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International audience; The mandarin horticultural group is an important component of world citrus production for the fresh fruit market. This group formerly classified as C. reticulata is highly polymorphic and recent molecular studies have suggested that numerous cultivated mandarins were introgressed by C. maxima (the pummelos). C. maxima and C. reticulata are also the ancestors of sweet and sour oranges, grapefruit, and therefore of all the "small citrus" modern varieties (mandarins, tangors, tangelos) derived from sexual hybridization between these horticultural groups. Recently, NGS technologies have greatly modified how plant evolution and genomic structure are analyzed, moving from phylogenetics to phylogenomics. The objective of this work was to develop a workflow for phylogenomic inference from Genotyping By Sequencing (GBS) data and to analyze the interspecific admixture along the nine citrus chromosomes for horticultural groups and recent varieties resulting from the combination of the C. reticulata and C. maxima gene pools. A GBS library was established from 55 citrus varieties, using the ApekI restriction enzyme and selective PCR to improve the read depth. Diagnostic polymorphisms (DPs) of C. reticulata/C. maxima differentiation were identified and used to decipher the phylogenomic structure of the 55 varieties. The GBS approach was powerful and revealed 30,289 SNPs and 8,794 Indels with 12.6% of missing data. 11,133 DPs were selected covering the nine chromosomes with a higher density in genic regions. GBS combined with the detection of DPs was powerful for deciphering the "phylogenomic karyotypes" of cultivars derived from admixture of the two ancestral species after a limited number of interspecific recombinations. All the mandarins, mandarin hybrids, tangelos and tangors analyzed displayed introgression of C. maxima in different parts of the genome. C. reticulata/C. maxima admixture should be a major component of the high phenotypic variability of this germplasm opening up the way for association studies based on phylogenomics.

International audience; Gene banks have been established to conserve the genetic diversity of crop species. Large germplasm collections lead to management problems (space, maintenance costs, etc.), especially in collections involving species with recalcitrant seeds that must be maintained as growing plants. Core collections (CCs) are thus developed to reduce the size of large germplasm collections while keeping the maximum variability. This also facilitates fine phenotypic evaluation. In this study, several software packages (DARwin, PowerMarker and MSTRAT) and methods (Max length subtree, M strategy, simulated annealing and MinSD) were compared to define a mandarin (Citrus reticulata) CC. One hundred and sixty-seven accessions were sampled from two germplasm collections, which were genotyped with 50 SSR, 24 InDel and 68 single nucleotide polymorphism markers. All the CC obtained were tested for the maintenance of the genetic variability parameters (Ho and He) of the initial collection, the level of linkage disequilibrium (LD) and the phenotypic diversity retention. The Max length subtree function from DARWin seemed to be the most appropriate method for establishing a CC in C. reticulata. It maintained 96.82% of the allelic richness and 17.96% of the size of the initial collection with only 30 accessions. Besides it did not increase the LD (r(2) value) of the initial collection and retained the vast majority of the phenotypic variability. However, a CC with 70 accessions would be more helpful for genetic association studies.

Previous studies with molecular markers (ISSR, RAPD, SCAR, AFLP and SSR) have shown that most of the genetic diversity of cultivated Citrus (except C. aurantifolia) comes from the recombination between three main species: C. medica (citron), C. reticulata (mandarin) and C. maxima (pummelo). However, the precise contribution of these basic species to the genome constitution of secondary species (C. sinensis, C. limon, C. aurantium, C. paradisi) and recent hybrids is not known. In this study, 58 nuclear and four mitochondrial markers were used to investigate the genetic diversity among 106 Citrus accessions, representing the three main ancestors groups, secondary species and several hybrids from the 20th century breeding programs. For the nuclear analysis, 50 simple sequence repeats (SSRs) developed from genomic libraries and ESTs databases were used. Moreover, 10 Insertion-Deletion (INDEL) markers were developed from genomic sequences of some primary and secondary metabolites determining the citrus fruit quality (sugars, acids, flavonoids and carotenoids). All the SSR markers and one INDEL are included in a consensus genetic map of clementine and pummelo Chandler and are distributed along the nine linkage groups, representing positively the global genome of Citrus. Genetic diversity statistics were calculated for each SSR and INDEL marker, within the entire population and within and between the different specified Citrus groups. The organizations of the genetic diversity among all the accessions were determined by constructing neighbor-joining trees for the different sets of primers. INDEL markers are less polymorphic than SSRs, display a higher organization of genetic diversity and appear to be better phylogenetic markers to trace the contribution of the three ancestral species. Population structure was studied using the Structure software, version 2.2.3, (http://cbsuapps.tc.cornell.edu/structure) which implements a model-based clustering method for inferring population structure using genotype data. The relative proportion of ancestral taxa genomes in the secondary species and recent hybrids was assigned. Mitochondrial markers revealed a maternal phylogeny of citrus germplasm accessions in agreement with previous studies with chloroplastic markers. This analysis allowed a better understanding of the genetic diversity organization among citrus cultivars, opening the way for a better management of citrus germplasm banks and breeding programs.(Texte intégral)

