GEVES

Molecular markers linked to phenotypically important traits are of great interest especially when traits are difficult and/or costly to be observed. In tomato where a strong focus on resistance breeding has led to the introgression of several resistance genes, resistance traits have become important characteristics in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications. Evaluation of disease traits in biological assays is not always straightforward because assays are often influenced by environmental factors, and difficulties in scoring exist. In this study, we describe the development and/or evaluation of molecular marker assays for the Verticillium genes Ve1 and Ve2, the tomato mosaic virus Tm1 (linked marker), the tomato mosaic virus Tm2 and Tm2 ( 2 ) genes, the Meloidogyne incognita Mi1-2 gene, the Fusarium I (linked marker) and I2 loci, which are obligatory traits in PBR testing. The marker assays were evaluated for their robustness in a ring test and then evaluated in a set of varieties. Although in general, results between biological assays and marker assays gave highly correlated results, marker assays showed an advantage over biological tests in that the results were clearer, i.e., homozygote/heterozygote presence of the resistance gene can be detected and heterogeneity in seed lots can be identified readily. Within the UPOV framework for granting of PBR, the markers have the potential to fulfil the requirements needed for implementation in DUS testing of candidate varieties and could complement or may be an alternative to the pathogenesis tests that are carried out at present.

Seeds are involved in the transmission of microorganisms from one plant generation to the next and consequently act as reservoirs for the plant microbiota. The driving processes influencing seed microbiota assemblage have not been yet deciphered because of confounding factors related to environmental location, agricultural practices, and host genotype selection. Nine genotypes were chosen among a large panel of genetically diverse Brassica napus accessions. The taxonomic structure of the seed microbiota was monitored by amplification and subsequent high-throughput sequencing of gyrB and internal transcribed spacer 1 markers for two successive years on seed lots collected from self-pollinated plants. Seed germination capacities were compared between all seed lots. Although harvesting year was the main driver of seed microbiota composition, the host genotype also significantly altered the structure of seed microbial assemblages. The core microbiota of B. napus included nine fungal taxa shared between all the genotypes and years, while no bacterial taxa were conserved across all genotypes and years. The harvesting year had the major effect on seed germination but with some differences between genotypes. The study demonstrated the relative contribution of host- and environmental-filtering on the assemblage of the seed microbiota. It suggested some influence of these assemblages on seed germination.

Synthetic Communities (SynComs) are being developed and tested to manipulate plant microbiota and improve plant health. To date, only few studies proposed the use of SynCom on seed despite its potential for plant microbiota engineering. We developed and presented a simple and effective seedling microbiota engineering method using SynCom inoculation on seeds. The method was successful using a wide diversity of SynCom compositions and bacterial strains that are representative of the common bean seed microbiota. First, this method enables the modulation of seed microbiota composition and community size. Then, SynComs strongly outcompeted native seed and potting soil microbiota and contributed on average to 80% of the seedling microbiota. We showed that strain abundance on seed was a main driver of an effective seedling microbiota colonization. Also, selection was partly involved in seed and seedling colonization capacities since strains affiliated to Enterobacteriaceae and Erwiniaceae were good colonizers while Bacillaceae and Microbacteriaceae were poor colonizers. Additionally, the engineered seed microbiota modified the recruitment and assembly of seedling and rhizosphere microbiota through priority effects. This study shows that SynCom inoculation on seeds represents a promising approach to study plant microbiota assembly and its consequence on plant fitness.

