A Picea abies linkage map based on SNP markers identifies QTLs for four aspects of resistance to Heterobasidion parviporum infection.

A consensus linkage map of Picea abies, an economically important conifer, was constructed based on the segregation of 686 SNP markers in a F1 progeny population consisting of 247 individuals. The total length of 1889.2 cM covered 96.5% of the estimated genome length and comprised 12 large linkage groups, corresponding to the number of haploid P. abies chromosomes. The sizes of the groups (from 5.9 to 9.9% of the total map length) correlated well with previous estimates of chromosome sizes (from 5.8 to 10.8% of total genome size). Any locus in the genome has a 97% probability to be within 10 cM from a mapped marker, which makes the map suited for QTL mapping. Infecting the progeny trees with the root rot pathogen Heterobasidion parviporum allowed for mapping of four different resistance traits: lesion length at the inoculation site, fungal spread within the sapwood, exclusion of the pathogen from the host after initial infection, and ability to prevent the infection from establishing at all. These four traits were associated with two, four, four and three QTL regions respectively of which none overlapped between the traits. Each QTL explained between 4.6 and 10.1% of the respective traits phenotypic variation. Although the QTL regions contain many more genes than the ones represented by the SNP markers, at least four markers within the confidence intervals originated from genes with known function in conifer defence; a leucoanthocyanidine reductase, which has previously been shown to upregulate during H. parviporum infection, and three intermediates of the lignification process; a hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase, a 4-coumarate CoA ligase, and a R2R3-MYB transcription factor.

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.

A 2nd generation linkage map of Heterobasidion annosum s.l. based on in silico anchoring of AFLP markers.

In this study, we present a 2(nd) generation genetic linkage map of a cross between the North American species Heterobasidion irregulare and H. occidentale, based on the alignment of the previously published 1(st) generation map to the parental genomes. We anchored 216 of the original 308 AFLP markers to their respective restriction sites using an in silico-approach. The map resolution was improved by adding 146 sequence-tagged microsatellite markers and 39 sequenced gene markers. The new markers confirmed the positions of the anchored AFLP markers, fused the original 39 linkage groups together into 17, and fully expanded 12 of these to single groups covering entire chromosomes. Map coverage of the genome increased from 55.3% to 92.8%, with 96.3% of 430 markers collinearly aligned with the genome sequence. The anchored map also improved the H. irregulare assembly considerably. It identified several errors in scaffold arrangements and assisted in reducing the total number of major scaffolds from 18 to 15. This denser, more comprehensive map allowed sequence-based mapping of three intersterility loci and one mating type locus. This demonstrates the possibility to utilize an in silico procedure to convert anonymous markers into sequence-tagged ones, as well as the power of a sequence-anchored linkage map and its usefulness in the assembly of a whole genome sequence.

Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen.

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

The pathogenic white-rot fungus Heterobasidion parviporum triggers non-specific defence responses in the bark of Norway spruce.

Norway spruce [Picea abies (L.) Karst.] is one of the economically most important conifer species in Europe. The major pathogen on Norway spruce is Heterobasidion parviporum (Fr.) Niemelä & Korhonen. To achieve a better understanding of Norway spruce's defence mechanisms, transcriptional responses in bark to H. parviporum infection were compared with the response to wounding using cDNA-amplified fragment length polymorphism. The majority of the recovered transcript-derived fragments (TDFs) showed a similar expression pattern for infection and wounding treatment, although inoculated samples showed an enhanced reaction. Genes related to systemic acquired resistance, e.g., PR1, accumulated after H. parviporum infection. Simultaneously, several transcripts involved in various aspects of jasmonic acid (JA)- and ethylene (ET)-mediated signalling accumulated. Genes involved in the ubiquitin/proteasome system were also regulated. Expression patterns have been confirmed by quantitative polymerase chain reaction. The expression patterns of the isolated TDFs suggest that infection with H. parviporum in Norway spruce induces a broad defence, with many similarities to non-specific defence responses in angiosperms. The parallel induction of salicylic acid- and JA/ET-mediated pathways implies spatially separated responses in different cell layers, with and without hyphal contact. A set of TDFs were analysed in an independent experiment with unrelated material treated with wounding or with inoculation with H. parviporum or Phlebiopsis gigantea, verifying the original observations and underlining the non-specific defence responses. In addition, our data suggest that rerouting of carbon in secondary metabolism is an integral part of Norway spruce induced defence. We report the sequences of three 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase genes (PaDAHP1, PaDAHP2 and PaDAHP3) and their relative expression in response to wounding and infection with H. parviporum and P. gigantea. The results clearly indicate differential regulation of the three DAHPs in the induced defence responses in Norway spruce. This study gives insights into the central mechanisms in the induced defences in Norway spruce.

Prediction of stopping-power ratios in flattening-filter free beams.

PURPOSE: In recent years, there has been an increasing interest in flattening-filter free (FFF) beams. However, since the removal of the flattening filter will affect both the mean and the variance of the energy spectrum, current beam-quality specifiers may not be adequate for reference dosimetry in such beams. The purpose of this work was to investigate an alternative, more general beam-quality specifier. METHODS: The beam-quality specifier used in this work was a combination of the kerma-weighted mean and the coefficient of variation of the linear attenuation coefficient in water. These parameters can in theory be determined from narrow-beam transmission measurements using a miniphantom "in-air," which is a measurement condition well suited also to small and nonstandard fields. The relation between the Spencer-Attix stopping-power ratios and this novel beam-quality specifier was described by a simple polynomial. For reference, the authors used Monte Carlo calculated spectra and stopping-power data for nine different beams, with and without flattening filter. RESULTS: The polynomial coefficients were obtained by least-squares optimization. For all beams included in this investigation, the average of the differences between the predicted and the Monte Carlo calculated stopping-power ratios was 0.02 +/- 0.17% (1 SD) (including TomoTherapy and CyberKnife example beams). CONCLUSIONS: An alternative dual-parameter beam-quality specifier was investigated. The evaluation suggests that it can be used successfully to predict stopping-power ratios in FFF as well as conventional beams, regardless of filtration.

