Nejvíce citovaný článek - PubMed ID 27401132
Forward-directed genetic screens are extremely powerful in identifying novel genes involved in a specific biological process, including various chromatin regulatory pathways. However, the traditional ways of genetic mapping are time- and cost-demanding. Recently, the whole process was revolutionized by the development of mapping-by-sequencing (MBS) protocols. In MBS, the causal mutations and their positions within genes are identified directly by whole-genome sequencing and bioinformatics analysis of the bulk of mutant plants selected based on the mutant phenotype from a segregating population. MBS increases precision and economizes the mapping. Here, we describe a general protocol and provide practical tips on how to proceed with the mapping-by-sequencing on the example of Arabidopsis forward-directed genetic screen designed to identify mutants sensitive to a specific type of DNA damage. The described protocol is generally applicable to a wide range of genetic screens in various inbreeding species with a reference genome sequence.
- Klíčová slova
- DNA damage repair, DNA-protein crosslinks, Forward genetics, Genetic mapping, High-throughput sequencing, Mapping-by-sequencing, SNP calling, Zebularine,
- MeSH
- Arabidopsis * genetika MeSH
- fenotyp MeSH
- genom rostlinný MeSH
- mapování chromozomů * metody MeSH
- mutace MeSH
- sekvenování celého genomu metody MeSH
- výpočetní biologie metody MeSH
- vysoce účinné nukleotidové sekvenování * metody MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The Trichophyton mentagrophytes complex encompasses common dermatophytes causing superficial mycoses in humans and animals. The taxonomy of the complex is unstable, with conflicting views on the species status of some taxa, particularly T. indotineae and T. interdigitale. Due to the presence of intermediate genotypes, neither MALDI-TOF MS nor ITS rDNA sequencing can accurately distinguish all taxa in the complex, potentially contributing to clinical misdiagnoses. OBJECTIVES: This research resolves phylogenetic relationships within the T. mentagrophytes complex. Based on these data, the taxonomical recommendations are suggested. METHODS: In order to resolve the phylogenetic relationship of the T. mentagrophytes complex, we employed Restriction Site-Associated DNA Sequencing (RADseq) to produce a high-resolution single nucleotide polymorphism (SNP) dataset from 95 isolates. The SNP-based analyses indicated the presence of two major genetic clusters corresponding to T. mentagrophytes (including T. indotineae) and T. interdigitale. RESULTS: Our results challenge the species status of T. indotineae because of insufficient genetic divergence from T. mentagrophytes. Therefore, we propose designating T. indotineae as T. mentagrophytes var. indotineae (or T. mentagrophytes ITS genotype VIII) to avoid further splitting of the complex and taxonomic inflation. Although T. interdigitale shows clearer genetic differentiation, its separation is incomplete and identification of some isolates is ambiguous when using routine methods, leading us to consider it a variety as well: T. mentagrophytes var. interdigitale. CONCLUSIONS: We recommend using T. mentagrophytes as the overarching species name for all complex isolates. Where precise molecular identification is possible, the use of variety ranks is encouraged. Since identical resistance mechanisms are not specific to any genotype or dermatophyte species, identifying antifungal resistance is more important than differentiating closely related genotypes or populations.
- Klíčová slova
- Trichophyton interdigitale, Trichophyton mentagrophytes, anthropophilic dermatophytes, antifungal resistance, dermatophytosis, population structure, taxonomy, zoophilic dermatophytes,
- MeSH
- antifungální látky * farmakologie MeSH
- Arthrodermataceae * genetika klasifikace účinky léků MeSH
- DNA fungální genetika MeSH
- fylogeneze * MeSH
- genomika metody MeSH
- genotyp MeSH
- jednonukleotidový polymorfismus MeSH
- lidé MeSH
- sekvenční analýza DNA MeSH
- tinea mikrobiologie MeSH
- Trichophyton * genetika klasifikace účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antifungální látky * MeSH
- DNA fungální MeSH
Lotmaria passim is a ubiquitous trypanosomatid parasite of honey bees nestled within the medically important subfamily Leishmaniinae. Although this parasite is associated with honey bee colony losses, the original draft genome-which was completed before its differentiation from the closely related Crithidia mellificae-has remained the reference for this species despite lacking improvements from newer methodologies. Here, we report the updated sequencing, assembly, and annotation of the BRL-type (Bee Research Laboratory) strain (ATCC PRA-422) of Lotmaria passim. The nuclear genome assembly has been resolved into 31 complete chromosomes and is paired with an assembled kinetoplast genome consisting of a maxicircle and 30 minicircle sequences. The assembly spans 33.7 Mb and contains very little repetitive content, from which our annotation of both the nuclear assembly and kinetoplast predicted 10,288 protein-coding genes. Analyses of the assembly revealed evidence of a recent chromosomal duplication event within chromosomes 5 and 6 and provided evidence for a high level of aneuploidy in this species, mirroring the genomic flexibility employed by other trypanosomatids as a means of adaptation to different environments. This high-quality reference can therefore provide insights into adaptations of trypanosomatids to the thermally regulated, acidic, and phytochemically rich honey bee hindgut niche, which offers parallels to the challenges faced by other Leishmaniinae during the challenges they undergo within insect vectors, during infection of mammals, and exposure to antiparasitic drugs throughout their multi-host life cycles. This reference will also facilitate investigations of strain-specific genomic polymorphisms, their role in pathogenicity, and the development of treatments for pollinator infection.