Despite the generalized use of cultivars carrying the rym4 resistance gene, the impact of viral mosaic diseases on winter barleys increased in recent years in France. This change could reflect i) an increased prevalence of the rym4 resistance-breaking pathotype of Barley yellow mosaic virus Y (BaYMV-2), ii) the emergence of rym4 resistance-breaking pathotypes of Barley mild mosaic virus (BaMMV) or iii) the emergence of other viruses. A study was undertaken to determine the distribution and diversity of viruses causing yellow mosaic disease. A collection of 241 symptomatic leaf samples from susceptible, rym4 and rym5 varieties was gathered from 117 sites. The viruses present in all samples were identified by specific RT-PCR assays and, for selected samples, by double-stranded RNA next-generation sequencing (NGS). The results show that BaYMV-2 is responsible for the symptoms observed in varieties carrying the resistance gene rym4. In susceptible varieties, both BaYMV-1 and BaYMV-2 were detected, together with BaMMV. Phylogenetic analyses indicate that the rym4 resistance-breaking ability independently evolved in multiple genetic backgrounds. Parallel analyses revealed a similar scenario of multiple independent emergence events in BaMMV for rym5 resistance-breaking, likely involving multiple amino acid positions in the viral-linked genome protein. NGS analyses and classical techniques provided highly convergent results, highlighting and validating the power of NGS approaches for diagnostics and viral population characterization.

Four closely related durum wheat varieties were compared by computer-assisted analysis of two-dimensional electrophoretic maps of leaf proteins. A low inter-varietal polymorphism was revealed and seven reliable qualitatively varying proteins allowed rapid visual identification of genotypes. For numerous spots, presence/absence or quantitative variations were greatly affected by a batch effect. Several criteria that should be used to discard unreliable spots or gels a priori were reviewed. Nevertheless, it was shown that, provided that the experimental design allows the integration of the batch effect, screening for discriminant markers as well as computing distances based on protein quantity variations are possible and allow variety identification. Euclidean and Mahalanobis distances allowed variety discrimination and single gel classification with a minimum risk of error, not only by taking into account the quantitative variations in discriminant proteins selected by analysis of variance, but also by taking into account all reproducible spots. The possible applications of two-dimensional electrophoresis in variety identification are discussed.

This study aimed to set a computer-integrated multichannel spectral imaging system as a high-throughput phenotyping tool for the analysis of individual cowpea seeds harvested at different developmental stages. The changes in germination capacity and variations in moisture, protein and different sugars during twelve stages of seed development from 10 to 32 days after anthesis were non-destructively monitored. Multispectral data at 20 discrete wavelengths in the ultraviolet, visible and near infrared regions were extracted from individual seeds and then modelled using partial least squares regression and linear discriminant analysis (LDA) models. The developed multivariate models were accurate enough for monitoring all possible changes occurred in moisture, protein and sugar contents with coefficients of determination in prediction Rp2 of 0.93, 0.80 and 0.78 and root mean square errors in prediction (RMSEP) of 6.045%, 2.236% and 0.890%, respectively. The accuracy of PLS models in predicting individual sugars such as verbascose and stachyose was reasonable with Rp2 of 0.87 and 0.87 and RMSEP of 0.071% and 0.485%, respectively; but for the prediction of sucrose and raffinose the accuracy was relatively limited with Rp2 of 0.24 and 0.66 and RMSEP of 0.567% and 0.045%, respectively. The developed LDA model was robust in classifying the seeds based on their germination capacity with overall correct classification of 96.33% and 95.67% in the training and validation datasets, respectively. With these levels of accuracy, the proposed multichannel spectral imaging system designed for single seeds could be an effective choice as a rapid screening and non-destructive technique for identifying the ideal harvesting time of cowpea seeds based on their chemical composition and germination capacity. Moreover, the development of chemical images of the major constituents along with classification images confirmed the usefulness of the proposed technique as a non-destructive tool for estimating the concentrations and spatial distributions of moisture, protein and sugars during different developmental stages of cowpea seeds.

National audience

KEY MESSAGE: Phenomic prediction implemented on a large diversity set can efficiently predict seed germination, capture low-effect favorable alleles that are not revealed by GWAS and identify promising genetic resources. Oilseed rape faces many challenges, especially at the beginning of its developmental cycle. Achieving rapid and uniform seed germination could help to ensure a successful establishment and therefore enabling the crop to compete with weeds and tolerate stresses during the earliest developmental stages. The polygenic nature of seed germination was highlighted in several studies, and more knowledge is needed about low- to moderate-effect underlying loci in order to enhance seed germination effectively by improving the genetic background and incorporating favorable alleles. A total of 17 QTL were detected for seed germination-related traits, for which the favorable alleles often corresponded to the most frequent alleles in the panel. Genomic and phenomic predictions methods provided moderate-to-high predictive abilities, demonstrating the ability to capture small additive and non-additive effects for seed germination. This study also showed that phenomic prediction estimated phenotypic values closer to phenotypic values than GEBV. Finally, as the predictive ability of phenomic prediction was less influenced by the genetic structure of the panel, it is worth using this prediction method to characterize genetic resources, particularly with a view to design prebreeding populations.

