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Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.

The French National Reference Center for Staphylococci currently uses DNA arrays and spa typing for the initial epidemiological characterization of Staphylococcus aureus strains. We here describe the use of whole-genome sequencing (WGS) to investigate retrospectively four distinct and virulent S. aureus lineages [clonal complexes (CCs): CC1, CC5, CC8, CC30] involved in hospital and community outbreaks or sporadic infections in France. We used a WGS bioinformatics pipeline based on de novo assembly (reference-free approach), single nucleotide polymorphism analysis, and on the inclusion of epidemiological markers. We examined the phylogeographic diversity of the French dominant hospital-acquired CC8-MRSA (methicillin-resistant S. aureus) Lyon clone through WGS analysis which did not demonstrate evidence of large-scale geographic clustering. We analyzed sporadic cases along with two outbreaks of a CC1-MSSA (methicillin-susceptible S. aureus) clone containing the Panton–Valentine leukocidin (PVL) and results showed that two sporadic cases were closely related. We investigated an outbreak of PVL-positive CC30-MSSA in a school environment and were able to reconstruct the transmission history between eight families. We explored different outbreaks among newborns due to the CC5-MRSA Geraldine clone and we found evidence of an unsuspected link between two otherwise distinct outbreaks. Here, WGS provides the resolving power to disprove transmission events indicated by conventional methods (same sequence type, spa type, toxin profile, and antibiotic resistance profile) and, most importantly, WGS can reveal unsuspected transmission events. Therefore, WGS allows to better describe and understand outbreaks and (inter-)national dissemination of S. aureus lineages. Our findings underscore the importance of adding WGS for (inter-)national surveillance of infections caused by virulent clones of S. aureus but also substantiate the fact that technological optimization at the bioinformatics level is still urgently needed for routine use. However, the greatest limitation of WGS analysis is the completeness and the correctness of the reference database being used and the conversion of floods of data into actionable results. The WGS bioinformatics pipeline (EpiSeqTM) we used here can easily generate a uniform database and associated metadata for epidemiological applications.

Background Human endogenous retroviruses (HERVs) have received much attention for their implications in the etiology of many human diseases and their profound effect on evolution. Notably, recent studies have highlighted associations between HERVs expression and cancers (Yu et al., Int J Mol Med 32, 2013), autoimmunity (Balada et al., Int Rev Immunol 29:351–370, 2010) and neurological (Christensen, J Neuroimmune Pharmacol 5:326–335, 2010) conditions. Their repetitive nature makes their study particularly challenging, where expression studies have largely focused on individual loci (De Parseval et al., J Virol 77:10414–10422, 2003) or general trends within families (Forsman et al., J Virol Methods 129:16–30, 2005; Seifarth et al., J Virol 79:341–352, 2005; Pichon et al., Nucleic Acids Res 34:e46, 2006). Methods To refine our understanding of HERVs activity, we introduce here a new microarray, HERV-V3. This work was made possible by the careful detection and annotation of genomic HERV/MaLR sequences as well as the development of a new hybridization model, allowing the optimization of probe performances and the control of cross-reactions. Results HERV-V3 offers an almost complete coverage of HERVs and their ancestors (mammalian apparent LTR-retrotransposons, MaLRs) at the locus level along with four other repertoires (active LINE-1 elements, lncRNA, a selection of 1559 human genes and common infectious viruses). We demonstrate that HERV-V3 analytical performances are comparable with commercial Affymetrix arrays, and that for a selection of tissue/pathological specific loci, the patterns of expression measured on HERV-V3 is consistent with those reported in the literature. Conclusions Given its large HERVs/MaLRs coverage and additional repertoires, HERV-V3 opens the door to multiple applications such as enhancers and alternative promoters identification, biomarkers identification as well as the characterization of genes and HERVs/MaLRs modulation caused by viral infection. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3669-7) contains supplementary material, which is available to authorized users.

MotivationAntimicrobial resistance has become a major worldwide public health concern, calling for a better characterization of existing and novel resistance mechanisms. GWAS methods applied to bacterial genomes have shown encouraging results for new genetic marker discovery. Most existing approaches either look at SNPs obtained by sequence alignment or consider sets of kmers, whose presence in the genome is associated with the phenotype of interest. While the former approach can only be performed when genomes are similar enough for an alignment to make sense, the latter can lead to redundant descriptions and to results which are hard to interpret.ResultsWe propose an alignment-free GWAS method detecting haplotypes of variable length associated to resistance, using compacted De Bruijn graphs. Our representation is flexible enough to deal with very plastic genomes subject to gene transfers while drastically reducing the number of features to explore compared to kmers, without loss of information. It accomodates polymorphisms in core genes, accessory genes and noncoding regions. Using our representation in a GWAS leads to the selection of a small number of entities which are easier to visualize and interpret than fixed-length kmers. We illustrate the benefit of our approach by describing known as well as potential novel determinants of antimicrobial resistance in P. aeruginosa, a pathogenic bacteria with a highly plastic genome.Availability and implementationThe code and data used in the experiments will be made available upon acceptance of this manuscript.Contactmagali.dancette@biomerieux.com

