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Replication stress is a major source of genomic instability and oncogene-induced replication stress has been proposed to play a key role in cancer development. Accordingly, the checkpoint mechanisms that prevent cell cycle progression under conditions of replication stress are reported to be highly active in pre-neoplastic lesions, where they might act as a potent barrier to tumorigenesis. Yet, multiple works suggest that DNA damage checkpoints are not infallible and that following various genotoxic stresses, DNA alterations may persist up to mitosis despite functional checkpoints mechanisms. Most notably, it was shown that unreplicated DNA may persist at Common Fragile Sites up to late G2 or early mitosis, resulting in chromosomes breaks that affect the genetic stability of the cell lineage. The main question of my PhD was to determine how the DNA checkpoint behave in cells that enter mitosis despite replication stress, and thus help characterize the mechanisms through which checkpoint-proficient cells enter mitosis with persistent DNA alterations. To explore those questions, I developed a highly sensitive and specific FRET biosensor of Chk1 activity and stably expressed this tool in a non-transformed human cells background. This novel system allowed me to describe the precise kinetics of Chk1 activity with exceptional time resolution and exquisite sensitivity in single cell live imaging assays, both following genotoxic stress or under non-perturbed conditions. In a cell background where endogenous PCNA was tagged with a fluorescent protein, I characterized a mild but highly reproducible Chk1 activation pattern in non-perturbed S phase, which correlates tightly with bulk PCNA foci appearance at the G1/S transition and disappearance at the S/G2 transition. This basal activity depends on the constant firing of new replication origins. Following induction of replication stress in S phase, this basal activity is increased but a fraction of S phase cells manages to reach G2 phase, and some further reach mitosis. Even under conditions of replication stress, the decreasing count of PCNA foci in late S phase correlates tightly with progressive Chk1 inactivation. Additionally, the specific expression profile of G2-specific mitotic inducers following PCNA disappearance indicates this is an actual G2 phase. This G2 phase reproducibly lasts several hours before either bifurcating to mitosis or to a p53-dependent G2 arrest.Surprisingly, the kinetics of Chk1 activity in G2 display high heterogeneity and Chk1 can either remain at a minimal level or re-activate in G2 phase to prevent premature mitotic entry. Yet, following Chk1 re-activation in G2 phase, a fraction of cells enter mitosis with a persistent high level of Chk1 activity, providing the first ever observation of mammalian cells entering mitosis despite an active checkpoint. Finally, cells that re-activated Chk1 in G2 phase are less likely to re-enter S phase during the next cell cycle, compared to daughters of cells that did not re-activate Chk1 in G2 phase, suggesting that the structures that activated Chk1 in G2 phase are genotoxic and/or prompt a G1 arrest. All in all, my results provide new insights on the regulation of cell cycle progression under both perturbed and non-perturbed conditions, open previously unforeseen questions about the temporal aspects of ATR/Chk1 activation, and suggest that genotoxic DNA structures arising from replication stress are indeed detected by the DNA damage checkpoint in G2 phase, but can be bypassed by the mitotic entrance machinery., Le stress réplicatif est une source majeure d'instabilité génétique et pourrait jouer un rôle clé dans le développement tumoral précoce. Des mécanismes de surveillance (checkpoint) qui empêchent la progression du cycle cellulaire sont fortement actifs dans les lésions prénéoplasiques, où ils pourraient agir comme une barrière à la tumorigenèse. Pourtant, de nombreux travaux suggèrent que les checkpoints ne sont pas infaillibles et que suite à divers stress génotoxiques, des altérations de l'ADN peuvent persister jusqu'à la mitose malgré un checkpoint fonctionnel. De l'ADN non répliqué peut notamment persister au niveau des sites fragiles communs (CFS) jusqu'à la fin de la phase G2 ou au début de la mitose, entraînant des gaps et cassures chromosomiques pouvant affecter la stabilité génétique de la lignée cellulaire. Le but de mon doctorat était de déterminer comment le checkpoint se comporte dans les cellules qui entrent en mitose malgré un stress réplicatif et de caractériser les mécanismes par lesquels les cellules entrent en mitose malgré la persistance de structures génotoxiques. Pour explorer ces questions, j'ai développé un senseur FRET spécifique de l'activité Chk1 et ai exprimé cet outil de manière stable dans des cellules humaines non transformées. Ce nouveau système m'a permis de décrire la cinétique précise de l'activité Chk1 avec une forte résolution temporelle en vidéomicroscopie sur cellule unique, que ce soit à la suite d'un stress génotoxique ou dans des conditions non perturbées. Ainsi, dans des cellules où PCNA endogène a été marqué avec une protéine fluorescente, j'ai déterminé que Chk1 s’active faiblement en phase S mais que son profil d’activité dépend de l’activation constante de nouvelles origines de réplication et est fortement corrélé à la dynamique des foyers de réplication. Suite à l'induction d'un stress réplicatif en phase S, cette activité basale est augmentée, mais une fraction des cellules en phase S parvient à atteindre la phase G2. Au cours de cette transition S/G2, Chk1 s’inactive de façon conjointe à la disparition des foyers de réplication, ce qui est suivi par l’expression des inducteurs mitotiques. De manière reproductible, cette phase G2 dure plusieurs heures avant de bifurquer vers la mitose ou vers un arrêt en G2 dépendant de p53. De façon surprenante, la cinétique d'activité Chk1 à la phase G2 montre une forte hétérogénéité et peut soit rester à un niveau minimal, soit se réactiver en phase G2 pour empêcher une entrée en mitose prématurée. J’ai cependant pu déterminer qu’une fraction des cellules entre en mitose malgré la persistance d’un niveau élevé d'activité Chk1, démontrant de façon directe que les cellules de mammifères peuvent entrer en mitose malgré un checkpoint actif. Enfin, au cycle suivant, les cellules dont la mère a réactivé Chk1 à la phase G2 précédente sont moins susceptibles de ré-entrer en phase S au cours du cycle cellulaire suivant, ce qui suggère que les structures qui ont activé Chk1 en phase G2 sont génotoxiques et/ou provoquent un arrêt en G1. Mes résultats élargissent nos connaissances sur la régulation de la progression du cycle cellulaire en conditions normales ou perturbées, ouvrent des questions sur les aspects temporels de l'activation d'ATR/Chk1, et suggèrent que les structures génotoxiques de l'ADN résultant d’un stress réplicatif en phase S sont effectivement détectées par le checkpoint en phase G2, mais que cette activation du checkpoint peut être contournée par les mécanismes d'entrée en mitose.

