growth delay, after which the fungus resumes its growth. In this growth phase, while genes providing most of the primary protective biological functions were active, genes involved in degrading hydrophobic compounds were up-regulated. This suggests that, like G. clavigera, O. piceae may be able to modify terpenes into less toxic compounds. However, while G. clavigera has a gene cluster that specifically responds to terpenes and is potentially involved in metabolizing terpenes , in O. piceae we found no such gene cluster. Only five of the 30 genes in this G. clavigera cluster had homologs in O. piceae, and these five genes were dispersed through the O. piceae genome. In ongoing work we are characterizing O. piceae genes that are involved in modifying terpenes.
We compared the genomes of O. piceae and G. clavigera. While we found no large-scale synteny, the ecological niches of both fungi involve growing in pine wood, and both produce similar sets of diverse enzymes. Neither fungus produces a complete battery of cell-wall degrading enzymes, and neither affects the structural properties of wood. We began to clarify differences between the saprophyte and the pathogen, focusing on ABC transporters, CYP450s, genes that produce secondary metabolites like melanin, genes involved in lipid metabolism, and genes that detoxify terpenes and phenolics. G. clavigera, but not O. piceae, can use monoterpenes as a carbon source. However, both O. piceae and G. clavigera have a similar ABC-G transporter that, for both fungi, may play an important role in reducing the intracellular concentration of toxic compounds like monoterpenes. Similar specialized transporters may have evolved in other ophiostomatoid fungi that are vectored by insects and inhabit the phloem and sapwood of living or processed conifers.
Strain and growth conditions
Growth conditions for RNA-seq
Carbon sources or treatment
200 μl Terpene blend
200 μl Terpene blend
Mannose (1% w/v)
*TG: Olive Oil (1% v/v)
Oleic acid (0.5% v/v)
DNA was extracted from fungal hyphae grown on MEA using methods described by Haridas and Gantt . We used two sequencing technologies: Illumina HiSeq 2000, which generated 100 nt reads, and 454 Titanium, which generated reads with a mean length of 318 nt. The libraries for Illumina HiSeq 2000 had two different insert sizes, 200 and 700 nt, while the library for 454 had an insert size of 8000 nt. Illumina HiSeq sequencing was done at the BC Genome Sciences Centre in Vancouver, Canada and 454 sequencing was done at the Plate-forme d’Analyses Génomiques at Laval University in Québec, Canada.
Reads generated by the two platforms were used with no further processing for genome assembly using ABySS v1.3.0  with a kmer size of 60. This assembler filters the FASTQ sequences based on quality scores. In order to efficiently use the 454 reads for scaffolding, we used a minimum contig size (1000) and read pairs for building scaffolds (2) (SCAFFOLD_OPTIONS = ‘-s1000 -n2’). The assembly was scrubbed and gaps closed with Anchor (v0.3.0; http://www.bcgsc.ca/platform/bioinfo/software/anchor). When Abyss is unable to find overlaps between contigs where paired end data suggests that the contigs should overlap, it joins the contigs with a single lowercase ‘n’. Such overlaps were resolved using transcriptome assembly (described below) or by finding small overlaps at the ends of the contigs using exonerate v2.2.0 . Genome synteny was assessed by MUMmer .
RNA-seq was performed on eight RNA samples extracted from the mycelia of O. piceae hyphae grown under various conditions as shown in Table 5. For each RNA-seq library, we collected samples from three biological replicates, extracted RNA separately from each replicate, and pooled the samples for paired-end sequencing on an Illumina HiSeq (Canada’s Michael Smith Genome Science Center, Vancouver). Multiplexed sequencing with three libraries per lane was done using the Illumina HiSeq platform to obtain 100 bp paired end reads from 250 bp fragments. Reads were analysed using fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and showed read bias and in the first few bases of the reads and poor quality in the last few. Reads with minimum (Phred) quality scores less than 20 were removed and the first six and last four bases of all reads were trimmed using prinseq . Processed RNA-seq reads were assembled using Trinity  using the jaccard_clip option to minimize fusion transcripts. The best protein coding transcripts were identified using the included scripts and aligned back to the assembled genome using exonerate v2.2.0 est2genome .
We used the Maker annotation pipeline (v2.26) for genome annotation . In addition to the trinity assembled best candidates, we also used two additional sources of evidence in Maker. The first was transcripts predicted by the Core Eukaryotic Genes Mapping Approach , (CEGMA) and the second was coding sequences of transcripts assembled by cufflinks  from RNA-seq reads mapped to the assembled genome. Within the Maker framework, we trained SNAP v2006-07-28  using the Trinity assembled transcripts, gene models of Magnaporthe grisea for Augustus (v2.5.5)  and an hmm file for Genemark-ES (v2.3)  using an independent run. The UniProtKB/Swiss-Prot (release 2012_01) fasta file was provided as protein homology evidence and pred_flank was set to 50 to minimize fusion transcripts. Predicted genes smaller than 100 amino acids were removed unless they were at least 80 amino acids long and had transcript, protein or CEGMA evidence. Selected gene models were manually curated. Functional identification of predicted genes was done using Blast2go (v2.5.1) . tRNA’s were identified using tRNAscan-SE (v1.3.1) . Relative synonymous codon usage (RSCU) was calculated using a local installation of the graphical codon usage analyser . Secretome predictions were made with TargetP  and Phobius . A protein was considered to be secreted if either TargetP or Phobius suggested that it was secreted and this result was not in conflict with the other. Identification of secondary metabolism genes and clusters was done using the Secondary Metabolite Unique Regions Finder (SMURF) .
Quality trimmed RNA-seq reads were aligned to the O. piceae genome using Bowtie (v0.12.7), Tophat (v2.0.4), Cufflinks (v2.0.2) as described by Trapnell et al. . Because mapping the RNA-seq reads to the genome without providing fixed gene models resulted in an unacceptable number of predicted fusion transcripts, reads were mapped using the curated gene models predicted by the Maker pipeline.
The sequences reported in this paper are being deposited in NCBI gene bank as assembly and annotations, Project NO. PRJNA182071.
Mega base pair
Mountain pine beetle
Nonribosomal peptide synthase
Fragments per kilobase of exon per million fragments mapped
Major facilitator superfamily
Yeast nitrogen base
Malt extract agar
Core Eukaryotic Gene Mapping Approach
Relative synonymous codon usage
Secondary Metabolite Unique Regions Finder.
The authors would like to thank Anthony Raymond and Mack Yuen for stimulating discussions on bioinformatics issues. Simon Chan for uploading the sequences to NCBI. The work was funded by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC; grant to JB and CB), and funds from Genome Canada, Genome BC and Genome Alberta (grant to JB, CB) that supported the Tria project (http://www.thetriaproject.ca).
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