In perennial grasses, the reproductive development consists of major phenological stages which highly determine the seasonal variations of grassland biomass production in terms of quantity and quality. The reproductive development is regulated by climatic conditions through complex interactions subjected to high genetic diversity. Understanding these interactions and their impact on plant development and growth is essential to optimize grassland management and identify the potential consequences of climate change. Here, we review the main stages of reproductive development, from floral induction to heading, i.e., spike emergence, considering the effect of the environmental conditions and the genetic diversity observed in perennial grasses. We first describe the determinants and consequences of reproductive development at individual tiller scale before examining the interactions between plant tillers and their impact on grassland perenniality. Then, we review the available grassland models through their ability to account for the complexity of reproductive development and genetic × environmental interactions. This review shows that (1) The reproductive development of perennial grasses is characterized by a large intraspecific diversity which has the same order of magnitude as the diversity observed between species or environmental conditions. (2) The reproductive development is determined by complex interactions between the processes of floral induction and morphogenesis of the tiller. (3) The perenniality of a plant is dependent on the reproductive behavior of each tiller. (4) Published models only partly explain the complex interactions between morphogenesis and climate on reproductive development. (5) Introducing more explicitly the underlying processes involved in reproductive development in models would improve our ability to anticipate grassland behavior in future growth conditions.

International audience

Xylella fastidiosa (Xf) is a phytopathogenic bacterium with a repertoire of self-replicating genetic elements, including plasmids, pathogenicity islands, and prophages. These elements provide potential avenues for horizontal gene transfer both within and between species and have the ability to confer new virulence traits, including the ability to colonize new host plants. However, they can also serve as a ‘footprint’ to type plasmid-bearing strains. Genome sequencing of several strains of Xf subsp. fastidiosa sequence type (ST) 1 from Mallorca Island, Spain, revealed the presence of a 38 kb plasmid (pXFAS_5235). In this study, we developed a PCR-based typing approach using primers targeting the traC gene to determine the presence of pXFAS_5235 plasmid or other plasmids carrying this gene in a world-wide collection of 65 strains X. fastidiosa from different subspecies and STs or in 226 plant samples naturally infected by the bacterium obtained from the different outbreaks of Xf in Spain. The traC gene was amplified only in the plant samples obtained from Mallorca Island infected by Xf subsp. fastidiosa ST1 and from all Spanish strains belonging to this ST. Maximum-likelihood phylogenetic tree of traC revealed a close relatedness among Spanish and Californian strains carrying similar plasmids. Our results confirm previous studies, which suggested that a single introduction event of Xf subsp. fastidiosa ST1 occurred in the Balearic Islands. Further studies on the presence and role of plasmids in Xf strains belonging to the same or different subspecies and STs can provide important information in studies of epidemiology, ecology, and evolution of this plant pathogen.

The analytical procedure used for GM detection and quantification at the laboratory level is composed of different modules. Each module can qualitatively and/or quantitatively impact the accuracy of the analytical result. Within the Co-Extra project, we have investigated different aspects of reliability of GMO detection. We have also established a system for improving reliability of quantitative analysis in samples with low DNA content. Additionally, we present and discuss efforts to reducethe trade-off between reliability and analysis costs.

The use of genetically modified organisms (GMOs) is regulated in most countries of the world. These\nregulations require adequate enforcement measures and detection of GM materials in food and feed\nproducts. Given the diversity of GMO, their detection is a complex, multi-step process requiring the\ncombination and validation of various types of methods. The different analytical steps can be\nconsidered independent modules that can be validated separately. This so-called 'modular approach'\nhas been extensively evaluated within the Co-Extra project and the results are summarized here. The\nneed to define intra- and inter-module performance criteria is also discussed.