Complete and accurate DNA replication is crucial to transfer correct genetic information to the daughter cells. Various obstacles can interfere with the progression of the replication machinery, threatening genome integrity. Specialized error-prone translesion DNA polymerases (TLS polymerases) assist the replicative polymerases to replicate across DNA lesions. During my PhD I characterized the contribution of TLS pol eta (polη), best known for its role in preventing UV-induced mutagenesis, during unperturbed replication. Polη was shown to promote the stability of the common fragile sites and associates with the replisome in unchallenged S phase. However, the kind of replication barriers requiring pol eta and the consequences of its absence on the replication of these regions were unclear. My results show that polη is recruited at a subset of replication forks all along the S phase and that polη defect modifies the replication timing of genomic regions enriched in large transcribed genes, where transcription-replication conflicts (TRCs) are more likely to occur. Overall, I show that polη recruitment at the replication fork is transcription-dependent, and that pol eta plays a role in the coping with TRCs. Altogether, these results highlight a new role for an error-prone DNA polymerase in protecting the genome stability., La réplication complète et fidèle de l’ADN est cruciale pour transmettre l’information génétique de manière correcte aux cellules filles. Divers obstacles peuvent interférer avec la progression de la machinerie de réplication, et donc menacer l’intégrité du génome. Des ADN polymérases spécialisées, dites translésionnelles (polymérases TLS), assistent les ADN polymérases réplicatives pour la poursuite de la réplication malgré ces lésions. Elles peuvent répliquer de manière fidèle ou non ces entraves, mais sont mutagènes sur des séquences d’ADN non-endommagées. Au cours de ma thèse, j’ai pu caractériser davantage la contribution de l’ADN polymérase TLS eta (polη) au cours de la réplication non-perturbée. Cette polymérase permet principalement de prévenir la mutagénèse induite par les UV. Mais il a également été montré qu’elle promeut la stabilité des sites fragiles communs, et est associée au réplisome durant la phase S non-perturbée. Cependant, la nature des obstacles nécessitant polη et les conséquences de son absence pour la réplication de ces régions restaient à déterminer. Mes résultats montrent que polη est recrutée au niveau d’une fraction des fourches de réplication tout au long de la phase S et que l’absence de pol eta conduit à une modification du timing de réplication de régions génomiques riches en grands gènes transcrits, où les conflits entre réplication et transcription sont potentiellement plus fréquents. Plus généralement, je montre que le recrutement de pol eta à la fourche de réplication dépend de la transcription et qu’elle joue un rôle dans la prise en charge des conflits entre réplication et transcription. Ces résultats mettent en évidence un nouveau rôle de protection de la stabilité du génome pour cette ADN polymérase mutagène.

The occurrence of somaclonal variants (ca 5%) among populations of somatic embryo-derived oil palms (Elaeis guineensis Jacq.) currently hampers the scaling-up of clonal plant production. In order to investigate the relationship between the "mantled" somaclonal variant and possible alterations in genomic DNA methylation rate, two complementary approaches have been used. HPLC quantification of relative amounts of 5-methyl-deoxycytidine has shown that global methylation in leaf DNA of abnormal regenerants is 0.5-2.5% lower than in their normal counterparts (20.8% vs 22%, respectively). When comparing nodular compact calli and fast growing calli, yielding respectively 5% and 100% of "mantled" plantlets, this decrease was up to 4.5% (from 23.2 to 18.7%). An alternative method, the Sssl-methylase accepting assay, based on the enzymatic saturation of CG sites with methyl groups, gave convergent results. This work demonstrates that a correlation exists between DNA hypomethylation and the "mantled" somaclonal variation in oil palm.

