Metatranscriptomics sheds light on the links between the functional traits of fungal guilds and ecological processes in forest soil ecosystems.

Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.

Multiple rearrangements and low inter- and intra-species mitogenome sequence variation in the Heterobasidion annosum s.l. species complex.

Introduction: Mitochondria are essential organelles in the eukaryotic cells and responsible for the energy production but are also involved in many other functions including virulence of some fungal species. Although the evolution of fungal mitogenomes have been studied at some taxonomic levels there are still many things to be learned from studies of closely related species. Methods: In this study, we have analyzed 60 mitogenomes in the five species of the Heterobasidion annosum sensu lato complex that all are necrotrophic pathogens on conifers. Results and Discussion: Compared to other fungal genera the genomic and genetic variation between and within species in the complex was low except for multiple rearrangements. Several translocations of large blocks with core genes have occurred between the five species and rearrangements were frequent in intergenic areas. Mitogenome lengths ranged between 108 878 to 116 176 bp, mostly as a result of intron variation. There was a high degree of homology of introns, homing endonuclease genes, and intergenic ORFs among the five Heterobasidion species. Three intergenic ORFs with unknown function (uORF6, uORF8 and uORF9) were found in all five species and was located in conserved synteny blocks. A 13 bp long GC-containing self-complementary palindrome was discovered in many places in the five species that were optional in presence/absence. The within species variation is very low, among 48 H. parviporum mitogenomes, there was only one single intron exchange, and SNP frequency was 0.28% and indel frequency 0.043%. The overall low variation in the Heterobasidion annosum sensu lato complex suggests a slow evolution of the mitogenome.

Mitonuclear Genetic Interactions in the Basidiomycete Heterobasidion parviporum Involve a Non-conserved Mitochondrial Open Reading Frame.

The mitochondrial and nuclear genomes of Eukaryotes are inherited separately and consequently follow distinct evolutionary paths. Nevertheless, the encoding of many mitochondrial proteins by the nuclear genome shows the high level of integration they have reached, which makes mitonuclear genetic interactions all the more conceivable. For each species, natural selection has fostered the evolution of coadapted alleles in both genomes, but a population-wise divergence of such alleles could lead to important phenotypic variation, and, ultimately, to speciation. In this study in the Basidiomycete Heterobasidion parviporum, we have investigated the genetic basis of phenotypic variation among laboratory-designed heterokaryons carrying the same pair of haploid nuclei, but a different mitochondrial genome. Radial growth rate data of thirteen unrelated homokaryotic parents and of their heterokaryotic offspring were combined with SNP data extracted from parental genome sequences to identify nuclear and mitochondrial loci involved in mitonuclear interactions. Two nuclear loci encoding mitochondrial proteins appeared as best candidates to engage in a genetic interaction affecting radial growth rate with a non-conserved mitochondrial open reading frame of unknown function and not reported apart from the Russulales order of Basidiomycete fungi. We believe our approach could be useful to investigate several important traits of fungal biology where mitonuclear interactions play a role, including virulence of fungal pathogens.

Genetic evidence for sexual reproduction and multiple infections of Norway spruce cones by the rust fungus Thekopsora areolata.

Rust fungi are obligate parasites, of plants, with complex and in many cases poorly known life cycles which may include host alteration and up to five spore types with haploid, diploid, and dikaryotic nuclear stages. This study supports that Thekopasora areolata, the causal agent of cherry-spruce rust in Norway spruce, is a macrocyclic heteroecious fungus with all five spore stages which uses two host plants Prunus padus and Picea abies to complete its life cycle. High genotypic diversity without population structure was found, which suggests predominantly sexual reproduction, random mating and a high gene flow within and between the populations in Fennoscandia. There was no evidence for an autoecious life cycle resulting from aeciospore infection of pistillate cones that would explain the previously reported rust epidemics without the alternate host. However, within cones and scales identical multilocus genotypes were repeatedly sampled which can be explained by vegetative growth of the fertilized mycelia or repeated mating of mycelium by spermatia of the same genotype. The high genotypic diversity within cones and haplotype inference show that each pistillate cone is infected by several basidiospores. This study provides genetic evidence for high gene flow, sexual reproduction, and multiple infections of Norway spruce cone by the rust fungus T. areolata which expands the general understanding of the biology of rust fungi.

