SummaryLeptosphaeria maculans is the ascomycete responsible for one of the most damaging diseases of oilseed rape (Brassica napus), stem canker of crucifers. Both avirulence (AvrLm) genes in the fungus and resistance (Rlm) genes in the plant are genetically clustered. Using a map-based cloning strategy, we delineated a 238 kb region containing the AvrLm7 locus. Structural features of the region were reminiscent of those previously found on another chromosome for genomic regions encompassing AvrLm1 and AvrLm6, i.e. GC-equilibrated, gene-rich isochores alternating with AT-rich, recombinationdeficient, gene-poor isochores. These latter corresponded to mosaics of degenerated and truncated transposable elements. AvrLm7 is the only gene located within a 60 kb AT-rich isochore. It induced resistance responses in plants harbouring either Rlm7 or Rlm4, and was thus renamed AvrLm4-7. It encodes a 143-amino-acid cysteine-rich protein, predicted to be secreted, and strongly induced during early stages of plant infection. Sequencing and restriction analyses of AvrLm4-AvrLm7 or avrLm4-AvrLm7 alleles in L. maculans field isolates, and targeted point mutagenesis strongly suggested that one single base mutation, leading to the change of a glycine to an arginine residue, was responsible for the loss of AvrLm4 specificity whereas AvrLm7 recognition was unaltered.

SummaryExtending the durability of plant resistance genes towards fungal pathogens is a major challenge. We identified and investigated the relationship between two avirulence genes of Leptosphaeria maculans, AvrLm3 and AvrLm4-7. When an isolate possesses both genes, the Rlm3-mediated resistance of oilseed rape (Brassica napus) is not expressed due to the presence of AvrLm4-7 but virulent isolates toward Rlm7 recover the AvrLm3 phenotype.Combining genetic and genomic approaches (genetic mapping, RNA-seq, BAC (bacterial artificial chromosome) clone sequencing and de novo assembly) we cloned AvrLm3, a telomeric avirulence gene of L. maculans. AvrLm3 is located in a gap of the L. maculans reference genome assembly, is surrounded by repeated elements, encodes for a small secreted cysteinerich protein and is highly expressed at early infection stages.Complementation and silencing assays validated the masking effect of AvrLm4-7 on AvrLm3 recognition by Rlm3 and we showed that the presence of AvrLm4-7 does not impede AvrLm3 expression in planta. Y2H assays suggest the absence of physical interaction between the two avirulence proteins.This unusual interaction is the basis for field experiments aiming to evaluate strategies that increase Rlm7 durability.

Modern agriculture favours the selection and spread of novel plant diseases. Furthermore, crop genetic resistance against pathogens is often rendered ineffective within a few years of its commercial deployment. Leptosphaeria maculans, the cause of phoma stem canker of oilseed rape, develops gene-for-gene interactions with its host plant, and has a high evolutionary potential to render ineffective novel sources of resistance in crops. Here, we established a four-year field experiment to monitor the evolution of populations confronted with the newly released Rlm7 resistance and to investigate the nature of the mutations responsible for virulence against Rlm7. A total of 2551 fungal isolates were collected from experimental crops of a Rlm7 cultivar or a cultivar without Rlm7. All isolates were phenotyped for virulence and a subset was genotyped with neutral genetic markers. Virulent isolates were investigated for molecular events at the AvrLm4-7 locus. Whilst virulent isolates were not found in neighbouring crops, their frequency had reached 36% in the experimental field after four years. An extreme diversity of independent molecular events leading to virulence was identified in populations, with large-scale Repeat Induced Point mutations or complete deletion of AvrLm4-7 being the most frequent. Our data suggest that increased mutability of fungal genes involved in the interactions with plants is directly related to their genomic environment and reproductive system. Thus, rapid allelic diversification of avirulence genes can be generated in L. maculans populations in a single field provided that large population sizes and sexual reproduction are favoured by agricultural practices.

Switchgrass (Panicum virgatum L.) is a native perennial warm season (C 4 ) grass that has been identified as a promising species for bioenergy research and production. Consequently, biomass yield and feedstock quality improvements are high priorities for switchgrass research. The objective of this study was to develop a switchgrass genetic linkage map using a full-sib pseudo-testcross mapping population derived from a cross between two heterozygous genotypes selected from the lowland cultivar 'Alamo' (AP13) and the upland cultivar 'Summer' (VS16). The female parent (AP13) map consists of 515 loci in 18 linkage groups (LGs) and spans 1,733 cM. The male parent (VS16) map arranges 363 loci in 17LGs and spans 1,508 cM. No obvious cause for the lack of one LG in VS16 could be identified. Comparative analyses between the AP13 and VS16 maps showed that the two major ecotypic classes of switchgrass have highly colinear maps with similar recombination rates, suggesting that chromosomal exchange between the two ecotypes should be able to occur freely. The AP13 and VS16 maps are also highly similar with respect to marker orders and recombination levels to previously published switchgrass maps. The genetic maps will be used to identify quantitative trait loci associated with biomass and quality traits. The AP13 Desalegn Serba, Limin Wu, and Guillaume Daverdin contributed equally to the work. Bioenerg. Res. (2013) 6:953-965 DOI 10.1007 genotype was used for the whole genome-sequencing project and the map will thus also provide a tool for the anchoring of the switchgrass genome assembly. Electronic supplementary material

Switchgrass (Panicum virgatum L.) biomass yield and feedstock quality improvement are priority research areas for bioenergy feedstock development. Identification of quantitative trait loci (QTL) underlying these traits and of traitlinked markers for application in marker-assisted selection (MAS) is of paramount importance in facilitating switchgrass breeding. Detection of QTL for biomass yield and plant height was conducted on parental linkage maps constructed using a heterozygous pseudo-F 1 population derived from a cross between lowland Alamo genotype AP13 and upland Summer genotype VS16. QTL analysis was performed with composite interval mapping. Four QTL for biomass yield and five QTL for plant height were identified using best linear unbiased predictors across ten and eight environments, respectively. The phenotypic variability explained (PVE) by QTL detected in the across environments analysis ranged from 4.9 to 12.4 % for biomass yield and 5.1 to 12.0 % for plant height. A total of 34 and 38 main effect QTL were detected for biomass yield and plant height, respectively, when data from each environment were analyzed separately. The PVE by individual environment QTL ranged from 3.3 to 15.3 % for biomass yield and from 4.3 to 17.4 % for plant height. In addition, 60 and 51 epistatic QTL were detected for biomass yield and plant height, respectively. Significant QTL by environment interactions were detected for QTL mapped in eight genomic regions for each of the two traits. Seven QTL affected both traits and may represent pleiotropic loci. Overall, 11 genomic regions were identified that were important in controlling biomass yield and/or plant height in switchgrass. The markers linked to the main effect and epistatic QTL may be used in MAS to maximize selection gain in switchgrass breeding, leading to a faster development of better biofuel cultivars.Electronic supplementary material The online version of this article (

Summary The avirulence gene AvrLm4–7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4–7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4–7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well‐conserved among AvrLm4–7 homologs. Loss of recognition of AvrLm4–7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well‐conserved C–terminal motif or close to the glycine involved in Rlm4‐mediated recognition, resulting in the loss of Rlm7‐mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4–7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4–7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.

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A switchgrass (Panicum virgatum L.) consensus map was developed that combined data from two mapping populations and integrated recombination data from both parents of this largely obligate outcrossing species. The consensus map consisted of 1,321 loci and spanned 2,122 cM. An analysis of the distribution of genic and genomic markers across the length of a linkage group showed that genic markers were relatively overrepresented in distal regions, while genomic markers were relatively overrepresented in pericentromeric regions. Furthermore, genic markers located in distal and pericentromeric regions identified orthologs in the genome of the closely related species foxtail millet (Setaria italica (L.) P. Beauv.) at a similar rate, but fewer orthologs were found for genomic markers that mapped to pericentromeric regions compared to distal regions. Because cross-species sequence conservation is largely limited to genes, our data suggest that genomic markers that map to distal regions are more likely to be derived from genic regions than those that map to pericentromeric regions. A comparative analysis between loci on the switchgrass consensus map and their orthologs in the foxtail millet genome showed that the two species varied by at least nine inversions and one reciprocal translocation. Extending the comparative analysis to sorghum demonstrated that the majority of the rearrangements occurred in the foxtail millet genome in the past 13 million years. The reduction in chromosome number from 10 in the Andropogoneae to 9 in the Paniceae was achieved through a complex set of rearrangements involving three ancestral chromosomes orthologous to sorghum chromosomes 6, 8, and 9 and chromosomes III and VII in the lineage leading to switchgrass and foxtail millet. Upon insertion of ancestral chromosome 9 into ancestral chromosome 8, the centromere of the recipient chromosome was lost, and this was accompanied by a loss of repetitive DNA. The switchgrass-foxtail millet comparative map provides a guide of the rearrangements that need to be taken into account when using foxtail millet as a surrogate for switchgrass in genetic analyses.