Salmonella is the second leading cause of human zoonoses in the European Union and represents a major challenge for the pork industry. Serovars S. Typhimurium, S. Derby and the monophasic variant of S. Typhimurium, implicated in human salmonellosis, are highly prevalent in pigs and also in human. The objectives of this research were to establish the infection dynamics in pigs by Salmonella under conventional and experimental rearing conditions, to evaluate pig colonization ability and pathogenicity in humans of the three serovars. In conventional farms, the serological monitoring of Salmonella antibodies allowed to evaluate the average age of seroconversion at 137 days and to identify a "farm effect" on the age of seroconversion. The first trial under experimental conditions revealed that pigs inoculated with the monophasic variant of S. Typhimurium excreted this serovar continuously in the feces. At autopsy dates 21, 49 or 84 days after inoculation, Salmonella were found in the different parts of the intestine, in the lymph nodes and at very high levels in the tonsils. After passage through the digestive tract, different MLVA profiles from that of the inoculated strain were identified suggesting that the genome of the strain has evolved. The second attempt to compare the colonizing ability in the pigs of the three serovars showed that the dynamics of excretion and colonization were similar regardless of the serovar. However, the amount excreted was significantly different: higher with the monophasic variant compared to S. Typhimurium. The pathogenicity in humans of 15 strains of porcine origin belonging to the 3 serovars was evaluated in vivo on an insect model Galleria mellonella, and in vitro on Caco-2 cells. The strains were found to be potentially virulent. On Caco-2, the monophasic variant had the highest cell adhesion percentage and Derby, the lowest. Different levels of virulence were observed between strains of the same serovar. This work brought new knowledge on the Salmonella issue in the pig sector.; Salmonella est la deuxième cause de zoonoses humaines dans l’Union Européenne et représente un enjeu majeur pour la filière porcine. Les sérovars, S. Typhimurium, S. Derby et le variant monophasique de S. Typhimurium sont très prévalents chez le porc et également chez les humains. Les objectifs de ces travaux étaient d’établir la dynamique d’infection chez le porc par Salmonella en condition d’élevage conventionnel et expérimental, d’évaluer le pouvoir colonisateur chez le porc et le pouvoir pathogène chez les humains des trois sérovars. En élevage conventionnel, le suivi sérologique des anticorps anti-Salmonella a permis d’évaluer l’âge moyen de séroconversion à 137 jours et d’identifier un « effet ferme » sur l’âge de séroconversion. Le premier essai en condition expérimentale a mis en évidence que les porcs inoculés avec le variant monophasique de S. Typhimurium excrétaient ce sérovar de façon continue dans les fèces. Aux dates d’autopsies 21, 49 ou 84 jours après inoculation, les salmonelles ont été retrouvées dans les différentes parties de l’intestin, dans les nœuds lymphatiques et à des niveaux très élevés dans les amygdales. Après passage dans le tractus digestif, des profils MLVA différents de celui de la souche inoculée ont été identifiés suggérant que le génome de la souche a évolué. Le deuxième essai visant à comparer le pouvoir colonisateur chez le porc des trois sérovars a montré que la dynamique d’excrétion et de colonisation était similaire quel que soit le sérovar. Cependant, la quantité excrétée était significativement différente ; plus élevée avec le variant monophasique par rapport à S.Typhimurium. Le pouvoir pathogène chez l’homme de 15 souches d’origine porcine appartenant aux 3 sérovars a été évalué in vivo sur un modèle insecte Galleria mellonella, et in vitro sur cellules Caco-2. Les souches se sont révélées être potentiellement virulentes. Sur Caco-2, le variant monophasique avait un pourcentage d’adhésion aux cellules le plus élevé et Derby le plus bas. Différents niveaux de virulence ont été observés entre souches d’un même sérovar. Ce travail a apporté des nouvelles connaissances sur la problématique Salmonella en filière porcine.