A genetic map is a necessary first step to guide plant breeding programs and to explore the genome structure by localizing genes involved in important agronomic traits expression. For citrus, some genetic maps have been published but more often they were unsaturated or they employed dominant markers, poorly transferable to other labs. We have initiated a collaborative action between INRA (France), CIRAD (France), IVIA (Spain) and the University of Florida (USA) to generate a reliable and saturated genetic map of the Clementine genome with co-dominant markers localized in coding sequences. For this goal, we have investigated the presence of microsatellite sequences (SSRs) inside EST sequences from different libraries developed for sweet orange and Clementine. Nearly 10% of the ESTs include at least one SSR. By this way we expect to map directly genes whose function is identified by sequence comparison with GenBank or NCBI accessions. A segregating population of 88 individuals was created by INRA at San Giuliano by backcrossing an intergeneric hybrid (Clementine x Poncirus trifoliata) with Clementine. Due to the heterozygosity of the two parents (62% and 84% for Clementine and the intergeneric hybrid respectively), the efficiency of polymorphic EST-SSR markers tends to 45%. We have studied the segregation of more than 200 EST-SSR markers, and complemented with 75 genomic SSR markers previously developed we have constructed a genetic map. (Texte intégral)

We have isolated and characterised microsatellite loci from citrus, to explore the potential of thes markers for cultivar identification, for genetic diversity evaluation and for genome mapping. Small insert library from an homozygous variety Cleopatra mandarin (Citrus reshni) was enriched for (GA) repeats. 960 clones have been sequenced. Pairs of primers for PCR amplification were designed to the flanking region of 250 microsatellites and until now, 100 have been tested for polymorphism and quality of amplification. Considering , the expected sizes of amplified DNA fragments, interpretable amplification products, mendelian segregation and monolocus position, 40 primers pairs have been used to fingerprint 50 accessions of a wide representation of Aurantioideae sub-family. Amplifications were always succefull for true citrus (Poncirus, Fortunella and Citrus) Severinia buxifolia and Eremocitrus glauca while the other genus have less sucess. Cleopatre and Sunki mandarins, known to be very homozygous with other types of markers (RFLP, isozymes) were characterised by 25% of heterozygous loci. A principal co-ordinate analysis and representation of pairwise distance matrices revealed a genetic diversity structure in accordance with the well known relationship and evolution of citrus genera and species. Otherwise, we have analysed the segregation among an inter-generic backcross progeny and mapped 45 microsatellite loci , in addition with 150 AFLP markers. Utilities of these microsatellites markers in our french genetic research program have been discussed. (Texte intégral)

Les nombreuses collections d'agrumes ont pour vocation de préserver à long terme la diversité des agrumes et des genres apparentés, et de constituer des parcs à bois pour fournir des greffons de variétés commerciales. Une étude de la structuration de la diversité morphologique dans le genre #Citrus# est réalisée à partir de 20 descripteurs de l'appareil végétatif observés sur 74 cultivars. Elle sert de support à l'analyse des relations entre la diversité morphologique et la diversité moléculaire présentée dans ce chapitre. Afin d'étudier les paramètres de structure des populations, l'analyse de la diversité allélique repose sur le polymorphisme de 9 systèmes isoenzymatiques. La diversité structurelle nucléaire est pour sa part examinée en évaluant la taille des génomes par cytométrie en flux. L'échantillonnage variétal des formes cultivées est le même que pour l'étude de la diversité morphologique. Dix-sept #Citrus# non cultivés complètent l'analyse. L'analyse de la diversité morphologique permet de retrouver la structuration globale autour des trois pôles identifiés d'après les données isoenzymatiques : le premier regroupant les mandariniers, le deuxième associant les pamplemoussiers et les pomelos, le troisième étant formé par les cédratiers, qui présentent un apparentement marqué avec les limettiers. Les positions relatives des espèces cultivées autour de ces trois pôles sont dans l'ensemble conservées. En revanche, les espèces monomorphes du point de vue enzymatique présentent une dispersion morphologique équivalente à celle des espèces polymorphes sur le plan moléculaire. Deux niveaux coexistent dans la structuration de la diversité morphologique : un niveau majeur, qui répond à des contraintes affectant l'évolution du génome dans son ensemble, et un niveau secondaire, dissocié de l'évolution moléculaire visualisée par les isoenzymes

