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In allopolyploids, correct chromosome segregation requires suppression of non-homologous crossovers while levels of homologous crossovers are ensured. To date, no mechanism able to specifically inhibit non-homologous crossovers has been described in allopolyploids other than in bread wheat. Here, we show that reducing the number of functional copies of MSH4, an essential gene for the main crossover pathway, prevents non-homologous crossovers in allotetraploid Brassica napus. We show that non-homologous crossovers originate almost exclusively from the MSH4-dependent recombination pathway and that their numbers decrease when MSH4 returns to single copy in B. napus; by contrast, homologous crossovers remain unaffected by MSH4 duplicate loss. We also demonstrate that MSH4 systematically returns to single copy following numerous independent polyploidy events, a pattern that is probably not by chance. These results suggest that stabilization of allopolyploid meiosis can be enhanced by loss of a key meiotic recombination gene., Non-homologous crossovers impair correct chromosome segregation in allopolyploids. Here the authors show that most non-homologous crossovers in Brassica napus arise from MSH4-dependent recombination and provide evidence that post-polyploidization reduction of MSH4 duplicate stabilizes meiosis.

National audience; Les derniers acquis de la recherche sur les mécanismes de la recombinaison génétique, lors de la méiose, ouvrent de nouvelles voies de sélection des caractères d’intérêts chez les plantes cultivées.

International audience; The crossovers (COs) that occur during meiotic recombination lead to genetic diversity upon which natural and artificial selection can act. The potential of tinkering with the mechanisms of meiotic recombination to increase the amount of genetic diversity accessible for breeders has been under the research spotlight for years. A wide variety of approaches have been proposed to increase CO frequency, alter CO distribution and induce COs between non-homologous chromosomal regions. For most of these approaches, translational biology will be crucial for demonstrating how these strategies can be of practical use in plant breeding. In this review, we describe how tinkering with meiotic recombination could benefit plant breeding and give concrete examples of how these strategies could be implemented into breeding programs.

International audience; Meiotic crossovers (COs) are essential for proper chromosome segregation and the reshuffling of alleles during meiosis. In WT plants, the number of COs is usually small, which limits the genetic variation that can be captured by plant breeding programs. Part of this limitation is imposed by proteins like FANCM, the inactivation of which results in a 3-fold increase in COs in Arabidopsis thaliana. Whether the same holds true in crops needed to be established. In this study, we identified EMS induced mutations in FANCM in two species of economic relevance within the genus Brassica. We showed that CO frequencies were increased in fancm mutants in both diploid and tetraploid Brassicas, Brassica rapa and Brassica napus respectively. In B. rapa, we observed a 3-fold increase in the number of COs, equal to the increase observed previously in Arabidopsis. In B. napus we observed a lesser but consistent increase (1.3-fold) in both euploid (AACC) and allohaploid (AC) plants. Complementation tests in A. thaliana suggest that the smaller increase in crossover frequency observed in B. napus reflects residual activity of the mutant C copy of FANCM. Altogether our results indicate that the anti-CO activity of FANCM is conserved across the Brassica, opening new avenues to make a wider range of genetic diversity accessible to crop improvement.

Meiosis is the cell division that reshuffles genetic information between generations. Recently, much progress has been made in understanding this process; in particular, the identification and functional analysis of more than 80 plant genes involved in meiosis have dramatically deepened our knowledge of this peculiar cell division. In this review, we provide an overview of advancements in the understanding of all aspects of plant meiosis, including recombination, chromosome synapsis, cell cycle control, chromosome distribution, and the challenge of polyploidy.

Book review: Polyploidy and genome evolution. Pamela S. Soltis, Douglas E. Soltis. eds. Springer

International audience; Chromosome rearrangements are common, but their dynamics over time, mechanisms of occurrence and the genomic features that shape their distribution and rate are still poorly understood. We used allohaploid Brassica napus (AC, n = 19) as a model to analyze the effect of genomic features on the formation and diversity of meiotically driven chromosome rearrangements. We showed that allohaploid B. napus meiosis leads to extensive new structural diversity. Almost every allohaploid offspring carried a unique combination of multiple rearrangements throughout the genome, and was thus structurally differentiated from both its haploid parent and its sister plants. This large amount of genome reshuffling was remarkably well-tolerated in the heterozygous state, as neither male nor female fertility were strongly reduced, and meiosis behavior was normal in most cases. We also used a quantitative statistical model, which accounted for 75% of the observed variation in rearrangement rates, to show that the distribution of meiotically driven chromosome rearrangements was not random but was shaped by three principal genomic features. In descending order of importance, the rate of marker loss increased strongly with genetic distance from the centromere, the degree of collinearity between chromosomes, and the genome of origin (A < C). Overall, our results demonstrate that B. napus accumulates a large number of genetic changes, but these rearrangements are not randomly distributed in the genome. The structural genetic diversity produced by the allohaploid pathway and its role in the evolution of polyploid species compared to diploid meiosis are discussed.