Salmonella is the second leading cause of human zoonoses in the European Union and represents a major challenge for the pork industry. Serovars S. Typhimurium, S. Derby and the monophasic variant of S. Typhimurium, implicated in human salmonellosis, are highly prevalent in pigs and also in human. The objectives of this research were to establish the infection dynamics in pigs by Salmonella under conventional and experimental rearing conditions, to evaluate pig colonization ability and pathogenicity in humans of the three serovars. In conventional farms, the serological monitoring of Salmonella antibodies allowed to evaluate the average age of seroconversion at 137 days and to identify a "farm effect" on the age of seroconversion. The first trial under experimental conditions revealed that pigs inoculated with the monophasic variant of S. Typhimurium excreted this serovar continuously in the feces. At autopsy dates 21, 49 or 84 days after inoculation, Salmonella were found in the different parts of the intestine, in the lymph nodes and at very high levels in the tonsils. After passage through the digestive tract, different MLVA profiles from that of the inoculated strain were identified suggesting that the genome of the strain has evolved. The second attempt to compare the colonizing ability in the pigs of the three serovars showed that the dynamics of excretion and colonization were similar regardless of the serovar. However, the amount excreted was significantly different: higher with the monophasic variant compared to S. Typhimurium. The pathogenicity in humans of 15 strains of porcine origin belonging to the 3 serovars was evaluated in vivo on an insect model Galleria mellonella, and in vitro on Caco-2 cells. The strains were found to be potentially virulent. On Caco-2, the monophasic variant had the highest cell adhesion percentage and Derby, the lowest. Different levels of virulence were observed between strains of the same serovar. This work brought new knowledge on the Salmonella issue in the pig sector.; Salmonella est la deuxième cause de zoonoses humaines dans l’Union Européenne et représente un enjeu majeur pour la filière porcine. Les sérovars, S. Typhimurium, S. Derby et le variant monophasique de S. Typhimurium sont très prévalents chez le porc et également chez les humains. Les objectifs de ces travaux étaient d’établir la dynamique d’infection chez le porc par Salmonella en condition d’élevage conventionnel et expérimental, d’évaluer le pouvoir colonisateur chez le porc et le pouvoir pathogène chez les humains des trois sérovars. En élevage conventionnel, le suivi sérologique des anticorps anti-Salmonella a permis d’évaluer l’âge moyen de séroconversion à 137 jours et d’identifier un « effet ferme » sur l’âge de séroconversion. Le premier essai en condition expérimentale a mis en évidence que les porcs inoculés avec le variant monophasique de S. Typhimurium excrétaient ce sérovar de façon continue dans les fèces. Aux dates d’autopsies 21, 49 ou 84 jours après inoculation, les salmonelles ont été retrouvées dans les différentes parties de l’intestin, dans les nœuds lymphatiques et à des niveaux très élevés dans les amygdales. Après passage dans le tractus digestif, des profils MLVA différents de celui de la souche inoculée ont été identifiés suggérant que le génome de la souche a évolué. Le deuxième essai visant à comparer le pouvoir colonisateur chez le porc des trois sérovars a montré que la dynamique d’excrétion et de colonisation était similaire quel que soit le sérovar. Cependant, la quantité excrétée était significativement différente ; plus élevée avec le variant monophasique par rapport à S.Typhimurium. Le pouvoir pathogène chez l’homme de 15 souches d’origine porcine appartenant aux 3 sérovars a été évalué in vivo sur un modèle insecte Galleria mellonella, et in vitro sur cellules Caco-2. Les souches se sont révélées être potentiellement virulentes. Sur Caco-2, le variant monophasique avait un pourcentage d’adhésion aux cellules le plus élevé et Derby le plus bas. Différents niveaux de virulence ont été observés entre souches d’un même sérovar. Ce travail a apporté des nouvelles connaissances sur la problématique Salmonella en filière porcine.

International audience; Long-term cell cultures were used in coffee to study the cytological, genetic and epigenetic changes occurring during cell culture ageing. The objective was toidentify the mechanisms associated with somaclonal variation (SV). Three embryogenic cell lines were established in Coffea arabica (2n = 4x = 44) and somatic seedlings were regenerated after 4, 11 and 27 months. Phenotypingand AFLP, MSAP, SSAP molecular markers were performed on 199 and 124 plants, respectively. SV were only observed from the 11 and 27-month-old cultures, affecting30 and 94 % of regenerated plants, respectively. Chromosome counts performed on 15 plants showed that normal plants systematically displayed normal chromosome numbers and that, conversely, aneuploidy (monosomy) was systematically found in variants. The allopolyploid structure of C. arabica allowed aneuploid cells to survive and regenerate viable plants. No polymorphic fragments were observed between the AFLP and SSAP electrophoretic profiles of mother plants and those of the in vitro progeny.Methylation polymorphism was low and ranged between 0.087 and 0.149 % irrespective of the culture age. The number of methylation changes per plant—normal or variant—was limited and ranged from 0 (55–80 % of the plants) to 4. The three cell lines showed similar SV rate increases during cell culture ageing and produced plantswith similar molecular patterns indicating a non randomprocess. The results showed that cell culture ageing ishighly mutagenic in coffee and chromosomal rearrangements are directly linked to SV. Conversely, the analysis of methylation and transposable elements changes did notreveal any relation between the epigenetic patterns and SV.