OBJECTIVE: Black poplar (Populus nigra L.) is a species native to Eurasia with a wide distribution area. It is an ecologically important species from riparian ecosystems, that is used as a parent of interspecific (P. deltoides x P. nigra) cultivated poplar hybrids. Variant detection from transcriptomics sequences of 241 P. nigra individuals, sampled in natural populations from 11 river catchments (in four European countries) is described here. These data provide new valuable resources for population structure analysis, population genomics and genome-wide association studies. DATA DESCRIPTION: We generated transcriptomics data from a mixture of young differentiating xylem and cambium tissues of 480 Populus nigra trees sampled in a common garden experiment located at Orléans (France), corresponding to 241 genotypes (2 clonal replicates per genotype, at maximum) by using RNAseq technology. We launched on the resulting sequences an in-silico pipeline that allowed us to obtain 878,957 biallelic polymorphisms without missing data. More than 99% of these positions are annotated and 98.8% are located on the 19 chromosomes of the P. trichocarpa reference genome. The raw RNAseq sequences are available at the NCBI Sequence Read Archive SPR188754 and the variant dataset at the Recherche Data Gouv repository under https://doi.org/10.15454/8DQXK5 .

The use of genetically modified organisms (GMOs) is regulated in most countries of the world. These regulations require adequate enforcement measures and detection of GM materials in food and feed products. Given the diversity of GMO, their detection is a complex, multi-step process requiring the combination and validation of various types of methods. The different analytical steps can be considered independent modules that can be validated separately. This so-called “modular approach” has been extensively evaluated within the Co-Extra project and the results are summarized here. The need to define intra- and inter-module performance criteria is also discussed.

Within the framework of the agroecological transition of agricultural systems, it will be necessary to think, create, and thus evaluate, a variety in its production system, leading to a more refined combination of genetic improvement, agronomy, but also and more broadly, all the modalities and resources for the management and protection of these agroecological crops.The field of possible successes is considerable, and the CTPS, through the richness of its contributing communities, will have a key role to play in achieving them. Droulement de la saisineCette saisine s'est droule de septembre 2020 octobre 2021.Une journe de rflexion du Comit scientifique a t consacre aux aspects relatifs aux besoins en termes d'espces, varits, semences et plants, diversit gntique, et la place de la slection participative en agrocologie.Des groupes de travail impliquant les membres du Comit Scientifique du CTPS ont t constitus, afin de traiter les diffrents aspects de cette saisine (Figure 1).Les groupes de travail consacrs aux besoins en termes d'espces, varits, semences et plants, diversit gntique, la place des dmarches participatives en agrocologie, et modalits d'valuation des varits se sont runis l'automne 2020.Une journe commune de rflexion a t organise en dcembre 2020, afin d'changer sur les rsultats obtenus et sur les premires propositions de modalits d'valuation des varits.

*INRA, Centre de Versailles Diffusion du document : INRA, Centre de Versailles

Abstract Synthetic Communities (SynComs) are being developed and tested to manipulate plant microbiota and improve plant health. To date, only few studies proposed the use of SynCom on seed despite its potential for plant microbiota engineering. We developed and presented a simple, reproducible and effective seedling microbiota engineering method using SynCom inoculation on seeds. The method was successful using a wide diversity of SynCom compositions and bacterial strains that are representative of the common bean seed microbiota. First, this method enables the modulation of seed microbiota composition and community size. Then, SynComs strongly outcompeted native seed and potting soil microbiota and contributed on average to 80% of the seedling microbiota. We showed that strain abundance on seed was a main driver of an effective seedling microbiota colonization. Also, selection was partly involved in seed and seedling colonization capacities since strains affiliated to Enterobacteriaceae and Erwiniaceae were good colonizers while Bacillaceae and Microbacteriaceae were poor colonizers. Additionally, the engineered seed microbiota modified the recruitment and assembly of seedling and rhizosphere microbiota through priority effects. This study shows that SynCom inoculation on seeds represents a promising approach to study plant microbiota assembly and its consequence on plant fitness.

International audience

National audience