QTL mapping in Fusarium graminearum identified an allele of FgVe1 involved in reduced aggressiveness.

Fusarium graminearum is one of the most frequent causal agents of the Fusarium Head Blight, a cereal disease spread throughout the world, reducing grain production and quality. F. graminearum isolates are genetically and phenotypically highly diverse. Notably, remarkable variations of aggressiveness between isolates have been observed, which could reflect an adaptive potential of this pathogen. In this study, we aimed to characterize the genetic basis of aggressiveness variation observed in an F1 population (n = 94), for which genome sequences of both parental strains are available. Aggressiveness was assessed by a panel of in planta and in vitro proxies during two phenotyping trials including, among others, disease severity and mycotoxin accumulation in wheat spike. One major and single QTL was mapped for all the traits measured, on chromosome I, that explained up to 90% of the variance for disease severity. The confidence interval at the QTL spanned 1.2 Mb and contained 428 genes on the reference genome. Of these, four candidates were selected based on the postulate that a non-synonymous mutation affecting protein function may be responsible for phenotypic differences. Finally, a new mutation was identified and functionally validated in the gene FgVe1, coding for a velvet protein known to be involved in pathogenicity and secondary metabolism production in several fungi.

Landscape of genomic diversity and host adaptation in Fusarium graminearum.

BACKGROUND: Fusarium graminearum is one of the main causal agents of the Fusarium Head Blight, a worldwide disease affecting cereal cultures, whose presence can lead to contaminated grains with chemically stable and harmful mycotoxins. Resistant cultivars and fungicides are frequently used to control this pathogen, and several observations suggest an adaptation of F. graminearum that raises concerns regarding the future of current plant disease management strategies. To understand the genetic basis as well as the extent of its adaptive potential, we investigated the landscape of genomic diversity among six French isolates of F. graminearum, at single-nucleotide resolution using whole-genome re-sequencing. RESULTS: A total of 242,756 high-confidence genetic variants were detected when compared to the reference genome, among which 96% are single nucleotides polymorphisms. One third of these variants were observed in all isolates. Seventy-seven percent of the total polymorphism is located in 32% of the total length of the genome, comprising telomeric/subtelomeric regions as well as discrete interstitial sections, delineating clear variant enriched genomic regions- 7.5 times in average. About 80% of all the F. graminearum protein-coding genes were found polymorphic. Biological functions are not equally affected: genes potentially involved in host adaptation are preferentially located within polymorphic islands and show greater diversification rate than genes fulfilling basal functions. We further identified 29 putative effector genes enriched with non-synonymous effect mutation. CONCLUSIONS: Our results highlight a remarkable level of polymorphism in the genome of F. graminearum distributed in a specific pattern. Indeed, the landscape of genomic diversity follows a bi-partite organization of the genome according to polymorphism and biological functions. We measured, for the first time, the level of sequence diversity for the entire gene repertoire of F. graminearum and revealed that the majority are polymorphic. Those assumed to play a role in host-pathogen interaction are discussed, in the light of the subsequent consequences for host adaptation. The annotated genetic variants discovered for this major pathogen are valuable resources for further genetic and genomic studies.

Deciphering the ability of Agaricus bisporus var. burnettii to produce mushrooms at high temperature (25 °C).

The button mushroom Agaricus bisporus is cultivated almost worldwide. Its cultivation is standardized and a temperature of 16-19 °C is needed during the fruiting period. The development of A. bisporus cultivars able to fruit at higher temperature (FHT) represents a promising alternative to reduce energy costs during cultivation in hot countries as well as in temperate countries during the hot season. A. bisporus var. burnettii is able to fruit at 25 °C. Understanding the biological mechanisms that underlie such a thermo-tolerance is a prerequisite to further development of breeding strains. The foundation of the FHT ability of the var. burnettii was dissected using a combination of candidate gene approaches and genetic tools. Transcript profiling of A. bisporus var. burnettii at two developmental stages (primordium P and sporophore SP) under two fruit-producing temperature conditions (17 °C and 25 °C) were established by cDNA-AFLP. The expression patterns were more similar within the same stage at the two different temperatures rather than between stages under the same temperature. Only one transcript-derived fragment (TDF) sequence differentially expressed between temperatures was recovered but it could not be further characterized. Twenty-nine TDF sequences differentially expressed between development stages were obtained. The phenotypic assessment of an intervarietal A. bisporus var. bisporus×A. bisporus var. burnettii progeny demonstrated the complex inheritance of the FHT trait. Two quantitative trait loci (QTL) involved in the number of fruit bodies yielded at 25 °C were found on LG II and LG VI. Two functional candidate genes known to be potentially involved in A. bisporus thermo-tolerance, a heat shock protein (HSP70) gene and a gene coding for a para-aminobenzoic acid synthase (PABA), were found in the vicinity of the QTL on LG II. Several positional candidate genes have been also identified in the confidence interval of the QTL on LG VI and are promising for further fine mapping purpose.

The first set of expressed sequence tags (EST) from the medicinal mushroom Agaricus subrufescens delivers resource for gene discovery and marker development.