International audience; In plants, undifferentiated or totally differentiated cells can be easily in vitro cultured to generate undifferentiated embryogenic cells that can regenerate complete plants. This is the most spectacular expression of totipotency. Embryogenic cells represent a key material in plant biotechnologies as they are used in many processes aimingat asexual reproduction by somatic embryogenesis (SE), genetic transformation, protoplast regeneration and cryopreservation. Nevertheless, tissue culture systems that involve the acquisition of competence for totipotency and extensive cell division remain risky with respect to genome and epigenome instabilities. Particularly, the use ofembryogenic cell suspensions has frequently been associated with an increased likelihood of genetic instability and somaclonal variation (SV) in the regenerated plants. SV is a major concern in all in vitro vegetative plant propagation systems because it leads to the loss of genetic fidelity. Since 20 years, embryogenic cell suspension based SE techniques have been applied to coffee for the large-scale dissemination of exceptional Arabica hybrids. Here, we present our production and research experience showing that SE is efficient and reliable for true-to-type propagation. Over 99% of coffee trees regenerated fully conform to the mother plant, both morphologically - they grow, flower and produce normally. Hence strong genetic and epigenetic changes in proliferating embryogenic cells are not a fatality. The results also demonstrated the importance of embryogenic culture age on SV and hence the non-random natureof this phenomenon. The genetic and epigenetic alterations are particularly limited during SE. The main change in most of phenotypic variants was aneuploidy showing that mitotic aberrations play a major role in SV in coffee. These results provide a proof of concept for the use of embryogenic cell suspensions with other plant species: a revolution in the world of plant micropropagation on an industrial scale.

In order to better understand somaclonal variant rate evolution in plant tissue culture, a statistical approach has been adopted. According to this approach, the variant percentage could be calculated by: %V = [1 - (1 - p)n] X 100, where %V is the percentage of variant, p the probability of variation and n the number of multiplication cycles. A numerical estimation was performed to characterize the variance of this function. It has been demonstrated that a wide scale of variance is associated with '%V', due to the occurrence of variations after a variable number of multiplication cycles in the different lines of culture. Two main conclusions can be drawn from this model: (1) a variant rate increase can be expected as an exponential function of the number of multiplication cycles; (2) after a given number of multiplication cycles, variable off-types percentages can be expected. Due to the complexity of biological systems, this statistical approach could obviously not be applied directly for the calculation and forecasting of variant rates in tissue culture. However, this approach results in a better understanding of two apparently confusing experimental features often reported in tissue culture: the increase of the variant rate as a function of the length of the culture period on the one hand, and, on the other hand, the observations of different variant rates among lines cultured for the same lengths of time under strictly identical culture conditions. This approach also underlined that the comparison of somaclonal variant percentage between batches of plants from different #in vitro# treatments could be, in some cases, insufficient for ascertaining a difference of variability generated by tissue culture.

In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1ºC min-1 from 25ºC to -30ºC followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30ºC water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol, we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid and triploid cytotypes. No morphological abnormalities were observed in any of the plants regenerated from cryopreserved embryos and their genetic stability was confirmed with 10 isozyme systems and nine RAPD profiles. Fil: Rey, Hebe Yolanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina; Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina; Fil: Faloci, Mirta Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina; Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina; Fil: Medina, Ricardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina; Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina; Fil: Dolce, Natalia Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina; Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina; Fil: Engelmann, Florent. Centre National de la Recherche Scientifique. Institut de Recherche Pour Le Developpement; Francia; Fil: Mroginski, Luis Amado. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Nordeste. Instituto de Botánica del Nordeste (i); Argentina; Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina

KEY MESSAGE : The long-term proliferation of embryogenic cell suspensions of oil palm is associated with changes in both genomic methylation rates and embryogenic capacities. In the aim of exploring the relationship between epigenetic stability and the long-term in vitro proliferation of plant tissues, we have studied changes in genomic DNA methylation levels in embryogenic suspensions of oil palm (Elaeis guineensis Jacq.). Five embryogenic callus lines were obtained from selected hybrid seeds and then proliferated as suspension cultures. Each clonal line obtained from a single genotype was subdivided into three independent subclonal lines. Once established, cultures proliferated for 12 months and genomic DNA was sampled at 4 months intervals for the estimation of global DNA methylation rates through high performance liquid chromatography (HPLC) quantitation of deoxynucleosides. Our results show that in vitro proliferation induces DNA hypermethylation in a time-dependent fashion. Moreover, this trend is statistically significant in several clonal lines and shared between subclonal lines originating from the same genotype. Interestingly, the only clonal line undergoing loss of genomic methylation in the course of proliferation has been found unable to generate somatic embryos. We discuss the possible implications of genome-wide DNA methylation changes in proliferating cells with a view to the maintenance of genomic and epigenomic stability.

