Flow cytometry distinction between species and between landraces within Lathyrus species and assessment of true-to-typeness of in vitro regenerants

International audienceThe genus Lathyrus includes a number of neglected wild relatives of pea with potential as genetic resources for acquisition of stress resistance traits, but, due to little breeding, genotypes under culture are mainly landraces and seldom true varieties. Development of in vitro approaches for Lathyrus is also limited, and assessments of nuclear DNA content, for taxonomical or breeding purposes, are sparse. Genome size and AT/GC ratio were determined by flow cytometry, allowing for distinction between protein and forage L. sativus, L. cicera, L. ochrus and L. clymenum and the ornamental sweet pea (L. odoratus), and also between landraces within L. sativus L. and L. cicera L. In addition, explants from in vitro seedlings of eight genotypes from the five Lathyrus species above were cultivated in vitro, plant regeneration was achieved for all landraces and species, and the nuclear DNA content of the regenerants was compared with that of their mother plants, whereby the true-to-typeness of such regenerants was confirmed

Structure and diversity of ssDNA Microviridae viruses in two peri-alpine lakes (Annecy and Bourget, France)

International audienceMicroviridae is a subset of single-stranded DNA (ssDNA) viruses infecting bacteria. This group of phages has been previously observed to be very abundant (representing >90% of the total known viral metagenomic sequences) in Lake Bourget. However, this observation was made only during one period (in summer) and from a single sample collected at a single depth (near surface). This result suggests the importance of these viruses, poorly examined thus far, especially in fresh waters. In this study, performed on the two largest natural lakes in France (e.g. Lakes Annecy and Bourget), Microviridae structure was determined each month throughout the year (2011) using PCR-DGGE, with primers that target the major-capsid-protein-encoding gene VP1; cloning/sequencing was used to investigate their diversity. Our results confirm that Microviridae are diverse in peri-alpine lakes and are mainly represented by gokushoviruses. We also found for the first time ssDNA viruses belonging to Alpavirinae, another subfamily within Microviridae recently proposed by Krupovic and Forterre (2011), generally prophages infecting members of the Phylum Bacteroidetes. Our data also support highly variable community composition and dynamics of individual components whose patterns were different between lakes, suggesting distinct host communities and/or abiotic influences between the two ecosystems. We point out that most of the major observed ssDNA Microviridae viruses display boom-bust patterns (with a sharp increase/decline) in their dynamics, with high relative abundances, suggesting brutal control of hosts and rapid regulation of the host community structure

Bdellovibrio and Like Organisms in Lake Geneva: An Unseen Elephant in the Room?

International audienceWhen considering microbial biotic interactions, viruses as well as eukaryotic grazers are known to be important components of aquatic microbial food webs. It might be the same for bacterivorous bacteria but these groups have been comparatively less studied. This is typically the case of the Bdellovibrio and like organisms (BALOs), which are obligate bacterial predators of other bacteria. Recently, the abundance and distribution of three families of this functional group were investigated in perialpine lakes, revealing their presence and quantitative importance. Here, a more in-depth analysis is provided for Lake Geneva regarding the diversity of these bacterial predators at different seasons, sites and depths. We reveal a seasonal and spatial (vertical) pattern for BALOs. They were also found to be relatively diverse (especially Bdellovibrionaceae) and assigned to both known and unknown phylogenetic clusters. At last we found that most BALOs were positively correlated to other bacterial groups, mainly Gram-negative, in particular Myxococcales (among which many are predators of other microbes). This study is the first shedding light on this potentially important bacterial killing group in a large and deep lake

Flow cytometric analysis and molecular characterization of Agrobacterium tumefaciens-mediated transformants of Medicago truncatula

Sur la publication, l'auteur S. Djennane est mal orthographié (Djenanne).International audienceLeaf explants from leaflets collected from either in vivo grown or in vitro grown seedlings of Medicago truncatula genotype R108-1 were co-cultivated with bacterial cells of Agrobacterium tumefaciens strains EHA105 or C58pMP90. Each of these strains was carrying the pCambia 1390 plasmid harbouring a hygromycin resistance gene cassette. Explants were then incubated on a medium containing 10 mg/l hygromycin and 800 mg/l augmentin to suppress Agrobacterium growth, and subcultured 4-5 times every 2 weeks for the proliferation of calli. After 8-10 weeks, callusing explants were transferred to hormone-free medium with 10 mg/l hygromycin and 400 mg/l augmentin for shoot regeneration. After rooting, a total of about 300 putative transformants were grown into plantlets, transferred to soil, acclimatized, and then moved to the greenhouse. Of these, a total of 43 independent PCR positive primary transformants and their T1 and T2 progeny were subjected to flow cytometric analysis, to assessing their trueness-to-type, as well as to southern blot analysis

Competence versus Recalcitrance for in Vitro Regeneration

Plant regeneration from tissue cultures of many species has been reported so far, but various groups. families and genera are still regarded as recalcitant, and tools susceptible to distinguish as early in culture as possible the regeneration-competent cells and tissues from those that will never regenerate would be of interest, whatever the regeneration pathway We have examined various cytological parameters to identify those that might serve as early indicators of cell competence to undergo somatic embryogenesis and/or organogenesis Water potential of the culture medium declined preceding the onset of embryogenesis while intracellular osmolarity and cell sui face increased concomitantly in embryogenic cells In addition, cellulose accumulated us the walls of non-embryogenic cells while cell walls became thinner with onset of embryogenesis, and diminished further as embryos matured In parallel experiments, rise cytogenetic, proteomic and molecular processes governing the competence for embryogenesis and/or organogenesis from plant cells were studied with Arabidopsis thaliana, and more recently also with Medicago truncatula and pea, both by flow cytometry, immunocytohistology, mono and bidimensional electrophoresis and comparing insertion mutants and RNA(is) with their respective wild types The implications of these results when applying biotechnology approaches for grain legume breeding, and in terms of plant regeneration competence in general arc discussed