First meioses of Brassica napus are genome blenders. Plant and Animal Genomes Conference

Chromosomal reshuffling occurs during meiosis in newly created polyploid species in Brassica, contributing to differentiation of parental genomes in the hybrid. In newly synthesized Brassica napus, studies have shown little effect of the very first meiosis in genomic restructuring. However, the frequent and non random fixation of translocations (HNRTs) in early generations of re-synthesized B. napus suppose a major role of the first meiosis in chromosome rearrangements. To study homeologous pairing and remodelling at the creation of the polyploidy, homeologous pair of chromosomes A1-C1 provide an appropriate model, being completely collinear in macrosynteny in B. napus and its progenitors and being the most rearranged pair in natural B. napus haploids.On multiple lineages of synthetic B.napus, we aimed to : (i) Establish the frequent pairing of A1-C1 during the first meiosis of neo-polyploids; (ii) Assess precisely the impact of this meiosis on the nature, the size and the frequency of rearrangements generated on A1 and C1; (iii) Determine the effect of A1-C1 rearrangements on the perturbation of regular meiotic behaviour in contrasted progeny. (i) Various S0 (colchicines doubled hybrids) lineages of resynthesized B. napus have been created from 4 different diploid parents to test genetic background as long as a reciprocal cross to test maternal cytoplasmic effect. By using BAC-FISH (Fluorescent In Situ Hybridisation) approach at meiosis of the first generation S0, it is possible to detect implication of A1-C1 in abnormal chromosome pairing. Evidence for A-C pairing at first meiosis exist, and this work will determine the A1-C1 pairing ability of the progenitors structure. (ii) To assess the impact of first meiosis on genome remodelling in gametes, crosses were performed between the 4 amphidiploids (S0) and a natural B. napus (92 ind. for each). We identified a contrasted behaviour of the B. rapa cytoplasm population, but all had highly frequent inherited chromosomes rearrangements using molecular markers from A1 and C1, and their nature (translocation vs. deletion) will be discussed on a subset of contrasted individuals using BAC FISH differentiating homeologous regions. (iii) By establishing the meiotic behaviour of this subset of plants, we will validate the effect of A1-C1 homogenisation on further genome instability. These data will bring new insight on genome restructuration in polyploids after genome duplication.

Polyploid formation ways in Brassica napus: The Tortoise and the Hare. Réunion du groupe « Cytogénétique et Polyploïdie »

Polyploidy (whole-genome duplication) has played a pervasive role in the evolution of fungi and animals, and is particularly prominent in plants (Wendel & Doyle, 2005; Cui et al., 2006; Otto, 2007; Wood et al., 2009). This important evolutionary phenomenon has attracted renewed and growing interest from the scientific community in the last decade since it was discovered that even the smallest plant genomes considered to be ‘diploid' (e.g. Arabidopsis thaliana, reviewed in Henry et al., 2006) have incurred at least one round of whole-genome duplication, possibly predating the origins of the angiosperms (Soltis et al., 2009). Polyploidy is an important speciation mechanism for all eukaryotes and has profound impacts on biodiversity dynamics and ecosystem functioning. Newly formed polyploids, and particularly those of hybrid origin (allopolyploids), frequently exhibit rapid range expansion (Ainouche et al., 2009), and over long periods of evolutionary time, polyploidy has increased morphological complexity and probably reduced the risk of species extinction (Fawcett et al., 2009). Last, but not least, genome duplication has often provided the raw material for plant domestication (e.g. wheat, Dubkovsky & Dvorak, 2007) and thus has had a major impact on human societies and the development of an agrarian lifestyle.

