Melanin plays an important role in virulence and antimicrobial resistance in several fungal pathogens. The wheat pathogen Zymoseptoria tritici is important worldwide, but little is known about the genetic architecture of pathogenicity, including the production of melanin. Because melanin production can exhibit complex inheritance, we used quantitative trait locus (QTL) mapping in two crosses to identify the underlying genes. Restriction site−associated DNA sequencing was used to genotype 263 (cross 1) and 261 (cross 2) progeny at ~8500 single-nucleotide polymorphisms and construct two dense linkage maps. We measured gray values, representing degrees of melanization, for single-spore colonies growing on Petri dishes by using a novel image-processing approach that enabled high-throughput phenotyping. Because melanin production can be affected by stress, each offspring was grown in two stressful environments and one control environment. We detected six significant QTL in cross 1 and nine in cross 2, with three QTL shared between the crosses. Different QTL were identified in different environments and at different colony ages. By obtaining complete genome sequences for the four parents and analyzing sequence variation in the QTL confidence intervals, we identified 16 candidate genes likely to affect melanization. One of these candidates was PKS1, a polyketide synthase gene known to play a role in the synthesis of dihydroxynaphthalene melanin. Three candidate quantitative trait nucleotides were identified in PKS1. Many of the other candidate genes were not previously associated with melanization.

Recombination has an impact on genome evolution by maintaining chromosomal integrity, affecting the efficacy of selection, and increasing genetic variability in populations. Recombination rates are a key determinant of the coevolutionary dynamics between hosts and their pathogens. Historic recombination events created devastating new pathogens, but the impact of ongoing recombination in sexual pathogens is poorly understood. Many fungal pathogens of plants undergo regular sexual cycles, and sex is considered to be a major factor contributing to virulence. We generated a recombination map at kilobase-scale resolution for the haploid plant pathogenic fungus Zymoseptoria tritici. To account for intraspecific variation in recombination rates, we constructed genetic maps from two independent crosses. We localized a total of 10,287 crossover events in 441 progeny and found that recombination rates were highly heterogeneous within and among chromosomes. Recombination rates on large chromosomes were inversely correlated with chromosome length. Short accessory chromosomes often lacked evidence for crossovers between parental chromosomes. Recombination was concentrated in narrow hotspots that were preferentially located close to telomeres. Hotspots were only partially conserved between the two crosses, suggesting that hotspots are short-lived and may vary according to genomic background. Genes located in hotspot regions were enriched in genes encoding secreted proteins. Population resequencing showed that chromosomal regions with high recombination rates were strongly correlated with regions of low linkage disequilibrium. Hence, genes in pathogen recombination hotspots are likely to evolve faster in natural populations and may represent a greater threat to the host.KEYWORDS recombination hotspots; pathogen evolution; restriction site-associated DNA sequencing; population genomics; linkage disequilibrium R ECOMBINATION is a fundamental process that shapes the evolution of genomes. Crossover between homologous chromosomes ensures proper segregation during meiosis (Mather 1938;Baker et al. 1976;Hassold and Hunt 2001), and the integrity of chromosomal structure over evolutionary time is affected by the frequency of recombination. Sex chromosomes in plants and animals and mating-type regions of fungi experience degeneration, including significant gene loss and sequence rearrangements, after cessation of recombination between homologs (Bull 1983;Charlesworth and Charlesworth 2000;Brown et al. 2005;Menkis et al. 2008;Wilson and Makova 2009). Recombination breaks up linkage between alleles of different loci and creates novel haplotypes and phenotypic diversity. Through this process, recombination promotes adaptation because selection acting across multiple loci becomes more efficient as the linkage between loci decreases (Hill and Robertson 1966;Otto and Barton 1997;Otto and Lenormand 2002).Recombination also plays a major role in the coevolutionary dynamics of hosts and their pathogens. A major driver to maintain sexual...

SUMMARYWe conducted a comprehensive analysis of virulence in the fungal wheat pathogen Zymoseptoria tritici using quantitative trait locus (QTL) mapping. High-throughput phenotyping based on automated image analysis allowed the measurement of pathogen virulence on a scale and with a precision that was not previously possible. Across two mapping populations encompassing more than 520 progeny, 540 710 pycnidia were counted and their sizes and grey values were measured. A significant correlation was found between pycnidia size and both spore size and number. Precise measurements of percentage leaf area covered by lesions provided a quantitative measure of host damage. Combining these large and accurate phenotypic datasets with a dense panel of restriction site-associated DNA sequencing (RADseq) genetic markers enabled us to genetically dissect pathogen virulence into components related to host damage and those related to pathogen reproduction. We showed that different components of virulence can be under separate genetic control. Large-and small-effect QTLs were identified for all traits, with some QTLs specific to mapping populations, cultivars and traits and other QTLs shared among traits within the same mapping population. We associated the presence of four accessory chromosomes with small, but significant, increases in several virulence traits, providing the first evidence for a meaningful function associated with accessory chromosomes in this organism. A large-effect QTL involved in host specialization was identified on chromosome 7, leading to the identification of candidate genes having a large effect on virulence.

Many studies have scrutinized the nutritional benefits of arbuscular mycorrhizal associations to their host plants, while the carbon (C) balance of the symbiosis has often been neglected. Here, we present quantification of both the C costs and the phosphorus (P) uptake benefits of mycorrhizal association between barrel medic (Medicago truncatula) and three arbuscular mycorrhizal fungal species, namely Glomus intraradices, Glomus claroideum, and Gigaspora margarita. Plant growth, P uptake and C allocation were assessed 7 weeks after sowing by comparing inoculated plants with their non-mycorrhizal counterparts, supplemented with different amounts of P. Isotope tracing ³³P and ¹³C) was used to quantify both the mycorrhizal benefits and the costs, respectively. G. intraradices supported greatest plant P acquisition and incurred high C costs, which lead to similar plant growth benefits as inoculation with G. claroideum, which was less efficient in supporting plant P acquisition, but also required less C. G. margarita imposed large C requirement on the host plant and provided negligible P uptake benefits. However, it did not significantly reduce plant growth due to sink strength stimulation of plant photosynthesis. A simple experimental system such as the one established here should allow quantification of mycorrhizal costs and benefits routinely on a large number of experimental units. This is necessary for rapid progress in assessment of C fluxes between the plants and different mycorrhizal fungi or fungal communities, and for understanding the dynamics between mutualism and parasitism in mycorrhizal symbioses.

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