Agaricus subrufescens is one of the most important culinary-medicinal cultivable mushrooms with potentially high-added-value products and extended agronomical valorization. The development of A. subrufescens-related technologies is hampered by, among others, the lack of suitable molecular tools. Thus, this mushroom is considered as a genomic orphan species with a very limited number of available molecular markers or sequences. To fill this gap, this study reports the generation and analysis of the first set of expressed sequence tags (EST) for A. subrufescens. cDNA fragments obtained from young sporophores (SP) and vegetative mycelium in liquid culture (CL) were sequenced using 454 pyrosequencing technology. After assembly process, 4,989 and 5,125 sequences were obtained in SP and CL libraries, respectively. About 87% of the EST had significant similarity with Agaricus bisporus-predicted proteins, and 79% correspond to known proteins. Functional categorization according to Gene Ontology could be assigned to 49% of the sequences. Some gene families potentially involved in bioactive compound biosynthesis could be identified. A total of 232 simple sequence repeats (SSRs) were identified, and a set of 40 EST-SSR polymorphic markers were successfully developed. This EST dataset provides a new resource for gene discovery and molecular marker development. It constitutes a solid basis for further genetic and genomic studies in A. subrufescens.

Comparative linkage mapping in the white button mushroom Agaricus bisporus provides foundation for breeding management.

To assess the feasibility of marker-assisted selection in mushrooms, a comparative mapping study between two connected populations of the white button mushroom Agaricus bisporus was performed. The first mapping population had been used already for the construction of the A. bisporus reference linkage map. In the present study, a new linkage map based on the segregation analysis of a second generation hybrid progeny was developed. In order to increase the number of available anchor markers, we developed a conversion procedure of an amplified fragment length polymorphism (AFLP) fragment into sequence-specific PCR marker. Seventeen AFLP-converted markers (ACM) were then used for mapping purpose, among which 14 were common to the two maps. The linkage map presented herein consists of 183 markers (53 cleaved amplified polymorphic sequence, 16 SSR, 17 ACM, 96 AFLP and PPC1 locus), distributed among 13 linkage groups (LG), and covering 851 cM. Thanks to 84 common markers, we have stated that marker order was well conserved, except for LG I; significant unequal recombination rates occurred over the whole genome; regions with markers showing skewed segregation patterns differed between the two maps. Our results suggested a strong impact of the genetic background on recombination ability. Consequences for mushroom breeding are discussed. These maps will facilitate further comparative mapping studies of quantitative trait locus detection.

An expanded genetic linkage map of an intervarietal Agaricus bisporus var. bisporusxA. bisporus var. burnettii hybrid based on AFLP, SSR and CAPS markers sheds light on the recombination behaviour of the species.

A genetic linkage map for the edible basidiomycete Agaricus bisporus was constructed from 118 haploid homokaryons derived from an intervarietal A. bisporus var. bisporus x A. bisporus var. burnettii hybrid. Two hundred and thirty-one AFLP, 21 SSR, 68 CAPS markers together with the MAT, BSN, PPC1 loci and one allozyme locus (ADH) were evenly spread over 13 linkage groups corresponding to the chromosomes of A. bisporus. The map covers 1156cM, with an average marker spacing of 3.9cM and encompasses nearly the whole genome. The average number of crossovers per chromosome per individual is 0.86. Normal recombination over the entire genome occurs in the heterothallic variety, burnettii, contrary to the homothallic variety, bisporus, which showed adaptive genome-wide suppressed recombination. This first comprehensive genetic linkage map for A. bisporus provides foundations for quantitative trait analyses and breeding programme monitoring, as well as genome organisation studies.

Novel microsatellite markers suitable for genetic studies in the white button mushroom Agaricus bisporus.

Co-dominant microsatellite molecular markers were obtained from the Agaricus bisporus cultivated mushroom. Their potential for both the molecular characterisation of commercial strains and the monitoring of the intraspecific genetic variation was demonstrated. The analysis of 673 unique sequences issued from public database and 59 from an enriched A. bisporus genomic library resulted in the development of a total of 33 single sequence repeat or microsatellite (SSR) markers. Their usefulness for genetic analysis was assessed on 28 strains, which include six cultivars representative of traditional lineage, two hybrids and 20 strains originating from wild populations. A. bisporus SSR markers displayed each from two to ten alleles, with an average of 5.6 alleles per locus. The observed heterozygosity ranged from 0 to 0.88. Cluster analysis resulting from SSR fingerprintings was in agreement with published A. bisporus population structure. A combination of only three selected SSR markers was sufficient to discriminate unambiguously 27 out of 28 distinct genotypes. However, the two genetically related hybrids were not distinguishable. Multiplexing was tested, and up to seven loci could be genotyped simultaneously. We are therefore reporting the first development in A. bisporus of a set of microsatellite markers powerful and suitable for genetic analysis.

High-resolution mapping of the recombination landscape of the phytopathogen Fusarium graminearum suggests two-speed genome evolution.