International audience; Polyploidy (whole-genome duplication) has played a pervasive role in the evolution of fungi and animals, and is particularly prominent in plants (Wendel & Doyle, 2005; Cui et al., 2006; Otto, 2007; Wood et al., 2009). This important evolutionary phenomenon has attracted renewed and growing interest from the scientific community in the last decade since it was discovered that even the smallest plant genomes considered to be ‘diploid' (e.g. Arabidopsis thaliana, reviewed in Henry et al., 2006) have incurred at least one round of whole-genome duplication, possibly predating the origins of the angiosperms (Soltis et al., 2009). Polyploidy is an important speciation mechanism for all eukaryotes and has profound impacts on biodiversity dynamics and ecosystem functioning. Newly formed polyploids, and particularly those of hybrid origin (allopolyploids), frequently exhibit rapid range expansion (Ainouche et al., 2009), and over long periods of evolutionary time, polyploidy has increased morphological complexity and probably reduced the risk of species extinction (Fawcett et al., 2009). Last, but not least, genome duplication has often provided the raw material for plant domestication (e.g. wheat, Dubkovsky & Dvorak, 2007) and thus has had a major impact on human societies and the development of an agrarian lifestyle.

Le colza et le rutabaga appartiennent à la même espèce, Brassica napus (AACC, 2n=38). Cette espèce allotétraploïde est issue de croisements naturels entre le chou B. oleracea (CC, 2n = 18) et la navette B. rapa (AA, 2n = 20). Aucun colza sauvage n’a été décrit ce qui conduit à penser qu’il est probablement apparue dans des jardins où du chou était cultivé pour la consommation humaine à côté de la navette servant à produire de l'huile d'éclairage ou du navet. Le colza aurait alors été sélectionné comme une navette capable de produire encore plus d'huile et le rutabaga comme un navet produisant des racines plus volumineuses. Les débuts de la véritable culture du colza en Europe datent probablement du 15ème siècle.Il existe deux stratégies pour mettre en évidence les modifications structurales et fonctionnelles des génomes lors de la stabilisation de l’espèce ; (i) la création de lignées synthétiques à partir du croisement des progéniteurs pour observer les événements qui se mettent en place lors de la stabilisation ; (ii) l’extraction des composantes diploïdes du tétraploïde et leur comparaison avec les diploïdes actuels. C’est cette deuxième stratégie que nous avons mise en place.Schéma d’extraction 1 : Nous avons croisé le colza avec la navette pour obtenir un hybride triploïde AAC. Cet hybride a été recroisé par le colza et seule une plante à 29 chromosomes (AAC) a été sélectionnée pour être à nouveau croisée par le colza. Nous avons réalisé ce cycle de croisement trois fois afin d’obtenir un génome A proche de celui du colza dans l’hybride AAC. L’autofécondation de ce dernier devait nous permettre de sélectionner une plante AA, composante diploïde de notre tétraploïde de départ. Cependant la stérilité de l’hybride AAC ne nous a pas permis d’obtenir une descendance. Le génome A du colza a sans doute subi trop de modifications dans son contexte trétraploïde pour être extrait aussi directement. Nous avons donc repensé notre schéma d’extraction.Schéma d’extraction 2 : Nous sommes repartir de notre schéma précédent en croisant le premier hybride AAC avec de la navette, dans la descendance nous avons sélectionné une plante AA à 2n=20. Cette plante à 20 chromosomes nous a servi pour produire le deuxième hybride AAC du schéma 1. Nous avons recommencé ce cycle de croisement trois fois et nous allons continuer pour nous rapprocher le plus possible du génome A du colza.Lignées d’addition monosomiques : A chaque cycle de croisement avec la navette nous avons sélectionné des lignées d’addition portant chacune un chromosome du génome C (C1, C2, C3, C5, C7 ou C8) en utilisant la cytomètrie en flux puis des observations cytogénétiques en les combinant à deux marqueurs moléculaires par chromosome C du colza. D’autres lignées d’addition sont à extraire pour avoir le set complet des chromosomes C du colza. Ces lignées constitueront un outil performant pour identifier l’impact de chaque chromosome C sur le phénotype et pour assigner sans ambigüité des marqueurs monomorphiques correspondant à des gènes candidats.

Precise chromosome segregation is vital for polyploid speciation. Here, we highlight recent findings that revitalize the old question of the genetic control of diploid-like meiosis behaviour in polyploid species. We first review new nformation on the genetic control of autopolyploid and allopolyploid cytological diploidization, notably in wheat and Brassica. These major advances provide new opportunities for speculating about the adaptation of meiosis during polyploid evolution. Some of these advances are discussed, and it is suggested that research on polyploidy and on meiosis should no longer be unlinked.