Embryogenic suspensions that involve extensive cell division are risky in respect to genome and epigenome instability. Elevated frequencies of somaclonal variation in embryogenic suspension-derived plants were reported in many species, including coffee. This problem could be overcome by using culture conditions that allow moderate cell proliferation. In view of true-to-type large-scale propagation of C. arabica hybrids, suspension protocols based on low 2,4-D concentrations and short proliferation periods were developed. As mechanisms leading to somaclonal variation are often complex, the phenotypic, genetic and epigenetic changes were jointly assessed so as to accurately evaluate the conformity of suspension-derived plants. The effects of embryogenic suspensions and secondary embryogenesis, used as proliferation systems, on the genetic conformity of somatic embryogenesis-derived plants (emblings) were assessed in two hybrids. When applied over a 6 month period, both systems ensured very low somaclonal variation rates, as observed through massive phenotypic observations in field plots (0.74% from 200 000 plant). Molecular AFLP and MSAP analyses performed on 145 three year-old emblings showed that polymorphism between mother plants and emblings was extremely low, i.e. ranges of 0-0.003% and 0.07-0.18% respectively, with no significant difference between the proliferation systems for the two hybrids. No embling was found to cumulate more than three methylation polymorphisms. No relation was established between the variant phenotype (27 variants studied) and a particular MSAP pattern. Chromosome counting showed that 7 of the 11 variant emblings analyzed were characterized by the loss of 1-3 chromosomes. This work showed that both embryogenic suspensions and secondary embryogenesis are reliable for true-to-type propagation of elite material. Molecular analyses revealed that genetic and epigenetic alterations are particularly limited during coffee somatic embryogenesis. The main change in most of the rare phenotypic variants was aneuploidy, indicating that mitotic aberrations play a major role in somaclonal variation in coffee.

The stability of agronomic and resistance traits for the International Clone Trial over eight countries was compared through linear regression analyses. The number of clones compared varied over the sites from 8 to 18. For average yield, the majority of the coefficients of correlation were positive and significant, showing reasonable stability of this trait over sites. For plant vigour measured by stem girth, the coefficients of correlation were generally positive but rarely significant, indicating interaction between sites and clones. Incidence of witches' broom (WB) disease was compared for four sites (Brazil, Ecuador, Trinidad and Venezuela). Correlations for the total number of brooms, for the number of vegetative brooms and for the number of cushion brooms per tree were positive but not always significant, suggesting some interaction between clones and sites for resistance to WB. In Ecuador, the total number of brooms per tree was significantly correlated with the average weight of the brooms. The numbers of cushion brooms and vegetative brooms were significantly correlated for three out of four sites. For vascular streak dieback (VSD) severity, the coefficient of correlation between two sites in Papua New Guinea was significant, but the correlations between these sites and Malaysia were not significant. The consequences of these findings for breeding are discussed.

National audience

For correspondence fax +33 467 615 605 ; email dhont@cirad.fr; International audience; Modern sugarcane (Saccharum spp.) is an important grass that contributes 60% of the raw sugar produced worldwide and has a high biofuel production potential. It was created about a century ago through hybridization of two highly polyploid species, namely S. officinarum and S. spontaneum. We investigated genome dynamics in this highly polyploid context by analyzing two homoeologous sequences (97 and 126 kb) in a region that has already been studied in several cereals. Our findings indicate that the two Saccharum species diverged 1.5–2 million years ago from one another and 8–9 million years ago from sorghum. The two sugarcane homoeologous haplotypes show perfect colinearity as well as high gene structure conservation. Apart from the insertion of a few retrotransposable elements, high homology was also observed for the ontranscribed regions. Relative to sorghum, the sugarcane sequences displayed colinearity, with the exception of two genes present only in sorghum, and striking homology in most non-coding parts of the genome. The gene distribution highlighted high synteny and colinearity with rice, and partial colinearity with each homoeologous maize region, which became perfect when the sequences were combined. The haplotypes observed in sugarcane may thus closely represent the ancestral Andropogoneae haplotype. This analysis of sugarcane haplotype organization at the sequence level suggests that the high ploidy in sugarcane did not induce generalized reshaping of its genome, thus challenging the idea that polyploidy quickly induces generalized rearrangement of genomes. These results also confirm the view that sorghum is the model of choice for sugarcane

Epigenetics is the study of heritable changes in gene expression that occur without a change in DNA sequence. In plants, although epigenetic mechanisms help to protect cells from parasitic elements, this defense can complicate the genetic engineering process through transcriptional gene silencing. Furthermore, these phenomena have economic relevance, for example, somaclonal variation: a genetic and phenotypic variation among clonally propagated plants from a single donor genotype. In oil palm (Elaeis guineensis Jacq), approximately 5% of somatic embryo-derived palms show abnormalities in their floral development. This variant phenotype offers a very interesting model, because somaclonal variation alters the expression of genes coding, directly or indirectly for the flower structure and DNA hypomethylation has already been clearly demonstrated in somaclonal variants. Methylation sensitive DNA markers have been investigated using MSAP and MS-RFLP techniques, but to date no genotype-independent markers linked to the "mantled" phenotype have been identified. Differential display and microarray approaches are currently developed in order to provide a range of putative candidate genes displaying expression which might be regulated through DNA methylation and/or chromatin remodeling. We have identified floral homeotic genes of the "MADS box" transcription factor family which are affected by the chain of events resulting in the "mantled" abnormality. Our aim is to describe specific molecular events which could be used for the development of markers of epigenetic instability in plants. These markers will be integrated in a strategy aimed at the identification of in vitro treatments which are prone to generate epigenetic variability in somatic embryogenesis-based micropropagation processes. (Texte intégral)

