RESUMO
Repeated herbicide applications in agricultural fields exert strong selection on weeds such as blackgrass (Alopecurus myosuroides), which is a major threat for temperate climate cereal crops. This inadvertent selection pressure provides an opportunity for investigating the underlying genetic mechanisms and evolutionary processes of rapid adaptation, which can occur both through mutations in the direct targets of herbicides and through changes in other, often metabolic, pathways, known as non-target-site resistance. How much target-site resistance (TSR) relies on de novo mutations vs. standing variation is important for developing strategies to manage herbicide resistance. We first generated a chromosome-level reference genome for A. myosuroides for population genomic studies of herbicide resistance and genome-wide diversity across Europe in this species. Next, through empirical data in the form of highly accurate long-read amplicons of alleles encoding acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) variants, we showed that most populations with resistance due to TSR mutations-23 out of 27 and six out of nine populations for ACCase and ALS, respectively-contained at least two TSR haplotypes, indicating that soft sweeps are the norm. Finally, through forward-in-time simulations, we inferred that TSR is likely to mainly result from standing genetic variation, with only a minor role for de novo mutations.
Assuntos
Resistência a Herbicidas , Herbicidas , Resistência a Herbicidas/genética , Poaceae/genética , Poaceae/metabolismo , Mutação , Haplótipos , Europa (Continente) , Herbicidas/farmacologia , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismoRESUMO
Rapid adaptation of weeds to herbicide applications in agriculture through resistance development is a widespread phenomenon. In particular, the grass Alopecurus myosuroides is an extremely problematic weed in cereal crops with the potential to manifest resistance in only a few generations. Target-site resistances (TSRs), with their strong phenotypic response, play an important role in this rapid adaptive response. Recently, using PacBio's long-read amplicon sequencing technology in hundreds of individuals, we were able to decipher the genomic context in which TSR mutations occur. However, sequencing individual amplicons are costly and time-consuming, thus impractical to implement for other resistance loci or applications. Alternatively, pool-based approaches overcome these limitations and provide reliable allele frequencies, although at the expense of not preserving haplotype information. In this proof-of-concept study, we sequenced with PacBio High Fidelity (HiFi) reads long-range amplicons (13.2 kb), encompassing the entire ACCase gene in pools of over 100 individuals, and resolved them into haplotypes using the clustering algorithm PacBio amplicon analysis (pbaa), a new application for pools in plants and other organisms. From these amplicon pools, we were able to recover most haplotypes from previously sequenced individuals of the same population. In addition, we analysed new pools from a Germany-wide collection of A. myosuroides populations and found that TSR mutations originating from soft sweeps of independent origin were common. Forward-in-time simulations indicate that TSR haplotypes will persist for decades even at relatively low frequencies and without selection, highlighting the importance of accurate measurement of TSR haplotype prevalence for weed management.
Assuntos
Acetil-CoA Carboxilase , Resistência a Herbicidas , Poaceae , Acetil-CoA Carboxilase/genética , Agricultura , Frequência do Gene/genética , Haplótipos/genética , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Mutação , Poaceae/genéticaRESUMO
BACKGROUND: Patient-directed knowledge tools such as patient versions of guidelines and patient decision aids are increasingly developed to facilitate shared decision making. In this paper, we report how consensus was reached within the Netherlands on quality criteria for development, content and governance of these tools. METHOD: A 12-month development and consensus study. The consortium worked on four work packages: (a) reviewing existing criteria; (b) drafting the quality criteria; (c) safe-guarding the acceptability and feasibility of the draft criteria by participatory research in on-going tool development projects; and (d) gaining formal support from national stakeholders on the quality criteria. RESULTS: We reached consensus on a 8-step guidance; describing minimal quality criteria for (a) the team composition; (b) setting the scope; (c) identifying needs; (d) the content and format; (e) testing the draft; (f) finalizing and approval; (g) dissemination and application, and (h) ownership and revision. The participants of the on-going tool development projects were positive about the quality criteria in general, but divided as to the degree of detail. Whereas some expressed a clear desire for procedural standards, others felt that it would be sufficient to provide only general directions. Despite the different views as to the degree of detail, consensus was reached in three stakeholder meetings. DISCUSSION: We successfully collaborated with all stakeholders and achieved formal support from national stakeholders on a set of minimum criteria for the development process, content and governance of patient-directed knowledge tools.