- Klíčová slova
- Lotmaria passim strain BRL, ATCC PRA-422, Hi-C, Leishmaniinae, PacBio, Trypanosomatidae, aneuploidy, monoxenous, polyploidy, trypanosomatid,
- MeSH
- anotace sekvence MeSH
- fylogeneze MeSH
- genom protozoální MeSH
- genomika metody MeSH
- molekulární evoluce * MeSH
- Trypanosomatina * genetika klasifikace MeSH
- včely parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND AND AIMS: The Greater Cape Floristic Region is one of the world's biodiversity hotspots and is considered poor in polyploids. To test this assumption, ploidy variation was investigated in a widespread Cape shrub, Dicerothamnus rhinocerotis (renosterbos, Asteraceae). The aim was to elucidate the cytotype distribution and population composition across the species range, and to assess differences in morphology, environmental niches and genetics. METHODS: Ploidy level and genome size were determined via flow cytometry and cytotype assignment was confirmed by chromosome counting. Restriction site-associated DNA sequencing (RADseq) analyses were used to infer genetic relationships. Cytotype climatic and environmental niches were compared using a range of environmental layers and a soil model, while morphological differences were examined using multivariate methods. KEY RESULTS: The survey of 171 populations and 2370 individuals showed that the species comprises diploid and tetraploid cytotypes, no intermediates and only 16.8 % of mixed populations. Mean 2C values were 1.80-2.06 pg for diploids and 3.48-3.80 pg for tetraploids, with very similar monoploid genome sizes. Intra-cytotype variation showed a significant positive correlation with altitude and longitude in both cytotypes and with latitude in diploids. Although niches of both cytotypes were highly equivalent and similar, their optima and breadth were shifted due to differences mainly in isothermality and available water capacity. Morphometric analyses showed significant differences in the leaves and corolla traits, the number of florets per capitulum, and cypsela dimensions between the two cytotypes. Genetic analyses revealed four groups, three of them including both cytotypes. CONCLUSIONS: Dicerothamnus rhinocerotis includes two distinct cytotypes that are genetically similar. While tetraploids arise several times independently within different genetic groups, morphological and ecological differences are evident between cytotypes. Our results open up new avenues for questions regarding the importance of ploidy in the megadiverse Cape flora, and exemplify the need for population-based studies focused on ploidy variation.
- Klíčová slova
- Elytropappus rhinocerotis, Stoebe clade, Asteraceae, Compositae, Gnaphalieae, RADseq, South Africa, flow cytometry, ploidy level, renosterbos, renosterveld,
- MeSH
- Asteraceae * genetika MeSH
- délka genomu MeSH
- diploidie * MeSH
- ekosystém * MeSH
- genetická variace MeSH
- genom rostlinný MeSH
- tetraploidie * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phytophthora pseudosyringae is a self-fertile pathogen of woody plants, particularly associated with tree species from the genera Fagus, Notholithocarpus, Nothofagus and Quercus, which is found across Europe and in parts of North America and Chile. It can behave as a soil pathogen infecting roots and the stem collar region, as well as an aerial pathogen infecting leaves, twigs and stem barks, causing particular damage in the United Kingdom and western North America. The population structure, migration and potential outcrossing of a worldwide collection of isolates were investigated using genotyping-by-sequencing. Coalescent-based migration analysis revealed that the North American population originated from Europe. Historical gene flow has occurred between the continents in both directions to some extent, yet contemporary migration is overwhelmingly from Europe to North America. Two broad population clusters dominate the global population of the pathogen, with a subgroup derived from one of the main clusters found only in western North America. Index of association and network analyses indicate an influential level of outcrossing has occurred in this preferentially inbreeding, homothallic oomycete. Outcrossing between the two main population clusters has created distinct subgroups of admixed individuals that are, however, less common than the main population clusters. Differences in life history traits between the two main population clusters should be further investigated together with virulence and host range tests to evaluate the risk each population poses to natural environments worldwide.
- Klíčová slova
- forest pathogen, homothallic, invasive pathogen, migration, outcrossing, population genetics,
- MeSH
- fylogeografie MeSH
- lidé MeSH
- nemoci rostlin MeSH
- Phytophthora * genetika MeSH
- rostliny MeSH
- stromy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.