International audience; Cloning of trees using somatic embryogenesis (SE) could have a major impact on tree breeding and commercial plantation forestry. In conjunction with cryopreservation, SE offers an opportunity to develop highly valuable clone lines. Commercial deployment of such genetically tested clone lines in forestry will dramatically increase forest productivity over any available conventional tree improvement techniques. However, sufficient technical advances must be made to use SE in clonal forestry. Progress in SE techniques and genetic stability of clones is reviewed, using white spruce (Picea glauca) and eastern white pine (Pinus strobus) as model species. There are several other issues in implementing clonal forestry. The main concern is managing clonal plantations for optimal genetic gain and diversity. The issues and considerations for selecting appropriate numbers of clones and deployment strategies are discussed. A clonal deployment strategy using a "mixture of clones and seedlings" is proposed for eastern Canada.; Mise en œuvre de l'embryogenèse somatique des conifères en foresterie multiclonale : exigences techniques et considérations sur son utilisation. Le clonage des arbres réalisé à l'aide de l'embryogenèse somatique (ES) pourrait avoir un impact majeur sur l'amélioration des arbres et la foresterie commerciale de plantation. L'ES, utilisée de pair avec la cryoconservation, offre la possibilité de développer des lignées multiclonales de grande valeur. L'utilisation commerciale de telles lignées multiclonales testées génétiquement permettra d'augmenter de façon radicale la productivité forestière comparativement aux autres techniques d'amélioration génétique classique. L'utilisation de l'ES en foresterie multiclonale exige toutefois certaines avancées techniques. Les progrès réalisés au niveau des techniques d'ES et de la stabilité des clones sont présentés en utilisant comme espèces modèles l'épinette blanche (Picea glauca) et le pin blanc ( Pinus strobus). De plus, lors de l'instauration d'un programme de foresterie multiclonale, il y a de nombreux obstacles à surmonter. Le principal point à surveiller est l'aménagement des plantations multiclonales afin de maximiser à la fois le gain et la diversité génétiques. Les problèmes rencontrés et les considérations à prendre en compte lors du choix du nombre approprié de clones et de la stratégie de déploiement sont également discutés. Une stratégie de déploiement multiclonal basée sur le recours à un mélange de clones et de semis est proposée pour l'est du Canada.

The genetic continuity of the potato spindle tuber viroid (PSTVd) genome was analysed after infection of tomato plants with cloned cDNAs of parental strains. During the six weeks of the experiment, several new sequence variants appeared. The sequence variants detected in the progeny population induced sequence-specific disease symptoms. The PSTVd genome therefore follows the pattern expected for typical pseudo-strains propagating in plants as a population of similar sequences. Assessing further the replicon continuity, a PSTVd cDNA mutant with a deletion in the central conserved region was constructed and proven to be non-infectious. Surprisingly, in a sub-population of potato transformants expressing the same deleted PSTVd RNA an infectious viroid was detected. This suggests specific transcript conversion followed by recovery of the full-length pathogen genome.

Un essai multilocal comprenant 16 clones et 9 lieux en Cote d'Ivoire a permis d'etudier l'interaction clone-lieu pour le rendement de Coffea canephora. En plus de l'ANOVA a 2 facteurs, les ecovalences, la regression lineaire des clones sur les lieux et le test de Nassar et Huhn ont ete calcules. Une interaction a ete detectee, bien que plusieurs clones se soient reveles stables. Les methodes utilisees ont demontre leur complementarite. En particulier des interactions avec ou sans modification du rang ont ete mises en evidence. Une analyse multivariee a ete realisee pour decrire la structuration de l'interaction et pour regrouper les lieux en fonction de la reponse des clones. Les consequences pour l'amelioration sont discutees. (Resume d'auteur)

The objective of the International Clone Trial is to compare a common set of about 20 clones in ten different countries. This will permit us to verify the stability of selection traits (yield, resistance and quality) in different environments. These clones will be compared to 20 local clones, either in the same trial or in a second trial, by using common control clones. The proposed method of multiplication of the clones is by hypocotyledonary budding onto a rootstock variety. The choice of the clones and type of rootstock is described here.

Ce document a pour but, à partir d'un bilan des travaux de recherche mené pendant la première phase du Projet Maïs des Birunga, de faire l'inventaire des technologies actuellement disponibles pour l'amélioration de la culture du maïs dans ce paysannat traditionnel, à très faible intrant mais très intensif, que constituent les terres de laves du Nord-Rwanda. Partant de quasiment rien sur le plan de la recherche, et qui plus est, dans un contexte très variable quant aux conditions physiques et agro-économiques, quatre années de recherches thématiques ne sauraient suffire pour résoudre les problèmes et dégager des recommandations sûres aux agriculteurs. Néanmoins, si les objectifs sont rapidement définis et bien ciblés, les acquis dans les domaines varietal et agronomique, après quelques années d'expérimentation bien conduites, doivent déja permettre de définir une première série de paquets technologiques recouvrant la plupart des situations; une seconde phase, dite de vulgarisation, doit logiquement suivre le travail de recherche et avoir pour objet d'améliorer le contexte économique et financier du paysan (disponibilité des intrants, prêts agricoles). A ce titre, on peut penser que les tests des paquets technologiques en milieu réel et les mesures agro-techniques et économiques de leur impact par le paysan lui-même tiendront lieu de procédure de vulgarisation

