RESUMEN
Arbuscular mycorrhizal fungi (AMF) are obligate mutualists that can enhance nutrition and growth of their plant hosts while providing protection against pathogens. AMF produce spores and hyphal networks that can carry thousands of nuclei in a continuous cytoplasm, with no evidence of sexual reproduction. This review examines the impact of genomic technologies on our view of AMF genetics and evolution. We highlight how the genetics, nuclear dynamics, and epigenetics of these prominent symbionts follow trends preserved in distant multinucleate fungal relatives. We also propose new avenues of research to improve our understanding of their nuclear biology and their intricate genetic interactions with plant hosts.
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Geosiphon pyriformis, a representative of the fungal sub-phylum Glomeromycotina, is unique in its endosymbiosis with cyanobacteria within a fungal cell. This symbiotic relationship occurs in bladders containing nuclei of G. pyriformis, Mollicutes-like bacterial endosymbionts (MRE), and photosynthetically active and dividing cells of Nostoc punctiforme. Recent genome analyses have shed light on the biology of G. pyriformis, but the genome content and biology of its endosymbionts remain unexplored. To fill this gap, we gathered and examined metagenomic data from the bladders of G. pyriformis, where N. punctiforme and MRE are located. This ensures that our analyses are focused on the organs directly involved in the symbiosis. By comparing this data with the genetic information of related cyanobacteria and MREs from other species of Arbuscular Mycorrhizal Fungi, we aimed to reveal the genetic content of these organisms and understand how they interact at a genetic level to establish a symbiotic relationship. Our analyses uncovered significant gene expansions in the Nostoc endosymbiont, particularly in mobile elements and genes potentially involved in xenobiotic degradation. We also confirmed that the MRE of Glomeromycotina are monophyletic and possess a highly streamlined genome. These genomes show dramatic differences in both structure and content, including the presence of enzymes involved in environmental sensing and stress response.
Asunto(s)
Metagenoma , Simbiosis , Filogenia , Cianobacterias/genética , Cianobacterias/clasificación , Cianobacterias/metabolismo , Nostoc/genética , Nostoc/metabolismo , Metagenómica , Genoma Fúngico , Genoma BacterianoRESUMEN
Optimizing agroecosystems and crops for micronutrient uptake while reducing issues with inorganic contaminants (metal(loid)s) is a challenging task. One promising approach is to use arbuscular mycorrhizal fungi (AMF) and investigate the physiological, molecular and epigenetic changes that occur in their presence and that lead to changes in plant metal(loid) concentration (biofortification of micronutrients or mitigation of contaminants). Moreover, it is important to understand these mechanisms in the context of the soil microbiome, particularly those interactions of AMF with other soil microbes that can further shape crop nutrition. To address these challenges, a two-pronged approach is recommended: exploring molecular mechanisms and investigating microbiome management and engineering. Combining both approaches can lead to benefits in human health by balancing nutrition and contamination caused by metal(loid)s in the agro-ecosystem.
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Rhizophagus irregularis is the model species for arbuscular mycorrhizal fungi (AMF) research and the most widely propagated species for commercial plant biostimulants. Using asymbiotic and symbiotic cultivation systems initiated from single spores, advanced microscopy, Sanger sequencing of the glomalin gene, and PacBio sequencing of the partial 45S rRNA gene, we show that four strains of R. irregularis produce spores of two distinct morphotypes, one corresponding to the morphotype described in the R. irregularis protologue and the other having the phenotype of R. fasciculatus. The two spore morphs are easily distinguished by spore colour, thickness of the subtending hypha, thickness of the second wall layer, lamination of the innermost layer, and the dextrinoid reaction of the two outer spore wall layers to Melzer's reagent. The glomalin gene of the two spore morphs is identical and that of the PacBio sequences of the partial SSU-ITS-LSU region (2780 bp) obtained from single spores of the R. cf fasciculatus morphotype has a median pairwise similarity of 99.8% (SD = 0.005%) to the rDNA ribotypes of R. irregularis DAOM 197198. Based on these results, we conclude that the model AMF species R. irregularis is dimorphic, which has caused taxonomic confusion in culture collections and possibly in AMF research.
