Génomique de la spéciation chez le grand corégone (Coregonus clupeaformis) : divergence adaptive et isolement reproducteur

La mise en place de barrières à la reproduction et, par conséquent, la spéciation elle-même, engendrent et maintiennent la biodiversité. Les principaux objectifs de mes travaux étaient premièrement d'identifier l'ampleur des différences d'expression de gènes entre des jeunes espèces de grand corégones nains, normaux, ainsi que leurs hybrides. Par la suite, grâce à une technique révolutionnaire de séquençage, nous avons obtenu de l'information de séquence et de polymorphisme pour plusieurs milliers de gènes. Finalement, le génotypage de marqueurs SNPs en populations naturelles et simultanément dans une famille de poissons hybrides a permis d'évaluer l'effet de la sélection naturelle sur la divergence génétique des corégones. Ainsi, cette thèse apporte une meilleure compréhension de la divergence adaptive et de l'isolement reproducteur chez le corégone, tout en identifiant spécifiquement des gènes candidats impliqués dans le processus de spéciation

Divergence in gene regulation at young life history stages of whitefish (Coregonus sp.) and the emergence of genomic isolation

<p>Abstract</p> <p>Background</p> <p>The evolution of barriers to reproduction is of key interest to understand speciation. However, there may be a current bias towards studying intrinsic postzygotic isolation in old species pairs as compared to the emergence of barriers to gene flow through adaptive divergence. This study evaluates the relative importance of both processes in the evolution of genomic isolation in incipient species of whitefish (<it>Coregonus clupeaformis</it>) for which preliminary data suggest that postzygotic isolation emerges with intrinsic factors acting at embryo stages but also due to extrinsic factors during adult life.</p> <p>Results</p> <p>Gene expression data were screened using cDNA microarrays to identify regulatory changes at embryo and juvenile stages that provide evidence for genomic divergence at the underlying genetic factors. A comparison of different life history stages shows that 16-week old juvenile fish have 14 times more genes displaying significant regulatory divergence than embryos. Furthermore, regulatory changes in juvenile fish match patterns in adult fish suggesting that gene expression divergence is established early in juvenile fish and persists throughout the adult phase. Comparative analyses with results from previous studies on dwarf-normal species pairs show that at least 26 genetic factors identified in juvenile fish are candidate traits for adaptive divergence in adult fish. Eight of these show parallel directions of gene expression divergence independent of tissue type or age of the fish. The latter are associated with energy metabolism, a complex trait known to drive adaptive divergence in dwarf and normal whitefish.</p> <p>Conclusion</p> <p>Although experimental evidence suggests the existence of genetic factors that cause intrinsic postzygotic isolation acting in embryos, the analysis presented here provided few candidate genes in embryos, which also corroborate previous studies showing a lack of ecological divergence between sympatric dwarf and normal whitefish at the larval stage. In contrast, gene expression divergence in juveniles can be linked to adaptive traits and seems to be driven by positive selection. The results support the idea that adaptive differentiation may be more important in explaining the emergence of barriers to gene flow in an early phase of speciation by providing a broad genomic basis for extrinsic postzygotic isolation rather than intrinsic barriers.</p

Maize Allergens during seed development: transgenic versus non-transgenic

Recombinant DNA technology, also known as genetic engineering, allows the transfer of genes between unrelated species. As a result, a genetically modified organism (GMO) may contain one or more proteins coming from other organism/s. The application of genetic engineering to plants improvement and food production is becoming a common practice. New and diverse plant varieties have been obtained which are pest and disease resistant, more productive and with improved nutritional quality, flavour and shelf life. In spite of all these potential benefits, some apprehension persist regarding genetically modified organisms putative effects over human health and environment. One of the main concerns regards GMO’s potential allergenicity. One of the possibilities is that the introduction of a new/ altered gene may putatively alter the expression of others, namely endogenous allergens. In maize, there are already some proteins characterized as allergens, namely a lipid transport protein (Pastorello et al. 2000), a tioredoxin Zea m25 (Weichel et al. 2006), two glutelins Zea m27kD (Frisner et al. 2000) and 50kD Zein (Pasini et al. 2002) and one trypsin inhibitor (Pastorello et al. 2000). In this study, we have evaluated the expression of these 5 allergens, throughout MON 810 vs its non-transgenic counterpart seed development (10, 16 and 23 days after pollination). The expression profile of each one of these allergens varies during seed development, although the observed differences between Transgenic and Non-Transgenic maize were not statistical significant (t-test). Since there is a lack of experimental data regarding the correlation of allergen expression with food allergy clinical relevance, we are now comparing the immunologic response of maize allergic individuals against MON 810 maize vs its non transgenic control. We also intend to characterize the encountered potential maize allergens by mass spectrometry

Environmental stress is the major cause of transcriptomic and proteomic changes in GM and non-GM plants