We propose a general theory of clonal reproduction for parasitic protozoa, which has important medical and biological consequences. Many parasitic protozoa have been assumed to reproduce sexually, because of diploidy and occasional sexuality in the laboratory. However, a population genetic analysis of extensive data on biochemical polymorphisms indicates that the two fundamental consequences of sexual reproduction (i.e., segregation and recombination) are apparently rare or absent in natural populations of the parasitic protozoa. Moreover, the clones recorded appear to be stable over large geographical areas and long periods of time. A clonal population structure demands that the medical attributes of clones be separately characterized; ubiquitous clones call for priority characterization. Uniparental reproduction renders unsatisfactory Linnean taxonomy; this needs to be supplemented by the "natural clone" as an additional taxonomic unit, which is best defined by means of genetic markers.

Cette étude se propose de suivre l’évolution des composantes de la variance phénotypique et génotypique de l’accroissement en hauteur en utilisant le coefficient de variation exprimé au niveau de chaque composante, l’accroissement en hauteur étant estimé par le cumul des accroissements successifs. Le coefficient de variation de l’erreur, de l’effet station, de l'interaction famille X station et de l’interaction mère X père décroît avec le temps alors que la variance des effets principaux reste stable. Jusqu’à 8 ans, cette évolution semble due à l’effet d’âge et s’explique par le changement des conditions du milieu au cours du temps. Les conséquences au niveau des paramètres génétiques sont immédiates : augmentation de l’héritabilité avec le cumul et diminution du rapport Vd/Vg à tel point que tout accroissement du cumul d’ordre n est meilleur prédicteur d’un accroissement de cumul d’ordre inférieur à n que ce dernier lui-même. Par ailleurs, au niveau de la décomposition de la variance génotypique, la variance d’additivité est plus importante que la variance de dominance. Enfin, la possibilité de prévoir l’aptitude spécifique à la combinaison (ASC) à partir de l'aptitude générale à la combinaison (AGC) des deux parents est envisagée., Components of the phenotypic and the genotypic variances of height growth are followed by using the coefficient of variation expressed at each level. Height growth is measured by successive increments between 5 and 12 years. Residual variance, that means error variance,environmental variance family X environmental variance and mother X father interaction decrease with age while main effect variances remain stable. Some of this results are explained by changes in environmental conditions with time. The heritability estimates increase with age while the ratio Vd/Vg decreases. This is true in so far that height increments of n successive years is more predictive of height increments of m successive years (m < n) than this latest trait itself. Height growth is affected more by additive genes effects than by non additive genes effects. Possibility to predict specific combining ability (ASC) by using general combining ability (AGC) of both parents is discussed by using a general model used in genotype - environment problems.

We evaluated the stability of trangene expression in a hybrid poplar (Populus tremula x P. alba) clone transformed with constructs carrying a reporter gene (uidA) under the control of either a constitutive (35S) or a vascular-specific promoter. Analyses of transgene expression by GUS fluorometry and histochemistry was performed on several hundreds of trees, originating from 44 different transgenic lines, grown under in vitro, greenhouse and field conditions. While important variations in expression levels occurred, the transgene appeared to be stably expressed throughout a 6-year period. Only one silenced transgenic line was detected under in vitro conditions: molecular analyses indicated that this line contained an elevated number of transgene copies and was probably silenced from the beginning, at the post-transcriptional level. Overall, these results suggest that transgene expression in perennial species such as trees remains stable over an extended period., Notre étude a pour but d'évaluer la stabilité de l'expression d'un transgène chez un peuplier hybride (Populus tremula x P. alba) transformé avec le gène reporter uidA sous le contrôle soit du promoteur 35S, soit du promoteur du gène cad2 de l'eucalyptus (EuCAD) à spécificité vasculaire. L'expression du transgène a été suivie quantitativement par fluorimétrie et qualitativement par histochimie à partir de matériel collecté in vitro, en serre ou en champ sur quelques centaines d'arbres, issus de 44 lignées transgéniques. Tandis qu'il existe d'importantes variations dans les niveaux d'expression du transgène selon les lignées, les arbres d'une même lignée, l'organe analysé ou la date de prélèvement, l'expression du transgène s'est révélée être stable sur une période de 6 ans. L'inactivation de l'expression d'un transgène n'a été observée que chez une seule lignée dès le stade in vitro. La caractérisation de cette lignée a permis de montrer qu'elle possédait un nombre élevé de copies du transgène, ce qui suggère qu'un phénomène de suppression post-transcriptionnel s'est produit dans cette lignée peu de temps après l'événement de transformation. Ainsi, dans l'ensemble, nos résultats suggèrent que l'expression des transgènes dans les arbres reste stable dans le temps.