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International audience; Modern cultivated Citrus species and varieties result from interspecific hybridization between four ancestral taxa. Among them, Citrus maxima and Citrus reticulata, closely associated with the pummelo and mandarin horticultural groups, respectively, were particularly important as the progenitors of sour and sweet oranges (Citrus aurantium and Citrus sinensis), grapefruits (Citrus paradisi), and hybrid types resulting from modern breeding programs (tangors, tangelos, and orangelos). The differentiation between the four ancestral taxa and the phylogenomic structure of modern varieties widely drive the phenotypic diversity’s organization. In particular, strong phenotypic differences exist in the coloration and sweetness and represent important criteria for breeders. In this context, focusing on the genes of the sugar, carotenoid, and chlorophyll biosynthesis pathways, the aim of this work was to develop a set of diagnostic single-nucleotide polymorphism (SNP) markers to distinguish the ancestral haplotypes of C. maxima and C. reticulata and to provide information at the intraspecific diversity level (within C. reticulata or C. maxima). In silico analysis allowed the identification of 3,347 SNPs from selected genes. Among them, 1,024 were detected as potential differentiation markers between C. reticulata and C. maxima. A total of 115 SNPs were successfully developed using a competitive PCR technology. Their transferability among all Citrus species and the true citrus genera was very good, with only 0.87% of missing data. The ancestral alleles of the SNPs were identified, and we validated the usefulness of the developed markers for tracing the ancestral haplotype in large germplasm collections and sexually recombined progeny issued from the C. reticulata/C. maxima admixture gene pool. These markers will pave the way for targeted association studies based on ancestral haplotypes.

Key message Based on their impact on many ecosystems, we review the relevance of mosses in research regarding stress tolerance, metabolism, and cell biology. We introduce the potential use of mosses as complementary model systems in molecular forest research, with an emphasis on the most developed model mossPhyscomitrella patens. Context and aims Mosses are important components of several ecosystems. The moss P. patens is a well-established non-vascular model plant with a high amenability to molecular biology techniques and was designated as a JGI plant flagship genome. In this review, we will provide an introduction to moss research and highlight the characteristics of P. patens and other mosses as a potential complementary model system for forest research. Methods Starting with an introduction into general moss biology, we summarize the knowledge about moss physiology and differences to seed plants. We provide an overview of the current research areas utilizing mosses, pinpointing potential links to tree biology. To complement literature review, we discuss moss advantages and available resources regarding molecular biology techniques. Results and conclusion During the last decade, many fundamental processes and cell mechanisms have been studied in mosses and seed plants, increasing our knowledge of plant evolution. Additionally, moss-specific mechanisms of stress tolerance are under investigation to understand their resilience in ecosystems. Thus, using the advantages of model mosses such as P. patens is of high interest for various research approaches, including stress tolerance, organelle biology, cell polarity, and secondary metabolism.

Aandacht voor het demonstratieproject "Risicoanalyse van genetisch gemodificeerde voedingsmiddelen als basis voor marktintroductie". Verslag van een workshop

Bram Kuijper deed onderzoek naar de relatie tussen seksuele selectie en sekse-allocatie en de onderliggende mechanismen. Hij concludeert onder meer: • Het maken van voorspellingen gebaseerd op slechts één modelleertechniek is vragen om problemen. Een pluralistische aanpak waarbij meerdere technieken met elkaar worden vergeleken geeft de meest robuuste voorspellingen met betrekking tot evolutionaire processen. • Nieuwe theoretische modellen zijn nodig om te onderzoeken of conclusies van bestaande modellen over seksuele selectie inderdaad zo algemeen geldend zijn als wordt gedacht: hierbij moet vooral worden gedacht aan mechanistische processen met betrekking tot paringskeuze, overerving en interacties met andere evolutionaire processen (zorg voor nakomelingen, sekse-allocatie). • Seksuele selectie in hermafrodieten (beide seksen aanwezig in hetzelfde individu) komt over het algemeen moeilijker van de grond dan in gonochoristen (seksen in aparte individuen). Dit geldt echter niet wanneer beide partners elkaar kiezen (in plaats van één partner de ander, welke dan altijd instemt): dan is seksuele selectie even plausibel in gonochoristen als in hermafrodieten. • De evolutie van haplodiploïdie door toedoen van ‘male-killers’ lijkt veel specifieker te zijn dan eerder werd voorspeld (bijvoorbeeld Normark 2004a). Haplodiploïdie komt daarom alleen onder specifieke omstandigheden van de grond, bijvoorbeeld wanneer ‘male-killers’ mutualistisch zijn of wanneer populaties gekenmerkt worden door een hoge mate van inteelt. • Twee of meerdere CSD loci liggen waarschijnlijk ten grondslag aan de geslachtsbepaling van de wesp Cotesia glomerata, waardoor het één van de weinige bekende soorten binnen de Hymenoptera is met multi-locus CSD. • Conflict tussen ouders en nakomelingen over sekse-allocatie kan leiden tot een stabiele mix van verschillende seksedeterminatiesystemen. Een dergelijk conflict kan bijvoorbeeld verklaren waarom sekschromosomen aanwezig zijn in sommige soorten met temperatuurafhankelijke geslachtsbepaling. • Flexibele seksratio’s gebaseerd op de aantrekkelijkheid van de partner evolueren vaak naar een patroon waarbij vrouwtjes gepaard met aantrekkelijke mannetjes zonen produceren, en vrouwtjes met onaantrekkelijke mannetjes dochters. Desalniettemin is selectie voor flexibele seksratios gebaseerd op de aantrekkelijkheid van de partner vaak zwak en afhankelijk van het mechanisme dat seksuele selectie veroorzaakt. • De evolutie van sekse-allocatie gebaseerd op aantrekkelijkheid van partners samen met de evolutie van een vrouwelijke voorkeur voor aantrekkelijke mannetjes leidt uiteindelijk tot het verlies van deze voorkeur. Daarmee gaat ook mannelijke aantrekkelijkheid verloren, waarmee sekseallocatie zichzelf uiteindelijk ondermijnt.

Background Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (array-CGH). Results Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control. Conclusions The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes.

Bram Kuijper deed onderzoek naar de relatie tussen seksuele selectie en sekse-allocatie en de onderliggende mechanismen. Hij concludeert onder meer: • Het maken van voorspellingen gebaseerd op slechts één modelleertechniek is vragen om problemen. Een pluralistische aanpak waarbij meerdere technieken met elkaar worden vergeleken geeft de meest robuuste voorspellingen met betrekking tot evolutionaire processen. • Nieuwe theoretische modellen zijn nodig om te onderzoeken of conclusies van bestaande modellen over seksuele selectie inderdaad zo algemeen geldend zijn als wordt gedacht: hierbij moet vooral worden gedacht aan mechanistische processen met betrekking tot paringskeuze, overerving en interacties met andere evolutionaire processen (zorg voor nakomelingen, sekse-allocatie). • Seksuele selectie in hermafrodieten (beide seksen aanwezig in hetzelfde individu) komt over het algemeen moeilijker van de grond dan in gonochoristen (seksen in aparte individuen). Dit geldt echter niet wanneer beide partners elkaar kiezen (in plaats van één partner de ander, welke dan altijd instemt): dan is seksuele selectie even plausibel in gonochoristen als in hermafrodieten. • De evolutie van haplodiploïdie door toedoen van ‘male-killers’ lijkt veel specifieker te zijn dan eerder werd voorspeld (bijvoorbeeld Normark 2004a). Haplodiploïdie komt daarom alleen onder specifieke omstandigheden van de grond, bijvoorbeeld wanneer ‘male-killers’ mutualistisch zijn of wanneer populaties gekenmerkt worden door een hoge mate van inteelt. • Twee of meerdere CSD loci liggen waarschijnlijk ten grondslag aan de geslachtsbepaling van de wesp Cotesia glomerata, waardoor het één van de weinige bekende soorten binnen de Hymenoptera is met multi-locus CSD. • Conflict tussen ouders en nakomelingen over sekse-allocatie kan leiden tot een stabiele mix van verschillende seksedeterminatiesystemen. Een dergelijk conflict kan bijvoorbeeld verklaren waarom sekschromosomen aanwezig zijn in sommige soorten met temperatuurafhankelijke geslachtsbepaling. • Flexibele seksratio’s gebaseerd op de aantrekkelijkheid van de partner evolueren vaak naar een patroon waarbij vrouwtjes gepaard met aantrekkelijke mannetjes zonen produceren, en vrouwtjes met onaantrekkelijke mannetjes dochters. Desalniettemin is selectie voor flexibele seksratios gebaseerd op de aantrekkelijkheid van de partner vaak zwak en afhankelijk van het mechanisme dat seksuele selectie veroorzaakt. • De evolutie van sekse-allocatie gebaseerd op aantrekkelijkheid van partners samen met de evolutie van een vrouwelijke voorkeur voor aantrekkelijke mannetjes leidt uiteindelijk tot het verlies van deze voorkeur. Daarmee gaat ook mannelijke aantrekkelijkheid verloren, waarmee sekseallocatie zichzelf uiteindelijk ondermijnt.

Invloed man zijn en DNA-hoeveelheid bij soortvorming parasitaire wespen Sinds Darwin’s The Origin of Species is er veel onderzoek gedaan naar de processen die een rol spelen bij soortvorming. Het promotieonderzoek van Tosca Koevoets richt zich op het vroege stadium in het proces van soortvorming. Wanneer groepen niet meer in contact staan met elkaar, zullen ze langzaam ten gaan veranderen ten opzichte van elkaar, totdat zij verschillende soorten zijn geworden. Uiteindelijk zullen deze soorten genetisch zo veranderd zijn, dat het combineren van DNA van de verschillende soorten niet meer mogelijk is, wat tot uiting komt in allerlei problemen in de hybride nakomelingen. Deze problemen blijken in het beginstadium van soortvorming anders te zijn in mannen dan in vrouwen. Om te onderzoeken waar dit door komt, maakte Koevoets gebruik van parasitaire wespensoorten uit het geslacht Nasonia. Wespen hebben geen geslachtschromosomen, maar vrouwen ontstaan uit bevruchte eitjes en mannen uit onbevruchte eitjes. Vrouwen hebben dus twee keer zoveel DNA als mannen. Ook bij wespen bleken mannen kwetsbaarder voor hybridisatie dan vrouwen. Deze kwetsbaarheid werd zelfs nog groter onder stressomstandigheden. Dit werd veroorzaakt doordat mannen minder genetische variatie hebben, maar ook doordat ze überhaupt minder DNA hebben. Via een genetische techniek kunnen we er voor zorgen dat bevruchte eitjes (dus eigenlijk bestemd om vrouw te worden) zich tot man ontwikkelen. Daardoor kon onderzocht worden wat belangrijker is: man zijn of de hoeveelheid DNA. Natuurlijk lag het antwoord in het midden; beide facetten bleken belangrijk. Dit onderzoek toont aan dat genetische interacties, de hoeveelheid DNA en de genetische variatie belangrijke aspecten zijn bij het ontstaan van soorten.

Invloed man zijn en DNA-hoeveelheid bij soortvorming parasitaire wespen Sinds Darwin’s The Origin of Species is er veel onderzoek gedaan naar de processen die een rol spelen bij soortvorming. Het promotieonderzoek van Tosca Koevoets richt zich op het vroege stadium in het proces van soortvorming. Wanneer groepen niet meer in contact staan met elkaar, zullen ze langzaam ten gaan veranderen ten opzichte van elkaar, totdat zij verschillende soorten zijn geworden. Uiteindelijk zullen deze soorten genetisch zo veranderd zijn, dat het combineren van DNA van de verschillende soorten niet meer mogelijk is, wat tot uiting komt in allerlei problemen in de hybride nakomelingen. Deze problemen blijken in het beginstadium van soortvorming anders te zijn in mannen dan in vrouwen. Om te onderzoeken waar dit door komt, maakte Koevoets gebruik van parasitaire wespensoorten uit het geslacht Nasonia. Wespen hebben geen geslachtschromosomen, maar vrouwen ontstaan uit bevruchte eitjes en mannen uit onbevruchte eitjes. Vrouwen hebben dus twee keer zoveel DNA als mannen. Ook bij wespen bleken mannen kwetsbaarder voor hybridisatie dan vrouwen. Deze kwetsbaarheid werd zelfs nog groter onder stressomstandigheden. Dit werd veroorzaakt doordat mannen minder genetische variatie hebben, maar ook doordat ze überhaupt minder DNA hebben. Via een genetische techniek kunnen we er voor zorgen dat bevruchte eitjes (dus eigenlijk bestemd om vrouw te worden) zich tot man ontwikkelen. Daardoor kon onderzocht worden wat belangrijker is: man zijn of de hoeveelheid DNA. Natuurlijk lag het antwoord in het midden; beide facetten bleken belangrijk. Dit onderzoek toont aan dat genetische interacties, de hoeveelheid DNA en de genetische variatie belangrijke aspecten zijn bij het ontstaan van soorten.

Invloed man zijn en DNA-hoeveelheid bij soortvorming parasitaire wespen Sinds Darwin’s The Origin of Species is er veel onderzoek gedaan naar de processen die een rol spelen bij soortvorming. Het promotieonderzoek van Tosca Koevoets richt zich op het vroege stadium in het proces van soortvorming. Wanneer groepen niet meer in contact staan met elkaar, zullen ze langzaam ten gaan veranderen ten opzichte van elkaar, totdat zij verschillende soorten zijn geworden. Uiteindelijk zullen deze soorten genetisch zo veranderd zijn, dat het combineren van DNA van de verschillende soorten niet meer mogelijk is, wat tot uiting komt in allerlei problemen in de hybride nakomelingen. Deze problemen blijken in het beginstadium van soortvorming anders te zijn in mannen dan in vrouwen. Om te onderzoeken waar dit door komt, maakte Koevoets gebruik van parasitaire wespensoorten uit het geslacht Nasonia. Wespen hebben geen geslachtschromosomen, maar vrouwen ontstaan uit bevruchte eitjes en mannen uit onbevruchte eitjes. Vrouwen hebben dus twee keer zoveel DNA als mannen. Ook bij wespen bleken mannen kwetsbaarder voor hybridisatie dan vrouwen. Deze kwetsbaarheid werd zelfs nog groter onder stressomstandigheden. Dit werd veroorzaakt doordat mannen minder genetische variatie hebben, maar ook doordat ze überhaupt minder DNA hebben. Via een genetische techniek kunnen we er voor zorgen dat bevruchte eitjes (dus eigenlijk bestemd om vrouw te worden) zich tot man ontwikkelen. Daardoor kon onderzocht worden wat belangrijker is: man zijn of de hoeveelheid DNA. Natuurlijk lag het antwoord in het midden; beide facetten bleken belangrijk. Dit onderzoek toont aan dat genetische interacties, de hoeveelheid DNA en de genetische variatie belangrijke aspecten zijn bij het ontstaan van soorten.

Background Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW). Results Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M. balbisiana haplotypes. Conclusions A large RGA08 gene cluster identified in wild banana corresponds to a highly variable genomic region between haplotypes surrounded by conserved flanking regions. High level of sequence identity (70 to 99%) of the genic and intergenic regions suggests a recent and rapid evolution of this cluster in M. balbisiana.

L'article original est publié par The American Society of Plant Biologists; Meiotic crossovers are necessary to generate balanced gametes and to increase genetic diversity. Even if crossover number is usually constrained, recent results suggest that manipulating karyotype composition could be a new way to increase rossover frequency in plants. In this study, we explored this hypothesis by analyzing the extent of crossover variation in a set of related diploid AA, allotriploid AAC, and allotetraploid AACC Brassica hybrids. We first used cytogenetic methods to describe the meiotic behavior of the different hybrids. We then combined a cytogenetic estimation of class I crossovers in the entire genome by immunolocalization of a key protein, MutL Homolog1, which forms distinct foci on meiotic chromosomes, with genetic analyses to specifically compare crossover rates between one pair of chromosomes in the different hybrids. Our results showed that the number of crossovers in the allotriploid AAC hybrid was higher than in the diploid AA hybrid. Accordingly, the allotetraploid AACC hybrid showed an intermediate behavior. We demonstrated that this increase was related to hybrid karyotype composition (diploid versus allotriploid versus allotetraploid) and that interference was maintained in the AAC hybrids. These results could provide another efficient way to manipulate recombination in traditional breeding and genetic studies.

Background In recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants. The use of homozygous lines presents a significant advantage for the accomplishment of sequencing projects. Commercial citrus species are characterized by high heterozygosity, making it difficult to assemble large genome sequences. Thus, the International Citrus Genomic Consortium (ICGC) decided to establish a reference whole citrus genome sequence from a homozygous plant. Due to the existence of important molecular resources and previous success in obtaining haploid clementine plants, haploid clementine was selected as the target for the implementation of the reference whole genome citrus sequence. Results To obtain haploid clementine lines we used the technique of in situ gynogenesis induced by irradiated pollen. Flow cytometry, chromosome counts and SSR marker (Simple Sequence Repeats) analysis facilitated the identification of six different haploid lines (2n = x = 9), one aneuploid line (2n = 2x+4 = 22) and one doubled haploid plant (2n = 2x = 18) of 'Clemenules' clementine. One of the haploids, obtained directly from an original haploid embryo, grew vigorously and produced flowers after four years. This is the first haploid plant of clementine that has bloomed and we have, for the first time, characterized the histology of haploid and diploid flowers of clementine. Additionally a double haploid plant was obtained spontaneously from this haploid line. Conclusion The first haploid plant of 'Clemenules' clementine produced directly by germination of a haploid embryo, which grew vigorously and produced flowers, has been obtained in this work. This haploid line has been selected and it is being used by the ICGC to establish the reference sequence of the nuclear genome of citrus.

La sécheresse est un problème principal chez le riz pluvial. On a démontré que les racines profondes et larges contribuaient à la résistance à la sécheresse. Difficiles à mesurer, ces caractéristiques n'ont pas été prises en compte dans les programmes de sélection. La sélection assistée par les marqueurs pourrait nous aider à venir à bout du problème phénotypique. Nous avons évalué 105 lignées d'haploïdes doublés issues d'un croisement indica japonica au cours d'une expérimentation en serre, dans un schéma complètement aléatoire avec 4 répliques. Nous avons mesuré la largeur, la profondeur maximum, le poids sec des racines par talle, le poids sec des racines au-dessous de 30 cm par talle et la proportion racine/tige. Nous avons utilisé une carte par RFLP de cette population avec 135 marqueurs pour identifier les marqueurs liés avec les différentes caractéristiques. Les marqueurs associés à la morphologie des racines ont été localisés sur les chromosomes 1, 2, 7 et 9. La comparaison des locus quantitatifs détectés avec les résultats d'une autre équipe montre que pour chaque caractéristique, entre 1 et 3 locus quantitatifs sont communs aux différentes populations. Le nombre limité de locus quantitatifs communs peut être dû aux différences d'allèles entre les populations, au manque de polymorphisme dans les localisations des locus quantitatifs et/ou aux seuils choisis pour les tests statistiques

Conditions for induction of androgenesis in coconut cv. Sri Lanka Tall were studied. Anthers collected from inflorescences at four maturity stages were given heat (38 degrees C) or cold (4 degrees C) pretreatments for 1, 3, 6 and 14 days, either prior to or post inoculation. Three different basal media and different anther densities were also tested. Androgenesis was observed only in anthers collected from inflorescences 3 weeks before splitting (WBS) and after a heat pretreatment at 38 degrees C for 6 days. Modified Eeuwens Y-3 liquid medium supplemented with 100 mu M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% activated charcoal and 9% sucrose was effective in inducing an androgenic response. The lowest anther density tested, 10 per petri plate, was found to be the optimal density. When androgenic calli or embryos were subcultured to Y-3 medium containing 66 mu M 2,4-D, followed by transfer to Y-3 medium without plant growth regulators and finally to Y-3 medium containing 5 mu M 6-benzyladenine (BA) and 0.35 mu M gibberellic acid (GA(3)), plantlets regenerated at a frequency of 7%. Histological study indicated that the calli and embryos originated from the inner tissues of the anthers. Ploidy analysis of calli and embryos showed that they were haploid. This is the first report of successful androgenesis yielding haploid plants from coconut anthers.

Introduction. This protocol describes the production of haploid and dihaploid plants from in vitro anther cultures of M. balbisiana. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required laboratory materials, medium preparation, anther isolation and culture. Results. White, creamy calli emerge from anthers about (6 to 15) weeks after the beginning of the culture. France (Guadeloupe) / Musa balbisiana / methods / plant biotechnology / haploidy

International audience; This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Haploidization in onion (Allium cepa L.) using immature flower buds simulates zygotic embryogenesis with no fecundation. In order to know the involvement of polyamines (PAs) in this process, we determined the concentration of endogenous PAs in flower buds and experimented the addition of various combinations of PA molecules in the medium. At the inoculation stage, high levels of free and conjugated spermidine and low putrescine + hydroxyputrescine/spermidine + spermine ratio characterized the highest responsive varieties. During in vitro culture, high levels of putrescine and its derivatives characterized the lowest responsive varieties, whereas high levels of spermidine and spermine characterized responsive varieties. The putrescine + hydroxyputrescine + homospermidine/spermidine + spermine ratio remained low in responsive varieties. The addition of spermidine or spermine (2 x 10(-3) M) to the culture medium improved significantly the embryo production. Our results suggest that the arginine decarboxylase pathway is involved in PA biosynthesis during the in vitro culture of flower buds. Our study showed that specific ratios of PAs are required for successful gynogenesis in onion

The new resources developed are already used in many fine mapping projects. This integrative genetic map provides a common representation framework and is a potential tool for gene positional cloning, marker-assisted selection and in silico QTL/genes co-localisation. It can also be used for comparative genomic studies by using anchor probes as bridge points to test local synteny and candidate genes of other cereals. The next step will be the integration of markers from other major rice genetic maps (Nipponbare x Kasalath) to enlarge the compilation to other genes and QTLs.

Hot pepper (Capsicum annuumL.) is the most important vegetable inIndonesia, but the yield is low, and the breeding programs are confined to the conventional methods and not efficient. To improve the efficiency of the breeding programs by speeding up the production of homozygous lines, studies were aimed at the introduction of haploid technology, which includes the regeneration and the production of doubled haploid plants from gametes. This technique is well developed in the model speciesBrassica napusL. via microspore culture. The results of various investigations involving both applied and fundamental aspects on microspore embryogenesis are presented in this thesis. The main results of the applied part deal with the development of an efficient shed-microspore culture protocol for the production of doubled haploid plants in Indonesian hot pepper (C. annuum), and its implementation under the local conditions of Indonesia. With regard to the more fundamental part, we presented for the first time an entirely new developmental pathway of embryogenesis including suspensor formation in microspore culture ofB. napuscv. Topas that mimics zygotic embryogenesis from early stages of development onwards. These results will have significant impact for practical application in hot pepper breeding programs as well as for further fundamental research on unraveling of early plant embryogenesis.

Hot pepper (Capsicum annuumL.) is the most important vegetable inIndonesia, but the yield is low, and the breeding programs are confined to the conventional methods and not efficient. To improve the efficiency of the breeding programs by speeding up the production of homozygous lines, studies were aimed at the introduction of haploid technology, which includes the regeneration and the production of doubled haploid plants from gametes. This technique is well developed in the model speciesBrassica napusL. via microspore culture. The results of various investigations involving both applied and fundamental aspects on microspore embryogenesis are presented in this thesis. The main results of the applied part deal with the development of an efficient shed-microspore culture protocol for the production of doubled haploid plants in Indonesian hot pepper (C. annuum), and its implementation under the local conditions of Indonesia. With regard to the more fundamental part, we presented for the first time an entirely new developmental pathway of embryogenesis including suspensor formation in microspore culture ofB. napuscv. Topas that mimics zygotic embryogenesis from early stages of development onwards. These results will have significant impact for practical application in hot pepper breeding programs as well as for further fundamental research on unraveling of early plant embryogenesis.

Mme Chantal Billard, Pr, Université de Caen (directeur de thèse) Mr Daniel Vaulot, D.R. CNRS, UMR 7127, Roscoff (rapporteur) Mr Jeremy R. Young, Dr, Natural History Museum, Londres (rapporteur) Mme Marie-Josèphe Chrétiennot-Dinet, C.R. CNRS, UMR 7621 Banyuls/Mer Mr Michel Matthieu, Pr, Université de Caen Mr Christophe Destombe, M.C.F. H.D.R., UMR 7127, Paris VI; Coccolithophores, one of the most important groups of marine phytoplankton, are calcified microalgae. Our knowledge of this group is nevertheless limited, due in part to the lack of cultured species. This study was focused on the life-cycle and its ecological implications for four species of coccolithophores in culture (Coccolithus pelagicus, Calcidiscus leptoporus, Coronosphaera mediterranea and Emiliania huxleyi). An heteromorphic haplo-diploid life-cycle was first demonstrated using various techniques. Ecophysiological experiments on abiotic factors (temperature, light, turbulence, nutrients) were then carried out showing the influence of the heteromorphic life-cycle on the ecology of these sepcies and an ecological cycle was established. Lastly, a preliminary study on toxicity was conducted, demonstrating the toxicity of five coastal coccolithophores.; Les coccolithophores sont des microalgues calcifiées constituant un des principaux groupes phytoplanctoniques marins. Ces organismes restent cependant encore mal connus, du fait du peu d'espèces cultivées. Dans ce travail, les connaissances sur la biologie et l'écologie de ce groupe ont été approfondies à travers l'étude du cycle de vie et de ses implications écologiques pour quatre coccolithophores en culture : Coccolithus pelagicus, Calcidiscus leptoporus, Coronosphaera mediterranea et Emiliania huxleyi. Un cycle digénétique haplo-diplophasique hétéromorphe a d'abord été démontré par différentes techniques. Des expériences écophysiologiques sur des facteurs abiotiques (température, lumière, turbulence, nutriments) ont ensuite permis d'émettre des hypothèses sur l'influence du cycle digénétique sur l'écologie de ces espèces et d'établir un cycle écologique. Enfin, une étude préliminaire de la toxicité a été réalisée, démontrant la toxicité de cinq espèces côtières de coccolithophores.

Coccolithophores, one of the most important groups of marine phytoplankton, are calcified microalgae. Our knowledge of this group is nevertheless limited, due in part to the lack of cultured species. This study was focused on the life-cycle and its ecological implications for four species of coccolithophores in culture (Coccolithus pelagicus, Calcidiscus leptoporus, Coronosphaera mediterranea and Emiliania huxleyi). An heteromorphic haplo-diploid life-cycle was first demonstrated using various techniques. Ecophysiological experiments on abiotic factors (temperature, light, turbulence, nutrients) were then carried out showing the influence of the heteromorphic life-cycle on the ecology of these sepcies and an ecological cycle was established. Lastly, a preliminary study on toxicity was conducted, demonstrating the toxicity of five coastal coccolithophores., Les coccolithophores sont des microalgues calcifiées constituant un des principaux groupes phytoplanctoniques marins. Ces organismes restent cependant encore mal connus, du fait du peu d'espèces cultivées. Dans ce travail, les connaissances sur la biologie et l'écologie de ce groupe ont été approfondies à travers l'étude du cycle de vie et de ses implications écologiques pour quatre coccolithophores en culture : Coccolithus pelagicus, Calcidiscus leptoporus, Coronosphaera mediterranea et Emiliania huxleyi. Un cycle digénétique haplo-diplophasique hétéromorphe a d'abord été démontré par différentes techniques. Des expériences écophysiologiques sur des facteurs abiotiques (température, lumière, turbulence, nutriments) ont ensuite permis d'émettre des hypothèses sur l'influence du cycle digénétique sur l'écologie de ces espèces et d'établir un cycle écologique. Enfin, une étude préliminaire de la toxicité a été réalisée, démontrant la toxicité de cinq espèces côtières de coccolithophores.

We report here, for the first time, the production of haploid plants of banana #Musa balbisiana# (BB). Callus was induced from anthers in which the majority of the microspores were at the uninucleate stage. The frequency of callus induction was 77%. Callus proliferation usually preceded embryo formation. About 8% of the anthers developed androgenic embryos. Of the 147 plantlets obtained, 41 were haploids (n=x=11). The frequency of haploid production depended on genotypes used: 18 haploid plants were produced from genotype 'Pisang klutuk', 12 from 'Pisang batu', seven from 'Pisang klutuk wulung' and four from 'Tani'. The frequency of regeneration was 1.1%, which was based on the total number of anthers cultured. Diploid plants (2n=2x=22) were also observed in the regenerated plants. The haploid banana plants that were developed will be important material for the improvement of banana through breeding programmes.

In this thesis two approaches were used to induce structural mutations in potato starch biosynthesis genes in potato. First production of new monoploid amf genotypes through parthenogenesis made it possible to initiate mutation breeding for amfae double mutants. Two amf monoploids were selected which fulfilled most of the prerequisites. By inducing a mutation in one of the branching enzymes in an amf -mutant it is possible to select a double mutant having less branched amylopectin. This mutation can easily be identified by iodine staining. Amylose-free starch will stain red and less branched amylopectin will stain blue, like amylose containing starch. Mutations were induced by X-ray irradiation of leaf explants followed by adventitious shoot regeneration and microtuber induction or followed by several rounds of multiplication of axillary buds and microtuber induction. In both cases the starch of microtubers was stained with iodine to screen for aberrant types. In 56 tuber samples blue or otherwise aberrant starch granules were found. With this kind of observations, the concept of mutation breeding for starch variants in monoploid potatoes is proven. A second way to induce structural mutations was the use of the Ac ( Activator )/ Ds ( Dissociation ) transposase system of maize in potato where the Ds transposon is activated by a transposase source. In this study the Ds element was linked to the GBSS gene, of which the phenotypic effect of deactivation is known i.e. red staining starch after iodine staining. The known amf mutation was used as a model system to gather more information about the transposition frequency of the Ds transposable elements in potato and to test the tagging of the wildtype GBSS gene. To activate the Ds element four Ds transposon containing plants were combined with the Ac transposase via cross combination or double transformation. Excision rates ranged from 14.8-48.4 %. Three phenotypic starch mutants were found after screening by iodine staining of tuber cut surfaces. These amylose-free mutants were analyzed by in vitro tests, Southern blot hybridization and sequencing. Strong indications were found that inactivation of the GBSS gene was caused by a transposable element.

L'application des méthodes classiques d'amélioration génétique est souvent entravée chez le bananier par des phénomènes de stérilité. Parmi les méthodes non conventionnelles pouvant appuyer les progrès de sélection, l'hybridation somatique est envisagée afin de contourner les obstacles qui peuvent se présenter lors des croisements sexués. Le matériel végétal utilisé pour la production de protoplastes recouvre un panel de génotypes haploïdes, diploïdes et triploïdes. Les protoplastes utilisés proviennent soit des cals, soit des gaines foliaires, soit de vitroplants soit de suspensions cellulaires embryogènes. Deux méthodes de fusion de protoplastes ont été utilisées : une méthode chimique (PEG) et une méthode électrique. Dans les deux cas, il a été observé des fusions binaires en nombre suffisant pour qu'elles puissent être exploitées. Après le processus de fusion, les protoplastes sont mis en culture à forte densité (106/ml) sur couche nourricière, constituée de cellules de bananiers en division active durant trois semaines. Un transfert sur milieu de régénération augmente significativement le taux de formation d'embryons. Les taux de survie des combinaisons utilisant des protoplastes de feuilles ou de cals ne permettent pas pour l'instant leur exploitation. Des diverses fusions réalisées entre protoplastes issus de suspensions embryogènes, il a été obtenu des plantes tétraploïdes (2X + 2X) ; pentaploïdes (2X + 3X), hexaploïdes (3X + 3X). Leur détection a été réalisée essentiellement par cytométrie de flux. La nature hybride de ces plantes est en cours de caractérisation morphologique après plantation au champ et études moléculaires réalisées au Cirad. Outre l'intérêt pratique de la méthode, la fusion de protoplastes devrait permettre à terme une meilleure compréhension de la génétique cytoplasmique ainsi qu'une meilleure approche de l'interaction des compartiments nucléaires et cytoplasmiques chez les bananiers.

In this thesis two approaches were used to induce structural mutations in potato starch biosynthesis genes in potato. First production of new monoploid amf genotypes through parthenogenesis made it possible to initiate mutation breeding for amfae double mutants. Two amf monoploids were selected which fulfilled most of the prerequisites. By inducing a mutation in one of the branching enzymes in an amf -mutant it is possible to select a double mutant having less branched amylopectin. This mutation can easily be identified by iodine staining. Amylose-free starch will stain red and less branched amylopectin will stain blue, like amylose containing starch. Mutations were induced by X-ray irradiation of leaf explants followed by adventitious shoot regeneration and microtuber induction or followed by several rounds of multiplication of axillary buds and microtuber induction. In both cases the starch of microtubers was stained with iodine to screen for aberrant types. In 56 tuber samples blue or otherwise aberrant starch granules were found. With this kind of observations, the concept of mutation breeding for starch variants in monoploid potatoes is proven. A second way to induce structural mutations was the use of the Ac ( Activator )/ Ds ( Dissociation ) transposase system of maize in potato where the Ds transposon is activated by a transposase source. In this study the Ds element was linked to the GBSS gene, of which the phenotypic effect of deactivation is known i.e. red staining starch after iodine staining. The known amf mutation was used as a model system to gather more information about the transposition frequency of the Ds transposable elements in potato and to test the tagging of the wildtype GBSS gene. To activate the Ds element four Ds transposon containing plants were combined with the Ac transposase via cross combination or double transformation. Excision rates ranged from 14.8-48.4 %. Three phenotypic starch mutants were found after screening by iodine staining of tuber cut surfaces. These amylose-free mutants were analyzed by in vitro tests, Southern blot hybridization and sequencing. Strong indications were found that inactivation of the GBSS gene was caused by a transposable element.

Cette étude a permis de développer une technique d'androgenèse applicable aux génotypes diploïdes du bananier. Certains génotypes de bananier possèdent un réel potentiel de production de lignées pures grâce à cette technique. Après l'induction des cals organisés, la régénération des plantes entières directement par embryogenèse somatique ou par caulogenèse sur cals globulaires semble assez aisée. Les plantes obtenues sont fréquemment diploïdes, issues du dédoublement spontané des génomes haploïdes. Enfin, aucune des plantes observées n'était albinos. A l'inverse d'autres plantes comme le riz, aucun facteur ne semble susceptible de diminuer significativement le rendement de l'androgenèse chez le bananier. En revanche, la phase d'induction demande encore à être mieux maîtrisée

This thesis describes the selection and evaluation of diploid potato clones (2n=2x=24) that produce unreduced or 2n-gametes with 24 chromosomes instead of the normal reduced n-gametes with 12 chromosomes. To elucidate the modes of origin of the 2n-gametes, the progenies derived from such gametes were analysed for different marker loci of one chromosome. Besides the already known mechanisms of First and Second Division Restitution (FDR and SDR) in potato, the multilocus analysis showed the additional mechanism of Post-Meiotic Restitution (PMR) resulting in completely homozygous 2n-gametes. The FDR and SDR 2n-gametes were used for gene-centromere mapping. Multilocus analysis of SDR 2n-gametes showed the map position of the centromere of chromosome 8 in relation to the RFLP-loci. These genetic analyses also demonstrated the occurrence of a single crossover per chromosome arm (high level of chiasma interference). In the light of these findings, it is possible to construct more critical genetic maps of potato.

Review of techniques used at Angers to increase genetic diversity. 47. Annual Conference of international Plant Propagators' Society

This thesis describes the selection and evaluation of diploid potato clones (2n=2x=24) that produce unreduced or 2n-gametes with 24 chromosomes instead of the normal reduced n-gametes with 12 chromosomes. To elucidate the modes of origin of the 2n-gametes, the progenies derived from such gametes were analysed for different marker loci of one chromosome. Besides the already known mechanisms of First and Second Division Restitution (FDR and SDR) in potato, the multilocus analysis showed the additional mechanism of Post-Meiotic Restitution (PMR) resulting in completely homozygous 2n-gametes. The FDR and SDR 2n-gametes were used for gene-centromere mapping. Multilocus analysis of SDR 2n-gametes showed the map position of the centromere of chromosome 8 in relation to the RFLP-loci. These genetic analyses also demonstrated the occurrence of a single crossover per chromosome arm (high level of chiasma interference). In the light of these findings, it is possible to construct more critical genetic maps of potato.

Un schéma de carte par RFLP d'une population haploïde doublée issue d'un croisement entre la variété indica IR64 et la variété japonica Azucena a été développée à l'aide de 146 marqueurs RFLP. La population porte 50,2 % de loci, de IR64 et 49,8 % de loci d'Azucena, indiquant qu'une quantité égale de matériel génétique venant de chaque parent a été transmise à la descendance à travers la culture d'anthères. En utilisant cette carte, nous sommes capables de situer 8 isozymes, 14 RAPDs, 12 gènes classés, 1 gène pour la résistance à brown planthopper (BPH) et 12 QTLs pour la longueur, la largeur du grain et le rapport longueur/largeur sur des chromosomes de riz. Le schéma de carte par RFLP présenté ici sera utile pour cartographier des gènes de ségrégation dans cette population haploïde doublée. Ainsi la génération rapide de lignées haploïdes doublées et leur ségrégation impartiale le rende très attractif pour la cartographie génétique

A deep thick root system has been demonstrated to have a positive effect on yield of upland rice under water stress conditions. Molecular-marker-aided selection could be helpful for the improvement of root morphological traits, which are otherwise difficult to score. We studied a doubled-haploid population of 105 lines derived from an indica×japonica cross and mapped the genes controlling root morphology and distribution (root thickness, maximum root length, total root weight, deep root weight, deep root weight per tiller, and deep root to shoot ratio). Most putative QTL activity was concentrated in fairly compact regions on chromosomes 1, 2, 3, 6, 7, 8 and 9, but was widely spread on chromosome 5 and largely absent on chromosomes 4, 10, 11 and 12. Between three and six QTLs were identified on different chromosomes for each trait. Individual QTLs accounted for between 4 and 22% of the variation in the traits. Multiple QTL models accounted for between 14 and 49%. The main QTLs were common between traits, showing that it should be possible to modify several aspects of root morphology simultaneously. There was evidence of interaction between marker locations in determining QTL expression. Interacting locations were mostly on different chromosomes and showed antagonistic effects with magnitudes large enough to mask QTL detection. The comparison of QTL locations with another population showed that one to three common QTLs per trait were recovered, among which the most significant was in one or other population. These results will allow the derivation of isogenic lines introgressed with these common segments, separately in the indica and japonica backgrounds.

Aandacht voor het demonstratieproject "Risicoanalyse van genetisch gemodificeerde voedingsmiddelen als basis voor marktintroductie". Verslag van een workshop