International audience

Résumés des communications du 6.Blackleg Workshop; International audience

National audience; Because of the development of transgenic herbicide resistant oilseed rape (Brassica napus, AACC, 2n=38) varieties, studies for assessing gene flow from this crop to its weeds have been performed. Our previous work showed that wild radish (Raphanus raphanistrum, RrRr, 2n=18) is one of the main weed which has to be considered under French cultivation conditions. At the first generation of interspecific hybridisation, viable reduced and unreduced gametes allow the production of F1 hybrids with different genomic structures. Theoretically, 10 different structures can be obtained from reciprocal crosses and 5 have already been observed. The identified hybrids were grown in presence of wild radish as pollinator and the chromosome numbers of the plants in the following generations ranged from 18 to 56. So a large variability can be observed, taking into account that we are looking for a rare event, the pollen donor remaining unknown at the harvest under natural conditions. Combination of different tools for characterisation i.e. morphology, transgene expression, cytogenetic analyses, molecular makers is needed for monitoring interspecific gene flow.; Le développement des variétés transgéniques de colza résistantes à certains herbicides (Brassica napus, AACC, 2n=38) a conduit à s'interroger sur les possibilités de flux de gènes du colza vers ses adventices. Nos travaux antérieurs ont montré que la ravenelle (Raphanus raphanistrum, RrRr, 2n=18) était l'une des principales espèces à prendre en compte dans les conditions françaises de culture. L'ensemble des résultats acquis a montré que la production de gamètes réduits et non réduits pouvaient conduire aux différentes générations à une grande diversité de structures. Cette importante variabilité, combinée aux contraintes d'un suivi en conditions naturelles (recherche d'un événement rare, diversité des milieux et impossibilité d'identifier le pollinisateur), rend difficile la caractérisation du matériel produit. Lors de la production des hybrides F1, 10 combinaisons génomiques (dont 5 déjà observées) sont possibles, en considérant les deux sens possibles de croisements. Aux générations ultérieures, sous l'hypothèse d'hybrides mis en présence de ravenelles, le nombre de chromosomes peut varier de 18 à 56. L'analyse des différents outils de caractérisation (observations morphologiques, présence du transgène, caractérisation cytogénétique, marquage moléculaire) révèle que seule la combinaison de différentes méthodes peut permettre d'identifier les différents hybrides. La diversité des structures génomiques et la lourdeur des méthodes de caractérisation à mettre en œuvre conduit à s'interroger sur les possibilités d'un suivi au champ dès les premières générations.

Le développement des variétés transgéniques de colza résistantes à certains herbicides (Brassica napus, AACC, 2n=38) a conduit à s'interroger sur les possibilités de flux de gènes du colza vers ses adventices. Nos travaux antérieurs ont montré que la ravenelle (Raphanus raphanistrum, RrRr, 2n=18) était l'une des principales espèces à prendre en compte dans les conditions françaises de culture. L'ensemble des résultats acquis a montré que la production de gamètes réduits et non réduits pouvaient conduire aux différentes générations à une grande diversité de structures. Cette importante variabilité, combinée aux contraintes d'un suivi en conditions naturelles (recherche d'un événement rare, diversité des milieux et impossibilité d'identifier le pollinisateur), rend difficile la caractérisation du matériel produit. Lors de la production des hybrides F1, 10 combinaisons génomiques (dont 5 déjà observées) sont possibles, en considérant les deux sens possibles de croisements. Aux générations ultérieures, sous l'hypothèse d'hybrides mis en présence de ravenelles, le nombre de chromosomes peut varier de 18 à 56. L'analyse des différents outils de caractérisation (observations morphologiques, présence du transgène, caractérisation cytogénétique, marquage moléculaire) révèle que seule la combinaison de différentes méthodes peut permettre d'identifier les différents hybrides. La diversité des structures génomiques et la lourdeur des méthodes de caractérisation à mettre en œuvre conduit à s'interroger sur les possibilités d'un suivi au champ dès les premières générations., Because of the development of transgenic herbicide resistant oilseed rape (Brassica napus, AACC, 2n=38) varieties, studies for assessing gene flow from this crop to its weeds have been performed. Our previous work showed that wild radish (Raphanus raphanistrum, RrRr, 2n=18) is one of the main weed which has to be considered under French cultivation conditions. At the first generation of interspecific hybridisation, viable reduced and unreduced gametes allow the production of F1 hybrids with different genomic structures. Theoretically, 10 different structures can be obtained from reciprocal crosses and 5 have already been observed. The identified hybrids were grown in presence of wild radish as pollinator and the chromosome numbers of the plants in the following generations ranged from 18 to 56. So a large variability can be observed, taking into account that we are looking for a rare event, the pollen donor remaining unknown at the harvest under natural conditions. Combination of different tools for characterisation i.e. morphology, transgene expression, cytogenetic analyses, molecular makers is needed for monitoring interspecific gene flow.