Optimized metabarcoding with Pacific biosciences enables semi-quantitative analysis of fungal communities.

Recent studies have questioned the use of high-throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi-quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking. We used artificially assembled communities consisting of 10 ITS-like fragments of varying lengths and guanine-cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies. Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under-represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (> 447 bases) were lost when sequenced with Illumina MiSeq. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases related to GC content were observed. Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.

Association genetics identifies a specifically regulated Norway spruce laccase gene, PaLAC5, linked to Heterobasidion parviporum resistance.

It is important to improve the understanding of the interactions between the trees and pathogens and integrate this knowledge about disease resistance into tree breeding programs. The conifer Norway spruce (Picea abies) is an important species for the forest industry in Europe. Its major pathogen is Heterobasidion parviporum, causing stem and root rot. In this study, we identified 11 Norway spruce QTLs (Quantitative trait loci) that correlate with variation in resistance to H. parviporum in a population of 466 trees by association genetics. Individual QTLs explained between 2.1 and 5.2% of the phenotypic variance. The expression of candidate genes associated with the QTLs was analysed in silico and in response to H. parviporum hypothesizing that (a) candidate genes linked to control of fungal sapwood growth are more commonly expressed in sapwood, and; (b) candidate genes associated with induced defences are respond to H. parviporum inoculation. The Norway spruce laccase PaLAC5 associated with control of lesion length development is likely to be involved in the induced defences. Expression analyses showed that PaLAC5 responds specifically and strongly in close proximity to the H. parviporum inoculation. Thus, PaLAC5 may be associated with the lignosuberized boundary zone formation in bark adjacent to the inoculation site.

The conifer root rot pathogens Heterobasidion irregulare and Heterobasidion occidentale employ different strategies to infect Norway spruce.

Heterobasidion irregulare and H. occidentale are two closely related conifer root rot pathogens in the H. annosum sensu lato (s.l.) species complex. The two species H. irregulare and H. occidentale have different host preference with pine and non-pine tree species favored, respectively. The comparison of transcriptomes of H. irregulare and H. occidentale growing in Norway spruce bark, a susceptible host non-native to North America, showed large differences in gene expression. Heterobasidion irregulare induced more genes involved in detoxification of host compounds and in production of secondary metabolites, while the transcriptome induced in H. occidentale was more oriented towards carbohydrate degradation. Along with their separated evolutionary history, the difference might be driven by their host preferences as indicated by the differentially expressed genes enriched in particular Gene Ontology terms.

Genotypic variation in Norway spruce correlates to fungal communities in vegetative buds.

The taxonomically diverse phyllosphere fungi inhabit leaves of plants. Thus, apart from the fungi's dispersal capacities and environmental factors, the assembly of the phyllosphere community associated with a given host plant depends on factors encoded by the host's genome. The host genetic factors and their influence on the assembly of phyllosphere communities under natural conditions are poorly understood, especially in trees. Recent work indicates that Norway spruce (Picea abies) vegetative buds harbour active fungal communities, but these are hitherto largely uncharacterized. This study combines internal transcribed spacer sequencing of the fungal communities associated with dormant vegetative buds with a genome-wide association study (GWAS) in 478 unrelated Norway spruce trees. The aim was to detect host loci associated with variation in the fungal communities across the population, and to identify loci correlating with the presence of specific, latent, pathogens. The fungal communities were dominated by known Norway spruce phyllosphere endophytes and pathogens. We identified six quantitative trait loci (QTLs) associated with the relative abundance of the dominating taxa (i.e., top 1% most abundant taxa). Three additional QTLs associated with colonization by the spruce needle cast pathogen Lirula macrospora or the cherry spruce rust (Thekopsora areolata) in asymptomatic tissues were detected. The identification of the nine QTLs shows that the genetic variation in Norway spruce influences the fungal community in dormant buds and that mechanisms underlying the assembly of the communities and the colonization of latent pathogens in trees may be uncovered by combining molecular identification of fungi with GWAS.