Genetic information of plant chloroplastic DNA presents a great interest for phylogenic studies. Recently, in Citrus, a new interest for cytoplasmic genome analysis risen from the impediment of somatic hybridization. Indeed this technique allows to combine nuclear, chloroplastic and mitochondrial genomes in new patterns that must be characterized. The development of PCR markers appears very suitable for cytoplasmic characterization at an early in vitro step. The application of Cleaved Amplified Polymorphic Sequences method with universal primer has been demonstrated recently to be efficient at the inter-specific level but display very few diversity at the infra-specific one. In the present work, the usefulness of chloroplastic SSR for citrus chloroplast genomes characterization has been studied. Thirty two PCR primers from Oryza sativa and Nicotiana tabacum chloroplast genome have been tested among 8 cultivated Citrus species and 3 related genera. From 26 tobacco couples of primers, 15 have allowed amplification in all citrus species, 10 of them displaying polymorphism. For 6 rice couples of primer, we got successful amplification without any polymorphism. Height couples of primers from tobacco displayed polymorphism have been used for diversity analysis among 50 species of Aurantioideae sub-family. Phylogenic inference were done from a cluster analysis of chloroplastic SSR data. At Citrus genus level, the genetic organization displayed was in agreement with previous hypothesis in cultivated citrus evolution. At the end, we present an example of application of these markers for chloroplastic genome characterization from somatic hybrid. (Texte intégral)

Les nombreuses collections d'agrumes ont pour vocation de préserver à long terme la diversité des agrumes et des genres apparentés, et de constituer des parcs à bois pour fournir des greffons de variétés commerciales. Une étude de la structuration de la diversité morphologique dans le genre #Citrus# est réalisée à partir de 20 descripteurs de l'appareil végétatif observés sur 74 cultivars. Elle sert de support à l'analyse des relations entre la diversité morphologique et la diversité moléculaire présentée dans ce chapitre. Afin d'étudier les paramètres de structure des populations, l'analyse de la diversité allélique repose sur le polymorphisme de 9 systèmes isoenzymatiques. La diversité structurelle nucléaire est pour sa part examinée en évaluant la taille des génomes par cytométrie en flux. L'échantillonnage variétal des formes cultivées est le même que pour l'étude de la diversité morphologique. Dix-sept #Citrus# non cultivés complètent l'analyse. L'analyse de la diversité morphologique permet de retrouver la structuration globale autour des trois pôles identifiés d'après les données isoenzymatiques : le premier regroupant les mandariniers, le deuxième associant les pamplemoussiers et les pomelos, le troisième étant formé par les cédratiers, qui présentent un apparentement marqué avec les limettiers. Les positions relatives des espèces cultivées autour de ces trois pôles sont dans l'ensemble conservées. En revanche, les espèces monomorphes du point de vue enzymatique présentent une dispersion morphologique équivalente à celle des espèces polymorphes sur le plan moléculaire. Deux niveaux coexistent dans la structuration de la diversité morphologique : un niveau majeur, qui répond à des contraintes affectant l'évolution du génome dans son ensemble, et un niveau secondaire, dissocié de l'évolution moléculaire visualisée par les isoenzymes

The detection and analysis of DNA polymorphisms in crops is an essential component of marker-assisted selection and cultivar identification in plant breeding. We have explored several molecular techniques which allow to detect a DNA polymorphism : isozymes, amplification of microsatellite interspaced DNA sequences (AMIS), random PCR amplified polymorphic DNA (RAPD) and restriction fragment lenght polymorphism (RFLP). Number of problems in citrus breeding and systematics can be approached using these techniques : - molecular markers provide a more direct method to screen a large number of plants, at early development stages; - genetic origin identification (parental origin or nucellar/zygotic origin); - genetic linkage map construction to locate genes involved in the development of agronomically interesting characters; markers associated with these genes could then be used for early selection of progenies and possible progenitors for breeding programs; - in new breeding schemes employing somatic hybridization these molecular markers are essential to control the genetic composition of the vitroplantlets and calli arising from protoplast fusion. Presently, the microsatellite marker seems to be the more valuable tool in genetic studies and appears very promising in all breeding applications.