In human lymphoblastoid cell lines, 8,902 loci were identified at which genetic variation is significantly associated with local DNase I sensitivity; these variants are responsible for a large fraction of expression quantitative trait loci. Expression quantitative trait loci (eQTLs) are stretches of DNA that regulate gene transcription and expression and contribute to a particular phenotypic trait. eQTL mapping is an important tool for linking genetic variation to changes in gene regulation, but identifying the causal variants underlying eQTLs and the regulatory mechanisms involved remains a challenge. Degner et al. used DNaseI sequencing to measure genome-wide chromatin accessibility in 70 Yoruba lymphoblastoid cell lines to produce genome-wide maps of chromatin accessibility for each individual. They identify variants that they call DNaseI sensitivity quantitative trait loci (dsQTLs). The implication is that changes in chromatin accessibility or transcription-factor binding occur at many gene loci and are likely to be important contributors to phenotypic variation. The mapping of expression quantitative trait loci (eQTLs) has emerged as an important tool for linking genetic variation to changes in gene regulation1,2,3,4,5. However, it remains difficult to identify the causal variants underlying eQTLs, and little is known about the regulatory mechanisms by which they act. Here we show that genetic variants that modify chromatin accessibility and transcription factor binding are a major mechanism through which genetic variation leads to gene expression differences among humans. We used DNase I sequencing to measure chromatin accessibility in 70 Yoruba lymphoblastoid cell lines, for which genome-wide genotypes and estimates of gene expression levels are also available6,7,8. We obtained a total of 2.7 billion uniquely mapped DNase I-sequencing (DNase-seq) reads, which allowed us to produce genome-wide maps of chromatin accessibility for each individual. We identified 8,902 locations at which the DNase-seq read depth correlated significantly with genotype at a nearby single nucleotide polymorphism or insertion/deletion (false discovery rate = 10%). We call such variants ‘DNase I sensitivity quantitative trait loci’ (dsQTLs). We found that dsQTLs are strongly enriched within inferred transcription factor binding sites and are frequently associated with allele-specific changes in transcription factor binding. A substantial fraction (16%) of dsQTLs are also associated with variation in the expression levels of nearby genes (that is, these loci are also classified as eQTLs). Conversely, we estimate that as many as 55% of eQTL single nucleotide polymorphisms are also dsQTLs. Our observations indicate that dsQTLs are highly abundant in the human genome and are likely to be important contributors to phenotypic variation.

Changes in gene expression may represent an important mode of human adaptation. However, to date, there are relatively few known examples in which selection has been shown to act directly on levels or patterns of gene expression. In order to test whether single nucleotide polymorphisms (SNPs) that affect gene expression in cis are frequently targets of positive natural selection in humans, we analyzed genome-wide SNP and expression data from cell lines associated with the International HapMap Project. Using a haplotype-based test for selection that was designed to detect incomplete selective sweeps, we found that SNPs showing signals of selection are more likely than random SNPs to be associated with gene expression levels in cis. This signal is significant in the Yoruba (which is the population that shows the strongest signals of selection overall) and shows a trend in the same direction in the other HapMap populations. Our results argue that selection on gene expression levels is an important type of human adaptation. Finally, our work provides an analytical framework for tackling a more general problem that will become increasingly important: namely, testing whether selection signals overlap significantly with SNPs that are associated with phenotypes of interest.

The interaction between members of a gene network has an important impact on the variation of quantitative traits, and can influence the outcome of phenotype/genotype association studies. Three genes (Ppd-H1, HvCO1, HvFT1) known to play an essential role in the regulation of flowering time under long days in barley were subjected to an analysis of nucleotide diversity in a collection of 220 spring barley accessions. The coding region of Ppd-H1 was highly diverse, while both HvCO1 and HvFT1 showed a rather limited level of diversity. Within all three genes, the extent of linkage disequilibrium was variable, but on average only moderate. Ppd-H1 is strongly associated with flowering time across four environments, showing a difference of five to ten days between the most extreme haplotypes. The association between flowering time and the variation at HvFT1 and HvCO1 was strongly dependent on the haplotype present at Ppd-H1. The interaction between HvCO1 and Ppd-H1 was statistically significant, but this association disappeared when the analysis was corrected for the geographical origin of the accessions. No association existed between flowering time and allelic variation at HvFT1. In contrast to Ppd-H1, functional variation at both HvCO1 and HvFT1 is limited in cultivated barley. peerReviewed

An association study conducted on 375 maize inbred lines indicates a strong relationship between Vgt1 polymorphisms and flowering time, extending former quantitative trait loci (QTL) mapping results. Analysis of allele frequencies in a landrace collection supports a key role of Vgt1 in maize altilatitudinal adaptation.

Motivation: Metagenomics characterizes the taxonomic diversity of microbial communities by sequencing DNA directly from an environmental sample. One of the main challenges in metagenomics data analysis is the binning step, where each sequenced read is assigned to a taxonomic clade. Because of the large volume of metagenomics datasets, binning methods need fast and accurate algorithms that can operate with reasonable computing requirements. While standard alignment-based methods provide state-of-the-art performance, compositional approaches that assign a taxonomic class to a DNA read based on the k-mers it contains have the potential to provide faster solutions. Results: We propose a new rank-flexible machine learning-based compositional approach for taxonomic assignment of metagenomics reads and show that it benefits from increasing the number of fragments sampled from reference genome to tune its parameters, up to a coverage of about 10, and from increasing the k-mer size to about 12. Tuning the method involves training machine learning models on about 108 samples in 107 dimensions, which is out of reach of standard softwares but can be done efficiently with modern implementations for large-scale machine learning. The resulting method is competitive in terms of accuracy with well-established alignment and composition-based tools for problems involving a small to moderate number of candidate species and for reasonable amounts of sequencing errors. We show, however, that machine learning-based compositional approaches are still limited in their ability to deal with problems involving a greater number of species and more sensitive to sequencing errors. We finally show that the new method outperforms the state-of-the-art in its ability to classify reads from species of lineage absent from the reference database and confirm that compositional approaches achieve faster prediction times, with a gain of 2–17 times with respect to the BWA-MEM short read mapper, depending on the number of candidate species and the level of sequencing noise. Availability and implementation: Data and codes are available at http://cbio.ensmp.fr/largescalemetagenomics. Contact: pierre.mahe@biomerieux.com Supplementary information: Supplementary data are available at Bioinformatics online.

Many studies already investigated marker-assisted selection (MAS) efficiency but mainly in biparental populations. Connected multiparental populations address a broader diversity and confer a gain of power for QTL detection which must be beneficial for MAS. Our objective was to compare multiparental connected designs to biparental populations taken separately for MAS and phenotypic selection. We first detected QTL for flowering time and grain yield in an experimental maize design involving four parental inbred lines crossed to produce six different biparental populations and confirmed the advantage of multiparental connected designs over biparental populations for QTL detection. Based on these results we performed stochastic simulations to evaluate the expected efficiency of four generations of MAS and phenotypic selection. Different parameters were considered: trait heritability, genetic architecture and whether QTL were assumed to be known or have to be detected. Genetic gains were higher in the multiparental design than on average over the biparental populations considered separately, especially when favourable alleles were equally distributed among parental lines. When QTL detection was included in the simulation process, we found that type I error risk considered for declaring QTL as significant should be adapted to the design. Type I error risks leading to the best response were higher for the biparental populations than for the multiparental design. Besides addressing a broader diversity, multiparental designs increase the power of QTL detection, which reinforces their superiority over biparental designs for MAS. Application of MAS to multiparental designs therefore appears promising to accelerate genetic gain in plant breeding programs.

Inference of population structure from neutral marker loci is a key issue for association genetics. However, the presence of highly related individuals, commonly observed in breeders' panels, may lead to deviation from model hypotheses and therefore unreliable group assignment. The present note proposes a tool to help interpret Structure software outputs on populations that include highly related individuals, such as plant breeding material. We ran Structure software on simple sequence repeat (SSR) data from two maize (Zea mays L. ssp. mays) inbred panels. We propose a criterion to evaluate Structure stability based on Euclidian distance between outputs. This approach shows a high stability across runs for the panel composed of first cycle inbred lines. On the contrary, the presence of highly related individuals in the second panel induces strong instability in Structure outputs.

Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates., IMPORTANCE P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Genoplante is a partnership program between public French institutes (INRA, CIRAD, IRD and CNRS) and private companies (Biogemma, Bayer CropScience and Bioplante) that aims at developing genome analysis programs for crop species (corn, wheat, rapeseed, sunflower and pea) and model plants (Arabidopsis and rice). The outputs of these programs form a wealth of information (genomic sequence, transcriptome, proteome, allelic variability, mapping and synteny, and mutation data) and tools (databases, interfaces, analysis software), that are being integrated and made public at the public bioinformatics resource centre of Genoplante: GenoPlante-Info (GPI). This continuous flood of data and tools is regularly updated and will grow continuously during the coming two years. Access to the GPI databases and tools is available at http://genoplante-info.infobiogen.fr/.

For the last century, in vitro diagnostic process in microbiology has mainly relied on the growth of bacteria on the surface of a solid agar medium. Nevertheless, few studies focused in the past on the dynamics of microcolonies growth on agar surface before 8 to 10h of incubation. In this article, chromatic confocal microscopy has been applied to characterize the early development of a bacterial colony. This technology relies on a differential focusing depth of the white light. It allows one to fully measure the tridimensional shape of microcolonies more quickly than classical confocal microscopy but with the same spatial resolution. Placing the device in an incubator, the method was able to individually track colonies growing on an agar plate, and to follow the evolution of their surface or volume. Using an appropriate statistical modeling framework, for a given microorganism, the doubling time has been estimated for each individual colony, as well as its variability between colonies, both within and between agar plates. A proof of concept led on four bacterial strains of four distinct species demonstrated the feasibility and the interest of the approach. It showed in particular that doubling times derived from early tri-dimensional measurements on microcolonies differed from classical measurements in micro-dilutions based on optical diffusion. Such a precise characterization of the tri-dimensional shape of microcolonies in their late-lag to early-exponential phase could be beneficial in terms of in vitro diagnostics. Indeed, real-time monitoring of the biomass available in a colony could allow to run well established microbial identification workflows like, for instance, MALDI-TOF mass-spectrometry, as soon as a sufficient quantity of material is available, thereby reducing the time needed to provide a diagnostic. Moreover, as done for pre-identification of macro-colonies, morphological indicators such as three-dimensional growth profiles derived from microcolonies could be used to perform a first pre-identification step, but in a shorten time.

Background Construction and validation of a prognostic model for survival data in the clinical domain is still an active field of research. Nevertheless there is no consensus on how to develop routine prognostic tests based on a combination of RT-qPCR biomarkers and clinical or demographic variables. In particular, the estimation of the model performance requires to properly account for the RT-qPCR experimental design. Results We present a strategy to build, select, and validate a prognostic model for survival data based on a combination of RT-qPCR biomarkers and clinical or demographic data and we provide an illustration on a real clinical dataset. First, we compare two cross-validation schemes: a classical outcome-stratified cross-validation scheme and an alternative one that accounts for the RT-qPCR plate design, especially when samples are processed by batches. The latter is intended to limit the performance discrepancies, also called the validation surprise, between the training and the test sets. Second, strategies for model building (covariate selection, functional relationship modeling, and statistical model) as well as performance indicators estimation are presented. Since in practice several prognostic models can exhibit similar performances, complementary criteria for model selection are discussed: the stability of the selected variables, the model optimism, and the impact of the omitted variables on the model performance. Conclusion On the training dataset, appropriate resampling methods are expected to prevent from any upward biases due to unaccounted technical and biological variability that may arise from the experimental and intrinsic design of the RT-qPCR assay. Moreover, the stability of the selected variables, the model optimism, and the impact of the omitted variables on the model performances are pivotal indicators to select the optimal model to be validated on the test dataset. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0537-9) contains supplementary material, which is available to authorized users.

The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.

Motivation: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been broadly adopted by routine clinical microbiology laboratories for bacterial species identification. An isolated colony of the targeted microorganism is the single prerequisite. Currently, MS-based microbial identification directly from clinical specimens can not be routinely performed, as it raises two main challenges: (i) the nature of the sample itself may increase the level of technical variability and bring heterogeneity with respect to the reference database and (ii) the possibility of encountering polymicrobial samples that will yield a ‘mixed’ MS fingerprint. In this article, we introduce a new method to infer the composition of polymicrobial samples on the basis of a single mass spectrum. Our approach relies on a penalized non-negative linear regression framework making use of species-specific prototypes, which can be derived directly from the routine reference database of pure spectra. Results: A large spectral dataset obtained from in vitro mono- and bi-microbial samples allowed us to evaluate the performance of the method in a comprehensive way. Provided that the reference matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprints were sufficiently distinct for the individual species, the method automatically predicted which bacterial species were present in the sample. Only few samples (5.3%) were misidentified, and bi-microbial samples were correctly identified in up to 61.2% of the cases. This method could be used in routine clinical microbiology practice. Availability and implementation: The complete dataset including both the reference database and the mock-up mixture spectra is available at http://archive.ics.uci.edu/ml/datasets/MicroMass. Contact: pierre.mahe@biomerieux.com Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: Paired-end sequencing allows circumventing the shortness of the reads produced by second generation sequencers and is essential for de novo assembly of genomes. However, obtaining a finished genome from short reads is still an open challenge. We present an algorithm that exploits the pairing information issued from inserts of potentially any length. The method determines paths through an overlaps graph by using a constrained search tree. We also present a method that automatically determines suited overlaps cutoffs according to the contextual coverage, reducing thus the need for manual parameterization. Finally, we introduce an interactive mode that allows querying an assembly at targeted regions. Results: We assess our methods by assembling two Staphylococcus aureus strains that were sequenced on the Illumina platform. Using 100 bp paired-end reads and minimal manual curation, we produce a finished genome sequence for the previously undescribed isolate SGH-10-168. Availability and implementation: The presented algorithms are implemented in the standalone Edena software, freely available under the General Public License (GPLv3) at www.genomic.ch/edena.php. Contact: david.hernandez@genomic.ch Supplementary Information: Supplementary data are available at Bioinformatics online.

Natural populations are known to differ not only in DNA but also in their chromatin-associated epigenetic marks. When such inter-individual epigenomic differences (or “epi-polymorphisms”) are observed, their stability is usually not known: they may or may not be reprogrammed over time or upon environmental changes. In addition, their origin may be purely epigenetic, or they may result from regulatory variation encoded in the DNA. Studying epi-polymorphisms requires, therefore, an assessment of their nature and stability. Here we estimate the stability of yeast epi-polymorphisms of chromatin acetylation, and we provide a genome-by-epigenome map of their genetic control. A transient epi-drug treatment was able to reprogram acetylation variation at more than one thousand nucleosomes, whereas a similar amount of variation persisted, distinguishing “labile” from “persistent” epi-polymorphisms. Hundreds of genetic loci underlied acetylation variation at 2,418 nucleosomes either locally (in cis) or distantly (in trans), and this genetic control overlapped only partially with the genetic control of gene expression. Trans-acting regulators were not necessarily associated with genes coding for chromatin modifying enzymes. Strikingly, “labile” and “persistent” epi-polymorphisms were associated with poor and strong genetic control, respectively, showing that genetic modifiers contribute to persistence. These results estimate the amount of natural epigenomic variation that can be lost after transient environmental exposures, and they reveal the complex genetic architecture of the DNA–encoded determinism of chromatin epi-polymorphisms. Our observations provide a basis for the development of population epigenetics., Author Summary Chemical modifications of chromatin, such as DNA methylation, incorporation of histone variants, or post-translational modifications of histone proteins, constitute the “epigenome” and confer specific properties to genome functions. Epigenomes differ from one individual to another, opening the exciting perspective to decipher the origins of these differences and their impact on physiology. However, population epigenomics remains challenging because, unlike DNA mutations, epigenetic hallmarks are themselves regulated. They can change upon particular environmental conditions, they may be inherited epigenetically, or they may result from activities encoded in the DNA. Thus, estimating the stability of intra-species epigenomic variation and its dependence on DNA polymorphisms is essential. Using a chemical perturbation of yeast cells as an experimental model system, we found that acetylation variation was persistent at some nucleosomes and labile at other nucleosomes. By studying a segregating population, we mapped DNA polymorphisms that affected chromatin acetylation levels at numerous nucleosomes. Strikingly, nucleosomes showing persistent variation of acetylation corresponded to those for which acetylation was under genetic control. Thus, part of epigenomic variation is stabilized by a DNA–encoded determinism, and another part can be reprogrammed if environmental perturbations are experienced. These results provide a necessary basis for upcoming developments in population epigenomics.

Background Integrating QTL results from independent experiments performed on related species helps to survey the genetic diversity of loci/alleles underlying complex traits, and to highlight potential targets for breeding or QTL cloning. Potato (Solanum tuberosum L.) late blight resistance has been thoroughly studied, generating mapping data for many Rpi-genes (R-genes to Phytophthora infestans) and QTLs (quantitative trait loci). Moreover, late blight resistance was often associated with plant maturity. To get insight into the genomic organization of late blight resistance loci as compared to maturity QTLs, a QTL meta-analysis was performed for both traits. Results Nineteen QTL publications for late blight resistance were considered, seven of them reported maturity QTLs. Twenty-one QTL maps and eight reference maps were compiled to construct a 2,141-marker consensus map on which QTLs were projected and clustered into meta-QTLs. The whole-genome QTL meta-analysis reduced by six-fold late blight resistance QTLs (by clustering 144 QTLs into 24 meta-QTLs), by ca. five-fold maturity QTLs (by clustering 42 QTLs into eight meta-QTLs), and by ca. two-fold QTL confidence interval mean. Late blight resistance meta-QTLs were observed on every chromosome and maturity meta-QTLs on only six chromosomes. Conclusions Meta-analysis helped to refine the genomic regions of interest frequently described, and provided the closest flanking markers. Meta-QTLs of late blight resistance and maturity juxtaposed along chromosomes IV, V and VIII, and overlapped on chromosomes VI and XI. The distribution of late blight resistance meta-QTLs is significantly independent from those of Rpi-genes, resistance gene analogs and defence-related loci. The anchorage of meta-QTLs to the potato genome sequence, recently publicly released, will especially improve the candidate gene selection to determine the genes underlying meta-QTLs. All mapping data are available from the Sol Genomics Network (SGN) database.

International audience; Earliness is very important for the adaptation of wheat to environmental conditions and the achievement of high grain yield. A detailed knowledge of key genetic components of the life cycle would enable an easier control by the breeders. The objective of the study was to investigate the effect of candidate genes on flowering time. Using a collection of hexaploid wheat composed of 235 lines from diverse geographical origins, we conducted an association study for six candidate genes for flowering time and its components (vernalization sensitivity and earliness per se). The effect on the variation of earliness components of polymorphisms within the copies of each gene was tested in ANOVA models accounting for the underlying genetic structure. The collection was structured in five groups that minimized the residual covariance. Vernalization requirement and lateness tend to increase according to the mean latitude of each group. Heading date for an autumnal sowing was mainly determined by the earliness per se. Except for the Constans (CO) gene orthologous of the barley HvCO3, all gene polymorphisms had a significant impact on earliness components. The three traits used to quantify vernalization requirement were primarily associated with polymorphisms at Vrn-1 and then at Vrn-3 and Luminidependens (LD) genes. We found a good correspondence between spring/winter types and genotypes at the three homeologous copies of Vrn-1. Earliness per se was mainly explained by polymorphisms at Vrn-3 and to a lesser extent at Vrn-1, Hd-1 and Gigantea (GI) genes. Vernalization requirement and earliness as a function of geographical origin, as well as the possible role of the breeding practices in the geographical distribution of the alleles and the hypothetical adaptive value of the candidate genes, are discussed.

Epigenomes commonly refer to the sequence of presence/absence of specific epigenetic marks along eukaryotic chromatin. Complete histone-borne epigenomes have now been described at single-nucleosome resolution from various organisms, tissues, developmental stages, or diseases, yet their intra-species natural variation has never been investigated. We describe here that the epigenomic sequence of histone H3 acetylation at Lysine 14 (H3K14ac) differs greatly between two unrelated strains of the yeast Saccharomyces cerevisiae. Using single-nucleosome chromatin immunoprecipitation and mapping, we interrogated 58,694 nucleosomes and found that 5,442 of them differed in their level of H3K14 acetylation, at a false discovery rate (FDR) of 0.0001. These Single Nucleosome Epi-Polymorphisms (SNEPs) were enriched at regulatory sites and conserved non-coding DNA sequences. Surprisingly, higher acetylation in one strain did not imply higher expression of the relevant gene. However, SNEPs were enriched in genes of high transcriptional variability and one SNEP was associated with the strength of gene activation upon stimulation. Our observations suggest a high level of inter-individual epigenomic variation in natural populations, with essential questions on the origin of this diversity and its relevance to gene x environment interactions., Author Summary Nucleosomes are the basic units of chromatin, with part of the long DNA molecule wrapped around a multiprotein core, which makes unpacked chromatin often portrayed as a string of pearls. This string can carry three types of sequences: DNA, methyl groups on cytosines, and, on every pearl, the presence-or-absence of histone post-translational modifications such as acetylation of lysines (nucleosomal epigenome). These latter sequences can change dynamically, and the mechanisms involved are heavily studied as they participate in many physiological processes (pluripotency, disease…). However, nothing is known about the natural diversity of nucleosomal epigenomes in natural populations. As a model, we compared two unrelated yeast strains for their epigenome of one histone modification. We found a high divergence, which was enriched at regulatory sites and often carried on specific nucleosomes. Although this nucleosome modification is usually associated with high transcription, higher acetylation in one strain did not necessarily imply higher expression of the corresponding gene. However, one nucleosomal variation was associated with a stronger gene activation upon stimulation. These results suggest that nucleosomal epigenomes largely differ between individuals, raising essential questions on the origin of these differences and their contribution to personal responses to environmental changes (such as clinical treatments).

Understanding the genetic mechanisms underlying natural variation in gene expression is a central goal of both medical and evolutionary genetics, and studies of expression quantitative trait loci (eQTLs) have become an important tool for achieving this goal1. Although all eQTL studies so far have assayed messenger RNA levels using expression microarrays, recent advances in RNA sequencing enable the analysis of transcript variation at unprecedented resolution. We sequenced RNA from 69 lymphoblastoid cell lines derived from unrelated Nigerian individuals that have been extensively genotyped by the International HapMap Project2. By pooling data from all individuals, we generated a map of the transcriptional landscape of these cells, identifying extensive use of unannotated untranslated regions and more than 100 new putative protein-coding exons. Using the genotypes from the HapMap project, we identified more than a thousand genes at which genetic variation influences overall expression levels or splicing. We demonstrate that eQTLs near genes generally act by a mechanism involving allele-specific expression, and that variation that influences the inclusion of an exon is enriched within and near the consensus splice sites. Our results illustrate the power of high-throughput sequencing for the joint analysis of variation in transcription, splicing and allele-specific expression across individuals.