Dosimetric characteristics of 6 and 10MV unflattened photon beams.

PURPOSE: To determine dosimetric properties of unflattened megavoltage photon beams. MATERIALS AND METHODS: Dosimetric data including depth dose, profiles, output factors and phantom scatter factors from three different beam qualities provided by Elekta Precise linacs, operated with and without flattening filter were examined. Additional measurements of leaf transmission, leakage radiation and surface dose were performed. In flattening filter free (FFF) mode a 6-mm thick copper filter was placed into the beam to stabilize it. RESULTS: Depths of dose maxima for flattened and unflattened beams did not deviate by more than 2mm and penumbral widths agreed within 1mm. In FFF mode the collimator exchange effect was found to be on average 0.3% for rectangular fields. Between maximum and minimum field size head scatter factors of unflattened beams showed on average 40% and 56% less variation for 6 and 10MV beams than conventional beams. Phantom scatter factors for FFF beams differed up to 4% from the published reference data. For field sizes smaller than 15cm, surface doses relative to the dose at d(max) increased for unflattened beams with maximum differences of 7% at 6MV and 25% at 10MV for a 5x5cm(2) field. For a 30x30cm(2) field, relative surface dose decreased by about 10% for FFF beams. Leaf transmission on the central axis was 0.3% and 0.4% lower for unflattened 6 and 10MV beams, respectively. Leakage radiation was reduced by 52% for 6MV and by 65% for 10MV unflattened beams. CONCLUSIONS: The results of the study were independently confirmed at two radiotherapy centres. Phantom scatter reference data need to be reconsidered for medical accelerators operated without a flattening filter.

Identification of quantitative trait loci affecting virulence in the basidiomycete Heterobasidion annosum s.l.

Identification of virulence factors of phytopathogens is important for the fundamental understanding of infection and disease progress in plants and for the development of control strategies. We have identified quantitative trait loci (QTL) for virulence on 1-year-old Pinus sylvestris and 2-year-old Picea abies seedlings and positioned them on a genetic linkage map of the necrotrophic phytopathogen Heterobasidion annosum sensu lato (s.l.), a major root rot pathogen on conifers. The virulence of 102 progeny isolates was analysed using two measurements: lesion lengths and fungal growth in sapwood from a cambial infection site. We found negative virulence effects of hybridization although this was contradicted on a winter-hardened spruce. On P. abies, both measurements identified several partially overlapping QTLs on linkage group (LG) 15 of significant logarithm of odds (LOD) values ranging from 2.31 to 3.85. On P. sylvestris, the lesion length measurement also identified a QTL (LOD 3.09) on LG 15. Moreover, QTLs on two separate smaller LGs, with peak LOD values of 2.78 and 4.58 were identified for fungal sapwood growth and lesion lengths, respectively. The QTL probably represent loci important for specific as well as general aspects of virulence on P. sylvestris and P. abies.

Genetics and QTL mapping of somatic incompatibility and intraspecific interactions in the basidiomycete Heterobasidion annosum s.l.

Somatic incompatibility (SI) is a system by which filamentous fungi can distinguish self from non-self by delimiting the own mycelia from that of other individuals of the same species. In this study, we show that SI in the basidiomycete Heterobasidion annosum sensu lato is controlled by four loci by observing the frequency of somatically compatible pairings in two experiments where isolates were paired in all possible combination. The first experiment utilized 63 heterokaryons each with one unique nucleus chosen from an array of sibling homokaryons paired with one unrelated nucleus of homokaryotic isolate TC-39-7. The second experiment used 39 heterokaryons each with one unique nucleus from the array of sibling homokaryons backcrossed with one of the parental strains (TC-122-12). We observed that SI allelic differences in a pairing alone are not enough to determine the degree of somatic incompatibility. In the first experiment, we also observed other interactions such as hyphal walls in interaction zones, increased exudation of dark-coloured metabolites and increased production of aerial hyphae. QTLs for the respective traits were positioned to a genetic linkage map of the H. annosum genome. Map-based cloning of the corresponding loci will shed much new light on intraspecific interactions in basidiomycetes.

An AFLP-markers based genetic linkage map of Heterobasidion annosum locating intersterility genes.

A genetic linkage map of the basidiomycete Heterobasidion annosum, casual agent of root rot in conifers, was constructed from a compatible mating between isolates from the North American S and P intersterility groups. In a population consisting of 102 progeny isolates, 358 AFLP markers were scored. The linkage analysis generated 19 large linkage groups, containing 6 or more markers, which covered 1468 cM. The physical size to genetic distance was approximately 11.1 kbp/cM. Segregation of three intersterility gene loci were analysed through mating of the progeny isolates with three tester strains carrying known intersterility genotypes. The loci for the two intersterility genes (S and P) were successfully located in the map. Segregation of the mating type locus was analysed by backcrossing the progeny isolates with their parental strains. The mating type locus could not be located in the map.