Recombination is a major evolutionary force, increasing genetic diversity and permitting efficient coevolution of fungal pathogen(s) with their host(s). The ascomycete Fusarium graminearum is a devastating pathogen of cereal crops, and can contaminate food and feed with harmful mycotoxins. Previous studies have suggested a high adaptive potential of this pathogen, illustrated by an increase in pathogenicity and resistance to fungicides. In this study, we provide the first detailed picture of the crossover events occurring during meiosis and discuss the role of recombination in pathogen evolution. An experimental recombinant population (n = 88) was created and genotyped using 1306 polymorphic markers obtained from restriction site-associated DNA sequencing (RAD-seq) and aligned to the reference genome. The construction of a high-density linkage map, anchoring 99% of the total length of the reference genome, allowed the identification of 1451 putative crossovers, positioned at a median resolution of 24 kb. The majority of crossovers (87.2%) occurred in a relatively small portion of the genome (30%). All chromosomes demonstrated recombination-active sections, which had a near 15-fold higher crossover rate than non-active recombinant sections. The recombination rate showed a strong positive correlation with nucleotide diversity, and recombination-active regions were enriched for genes with a putative role in host-pathogen interaction, as well as putative diversifying genes. Our results confirm the preliminary analysis observed in other F. graminearum strains and suggest a conserved 'two-speed' recombination landscape. The consequences with regard to the evolutionary potential of this major fungal pathogen are also discussed.

The Genetic Linkage Map of the Medicinal Mushroom Agaricus subrufescens Reveals Highly Conserved Macrosynteny with the Congeneric Species Agaricus bisporus.

Comparative linkage mapping can rapidly facilitate the transfer of genetic information from model species to orphan species. This macrosynteny analysis approach has been extensively used in plant species, but few example are available in fungi, and even fewer in mushroom crop species. Among the latter, the Agaricus genus comprises the most cultivable or potentially cultivable species. Agaricus bisporus, the button mushroom, is the model for edible and cultivable mushrooms. We have developed the first genetic linkage map for the basidiomycete A. subrufescens, an emerging mushroom crop known for its therapeutic properties and potential medicinal applications. The map includes 202 markers distributed over 16 linkage groups (LG), and covers a total length of 1701 cM, with an average marker spacing of 8.2 cM. Using 96 homologous loci, we also demonstrated the high level of macrosynteny with the genome of A. bisporus The 13 main LG of A. subrufescens were syntenic to the 13 A. bisporus chromosomes. A disrupted synteny was observed for the three remaining A. subrufescens LG. Electronic mapping of a collection of A. subrufescens expressed sequence tags on A. bisporus genome showed that the homologous loci were evenly spread, with the exception of a few local hot or cold spots of homology. Our results were discussed in the light of Agaricus species evolution process. The map provides a framework for future genetic or genomic studies of the medicinal mushroom A. subrufescens.

Development of polymorphic microsatellite markers issued from pyrosequencing technology for the medicinal mushroom Agaricus subrufescens.

The recently described procedure of microsatellite-enriched library pyrosequencing was used to isolate microsatellite loci in the gourmet and medicinal mushroom Agaricus subrufescens. Three hundred and five candidate loci containing at least one simple sequence repeats (SSR) locus and for which primers design was successful, were obtained. From a subset of 95 loci, 35 operational and polymorphic SSR markers were developed and characterized on a sample of 14 A. subrufescens genotypes from diverse origins. These SubSSR markers each displayed from two to 10 alleles with an average of 4.66 alleles per locus. The observed heterozygosity ranged from 0 to 0.71. Several multiplex combinations can be set up, making it possible to genotype up to six markers easily and simultaneously. Cross-amplification in some closely congeneric species was successful for a subset of loci. The 35 microsatellite markers developed here provide a highly valuable molecular tool to study genetic diversity and reproductive biology of A. subrufescens.

Evidence for outcrossing via the Buller phenomenon in a substrate simultaneously inoculated with spores and mycelium of Agaricus bisporus.

In Agaricus bisporus, traditional cultivars and most of the wild populations belong to A. bisporus var. bisporus, which has a predominantly pseudohomothallic life cycle in which most meiospores are heterokaryons (n + n). A lower proportion of homokaryotic (n) meiospores, which typify the heterothallic life cycle, also are produced. In wild populations, pseudohomothallism was thought previously to play a major role, but recent analyses have found that significant outcrossing also may occur. We inoculated a standard substrate for A. bisporus cultivation simultaneously with homokaryotic mycelium from one parent and spores from a second parent. Culture trays produced numerous sporocarps that could theoretically have resulted from five different reproductive modes (pseudohomothallism, selfing or outcrossing via heterothallism, and selfing or outcrossing via the Buller phenomenon [i.e., between a homokaryon and a heterokaryon]). Most or all of the sporocarps resulted from outcrossing between the inoculated homokaryon and the inoculated heterokaryotic spores (or mycelia that grew from them). These data broaden our understanding of population dynamics under field conditions and provide an outcrossing method that could be used in commercial breeding programs.