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Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts of most land plants. In these organisms, thousands of nuclei that are either genetically similar (homokaryotic) or derived from two distinct parents (dikaryotic) co-exist in a large syncytium. Here, we investigated the impact of these two nuclear organizations on the mycorrhizal response of potatoes (Solanum tuberosum) by inoculating four potato cultivars with eight Rhizophagus irregularis strains individually (four homokaryotic and four dikaryotic). By evaluating plant and fungal fitness-related traits four months post inoculation, we found that AMF genetic organization significantly affects the mycorrhizal response of host plants. Specifically, homokaryotic strains lead to higher total, shoot, and tuber biomass and a higher number of tubers, compared to dikaryotic strains. However, fungal fitness-related traits showed no clear differences between homokaryotic and dikaryotic strains. Nucleotype content analysis of single spores confirmed that the nucleotype ratio of AMF heterokaryon spores can shift depending on host identity. Together, these findings continue to highlight significant ecological differences derived from the two distinct genetic organizations in AMF.
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Micorrizas , Solanum tuberosum , Micorrizas/genética , Fenotipo , Plantas/microbiología , Biomasa , HongosRESUMEN
Arbuscular mycorrhizal fungi (AMF) are widespread obligate root symbionts that assist plants in obtaining nutrients and protection against environmental stresses. In the model species Rhizophagus irregularis, heterokaryotic strains (AMF dikaryons) carry thousands of nuclei originating from two parental strains whose frequency varies depending on strains and host identity. Here, using digital droplet PCR, we demonstrate that surrounding abiotic factors (temperature, phosphorus, and pH) also change the nuclear dynamics of such strains in root organ cultures. Furthermore, when spatially separated portions of the AMF mycelium grow under different abiotic conditions, all the produced spores carry highly similar nuclear ratios. Overall, these findings demonstrate that abiotic stressors impact the nuclear organization of a widespread group of multinucleate plant symbionts, and reveal remarkable mechanisms of nuclear ratio harmonization across the mycelium in these prominent symbionts.
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Micorrizas , Hongos , Micelio/genética , Micorrizas/genética , Fósforo , Raíces de PlantasRESUMEN
Chromosome folding links genome structure with gene function by generating distinct nuclear compartments and topologically associating domains. In mammals, these undergo preferential interactions and regulate gene expression. However, their role in fungal genome biology is unclear. Here, we combine Nanopore (ONT) sequencing with chromatin conformation capture sequencing (Hi-C) to reveal chromosome and epigenetic diversity in a group of obligate plant symbionts: the arbuscular mycorrhizal fungi (AMF). We find that five phylogenetically distinct strains of the model AMF Rhizophagus irregularis carry 33 chromosomes with substantial within-species variability in size, as well as in gene and repeat content. Strain-specific Hi-C contact maps reveal a 'checkerboard' pattern that underline two dominant euchromatin (A) and heterochromatin (B) compartments. Each compartment differs in the level of gene transcription, regulation of candidate effectors and methylation frequencies. The A-compartment is more gene-dense and contains most core genes, while the B-compartment is more repeat-rich and has higher rates of chromosomal rearrangement. While the B-compartment is transcriptionally repressed, it has significantly more secreted proteins and in planta upregulated candidate effectors, suggesting a possible host-induced change in chromosome conformation. Overall, this study provides a fine-scale view into the genome biology and evolution of model plant symbionts, and opens avenues to study the epigenetic mechanisms that modify chromosome folding during host-microbe interactions.
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Glomeromycota , Micorrizas , Hongos , Genoma Fúngico , Glomeromycota/genética , Glomeromycota/metabolismo , Micorrizas/fisiología , Plantas/genéticaRESUMEN
Microsporidia are eukaryotic parasites of many animals that appear to have adapted to an obligate intracellular lifestyle by modifying the morphology and content of their cells. Living inside other cells, they have lost many, or all, metabolic functions, resulting in genomes that are always gene poor and often very small. The minute content of microsporidian genomes led many to assume that these parasites are biochemically static and uninteresting. However, recent studies have demonstrated that these organisms can be surprisingly complex and dynamic. In this review I detail the most significant recent advances in microsporidian genomics and discuss how these have affected our understanding of many biological aspects of these peculiar eukaryotic intracellular pathogens.