The non-mendelian mode of inheritance of cytoplasmic organelle genomes (mitochondrial and chloroplast) makes the information present in the plant mitochondrial DNA (mtDNA) and chloroplast DNA cpDNA) of great interest for phylogeny. Furthermore, in our protoplast fusion program, we need cytoplasmic molecular markers to study the genetic origin of plantlets obtained from different citrus somatic hybrids in inter-specific and inter-generic combinations, The cpDNA is know to be highly conserved in structure and the substitution rate of plant mitochondrial DNA genes is low and these conserved coding sequences are easily identified. Here we describe the use for #Citrus# of mitochondrial and chloroplast universal primers corresponding to conserved coding sequences previously selected for population genetic studies in oak (#Quercus robur#). These primers allow the amplification of interstitial non-coding regions. Variation in cytoplasmic organelles can be detected by coupling amplification and RFLP techniques : The amplified DNA fragments are digested by frequently cutting restriction enzymes and analysed by acrylamide gel electrophoresis.

L'accroissement des pressions phytosanitaires et la diversification variétale effectuée sur des critères de qualité attendue par le marché sont deux des justifications majeures des programmes d'amélioration des agrumes à travers le monde. Ces derniers se sont longtemps heurtés aux contraintes spécifiques du système de reproduction des agrumes. Le développement des techniques de marquage moléculaire et de biologie cellulaire, en particulier celles associées à l'embryogenèse somatique, ouvre aujourd'hui des perspectives très prometteuses pour apporter une solution variétale aux grands enjeux de l'agrumiculture. L'apport des biotechnologies dans le domaine de l'amélioration des agrumes, replacé dans le contexte de ses objectifs et des contraintes associées à l'amélioration conventionnelle, est présenté sur la base d'une synthèse bibliographique

La technique RFLP et les marqueurs génétiques ont été utilisés pour déterminer le mode de transmission de la diversité génétique chez les agrumes (Citrus, Poncirus et Fortunella). Trois groupes ont été définis dans le genre Citrus : oranger-mandarinier, pomelo et citronnier, limettier et cédratier. Ces résultats sont conformes aux études qui ont démontré que toutes les espèces d'agrumes dérivent de 3 espèces ancestrales (C. reticulata, C. grandis et C. medica). La comparaison du polymorphisme des ADN cytoplasmiques est effectuée

Des fusions ont été réalisées entre des protoplastes issus de cals embryogènes de mandariniers (Willow Leaf) d'une part et des protoplastes de feuilles de pomelo (Ducan) ou d'oranger (Hamlin) d'autre part. Contre toute attente, des plantules ayant des phénotypes de pomelo et d'oranger ont été régénérées, alors que les protoplastes de feuilles ne sont pas aptes à régénérer sur les milieux utilisés. La cytométrie en flux a permis de révéler la nature diploïde de l'ensemble des plantules issues de la fusion somatique. Une étude RFLP des différents génomes cellulaires a montré que les plantules de phénotype 'mandarinier' possèdent le cytoplasme et le noyau du mandarinier et sont probablement issues des cals non fusionnés. Les autres plantules sont des hybrides constitués par les organelles du cytoplasme du parent "cal" et du noyau du parent "feuille". Ces résultats semblent indiquer que l'aptitude à la régénération possède un déterminisme génétique

Présentation d'une méthode de cartographie qui prend en compte les déviations à partir de simples taux de ségrégation sur locus également nommés distorsions de ségrégation. La méthode est brièvement décrite pour les populations de backross et F2, avec une application à la cartographie génétique du genre Citrus

Une carte de différents marqueurs moléculaires a été établie sur la base d'un croisement intergénérique : un hybride entre la mandarine Cleopatra et le Poncirus trifoliata a été rétrocroisé avec le pamplemousse sans pépins (C. grandis). La grande différenciation génétique entre les 3 géniteurs a permis de déduire les structures gamétiques à partir des zygotes. Une cartographie partielle de l'hybride intergénérique a été établie