The approval of genetically modified (GM) crops is preceded by years of intensive research to demonstrate safety to humans and environment. We recently showed that in vitro culture stress is the major factor influencing proteomic differences of GM vs. non-GM plants. This made us question the number of generations needed to erase such “memory”. We also wondered about the relevance of alterations promoted by transgenesis as compared to environment-induced ones. Here we followed three rice lines (1-control, 1-transgenic and 1-negative segregant) throughout eight generations after transgenesis combining proteomics and transcriptomics, and further analyzed their response to salinity stress on the F6 generation. Our results show that: (a) differences promoted during genetic modification are mainly short-term physiological changes, attenuating throughout generations, and (b) environmental stress may cause far more proteomic/transcriptomic alterations than transgenesis. Based on our data, we question what is really relevant in risk assessment design for GM food crops.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Project PTDC/EBB-BIO/098983/2008 and research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013). S.N. acknowledges funding from King Abdullah University of Science and Technology (KAUST).info:eu-repo/semantics/publishedVersio

Mandated data archiving greatly improves access to research data

The data underlying scientific papers should be accessible to researchers both now and in the future, but how best can we ensure that these data are available? Here we examine the effectiveness of four approaches to data archiving: no stated archiving policy, recommending (but not requiring) archiving, and two versions of mandating data deposition at acceptance. We control for differences between data types by trying to obtain data from papers that use a single, widespread population genetic analysis, STRUCTURE. At one extreme, we found that mandated data archiving policies that require the inclusion of a data availability statement in the manuscript improve the odds of finding the data online almost a thousand-fold compared to having no policy. However, archiving rates at journals with less stringent policies were only very slightly higher than those with no policy at all. At one extreme, we found that mandated data archiving policies that require the inclusion of a data availability statement in the manuscript improve the odds of finding the data online almost a thousand fold compared to having no policy. However, archiving rates at journals with less stringent policies were only very slightly higher than those with no policy at all. We also assessed the effectiveness of asking for data directly from authors and obtained over half of the requested datasets, albeit with about 8 days delay and some disagreement with authors. Given the long term benefits of data accessibility to the academic community, we believe that journal based mandatory data archiving policies and mandatory data availability statements should be more widely adopted

Lettuce (Lactuca sativa L.) leaf-proteome profiles after exposure to cylindrospermopsin and a microcystin-LR/cylindrospermopsin mixture: A concentration-dependent response

The intensification of agricultural productivity is an important challenge worldwide. However, environmental stressors can provide challenges to this intensification. The progressive occurrence of the cyanotoxins cylindrospermopsin (CYN) and microcystin-LR (MC-LR) as a potential consequence of eutrophication and climate change is of increasing concern in the agricultural sector because it has been reported that these cyanotoxins exert harmful effects in crop plants. A proteomic-based approach has been shown to be a suitable tool for the detection and identification of the primary responses of organisms exposed to cyanotoxins. The aim of this study was to compare the leaf-proteome profiles of lettuce plants exposed to environmentally relevant concentrations of CYN and a MC-LR/CYN mixture. Lettuce plants were exposed to 1, 10, and 100 lg/l CYN and a MC-LR/CYN mixture for five days. The proteins of lettuce leaves were separated by twodimensional electrophoresis (2-DE), and those that were differentially abundant were then identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF/TOF MS). The biological functions of the proteins that were most represented in both experiments were photosynthesis and carbon metabolism and stress/defense response. Proteins involved in protein synthesis and signal transduction were also highly observed in the MC-LR/CYN experiment. Although distinct protein abundance patterns were observed in both experiments, the effects appear to be concentration-dependent, and the effects of the mixture were clearly stronger than those of CYN alone. The obtained results highlight the putative tolerance of lettuce to CYN at concentrations up to 100 lg/l. Furthermore, the combination of CYN with MC-LR at low concentrations (1 lg/l) stimulated a significant increase in the fresh weight (fr. wt) of lettuce leaves and at the proteomic level resulted in the increase in abundance of a high number of proteins. In contrast, many proteins exhibited a decrease in abundance or were absent in the gels of the simultaneous exposure to 10 and 100 lg/l MC-LR/CYN. In the latter, also a significant decrease in the fr. wt of lettuce leaves was obtained. These findings provide important insights into the molecular mechanisms of the lettuce response to CYN and MC-LR/CYN and may contribute to the identification of potential protein markers of exposure and proteins that may confer tolerance to CYN and MC-LR/CYN. Furthermore, because lettuce is an important crop worldwide, this study may improve our understanding of the potential impact of these cyanotoxins on its quality traits (e.g., presence of allergenic proteins)

Maize IgE binding proteins: each plant a different profile?

Background: Allergies are nearly always triggered by protein molecules and the majority of individuals with documented immunologic reactions to foods exhibit IgE hypersensitivity reactions. In this study we aimed to understand if natural differences, at proteomic level, between maize populations, may induce different IgE binding proteins profiles among maize-allergic individuals. We also intended to deepen our knowledge on maize IgE binding proteins. Results: In order to accomplish this goal we have used proteomic tools (SDS-PAGE and 2-D gel electrophoresis followed by western blot) and tested plasma IgE reactivity from four maize-allergic individuals against four different protein fractions (albumins, globulins, glutelins and prolamins) of three different maize cultivars. We have observed that maize cultivars have different proteomes that result in different IgE binding proteins profiles when tested against plasma from maize-allergic individuals. We could identify 19 different maize IgE binding proteins, 11 of which were unknown to date. Moreover, we found that most (89.5%) of the 19 identified potential maize allergens could be related to plant stress. Conclusions: These results lead us to conclude that, within each species, plant allergenic potential varies with genotype. Moreover, considering the stress-related IgE binding proteins identified, we hypothesise that the environment, particularly stress conditions, may alter IgE binding protein profiles of plant components

Selection of the best comparator for the risk assessment of GM plants- conventional counterpart vs. negative segregant

Abstract for 12th international Symposium on biosafety of genetically modified organisms The identification of similarities and differences between GM plants and derived food/feed and their comparators plays a central role in risk assessment strategy. Therefore, selecting the right comparators must be one of the top priorities. The question is which control would allow us to better evaluate the potential unintended effects related directly to the transgene and/or DNA rearrangements, discounting the potential effects caused by in vitro culture procedures; since, these are non-controversial procedures also used in conventional breeding. Aiming to answer the previous question we have used Multiplex fluorescence 2D gel electrophoresis technology (DyeAGNOSTICS Refraction-2D) coupled with MS to characterize the proteome of three different rice lines (Oriza sativa L. ssp. Japonica cv Nipponbare): A control conventional counterpart. An Agrobacterium transformed transgenic line. A negative segregant (homozygous negative progeny) from a different transgenic line. We have observed that transgenic and negative segregant plant lines grouped together (only 1 differentially regulated spot - fold difference > 1.5, ANOVA, P1.5, ANOVA, P<0.05, in both transgenic and negative segregant lines comparing to control line). Additionally, the 35 proteins identified (using MS) in this study, were already associated with stress response by other authors. The only feature in common between the transgenic and negative segregant lines is that they have both suffered in vitro culture procedures. Hence, the results obtained indicate that, in this study, different gene disruption and/or DNA rearrangements and the presence/absence of transgene were factors with less impact on rice proteome than the proteomic promoted differences caused by in vitro culture, and eventually the stress caused by this process. This work highlights the importance of continuous revision and upgrade of the guidance criteria to be followed for the selection of suitable comparators in GMO risk assessment

Flowering under stress: phenomics and proteomics approaches to chickpea grain yield and quality.

Chickpea (Cicer arietinum) is a major player in the FAO Zero Hunger program “toolkit”. It is the second most cultivated food legume, a source of sustainable protein (and other nutrients) and contributes to improved soil health and lower fertilization input. Grain yield, seed protein content and nutritional quality were found to be largely affected by Genotype x Environment. In the Mediterranean regions, higher frequency, intensity and duration of droughts, as well as hotter droughts are being registered, thus interfering with agroecosystems’ structure, composition, and functions. A controlled conditions assay (non-invasive phenotyping@PhenoLab) was undertaken in two genotypes from the Portuguese chickpea breeding program (Elvas, INIAV). Results revealed that high temperature (32º C during the reproductive phase) and water regime (40% vs 10% soil water content) play a significant role in chickpea development, seed production and composition, and protein content. As seed biochemical signatures allow to discriminate between genotypes, the power protein-based methodologies in genotype assessment are highlighted. Because some proteins can resist gastrointestinal digestion and influence human health, the processed seed proteome (i.e. following soaking, boiling and in-vitro digestion) in three consecutive years was further analysed in field-grown chickpeas (Elvas, INIAV). The seed proteome was found to be highly conserved, with minor changes being attributed to the seed development conditions. Furthermore, in vitro digestion efficiently removed many anti-nutritional proteins. Combined phenotyping and omic´s approaches contribute to the mechanistic knowledge of how severe terminal drought and high temperature modulate sink capacity and productivity (yield and quality). The integrated use of phenomics and omics methodologies has significant potential to increase our understanding of plant growth and development and, thus, an efficient, knowledge-based management of crops and resources.Work supported by EMPHASIS-GO (HORIZON-INFRA-2021-DEV-02 contract n. 101079772), by the European Plant Phenotyping Network 2020 (EXCELLENT SCIENCE - Research Infrastructures contract n. 731013) and by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P. namelly: UCIBIO – Research Unit on Applied Molecular Biosciences (FCT UIDP/04378/2020; FCT UIDB/04378/ 2020); 4HB - Associate Laboratory Institute for Health and Bioeconomy (FCT LA/P/0140/2020); LEAF – Linking Landscape, Environment, Agriculture and Food (FCT UID/AGR/04129/2020); PhD fellowship SFRH/BD/70345/2010.info:eu-repo/semantics/publishedVersio