National audience

National audience

International audience

National audience; Crop/weed gene flow is often of great concern for a better understanding of germplasm diversity. This paper provides an overview of the levels and partition of genetic diversity assessed between wild and cultivated populations of Medicago sativa L. in Spain for both molecular markers and agromorphological traits. We report that most wild populations preserve a great agronomic and morphological originality despite their occasional parapatry with cultivated forms and the occurrence of crop/weed gene flow in many locations. However, populations that display either a continuous range of intermediate plants between the typical wild and cultivated forms, or a mosaic of individuals that can be assigned to each gene pool respectively, were also identified. Providing that reproductive isolation is not strict, we hypothesise that contrasted selective pressure between wild and cultivated populations mainly accounted for the maintenance of their phenotypic integrity.; L’existence d’un flux génique entre des populations cultivées et sauvages joue souvent un rôle prépondérant dans l’évolution de la diversité génétique de ces dernières. Nous présentons ici une vue générale des niveaux, et de l’organisation de la diversité génétique mesurée sur la base de marqueurs neutres et de caractères agromorphologiques dans des populations sauvages et cultivées de luzerne (M. sativa L.)en Espagne. La plupart des populations sauvages maintiennent une claire originalité agronomique et morphologique bien qu’elles soient parapatriques de populations cultivées et qu’elles fassent parfois l’objet d’introgressions régulières de leur part. Néanmoins notre étude a permis de mettre en évidence un ensemble de populations sauvages moins typiques, présentant un continuum de formes intermédiaires ou une mosaïque d’individus caractéristiques de chaque forme. Notre étude suggère que l’isolement reproducteur entre populations sauvages et cultivées n’est pas total et qu’il est vraisemblable que de fortes pressions de sélection engendrées par le milieu jouent un rôle déterminant dans le maintien de l’intégrité phénotypique des formes sauvages.

International audience; Given their tremendous importance for correct chromosome segregation, the number and distribution of crossovers are tightly controlled during meiosis. In this review,we give an overview of crossover formation in polyploid Brassica hybrids and haploids that illustrates or underscores several aspects of crossover control. We first demonstrate that multiple targets for crossover formation (i.e. different but related chromosomes or duplicated regions) are sorted out during meiosis based on their level of relatedness. In euploid Brassica napus (AACC; 2n = 38), crossovers essentially occur between homologous chromosomes and only a few of them form between homeologues. The situation is different in B. napus haploids in which crossovers preferentially occur between homeologous chromosomes and a few can then form between more divergent duplicated regions. We then provide evidence that the frequency of crossovers between a given pair of chromosomes is influenced by the karyotypic and genetic composition of the plants that undergo meiosis. For instance, genetic evidence indicates that the number of crossovers between exactly the same pairs of homologous A chromosomes gets a boost in Brassica digenomic tetraploid (AACC) and triploid (AAC) hybrids. Increased autosyndesis within B. napus haploids as compared to monoploid B. rapa and B. oleracea is another illustration of this process. All these observations may suggest that polyploidization overall boosts up crossover machinery and/or that the number of crossovers is modulated through inter-bivalents or univalent-bivalent cross-talk effects. The last part of this review gives an upto-date account of what we know about the genetic control of homologous and homeologous crossover formation among Brassica species.

In allopolyploids, correct chromosome segregation requires suppression of non-homologous crossovers while levels of homologous crossovers are ensured. To date, no mechanism able to specifically inhibit non-homologous crossovers has been described in allopolyploids other than in bread wheat. Here, we show that reducing the number of functional copies of MSH4, an essential gene for the main crossover pathway, prevents non-homologous crossovers in allotetraploid Brassica napus. We show that non-homologous crossovers originate almost exclusively from the MSH4-dependent recombination pathway and that their numbers decrease when MSH4 returns to single copy in B. napus; by contrast, homologous crossovers remain unaffected by MSH4 duplicate loss. We also demonstrate that MSH4 systematically returns to single copy following numerous independent polyploidy events, a pattern that is probably not by chance. These results suggest that stabilization of allopolyploid meiosis can be enhanced by loss of a key meiotic recombination gene.