Estimating the Fitness Effect of Deleterious Mutations During the Two Phases of the Life Cycle: A New Method Applied to the Root-Rot Fungus Heterobasidion parviporum.

Many eukaryote species, including taxa such as fungi or algae, have a lifecycle with substantial haploid and diploid phases. A recent theoretical model predicts that such haploid-diploid lifecycles are stable over long evolutionary time scales when segregating deleterious mutations have stronger effects in homozygous diploids than in haploids and when they are partially recessive in heterozygous diploids. The model predicts that effective dominance-a measure that accounts for these two effects-should be close to 0.5 in these species. It also predicts that diploids should have higher fitness than haploids on average. However, an appropriate statistical framework to conjointly investigate these predictions is currently lacking. In this study, we derive a new quantitative genetic model to test these predictions using fitness data of two haploid parents and their diploid offspring, and genome-wide genetic distance between haploid parents. We apply this model to the root-rot basidiomycete fungus Heterobasidion parviporum-a species where the heterokaryotic (equivalent to the diploid) phase is longer than the homokaryotic (haploid) phase. We measured two fitness-related traits (mycelium growth rate and the ability to degrade wood) in both homokaryons and heterokaryons, and we used whole-genome sequencing to estimate nuclear genetic distance between parents. Possibly due to a lack of power, we did not find that deleterious mutations were recessive or more deleterious when expressed during the heterokaryotic phase. Using this model to compare effective dominance among haploid-diploid species where the relative importance of the two phases varies should help better understand the evolution of haploid-diploid life cycles.

Side-by-side biochemical comparison of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare on their activity against crystalline cellulose and glucomannan.

Our comparative studies reveal that the two lytic polysaccharide monooxygenases HiLPMO9B and HiLPMO9I from the white-rot conifer pathogen Heterobasidion irregulare display clear difference with respect to their activity against crystalline cellulose and glucomannan. HiLPMO9I produced very little soluble sugar on bacterial microcrystalline cellulose (BMCC). In contrast, HiLPMO9B was much more active against BMCC and even released more soluble sugar than the H. irregulare cellobiohydrolase I, HiCel7A. Furthermore, HiLPMO9B was shown to cooperate with and stimulate the activity of HiCel7A, both when the BMCC was first pretreated with HiLPMO9B, as well as when HiLPMO9B and HiCel7A were added together. No such stimulation was shown by HiLPMO9I. On the other hand, HiLPMO9I was shown to degrade glucomannan, using a C4-oxidizing mechanism, whereas no oxidative cleavage activity of glucomannan was detected for HiLPMO9B. Structural modeling and comparison with other glucomannan-active LPMOs suggest that conserved sugar-interacting residues on the L2, L3 and LC loops may be essential for glucomannan binding, where 4 out of 7 residues are shared by HiLPMO9I, but only one is found in HiLPMO9B. The difference shown between these two H. irregulare LPMOs may reflect distinct biological roles of these enzymes within deconstruction of different plant cell wall polysaccharides during fungal colonization of softwood.

Biochemical studies of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare and their roles in lignocellulose degradation.