Recent studies of the HapMap lymphoblastoid cell lines have identified large numbers of quantitative trait loci for gene expression (eQTLs). Reanalyzing these data using a novel Bayesian hierarchical model, we were able to create a surprisingly high-resolution map of the typical locations of sites that affect mRNA levels in cis. Strikingly, we found a strong enrichment of eQTLs in the 250 bp just upstream of the transcription end site (TES), in addition to an enrichment around the transcription start site (TSS). Most eQTLs lie either within genes or close to genes; for example, we estimate that only 5% of eQTLs lie more than 20 kb upstream of the TSS. After controlling for position effects, SNPs in exons are ∼2-fold more likely than SNPs in introns to be eQTLs. Our results suggest an important role for mRNA stability in determining steady-state mRNA levels, and highlight the potential of eQTL mapping as a high-resolution tool for studying the determinants of gene regulation., Author Summary Individual phenotypes within natural populations generally exhibit a large diversity resulting from a complex interplay of genes and environmental factors. Since the advent of molecular markers in the 1980s, quantitative genetics has made a significant step toward unraveling the genetic bases of such complex traits, in particular by developing sophisticated tools to map the genomic locations of genes that affect complex traits. These regions are known as quantitative trait loci (QTLs). More recently, these tools have been extended to the study of gene expression phenotypes on a massive scale. In this paper, we used a previously published dataset consisting of expression measurements of 11,446 genes in human cell lines derived from 210 unrelated human individuals that have been genetically characterized by the International HapMap Project. Our article develops and applies a framework for determining the genetic factors that impact gene regulation. We show that these factors cluster strongly near to the gene start and gene end and are enriched within the transcribed region. Our approach suggests a general framework for studying the genetic factors that affect variation in gene expression.

The objective of this study was to map and characterize QTLs for traits related to nitrogen utilization efficiency (NUE), grain N yield, N-remobilization and post-silking N-uptake. Furthermore, to examine whether QTLs detected with recombinant inbred lines (RILs) crossed to a tester are common to those detected with line per se evaluation, both types of evaluations were developed from the same set of RILs. The material was studied over two years at high N-input, and one year at low N-input. We used (15)N-labelling to evaluate with accuracy the proportion of N remobilized from stover to kernels and the proportion of postsilking N-uptake allocated to kernels. With 59 traits studied in three environments, 608 QTLs were detected. Using a method of QTL clustering, 72 clusters were identified, with few QTLs being specific to one environment or to the type of plant material (lines or testcross families). However, considering each trait separately, few QTLs were common to both line per se and testcross evaluation. This shows that genetic variability is expressed differently according to the type of progeny. Studies of coincidences among QTLs within the clusters showed an antagonism between N-remobilization and N-uptake in several QTL-clusters. QTLs for N-uptake, root system architecture and leaf greenness coincided positively in eight clusters. QTLs for remobilization mainly coincided in clusters with QTLs for leaf senescence. On the whole, sign of coincidences between QTLs underlined the role of a "stay-green" phenotype in favouring N-uptake capacity, and thus grain yield and N grain yield.

To investigate the genetic basis of maize adaptation to temperate climate, collections of 375 inbred lines and 275 landraces, representative of American and European diversity, were evaluated for flowering time under short- and long-day conditions. The inbred line collection was genotyped for 55 genomewide simple sequence repeat (SSR) markers. Comparison of inbred line population structure with that of landraces, as determined with 24 SSR loci, underlined strong effects of both historical and modern selection on population structure and a clear relationship with geographical origins. The late tropical groups and the early “Northern Flint” group from the northern United States and northern Europe exhibited different flowering times. Both collections were genotyped for a 6-bp insertion/deletion in the Dwarf8 (D8idp) gene, previously reported to be potentially involved in flowering time variation in a 102 American inbred panel. Among-group D8idp differentiation was much higher than that for any SSR marker, suggesting diversifying selection. Correcting for population structure, D8idp was associated with flowering time under long-day conditions, the deletion allele showing an average earlier flowering of 29 degree days for inbreds and 145 degree days for landraces. Additionally, the deletion allele occurred at a high frequency (>80%) in Northern Flint while being almost absent (

