SNP discovery and genetic mapping using genotyping by sequencing of whole genome genomic DNA from a pea RIL population

International audienceBackground - Progress in genetics and breeding in pea still suffers from the limited availability of molecular resources. SNP markers that can be identified through affordable sequencing processes, without the need for prior genome reduction or a reference genome to assemble sequencing data would allow the discovery and genetic mapping of thousands of molecular markers. Such an approach could significantly speed up genetic studies and marker assisted breeding for non-model species. Results - A total of 419,024 SNPs were discovered using HiSeq whole genome sequencing of four pea lines, followed by direct identification of SNP markers without assembly using the discoSnp tool. Subsequent filtering led to the identification of 131,850 highly designable SNPs, polymorphic between at least two of the four pea lines. A subset of 64,754 SNPs was called and genotyped by short read sequencing on a subpopulation of 48 RILs from the cross 'Baccara' x 'PI180693'. This data was used to construct a WGGBS-derived pea genetic map comprising 64,263 markers. This map is collinear with previous pea consensus maps and therefore with the Medicago truncatula genome. Sequencing of four additional pea lines showed that 33 % to 64 % of the mapped SNPs, depending on the pairs of lines considered, are polymorphic and can therefore be useful in other crosses. The subsequent genotyping of a subset of 1000 SNPs, chosen for their mapping positions using a KASP™ assay, showed that almost all generated SNPs are highly designable and that most (95 %) deliver highly qualitative genotyping results. Using rather low sequencing coverages in SNP discovery and in SNP inferring did not hinder the identification of hundreds of thousands of high quality SNPs. Conclusions - The development and optimization of appropriate tools in SNP discovery and genetic mapping have allowed us to make available a massive new genomic resource in pea. It will be useful for both fine mapping within chosen QTL confidence intervals and marker assisted breeding for important traits in pea improvement

RDR2 Partially Antagonizes the Production of RDR6-Dependent siRNA in Sense Transgene-Mediated PTGS

Background: RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and SUPPRESSOR of GENE SILENCING 3 (SGS3) are required for DNA methylation and post-transcriptional gene silencing (PTGS) mediated by 21-nt siRNAs produced by sense transgenes (S-PTGS). In contrast, RDR2, but not RDR6, is required for DNA methylation and TGS mediated by 24-nt siRNAs, and for cellto-cell spreading of IR-PTGS mediated by 21-nt siRNAs produced by inverted repeat transgenes under the control of a phloem-specific promoter. Principal Findings: In this study, we examined the role of RDR2 and RDR6 in S-PTGS. Unlike RDR6, RDR2 is not required for DNA methylation of transgenes subjected to S-PTGS. RDR6 is essential for the production of siRNAs by transgenes subjected to S-PTGS, but RDR2 also contributes to the production of transgene siRNAs when RDR6 is present because rdr2 mutations reduce transgene siRNA accumulation. However, the siRNAs produced via RDR2 likely are counteractive in wildtype plants because impairement of RDR2 increases S-PTGS efficiency at a transgenic locus that triggers limited silencing, and accelerates S-PTGS at a transgenic locus that triggers efficient silencing. Conclusions/Significance: These results suggest that RDR2 and RDR6 compete for RNA substrates produced by transgenes subjected to S-PTGS. RDR2 partially antagonizes RDR6 because RDR2 action likely results in the production of counteractiv

Etudes moléculaires de la voie de signalisation Hedgehog chez l'homme

Les hedgehogopathies regroupent un ensemble de syndromes dysmorphiques héréditaires caractérisés par une altération de la voie de signalisation Hedgehog (Hh). Dans cette voie, le gène patched code pour un récepteur qui agit en opposition à la protéine Hh. En l'absence d'une protéine PTCH fonctionnelle, les facteurs de transcription GLI sont surexprimés. Chez l'homme, ptch1 est muté dans le syndrome de Gorlin ou Naevomatose baso-cellulaire, et gli3 est muté dans les syndromes de Greig (GCPS) et de Pallister-Hall (PHS). Nous avons étudié le spectre des mutations géniques dans 151 échantillons de patients atteints du qyndrome de Gorlin et 52 échantillons de patients présentant les syndromes GCPS et PHS, par différentes techniques dont la dHPLC. 27 mutations du gène ptch1 ont été identifiées, dont une mutation récurrente dans l'exon 8, et 5 mutations du gène gli3. Alors qu'il semble exister une corrélation génotype/phénotype dans les syndromes de Greig et Pallister-Hall, aucune corrélation n'a été identifiée dans le syndrome de Gorlin. Les patients atteints du syndrome de Gorlin développent des carcinomes baso-cellulaires (CBC), dans lesquels la protéine GLI1 est surexprimée. Nous émettons l'hypothèse que des oligonucléotides leurres, contenant le site de fixation de GLI1 sur l'ADN, pourraient capturer GLI1, empêchant la transactivation de gènes essentiels de la voie Hh et inhiber ainsi la prolifération. Nous avons construit un système expérimental in vitro basé sur la transactivation d'un gène rapporteur, la luciférase, par l'expression des prtéines GLI1 et GLI3, permettant ainsi de visualiser la fixation de GLI sur l'ADN. Malheureusement les essais d'inactivation de la surexpression de GLI1 par les oligonucléotides leurres se sont révélés négatifs."Hedgehogopathy" defines in human a group of hereditary dysmorphic syndromes with an alteration of the Hedgehog (Hh) pathway. In this pathway, the patched gene encodes a receptor that acts in opposition to Hh protein. In the absence of a functional PTCH protein, GLI transcription factors are overexpressed.In human, ptch1 gene mutations are involved in Gorlin syndrome or Nevoid Basal Cell Carcinoma Syndrome, and gli3 is mutated in Greig syndrome (GCPS) and Pallister-Hall syndrome (PHS). We have studied gene mutations spectrum in 151 patients cases of Gorlin syndrome, 52 cases of Greig and Pallister-Hall syndromes, with different techniques including dHPLC. 27 ptch1 mutations have been identified, where one recurrent mutation in exon 8, and 5 gli3 mutations. While a phenotype-genotype correlation seems to exist in Greig and Pallister-Hall syndromes, no correlation was found in the Gorlin syndrome. Patients affected by Gorlin syndrome generally develop basal cell carcinoma (BCC), in which GLI1 protein is overexpressed. We hypothesize that decoy oligonucleotides containing the GLI1 DNA binding site would effectively bind GLI1, preventing the transactivation of essential genes of the Hh pathway and thereby inhibiting proliferation. In this way, we design an experimental in vitro system, based on the transactivation of a reporter gene, the luciferase, which allows the visualisation of GLI DNA binding. However we were unable to demonstrate any inactivation of GLI1 by using decoy oligonucleotides.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome of Turnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta

The RNA genome of Turnip mosaic virus is covalently linked at its 5′ end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication

Capture : Grégory Chatonsky

" Grégory Chatonsky numbers among the contemporary artists who emerged at the same time as the Web in the 1990s. Since then, he did not stop staging the flows generated by networks in order to built fictions he now refers to as “variables”. His online creations, installations or performances question the relations between private and public. This first monographic catalog gathers texts by Michael Joyce, Violaine Boutet de Monvel, Pau Waelder, Natalie Leleu and Jay Murphy... " -- Publisher's website

Le tutorat maître-élève et le tutorat par les pairs : deux mesures d'aide individualisée au collégial.

Bibliogr.Examen des programmes de tutorat maître-élève dans les collèges du réseau québécois [résultats d'un sondage] / Pour une approche développementale en tutorat maître-élève [paramètres à prendre en compte dans la mise en place d'un programme de tutorat] / Le tutorat : une modalité de la relation maître-élève [présentation du programme mis sur pied par le cégep de Rivière-du-Loup] / Le tutorat par les pairs au Collège Dawson

Le tutorat maître-élève et le tutorat par les pairs : deux mesures d'aide individualisée au collégial

Examen des programmes de tutorat maître-élève dans les collèges du réseau québécois [résultats d'un sondage] Pour une approche développementale en tutorat maître-élève [paramètres à prendre en compte dans la mise en place d'un programme de tutorat] Le tutorat : une modalité de la relation maître-élève [présentation du programme mis sur pied par le cégep de Rivière-du-Loup] Le tutorat par les pairs au Collège Dawso

PEPPER: Precise Privacy-Preserving Contact Tracing with Cheap, BLE/UWB Capable Tokens

International audienceContact Tracing (CT) is an old, recognized epidemiological tool, and since a digital variant is now within reach, a variety of smartphone-based solutions have been rapidly developed and deployed since 2020, with mixed results and amid controversies. Yet, achieving reliable and effective digital CT at large scale is still an open problem. In this work, we contribute with an open source software platform on top of which various CT solutions can be quickly developed and tested. More specifically, we design PEPPER, which jointly leverages Bluetooth Low Energy (BLE) and Ultra Wide Band (UWB) radios for contact detection, combined with the DESIRE privacy-preserving CT protocol. We show that PEPPER+DESIRE can operate on cheap physical tokens based on low-power microcontrollers, opening new use-cases with less personal, potentially disposable devices, that could be more widely used. We also evaluate the complementarity of Bluetooth and UWB in this context, via experiments mimicking various scenarios relevant for CT. Compared to BLE-only CT, we show that UWB can decrease false negatives (e.g., in presence of human body occlusion), meaning that more actual contacts will be found, a key benefit from an epidemiological viewpoint. Our results suggest that, while PEPPER+DESIRE improves precision over state-of-the-art, further research is required to harness UWB-BLE synergy for CT in practice. To this end, our open source platform (which can run on an open-access testbed) provides a useful playground for the research community

Reference-free high-throughput SNP detection in pea: an example of discoSnp usage for a non-model complex genome

International audienceBackground / Purpose:Detecting Single Nucleotide Polymorphisms (SNPs) between genomes is a routine task with Next Generation Sequencers (NGS) data. SNP detection methods generally need a reference genome. As non-model organisms are increasingly investigated, reference-free methods are needed. The discoSnp method detects SNPs directly from raw NGS data set(s) without using any third-party information. The pea non-model organism has a 4.5 GB complex genome without reference. We compared, on the same set of low depth pea sequences, the SNPs generated by discoSnp with those published with a previous SNP discovery pipeline, and those generated using classical mapping approach with the association of Bowtie2 and GATK tools.Main conclusion:The quality of discoSnp results in association with its very low memory needs and low time footprints led us to choose this software for a SNP discovery and direct Genotypin. By Sequencing project on a set of 48 pea genomic DNA libraries from a recombinant inbred lines subpopulation sequenced with Illumina HiSeq2000 technology. The analysis enabled to identify 88,851 SNP polymorphs on this population, from which around 60k SNPs will be genetically mapped