Invasive, exotic plant pathogens pose a major threat to native and agricultural ecosystems. Phytophthora × cambivora is an invasive, destructive pathogen of forest and fruit trees causing severe damage worldwide to chestnuts (Castanea), apricots, peaches, plums, almonds and cherries (Prunus), apples (Malus), oaks (Quercus), and beech (Fagus). It was one of the first damaging invasive Phytophthora species to be introduced to Europe and North America, although its origin is unknown. We determined its population genetic history in Europe, North and South America, Australia and East Asia (mainly Japan) using genotyping-by-sequencing. Populations in Europe and Australia appear clonal, those in North America are highly clonal yet show some degree of sexual reproduction, and those in East Asia are partially sexual. Two clonal lineages, each of opposite mating type, and a hybrid lineage derived from these two lineages, dominated the populations in Europe and were predominantly found on fagaceous forest hosts (Castanea, Quercus, Fagus). Isolates from fruit trees (Prunus and Malus) belonged to a separate lineage found in Australia, North America, Europe and East Asia, indicating the disease on fruit trees could be caused by a distinct lineage of P. × cambivora, which may potentially be a separate sister species and has likely been moved with live plants. The highest genetic diversity was found in Japan, suggesting that East Asia is the centre of origin of the pathogen. Further surveys in unsampled, temperate regions of East Asia are needed to more precisely identify the location and range of the centre of diversity.
- Klíčová slova
- Hybridization, Invasive pathogen, Polyploidy, Population genetics,
- Publikační typ
- časopisecké články MeSH
Recurrent polyploid formation and weak reproductive barriers between independent polyploid lineages generate intricate species complexes with high diversity and reticulate evolutionary history. Uncovering the evolutionary processes that formed their present-day cytotypic and genetic structure is a challenging task. We studied the species complex of Cardamine pratensis, composed of diploid endemics in the European Mediterranean and diploid-polyploid lineages more widely distributed across Europe, focusing on the poorly understood variation in Central Europe. To elucidate the evolution of Central European populations we analyzed ploidy level and genome size variation, genetic patterns inferred from microsatellite markers and target enrichment of low-copy nuclear genes (Hyb-Seq), and environmental niche differentiation. We observed almost continuous variation in chromosome numbers and genome size in C. pratensis s.str., which is caused by the co-occurrence of euploid and dysploid cytotypes, along with aneuploids, and is likely accompanied by inter-cytotype mating. We inferred that the polyploid cytotypes of C. pratensis s.str. are both of single and multiple, spatially and temporally recurrent origins. The tetraploid Cardamine majovskyi evolved at least twice in different regions by autopolyploidy from diploid Cardamine matthioli. The extensive genome size and genetic variation of Cardamine rivularis reflects differentiation induced by the geographic isolation of disjunct populations, establishment of triploids of different origins, and hybridization with sympatric C. matthioli. Geographically structured genetic lineages identified in the species under study, which are also ecologically divergent, are interpreted as descendants from different source populations in multiple glacial refugia. The postglacial range expansion was accompanied by substantial genetic admixture between the lineages of C. pratensis s.str., which is reflected by diffuse borders in their contact zones. In conclusion, we identified an interplay of diverse processes that have driven the evolution of the species studied, including allopatric and ecological divergence, hybridization, multiple polyploid origins, and genetic reshuffling caused by Pleistocene climate-induced range dynamics.
- Klíčová slova
- Brassicaceae, environmental niche, genome size, hybridization, microsatellites, phylogeography, polyploidy, target enrichment,
- Publikační typ
- časopisecké články MeSH
N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.
- Klíčová slova
- butanol efflux, butanol tolerance, genome sequence, random chemical mutagenesis, solventogenic Clostridium species,
- Publikační typ
- časopisecké články MeSH
Sustainable food production in the context of climate change necessitates diversification of agriculture and a more efficient utilization of plant genetic resources. Fonio millet (Digitaria exilis) is an orphan African cereal crop with a great potential for dryland agriculture. Here, we establish high-quality genomic resources to facilitate fonio improvement through molecular breeding. These include a chromosome-scale reference assembly and deep re-sequencing of 183 cultivated and wild Digitaria accessions, enabling insights into genetic diversity, population structure, and domestication. Fonio diversity is shaped by climatic, geographic, and ethnolinguistic factors. Two genes associated with seed size and shattering showed signatures of selection. Most known domestication genes from other cereal models however have not experienced strong selection in fonio, providing direct targets to rapidly improve this crop for agriculture in hot and dry environments.
- MeSH
- anotace sekvence MeSH
- Digitaria klasifikace genetika MeSH
- domestikace MeSH
- druhová specificita MeSH
- genetická variace MeSH
- genom rostlinný MeSH
- jedlá semena klasifikace genetika MeSH
- klimatické změny MeSH
- molekulární evoluce MeSH
- selekce (genetika) MeSH
- zemědělství metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Afrika MeSH