Recent gene expression QTL (eQTL) mapping studies have provided considerable insight into the genetic basis for inter-individual regulatory variation. However, a limitation of all eQTL studies to date, which have used measurements of steady-state gene expression levels, is the inability to directly distinguish between variation in transcription and decay rates. To address this gap, we performed a genome-wide study of variation in gene-specific mRNA decay rates across individuals. Using a time-course study design, we estimated mRNA decay rates for over 16,000 genes in 70 Yoruban HapMap lymphoblastoid cell lines (LCLs), for which extensive genotyping data are available. Considering mRNA decay rates across genes, we found that: (i) as expected, highly expressed genes are generally associated with lower mRNA decay rates, (ii) genes with rapid mRNA decay rates are enriched with putative binding sites for miRNA and RNA binding proteins, and (iii) genes with similar functional roles tend to exhibit correlated rates of mRNA decay. Focusing on variation in mRNA decay across individuals, we estimate that steady-state expression levels are significantly correlated with variation in decay rates in 10% of genes. Somewhat counter-intuitively, for about half of these genes, higher expression is associated with faster decay rates, possibly due to a coupling of mRNA decay with transcriptional processes in genes involved in rapid cellular responses. Finally, we used these data to map genetic variation that is specifically associated with variation in mRNA decay rates across individuals. We found 195 such loci, which we named RNA decay quantitative trait loci (“rdQTLs”). All the observed rdQTLs are located near the regulated genes and therefore are assumed to act in cis. By analyzing our data within the context of known steady-state eQTLs, we estimate that a substantial fraction of eQTLs are associated with inter-individual variation in mRNA decay rates., Author Summary Recent studies of functional genetic variation in humans have identified numerous loci that are associated with variation in gene expression levels, called expression quantitative trait loci (eQTLs). The mechanisms by which these loci affect gene expression, however, are still largely unknown. Specifically, since most studies rely on measures of steady-state gene expression levels, they are unable to distinguish between the relative influences of either transcriptional- or decay-related processes. To address this gap, we examined the specific impact of mRNA decay processes on steady-state gene expression levels for over 16,000 genes in human lymphoblastoid cell lines. By characterizing decay rates in 70 individuals, we show that steady-state expression levels are significantly influenced by variation in decay rates for 10% of genes. Yet, for roughly half of these genes, we find that individuals with higher expression levels also have faster decay rates. This pattern points to a non-simple mechanistic interplay between transcriptional and decay processes, especially for genes involved in rapid cellular responses. Finally, we identify 195 genetic variants that are significantly associated with both gene expression variation and variation in mRNA decay rates. Using these data, we estimate that that a substantial fraction of eQTLs are associated with inter-individual variation in mRNA decay rates.

Mapping of expression quantitative trait loci (eQTLs) is an important technique for studying how genetic variation affects gene regulation in natural populations. In a previous study using Illumina expression data from human lymphoblastoid cell lines, we reported that cis-eQTLs are especially enriched around transcription start sites (TSSs) and immediately upstream of transcription end sites (TESs). In this paper, we revisit the distribution of eQTLs using additional data from Affymetrix exon arrays and from RNA sequencing. We confirm that most eQTLs lie close to the target genes; that transcribed regions are generally enriched for eQTLs; that eQTLs are more abundant in exons than introns; and that the peak density of eQTLs occurs at the TSS. However, we find that the intriguing TES peak is greatly reduced or absent in the Affymetrix and RNA-seq data. Instead our data suggest that the TES peak observed in the Illumina data is mainly due to exon-specific QTLs that affect 3' untranslated regions, where most of the Illumina probes are positioned. Nonetheless, we do observe an overall enrichment of eQTLs in exons versus introns in all three data sets, consistent with an important role for exonic sequences in gene regulation.

Linkage disesquilibrium (LD) and association mapping is receiving considerable attention in the plant genetics community for its potential to use existing genetic resources collections to fine map quantitative trait loci (QTL), validate candidate genes, and idently alleles of interest. Based on investigations in maize (Zea mays L.) either published or recently conducted in our group, we discuss three elements of particular importance for conducting association mapping or interpreting the results (i) the analysis of population structure into sub-groups, (ii) its use to control for spurious associations and consequences in the specific case of differential selection among subgroup, and (iii) the analysis of the local structure of LD into haplotypes and its consequences on the resolution and the application of LD mapping. Consequences and perspectives for plant breeding are briefly discussed.

Advances in epigenomics are opening the exciting perspective to associate chromatin variation to the variation of physiological traits in natural populations. Yet, inter-individual epigenomic differences may or may not result from regulatory variation encoded in the DNA. If they do, association then resumes to mapping underlying genetic variants, as for any genetic study. If they don’t, association requires a framework where the epigenetic nature of the variation is taken into account, in particular its potential reversibility. The development of population epigenomics therefore requires an assessment of the nature of epigenomic variation. As a model experimental system, we have traced quantitative variation of acetylation at Lysine 14 of Histone H3 at all nucleosomes in a segregating population of S. cerevisiae. Treating acetylation of every nucleosome as a quantitative trait allowed the identification of hundreds of genetic loci underlying epigenomic divergence either locally (in cis) or distantly (in trans), and this genetic control overlapped only partially with the genetic control of gene expression. We also estimated the stability of epigenetic differences across environmentally induced reprogramming. Strikingly, ‘labile’ and ‘persistent’ nucleosomal variations were associated with poor and strong genetic control, respectively. The study reveals the dual nature of natural chromatin epi-polymorphisms (DNA-encoded vs. not), providing a basis for the development of population epigenomics. Supported by grant SiGHT no281359 from the E.U.