Transcription of two blue copper-binding protein isogenes is highly correlated with arbuscular mycorrhizal development in Medicago truncatula

International audienceExpression profiling of two paralogous arbuscular mycorrhizal (AM)-specific blue copper-binding gene (MtBcp1a and MtBcp1b) isoforms was performed by real-time quantitative polymerase chain reaction in wild-type Medicago truncatula Jemalong 5 (J5) during the mycorrhizal development with Glomus intraradices for up to 7 weeks. Time-course analysis in J5 showed that expression of both MtBcp1 genes increased continuously and correlated strongly with the colonization intensity and arbuscule content. MtPT4, selected as a reference gene of the functional plant-fungus association, showed a weaker correlation to mycorrhizal development. In a second experiment, a range of mycorrhizal mutants of the wild-type J5 was assessed. Strictly AM-penetration-defective TRV25-C and TRV25-D (dmi3, Mtsym13), hypomycorrhizal TR25 and TR89 (dmi2, Mtsym2) mutants, and a hypermycorrhizal mutant TRV17 (sunn, Mtsym12) were compared with J5 3 and 7 weeks after inoculation. No MtBcp1 transcripts were detected in the mutants blocked at the appressoria stage. Conversely, TR25, TR89, and J5 showed a gradual increase of the expression of both MtBcp1 genes in 3- and 7-week-old plants, similar to the increase in colonization intensity and arbuscule abundance. The strong correlation between the expression level of AM-specific blue copper-binding protein-encoding genes and AM colonization may imply a basic role in symbiotic functioning for these genes, which may serve as new molecular markers of arbuscule development in M. truncatula

Blue-copper binding proteins of <em>Medicago truncatula</em>: Characterization of the expression of two genes related to the arbuscular mycorrhizal symbiosis

International audienceExpression profiling of two paralogous arbuscular mycorrhizal (AM)-specific blue copper-binding gene (MtBcpla and MtBcp1b) isoforms was performed by real-time quantitative polymerase chain reaction in wild-type Medicago truncatula Jemalong 5 (J5) during the mycorrhizal development with Glomus intraradices for up to 7 weeks. Timecourse analysis in J5 showed that expression of both MtBcp1 genes increased continuously and correlated strongly with the colonization intensity and arbuscule content. MtPT4, selected as a reference gene of the functional plant-fungus association, showed a weaker correlation to mycorrhizal development. In a second experiment, a range of mycorrhizal mutants of the wild-type J5 was assessed. Strictly AMpenetration-defective TRV25-C and TRV25-D (dmi3, Mtsym13), hypomycorrhizal TR25 and TR89 (dmi2, Mtsym2) mutants, and a hypermycorrhizal mutant TRV17 (sunn, Mtsym12) were compared with J5 3 and 7 weeks after inoculation. No MtBcp1 transcripts were detected in the mutants blocked at the appressoria stage. Conversely, TR25, TR89, and J5 showed a gradual increase of the expression of both MtBcp1 genes in 3- and 7-week-old plants, similar to the increase in colonization intensity and arbuscule abundance. The strong correlation between the expression level of AM-specific blue copper-binding protein-encoding genes and AM colonization may imply a basic role in symbiotic functioning for these genes, which may serve as new molecular markers of arbuscule development in M. truncatula

Insertion mutagenesis in <em>Medicago truncatula</em>. The grain legume (FP6-GLIP) Tnt1 project

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The Fellowship of the Ring Test: DNAqua-Net WG2 initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardisation

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems.Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring

Development and implementation of eco-genomic tools for aquatic ecosystem biomonitoring: the SYNAQUA French-Swiss program

International audienceThe effectiveness of environmental protection measures is based on the early identification and diagnosis of anthropogenic pressures. Similarly, restoration actions require precise monitoring of changes in the ecological quality of ecosystems, in order to highlight their effectiveness. Monitoring the ecological quality relies on bioindicators, which are organisms revealing the pressures exerted on the environment through the composition of their communities. Their implementation, based on the morphological identification of species, is expensive because it requires time and experts in taxonomy. Recent genomic tools should provide access to reliable and high-throughput environmental monitoring by directly inferring the composition of bioindicators' communities from their DNA (metabarcoding). The French-Swiss program SYNAQUA (INTERREG France-Switzerland 2017-2019) proposes to use and validate the tools of environmental genomic for biomonitoring and aims ultimately at their implementation in the regulatory bio-surveillance. SYNAQUA will test the metabarcoding approach focusing on two bioindicators, diatoms, and aquatic oligochaetes, which are used in freshwater biomonitoring in France and Switzerland. To go towards the renewal of current biomonitoring practices, SYNAQUA will (1) bring together different actors: scientists, environmental managers, consulting firms, and biotechnological companies, (2) apply this approach on a large scale to demonstrate its relevance, (3) propose robust and reliable tools, and (4) raise public awareness and train the various actors likely to use these new tools. Biomonitoring approaches based on such environmental genomic tools should address the European need for reliable, higher-throughput monitoring to improve the protection of aquatic environments under multiple pressures, guide their restoration , and follow their evolution