Lytic polysaccharide monooxygenases (LPMO) are important redox enzymes produced by microorganisms for the degradation of recalcitrant natural polysaccharides. Heterobasidion irregulare is a white-rot phytopathogenic fungus that causes wood decay in conifers. The genome of this fungus encodes 10 putative Auxiliary Activity family 9 (AA9) LPMOs. We describe the first biochemical characterization of H. irregulare LPMOs through heterologous expression of two CBM-containing LPMOs from this fungus (HiLPMO9H, HiLPMO9I) in Pichia pastoris. The oxidization preferences and substrate specificities of these two enzymes were determined. The two LPMOs were shown to cleave different carbohydrate components of plant cell walls. HiLPMO9H was active on cellulose and oxidized the substrate at the C1 carbon of the pyranose ring at ß-1,4-glycosidic linkages, whereas HiLPMO9I cleaved cellulose with strict oxidization at the C4 carbon of glucose unit at internal bonds, and also showed activity against glucomannan. We propose that the two LPMOs play different roles in the plant-cell-wall degrading system of H. irregulare for degradation of softwood and that the lignocellulose degradation mediated by this white-rot fungus may require collective efforts from multi-types of LPMOs.

Different Alleles of a Gene Encoding Leucoanthocyanidin Reductase (PaLAR3) Influence Resistance against the Fungus Heterobasidion parviporum in Picea abies.

Despite the fact that fungal diseases are a growing menace for conifers in modern silviculture, only a very limited number of molecular markers for pathogen resistance have been validated in conifer species. A previous genetic study indicated that the resistance of Norway spruce (Picea abies) to Heterobasidion annosum s.l., a pathogenic basidiomycete species complex, is linked to a quantitative trait loci that associates with differences in fungal growth in sapwood (FGS) that includes a gene, PaLAR3, which encodes a leucoanthocyanidin reductase. In this study, gene sequences showed the presence of two PaLAR3 allelic lineages in P. abies. Higher resistance was associated with the novel allele, which was found in low frequency in the four P. abies populations that we studied. Norway spruce plants carrying at least one copy of the novel allele showed a significant reduction in FGS after inoculation with Heterobasidion parviporum compared to their half-siblings carrying no copies, indicating dominance of this allele. The amount of (+) catechin, the enzymatic product of PaLAR3, was significantly higher in bark of trees homozygous for the novel allele. Although we observed that the in vitro activities of the enzymes encoded by the two alleles were similar, we could show that allele-specific transcript levels were significantly higher for the novel allele, indicating that regulation of gene expression is responsible for the observed effects in resistance, possibly caused by differences in cis-acting elements that we observe in the promoter region of the two alleles.

Secondary metabolite comparison of the species within the Heterobasidion annosum s.l. complex.

The metabolite production of the five members of the fungal species complex Heterobasidion annosum s.l., i.e. H. annosum s.s., H. abietinum, H. parviporum, H. occidentale and H. irregulare, was analyzed by LC-HRMS. The five members are described to have differences in host preferences: H. annosum s.s. and H. irregulare are pine infecting species, and H. parviporum, H. occidentale and H. abietinum are non-pine infecting. Principal component analysis (PCA) of the LC-HRMS data showed that samples from the five species could be separated into five groups and in accordance with the differences in host preferences. Twenty-three compounds, important to the observed PCA grouping, were isolated and identified. The main contributor to the separation of the pine infecting species from the non-pine infecting species in PC1 was the benzohydrofuran fomannoxin, which was only detected in the pine infecting species H. annosum s.s. and H. irregulare. These two species were further separated in PC3, and one major contributor here was the sesquiterpene deoxyfomannosin A. The three non-pine infecting species were separated in PC2, by epoxydrimenol that was detected in only two of the species and further in PC4, where a few fomannoxin related compounds were important for the grouping. During the work, three unknown compounds were isolated and described: 3-hydroxy-2-(1,3-dihydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde, 3-hydroxy-2-(1,2,3-trihydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde and 3-hydroxy-2,3-dihydrobenzofuran-5-carboxylic acid.

Intronic and plasmid-derived regions contribute to the large mitochondrial genome sizes of Agaricomycetes.