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Microsporidios/fisiología , Animales , Ecosistema , Transferencia de Gen Horizontal , Humanos , Microsporidios/clasificación , Microsporidios/genética , Microsporidiosis/microbiología , Microsporidiosis/veterinariaRESUMEN
Glomeromycotina is a lineage of early diverging fungi that establish arbuscular mycorrhizal (AM) symbiosis with land plants. Despite their major ecological role, the genetic basis of their obligate mutualism remains largely unknown, hindering our understanding of their evolution and biology. We compared the genomes of Glomerales (Rhizophagus irregularis, Rhizophagus diaphanus, Rhizophagus cerebriforme) and Diversisporales (Gigaspora rosea) species, together with those of saprotrophic Mucoromycota, to identify gene families and processes associated with these lineages and to understand the molecular underpinning of their symbiotic lifestyle. Genomic features in Glomeromycotina appear to be very similar with a very high content in transposons and protein-coding genes, extensive duplications of protein kinase genes, and loss of genes coding for lignocellulose degradation, thiamin biosynthesis and cytosolic fatty acid synthase. Most symbiosis-related genes in R. irregularis and G. rosea are specific to Glomeromycotina. We also confirmed that the present species have a homokaryotic genome organisation. The high interspecific diversity of Glomeromycotina gene repertoires, affecting all known protein domains, as well as symbiosis-related orphan genes, may explain the known adaptation of Glomeromycotina to a wide range of environmental settings. Our findings contribute to an increasingly detailed portrait of genomic features defining the biology of AM fungi.
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Genoma Fúngico , Genómica , Glomeromycota/genética , Secuencia Conservada , Elementos Transponibles de ADN/genética , Genes Fúngicos , Lignina/metabolismo , Familia de Multigenes , Filogenia , Polisacáridos/metabolismo , Reproducción , Simbiosis/genética , Transcripción Genética , Regulación hacia Arriba/genéticaRESUMEN
The recent global decline in Western honeybee (Apis mellifera) populations is of great concern for pollination and honey production worldwide. Declining honeybee populations are frequently infected by the microsporidian pathogen Nosema ceranae. This species was originally described in the Asiatic honeybee (Apis cerana), and its identification in global A. mellifera hives could result from a recent host transfer. Recent genome studies have found that global populations of this parasite are polyploid and that humans may have fueled their global expansion. To better understand N. ceranae biology, we investigated its genetic diversity within part of their native range (Thailand) and among different hosts (A. mellifera, A. cerana) using both PCR and genome-based methods. We find that Thai N. ceranae populations share many SNPs with other global populations and appear to be clonal. However, in stark contrast with previous studies, we found that these populations also carry many SNPs not found elsewhere, indicating that these populations have evolved in their current geographic location for some time. Our genome analyses also indicate the potential presence of diploidy within Thai populations of N. ceranae.
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Abejas/microbiología , Genoma Fúngico , Nosema/genética , Polimorfismo de Nucleótido Simple , Animales , Genómica , Reacción en Cadena de la Polimerasa , TailandiaRESUMEN
Contents Summary 1129 I. Introduction 1129 II. Intraspecific phenotypic variation and the plant host 1130 III. High inter-isolate genetic diversity in model AMF 1130 IV. Genome diversity within the model AM fungus Rhizophagus irregularis 1131 V. Pangenomes and the future of AMF ecological genomics 1131 Acknowledgements 1133 References 1133 SUMMARY: Arbuscular mycorrhizal fungi (AMF) are ubiquitous plant symbionts with an intriguing population biology. Conspecific AMF strains can vary substantially at the genetic and phenotypic levels, leading to direct and quantifiable variation in plant growth. Recent studies have shown that high intraspecific diversity is very common in AMF, and not only found in model species. Studies have also revealed how the phenotype of conspecific isolates varies depending on the plant host, highlighting the functional relevance of intraspecific phenotypic plasticity for the AMF ecology and mycorrhizal symbiosis. Recent work has also demonstrated that conspecific isolates of the model AMF Rhizophagus irregularis harbor large and highly variable pangenomes, highlighting the potential role of intraspecific genome diversity for the ecological adaptation of these symbionts.
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Variación Genética , Genoma Fúngico , Micorrizas/genética , Fenotipo , Plantas/microbiología , Especificidad de la EspecieRESUMEN
A workshop at the recent International Conference on Mycorrhiza was focused on species recognition in Glomeromycotina and parts of their basic biology that define species. The workshop was motivated by the paradigm-shifting evidence derived from genomic data for sex and for the lack of heterokaryosis, and by published exchanges in Science that were based on different species concepts and have led to differing views of dispersal and endemism in these fungi. Although a lively discussion ensued, there was general agreement that species recognition in the group is in need of more attention, and that many basic assumptions about the biology of these important fungi including sexual or clonal reproduction, similarity or dissimilarity of nuclei within an individual, and species boundaries need to be re-examined and scrutinized with current techniques.
