Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping

Régulateurs de l'endoréduplication et croissance du fruit de tomate

La croissance du péricarpe de tomate peut être décrite comme une succession d'événements cellulaires qui se chevauchent et sont interconnectés, avec des taux et des durées différents en fonction des couches cellulaires : divisions cellulaires anticlinales, périclinales et obliques, et expansion cellulaire isotrope et anisotrope. En outre, pendant l'expansion cellulaire, un cycle cellulaire modifié, l'endocycle, au cours duquel la synthèse de l'ADN se produit indépendamment de la mitose et conduit à une augmentation du contenu en ADN des cellules, a lieu. La taille finale du fruit ne peut donc être atteinte que par un contrôle et une coordination spatiale et temporelle stricts de ces événements.Dans ce contexte, l'objectif de mon doctorat était d'accroître les connaissances sur la régulation de la division cellulaire et de l'endoréduplication au cours du développement du fruit de tomate. Pour ce faire, j'ai utilisé deux approches : des approches de génétique inverse et de génétique directe. La première approche était basée sur des travaux antérieurs réalisés sur Arabidopsis et dans l'équipe sur des régulateurs connus du cycle cellulaire et de l'endocycle : les protéines CCS52. J'ai produit et phénotypé des lignées KO de tomate par édition du génome pour les trois gènes CCS52 de tomate. En plus de la conservation partielle de la fonction de ces gènes entre les deux espèces, j'ai montré que SlCCS52B était impliqué dans le contrôle de la forme des organes par la régulation de l'orientation des divisions cellulaires. En plus de la fonction majeure décrite pour SlCCS52A dans la régulation de l'endoréduplication, j'ai observé que les mutants de tomate semblent montrer une reprise de la division cellulaire avec la formation de noyaux anormaux qui doivent être étudiés plus en détail. SlCCS52A-L semble être impliqué dans l'organisation du méristème. La deuxième approche visait à identifier de nouveaux régulateurs de l'endoréduplication par génétique directe. Pour ce faire, j'ai utilisé une collection de mutants EMS dans la variété microtom disponible au laboratoire et j'ai sélectionné des familles présentant une altération de la ploïdie et du développement du péricarpe. J’ai ensuite recherché la mutation causale par une approche d'analyse de population en ségrégation. Malgré l'identification de plusieurs loci candidats, la ou les mutations causales restent encore à être confirmées.The tomato fruit pericarp growth can be described as the succession of overlapping and interconnected cellular events with different onsets, different rates and duration in function of the cell layers: anticlinal, periclinal and oblique cell divisions, and isotropic and anisotropic cell expansion. In addition, during cell expansion, a modified cell cycle, the endocycle, during which DNA synthesis occurs independently from mitosis and lead to an increase of DNA content of the cells, takes place. Final fruit size can thus only be achieved through the strict spatial and temporal control and coordination of these events.In this context, the aim of my PhD was to increase the knowledge on the regulation of cell division and endoreduplication during tomato fruit development. To do so, I used two approaches: reverse and forward genetics approaches. The first approach was based on previous work done on Arabidopsis and in the team on known regulators of the cell cycle and the endocycle: the CCS52 proteins. I produced and phenotyped tomato KO-lines by gene editing for the three tomato CCS52 genes. In addition to partial conservation of the function of theses genes between the two species, I showed that SlCCS52B was involved in the control of organ shape through the regulation of cell division orientation. Beside the major function described for SlCCS52A in the regulation of endoreduplication, I observed that the tomato mutants seem to show a resumption of cell division with the formation of abnormal nuclei that need to be further studied. SlCCS52A-L seems to be involved in meristem organisation. The second approach aimed at identifying new regulators of endoreduplication by forward genetics. To do so, I made use of an EMS mutant microtom collection available in the lab and selected families with an alteration in ploidy and pericarp development and searched for the causal mutation by Bulk-segregant analysis approach. Despite the identification of several candidate loci, the causal muttion(s) still remain(s) to be confirmed

Complex cellular and molecular events determining fruit size

The understanding of plant organ-size determination represents an important challenge, especially because of the significant role of plants as food and renewable energy sources and the increasing need for plant-derived products. Most of the knowledge on the regulation of organ growth and the molecular network controlling cell division and cell expansion, the main drivers of growth, is derived from arabidopsis. The increasing use of crops such as tomato for research is now bringing essential information on the mechanisms underlying size control in agronomically important organs. This review describes our current knowledge, still very scarce, of the cellular and molecular mechanisms governing tomato fruit size and proposes future research to better understand the regulation of growth in this important crop

Endoreduplication in plant organogenesis: a means to boost fruit growth

Abstract Endoreduplication is the major source of somatic endopolyploidy in higher plants, and leads to variation in cell ploidy levels due to iterative rounds of DNA synthesis in the absence of mitosis. Despite its ubiquitous occurrence in many plant organs, tissues, and cells, the physiological meaning of endoreduplication is not fully understood, although several roles during plant development have been proposed, mostly related to cell growth, differentiation, and specialization via transcriptional and metabolic reprogramming. Here, we review recent advances in our knowledge of the molecular mechanisms and cellular characteristics of endoreduplicated cells, and provide an overview of the multi-scale effects of endoreduplication on supporting growth in plant development. In addition, the effects of endoreduplication in fruit development are discussed, since it is highly prominent during fruit organogenesis where it acts as a morphogenetic factor supporting rapid fruit growth, as illustrated by case of the model fleshy fruit, tomato (Solanum lycopersicum).Comment la communication de cellule à cellule régule-t'elle la croissance du fruit

Breeding for cuticle-associated traits in crop species: traits, targets, and strategies

UMR BFP - Equipe GFDFInternational audienc

SlCCS52B, an activator of the APC/C complex, regulates fruit shape in Tomato

Cell cycle progression is controlled by the tight regulation of the amount and activity of the main cell cycle regulators, the CYCLIN-DEPENDENT KINASEs and CYCLINs. Specific ubiquitination and targeting to degradation of these proteins participate in this regulation and are executed by the E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C). In plants, the CCS52 proteins are activators of the APC/C complex and allow the specific targeting of proteins to be degraded by the 26S proteasome. While the involvement of CCS52A in the regulation of the endocycle, a process triggering DNA replication without mitosis, and its targeted proteins are pretty well described in several species, including tomato, only little information is available about the function of CCS52B. We produced ccs52b mutants in tomato by CRISPR-CAS9 gene editing and studied their growth-related phenotypes. We observed that these mutants produced elongated fruit with heart-like shape, compared to control plants. Through a detailed cellular analysis overtime, we found that the altered shape is already present during ovary development and probably results from a change in cell division orientation.To identify SlCCS52B targets potentially involved in this cell division regulation, mutant and WT ovaries at stage 11 were harvested and used for proteomic analysis. We found 789 proteins differentially accumulating with 37 increased by more than two fold in the mutant, potentially due to a lack of degradation. We are currently studying the direct interaction between SlCCS52B and these candidate targets

NGS-TILTOM: Toward the TILLING by Next-Generation Sequencing

National audienc