Sizes of mitochondrial genomes vary extensively between fungal species although they typically contain a conserved set of core genes. We have characterised the mitochondrial genome of the conifer root rot pathogen Heterobasidion irregulare and compared the size, gene content and structure of 20 Basidiomycete mitochondrial genomes. The mitochondrial genome of H. irregulare was 114, 193 bp and contained a core set of 15 protein coding genes, two rRNA genes and 26 tRNA genes. In addition, we found six non-conserved open reading frames (ORFs) and four putative plasmid genes clustered in three separate regions together with 24 introns and 14 intronic homing endonuclease genes, unequally spread across seven of the core genes. The size differences among the 20 Basidiomycetes can largely be explained by length variation of intergenic regions and introns. The Agaricomycetes contained the nine largest mitochondrial genomes in the Basidiomycete group and Agaricomycete genomes are significantly (p < 0.001) larger than the other Basidiomycetes. A feature of the Agaricomycete mitochondrial genomes in this study was the simultaneous occurrence of putative plasmid genes and non-conserved ORFs, with Cantharellus cibarius as only exception, where no non-conserved ORF was identified. This indicates a mitochondrial plasmid origin of the non-conserved ORFs or increased mitochondrial genome dynamics of species harbouring mitochondrial plasmids. We hypothesise that two independent factors are the driving forces for large mitochondrial genomes: the homing endonuclease genes in introns and integration of plasmid DNA.

Ectomycorrhizal Cortinarius species participate in enzymatic oxidation of humus in northern forest ecosystems.

In northern forests, belowground sequestration of nitrogen (N) in complex organic pools restricts nutrient availability to plants. Oxidative extracellular enzymes produced by ectomycorrhizal fungi may aid plant N acquisition by providing access to N in macromolecular complexes. We test the hypotheses that ectomycorrhizal Cortinarius species produce Mn-dependent peroxidases, and that the activity of these enzymes declines at elevated concentrations of inorganic N. In a boreal pine forest and a sub-arctic birch forest, Cortinarius DNA was assessed by 454-sequencing of ITS amplicons and related to Mn-peroxidase activity in humus samples with- and without previous N amendment. Transcription of Cortinarius Mn-peroxidase genes was investigated in field samples. Phylogenetic analyses of Cortinarius peroxidase amplicons and genome sequences were performed. We found a significant co-localization of high peroxidase activity and DNA from Cortinarius species. Peroxidase activity was reduced by high ammonium concentrations. Amplification of mRNA sequences indicated transcription of Cortinarius Mn-peroxidase genes under field conditions. The Cortinarius glaucopus genome encodes 11 peroxidases - a number comparable to many white-rot wood decomposers. These results support the hypothesis that some ectomycorrhizal fungi--Cortinarius species in particular--may play an important role in decomposition of complex organic matter, linked to their mobilization of organically bound N.

Polyporales genomes reveal the genetic architecture underlying tetrapolar and bipolar mating systems.

The process of mating in Basidiomycota is regulated by homeodomain-encoding genes (HD) and pheromones and G protein-coupled pheromone receptor genes (P/R). Whether these genes are actually involved in determining mating type distinguishes mating systems that are considered tetrapolar (two locus) from bipolar (one locus). Polyporales are a diverse group of wood-decay basidiomycetes displaying high variability in mating and decay systems. Many of the bipolar species appear to be brown-rot fungi, and it has been hypothesized that there is a functional basis for this correlation. Here we characterize mating genes in recently sequenced Polyporales and other Agaricomycete genomes. All Agaricomycete genomes encode HD and pheromone receptor genes regardless of whether they are bipolar or tetrapolar. The HD genes are organized into a MAT-HD locus with a high degree of gene order conservation among neighboring genes, with the gene encoding mitochondrial intermediate peptidase consistently syntenic but no linkage to the P/R genes. To have a complete dataset of species with known mating systems we determined that Wolfiporia cocos appears to be bipolar, using the criterion that DNA polymorphism of MAT genes should be extreme. Testing the correlation of mating and decay systems while controlling for phylogenetic relatedness failed to identify a statistical association, likely due to the small number of taxa employed. Using a phylogenetic analysis of Ste3 proteins, we identified clades of sequences that contain no known mating type-specific receptors and therefore might have evolved novel functions. The data are consistent with multiple origins of bipolarity within the Agaricomycetes and Polyporales, although the alternative hypothesis that tetrapolarity and bipolarity are reversible states needs better testing.