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Glomeromycota/fisiología , Glomeromycota/clasificación , Glomeromycota/genética , Filogenia , Especificidad de la EspecieRESUMEN
Arbuscular mycorrhizal fungi (AMF) are known to improve plant fitness through the establishment of mycorrhizal symbioses. Genetic and phenotypic variations among closely related AMF isolates can significantly affect plant growth, but the genomic changes underlying this variability are unclear. To address this issue, we improved the genome assembly and gene annotation of the model strain Rhizophagus irregularis DAOM197198, and compared its gene content with five isolates of R. irregularis sampled in the same field. All isolates harbor striking genome variations, with large numbers of isolate-specific genes, gene family expansions, and evidence of interisolate genetic exchange. The observed variability affects all gene ontology terms and PFAM protein domains, as well as putative mycorrhiza-induced small secreted effector-like proteins and other symbiosis differentially expressed genes. High variability is also found in active transposable elements. Overall, these findings indicate a substantial divergence in the functioning capacity of isolates harvested from the same field, and thus their genetic potential for adaptation to biotic and abiotic changes. Our data also provide a first glimpse into the genome diversity that resides within natural populations of these symbionts, and open avenues for future analyses of plant-AMF interactions that link AMF genome variation with plant phenotype and fitness.
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Variación Genética , Genoma Fúngico , Glomeromycota/genética , Modelos Biológicos , Micorrizas/genética , Simbiosis/genética , Adaptación Fisiológica/genética , Elementos Transponibles de ADN/genética , Proteínas Fúngicas/química , Genes Fúngicos , Glomeromycota/aislamiento & purificación , Anotación de Secuencia Molecular , Filogenia , Dominios Proteicos , Especificidad de la EspecieRESUMEN
Microsporidia are highly successful parasites that infect virtually all known animal lineages, including the model Danio rerio (zebrafish). The widespread use of this aquatic model for biomedical research has resulted in an unexpected increase in infections from the microsporidium Pseudoloma neurophilia, which can lead to significant physical, behavioral, and immunological modifications, resulting in nonprotocol variation during experimental procedures. Here, we seek to obtain insights into the biology of P. neurophilia by investigating its genome content, which was obtained from only 29 nanograms of DNA using the MiSeq technology and paired-end Illumina sequencing. We found that the genome of P. neurophilia is phylogenetically and genetically related to other fish-microsporidians, but features unique to this intracellular parasite are also found. The small 5.25-Mb genome assembly includes 1,139 unique open-reading frames and an unusually high number of transposable elements for such a small genome. Investigations of intragenomic diversity also provided strong indications that the mononucleate nucleus of this species is diploid. Overall, our study provides insights into the dynamics of microsporidian genomes and a solid sequence reference to be used in future studies of host-parasite interactions using the zebrafish D. rerio and P. neurophilia as a model.
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ADN de Hongos/genética , Enfermedades de los Peces/microbiología , Microsporidios/genética , Microsporidiosis/veterinaria , Pez Cebra/microbiología , Animales , Secuencia de Bases , Biodiversidad , Elementos Transponibles de ADN , ADN de Hongos/análisis , ADN de Hongos/aislamiento & purificación , Proteínas Fúngicas/genética , Genoma Fúngico , Interacciones Huésped-Parásitos , Microsporidiosis/microbiología , Familia de Multigenes , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Interferencia de ARNRESUMEN
The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.
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Evolución Molecular , Genoma Fúngico/genética , Glomeromycota/genética , Micorrizas/genética , Plantas/microbiología , Simbiosis/genética , Secuencia de Bases , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed.
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Hongos/clasificación , Hongos/genética , Genoma Fúngico , FilogeniaRESUMEN
Nosema ceranae is a microsporidian pathogen whose infections have been associated with recent global declines in the populations of western honeybees (Apis mellifera). Despite the outstanding economic and ecological threat that N. ceranae may represent for honeybees worldwide, many aspects of its biology, including its mode of reproduction, propagation and ploidy, are either very unclear or unknown. In the present study, we set to gain knowledge in these biological aspects by re-sequencing the genome of eight isolates (i.e. a population of spores isolated from one single beehive) of this species harvested from eight geographically distant beehives, and by investigating their level of polymorphism. Consistent with previous analyses performed using single gene sequences, our analyses uncovered the presence of very high genetic diversity within each isolate, but also very little hive-specific polymorphism. Surprisingly, the nature, location and distribution of this genetic variation suggest that beehives around the globe are infected by a population of N. ceranae cells that may be polyploid (4n or more), and possibly clonal. Lastly, phylogenetic analyses based on genome-wide single-nucleotide polymorphism data extracted from these parasites and mitochondrial sequences from their hosts all failed to support the current geographical structure of our isolates.