Assuntos
Técnicas de Apoio para a Decisão , Participação do Paciente , Guias de Prática Clínica como Assunto , Consenso , Medicina Baseada em Evidências , Humanos , Países Baixos , Participação dos InteressadosRESUMO
Conflicts of interest (COIs) have been defined by the American Thoracic Society as "a divergence between an individual's private interests and his or her professional obligations such that an independent observer might reasonably question whether the individual's professional actions or decisions are motivated by personal gain, such as direct financial, academic advancement, clinical revenue streams, or community standing." In the context of guideline development, the concerns are not simply about identifying and disclosing direct financial or indirect COIs. Despite this recognition, the management of COIs in guidelines is often unsatisfactory. In response to requests from its international membership and informed by existing syntheses of the evidence and policies of international organizations, the Guidelines International Network Board of Trustees developed guidance on the disclosure of interests and management of COIs. Current approaches are relatively similar throughout the guideline development community, with an increasing recognition of the importance of disclosing and managing indirect COIs. Although there are differences in detail among the approaches, the similarities allow for the formulation of 9 core principles for managing COIs. In formulating these principles, the Guidelines International Network Board of Trustees recognizes that COIs cannot be totally avoided when panel members are being chosen for certain guidelines or in certain settings; thus, the important issue is the management of COIs in a fair, judicious, transparent manner.
Assuntos
Pesquisa Biomédica/ética , Conflito de Interesses , Revelação , Guias como Assunto , HumanosRESUMO
In this study, we aimed to systematically assess the frequency at which potentially deleterious phenotypes appear in natural populations of the outcrossing model plant Arabidopsis arenosa, and to establish their underlying genetics. For this purpose, we collected seeds from wild A. arenosa populations and screened over 2,500 plants for unusual phenotypes in the greenhouse. We repeatedly found plants with obvious phenotypic defects, such as small stature and necrotic or chlorotic leaves, among first-generation progeny of wild A. arenosa plants. Such abnormal plants were present in about 10% of maternal sibships, with multiple plants with similar phenotypes in each of these sibships, pointing to a genetic basis of the observed defects. A combination of transcriptome profiling, linkage mapping and genome-wide runs of homozygosity patterns using a newly assembled reference genome indicated a range of underlying genetic architectures associated with phenotypic abnormalities. This included evidence for homozygosity of certain genomic regions, consistent with alleles that are identical by descent being responsible for these defects. Our observations suggest that deleterious alleles with different genetic architectures are segregating at appreciable frequencies in wild A. arenosa populations.
Assuntos
Arabidopsis , Arabidopsis/genética , Fenótipo , Mapeamento Cromossômico , SementesRESUMO
Microorganisms from all domains of life establish associations with plants. Although some harm the plant, others antagonize pathogens or prime the plant immune system, support the acquisition of nutrients, tune plant hormone levels, or perform additional services. Most culture-independent plant microbiome research has focused on amplicon sequencing of the 16S rRNA gene and/or the internal transcribed spacer (ITS) of rRNA genomic loci, which show the relative abundance of the microbes to each other. Here, we describe shotgun sequencing of 275 wild Arabidopsis thaliana leaf microbiomes from southwest Germany, with additional bacterial 16S and eukaryotic ITS1 rRNA amplicon data from 176 of these samples. Shotgun data, which unlike the amplicon data capture the ratio of microbe to plant DNA, enable scaling of microbial read abundances to reflect the microbial load on the host. In a more cost-effective hybrid strategy, we show they also allow a similar scaling of amplicon data to overcome compositionality problems. Our wild plants were dominated by bacterial sequences, with eukaryotes contributing only a minority of reads. Microbial membership showed weak associations with both site of origin and plant genotype, both of which were highly confounded in this dataset. There was large variation among microbiomes, with one extreme comprising samples of low complexity and a high load of microorganisms typical of infected plants, and the other extreme being samples of high complexity and a low microbial load. Critically, considering absolute microbial load led to fundamentally different conclusions about microbiome assembly and the interaction networks among major taxa.
Assuntos
Microbiota , Genes de RNAr , Alemanha , Folhas de Planta , RNA Ribossômico 16S/genéticaRESUMO
Species' responses at the genetic level are key to understanding the long-term consequences of anthropogenic global change. Herbaria document such responses, and, with contemporary sampling, provide high-resolution time-series of plant evolutionary change. Characterizing genetic diversity is straightforward for model species with small genomes and a reference sequence. For nonmodel species-with small or large genomes-diversity is traditionally assessed using restriction-enzyme-based sequencing. However, age-related DNA damage and fragmentation preclude the use of this approach for ancient herbarium DNA. Here, we combine reduced-representation sequencing and hybridization-capture to overcome this challenge and efficiently compare contemporary and historical specimens. Specifically, we describe how homemade DNA baits can be produced from reduced-representation libraries of fresh samples, and used to efficiently enrich historical libraries for the same fraction of the genome to produce compatible sets of sequence data from both types of material. Applying this approach to both Arabidopsis thaliana and the nonmodel plant Cardamine bulbifera, we discovered polymorphisms de novo in an unbiased, reference-free manner. We show that the recovered genetic variation recapitulates known genetic diversity in A. thaliana, and recovers geographical origin in both species and over time, independent of bait diversity. Hence, our method enables fast, cost-efficient, large-scale integration of contemporary and historical specimens for assessment of genome-wide genetic trends over time, independent of genome size and presence of a reference genome.
Assuntos
DNA de Plantas/genética , Genética Populacional , Genômica , Plantas/genética , Arabidopsis , Cardamine , Hibridização de Ácido Nucleico , Análise de Sequência de DNARESUMO
All European countries are facing common challenges for delivering appropriate, evidence-based care to patients with cancer. Despite tangible improvements in diagnosis and treatment, marked differences in cancer survival exist throughout Europe. The reliable translation of new research evidence into consistent patient-oriented strategies is a key endeavour to overcome inequalities in healthcare. Clinical-practice guidelines are important tools for improving quality of care by informing professionals and patients about the most appropriate clinical practice. Guideline programmes in different countries use similar strategies to achieve similar goals. This results in unnecessary duplication of effort and inefficient use of resources. While different initiatives at the international level have attempted to improve the quality of guidelines, less investment has been made to overcome existing fragmentation and duplication of effort in cancer guideline development and research. To provide added value to existing initiatives and foster equitable access to evidence-based cancer care in Europe, CoCanCPG will establish cooperation between cancer guideline programmes. CoCanCPG is an ERA-Net coordinated by the French National Cancer Institute with 17 partners from 11 countries. The CoCanCPG partners will achieve their goal through an ambitious, stepwise approach with a long-term perspective, involving: 1. implementing a common framework for sharing knowledge and skills; 2. developing shared activities for guideline development; 3. assembling a critical mass for pertinent research into guideline methods; 4. implementing an appropriate framework for cooperation. Successful development of joint activities involves learning how to adopt common quality standards and how to share responsibilities, while taking into account the cultural and organisational diversity of the participating organisations. Languages barriers and different organisational settings add a level of complexity to setting up transnational collaboration. Through its activities, CoCanCPG will make an important contribution towards better access to evidence-based cancer practices and thus contribute to reducing inequalities and improving care for patients with cancer across Europe.
Assuntos
Medicina Baseada em Evidências , Disseminação de Informação , Cooperação Internacional , Neoplasias/terapia , Guias de Prática Clínica como Assunto , Qualidade da Assistência à Saúde , Benchmarking , Comunicação , Diversidade Cultural , Europa (Continente) , França , Disparidades em Assistência à Saúde , Humanos , IdiomaRESUMO
Crop disease outbreaks are often associated with clonal expansions of single pathogenic lineages. To determine whether similar boom-and-bust scenarios hold for wild pathosystems, we carried out a multi-year, multi-site survey of Pseudomonas in its natural host Arabidopsis thaliana. The most common Pseudomonas lineage corresponded to a ubiquitous pathogenic clade. Sequencing of 1,524 genomes revealed this lineage to have diversified approximately 300,000 years ago, containing dozens of genetically identifiable pathogenic sublineages. There is differentiation at the level of both gene content and disease phenotype, although the differentiation may not provide fitness advantages to specific sublineages. The coexistence of sublineages indicates that in contrast to crop systems, no single strain has been able to overtake the studied A. thaliana populations in the recent past. Our results suggest that selective pressures acting on a plant pathogen in wild hosts are likely to be much more complex than those in agricultural systems.