Evolution of RNA interference proteins dicer and argonaute in Basidiomycota.

RNA interference (RNAi) refers to a mechanism in which cells control gene expression, protect the genome against mobile repetitive DNA sequences, retro elements and transposons, and defend themselves against viruses. Two core components, dicer and argonaute, are central in the RNAi machinery. In this study the evolution of argonaute and dicer genes were analyzed with 43 fungal genomes, with the focus on Basidiomycota. Argonaute and dicer genes are widely represented in Basidiomycota as well as in other fungal groups, but the number of copies of them vary. However, in certain lineages, argonaute or dicer is missing. Our results suggest an ancient duplication of dicer and argonaute genes concurrently with early diversification of the Basidiomycota followed by additional species-specific duplications and losses of more recent origin. Several distinct RNAi pathways exist in fungi, based on structural similarity and phylogenetic relationship, our results indicate that quelling possibly exists in most Basidiomycota, while we could not find any evidence for the MSUD (meiotic-silencing) pathway in Basidiomycota. RNAi has been developed to be an important tool for reverse genetics studies. Because both argonaute and dicer are present in almost all Basidiomycota our results indicate that it should be possible to develop RNAi as a tool for functional studies of genes in most Basidiomycota species.

Extensive trans-specific polymorphism at the mating type locus of the root decay fungus Heterobasidion.

Incompatibility systems in which individuals bearing identical alleles reject each other favor the maintenance of a diversity of alleles. Mushroom mating type loci (MAT) encode for dozens or hundreds of incompatibility alleles whose loss from the population is greatly restricted through negative frequency selection, leading to a system of alleles with highly divergent sequences. Here, we use DNA sequences of homeodomain (HD) encoding genes at the MAT locus of five closely related species of the root rot basidiomycete Heterobasidion annosum sensu lato to show that the extended coalescence time of MAT alleles greatly predates speciation in the group, contrasting loci outside of MAT that show allele divergences largely consistent with the species phylogeny with those of MAT, which show rampant trans-species polymorphism. We observe a roughly 6-fold greater genealogical depth and polymorphism of MAT compared with non-MAT that argues for the maintenance of balanced polymorphism for a minimum duration of 24 My based on a molecular-clock calibrated species phylogeny. As with other basidiomycete HD genes, balancing selection appears to be concentrated at the specificity-determining region in the N-terminus of the protein based on identification of codons under selection and the absence of recombination within the region. However, the elevated polymorphism extends into the nonspecificity determining regions as well as a neighboring non-MAT gene, the mitochondrial intermediate peptidase (MIP). In doing so, increased divergence should decrease recombination among alleles and as a by-product create incompatibilities in the functional domains not involved in allele recognition but in regulating sexual development.

The Norway spruce genome sequence and conifer genome evolution.

Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

A genome-wide association study identifies genomic regions for virulence in the non-model organism Heterobasidion annosum s.s.

The dense single nucleotide polymorphisms (SNP) panels needed for genome wide association (GWA) studies have hitherto been expensive to establish and use on non-model organisms. To overcome this, we used a next generation sequencing approach to both establish SNPs and to determine genotypes. We conducted a GWA study on a fungal species, analysing the virulence of Heterobasidion annosum s.s., a necrotrophic pathogen, on its hosts Picea abies and Pinus sylvestris. From a set of 33,018 single nucleotide polymorphisms (SNP) in 23 haploid isolates, twelve SNP markers distributed on seven contigs were associated with virulence (P<0.0001). Four of the contigs harbour known virulence genes from other fungal pathogens and the remaining three harbour novel candidate genes. Two contigs link closely to virulence regions recognized previously by QTL mapping in the congeneric hybrid H. irregulare × H. occidentale. Our study demonstrates the efficiency of GWA studies for dissecting important complex traits of small populations of non-model haploid organisms with small genomes.