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Abejas/microbiología , ADN de Hongos/genética , Nosema/genética , Poliploidía , Animales , Secuencia de Bases , Geografía , Desequilibrio de Ligamiento/genética , Nosema/aislamiento & purificación , Filogenia , Polimorfismo Genético/genética , Análisis de Secuencia de ADNRESUMEN
Microsporidia of the genus Encephalitozoon are widespread pathogens of animals that harbor the smallest known nuclear genomes. Complete sequences from Encephalitozoon intestinalis (2.3 Mbp) and Encephalitozoon cuniculi (2.9 Mbp) revealed massive gene losses and reduction of intergenic regions as factors leading to their drastically reduced genome size. However, microsporidian genomes also have gained genes through horizontal gene transfers (HGT), a process that could allow the parasites to exploit their hosts more fully. Here, we describe the complete sequences of two intermediate-sized genomes (2.5 Mbp), from Encephalitozoon hellem and Encephalitozoon romaleae. Overall, the E. hellem and E. romaleae genomes are strikingly similar to those of Encephalitozoon cuniculi and Encephalitozoon intestinalis in both form and content. However, in addition to the expected expansions and contractions of known gene families in subtelomeric regions, both species also were found to harbor a number of protein-coding genes that are not found in any other microsporidian. All these genes are functionally related to the metabolism of folate and purines but appear to have originated by several independent HGT events from different eukaryotic and prokaryotic donors. Surprisingly, the genes are all intact in E. hellem, but in E. romaleae those involved in de novo synthesis of folate are all pseudogenes. Overall, these data suggest that a recent common ancestor of E. hellem and E. romaleae assembled a complete metabolic pathway from multiple independent HGT events and that one descendent already is dispensing with much of this new functionality, highlighting the transient nature of transferred genes.
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Cromosomas Fúngicos/genética , Encephalitozoon cuniculi/fisiología , Evolución Molecular , Transferencia de Gen Horizontal/fisiología , Genoma Fúngico/fisiología , Animales , Secuencia de Bases , Cromosomas Fúngicos/metabolismo , Ácido Fólico/genética , Ácido Fólico/metabolismo , Datos de Secuencia Molecular , Purinas/metabolismo , Telómero/genética , Telómero/metabolismoRESUMEN
Arbuscular mycorrhizal fungi (AMF) are important plant symbionts that have long been considered evolutionary anomalies because of their apparent long-term lack of sexuality, but recent explorations of available DNA sequence have challenged this notion by revealing the presence of homologues of fungal mating type-high-mobility group (MATA-HMG) and core meiotic genes in these organisms. To obtain more insights into the sexual potential of AMF, homologues of MATA-HMGs were sought in the transcriptome of three AMF isolates, and their functional and evolutionary trajectories were studied in genetically divergent strains of Rhizophagus irregularis using conventional and quantitative PCR procedures. Our analyses revealed the presence of at least 76 homologues of MATA-HMGs in R. irregularis isolates. None of these was found to be surrounded by genes generally found near other known fungal mating type loci, but here we report the presence of a 9-kb-long region in the AMF R. irregularis harbouring a total of four tandem-repeated MATA-HMGs; a feature that highlights a potentially elevated intragenomic diversity in this AMF species. The present study provides intriguing insights into the genome evolution of R. irregularis, and represents a stepping stone for understanding the potential of these fungi to undergo cryptic sex.
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ADN de Hongos , Evolución Molecular , Proteínas Fúngicas/genética , Genes del Tipo Sexual de los Hongos , Glomeromycota/genética , Proteínas del Grupo de Alta Movilidad/genética , Micorrizas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Glomeromycota/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Familia de Multigenes , Micorrizas/metabolismo , Filogenia , Reproducción , Secuencias Repetidas en Tándem , TranscriptomaRESUMEN
The genomes of microsporidia in the genus Encephalitozoon have been extensively studied for their minimalistic features, but they have seldom been used to investigate basic characteristics of the biology of these organisms, such as their ploidy or their mode of reproduction. In the present study, we aimed to tackle this issue by mapping Illumina sequence reads against the genomes of four strains of E. cuniculi. This approach, combined with more conventional molecular biology techniques, resulted in the identification of heterozygosity in all strains investigated, a typical signature of a diploid nuclear state. In sharp contrast with similar studies recently performed on a distant microsporidian lineage (Nematocida spp.), the level of heterozygosity that we identified across the E. cuniculi genomes was found to be extremely low. This reductive intraindividual genetic variation could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce.