New soybean lines have been developed with significantly higher oil, protein + oil and higher meal protein. These soybeans contain a VgD1 gene (highly active acyl-CoA:diacylglycerol acyltransferase, DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in soybean. Soybean with active DGAT from Vernonia galamensis (VgDGAT1A) has active TAG biosynthesis relative to other DGATs including from soybeans and Arabidopsis. DGATs catalyze the final step of TAG synthesis: DAG (diacylglycerol) + acyl-CoA → TAG + CoASH (Coenzyme A is notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle). A thorough analysis of the major components in VgD1 lines, especially those of nutritional or anti-nutritional value including what else changed (decreased); and what remained at normal levels was conducted. A field study was conducted in Spindletop and Princeton KY, reviled no reduction in yield nor protein, and about 4 % (DW) more oil was obtained in Princeton and 2% (DW) in Spindeltop. No consistent reduction in the other seed composition.VgDGAT1A soybean lines indicated noticeably early maturation compared to the parental line. This is associated with higher expression of the flowering genes FT2 (FLOWERING LOCUS T2) and FT5 (FLOWERING LOCUS T5), for the high oil lines. A single recessive mutation in soybean (MIPS) myo-inositol 1-phosphate synthase, confers a seed phenotype of increase inorganic phosphate (Pi) crossed with high oil lines expressing a DGAT from Vernonia galamensis (VgDGAT1A) (VgD). The oil and protein were maintained compart to VgD. VgD X MIPS (VM), had 21.2, and 22 % oil in 2015, and 23.3 and 24.0 oil in 2016, and protein 46, 49 in 2015, and 37 and 39 % in 2016. Phosphate results suggesting the cross MV is still segregating for MIPS and more selection and planting are needed. Measurement of seed phosphate levels is an established technique for screening for low phytate mutants but to date, it has not been performed non-destructively from single soybean seeds. A protocol was developed greatly reducing the sample size thereby reducing the cost and time and saving a generation in the selection of low phytate mutant seeds based on the high Pi phenotype. Genotyping single seeds are useful in breeding and genetics while maintaining high germination rates. Nondestructive single-seed genomic DNA extraction protocols using 12 mg cotyledon tissue with a modified cetyl trimethyl ammonium bromide (CTAB) technique and a commercial seed DNA extraction kit using 1 mg cotyledon tissue were developed for dry soybean seeds and cross-verified with leaf DNA analysis.

A laranjeira ,,x11\" é um mutante espontâneo de laranja doce, com seedlings florescendo a partir do primeiro ou segundo ano de cultivo e plantas adultas podendo florescer em várias épocas num mesmo ano. Estas características tornam este mutante um excelente material para estudos de genômica funcional relacionado ao florescimento e a frutificação. Os objetivos deste trabalho foram caracterizar o florescimento da laranjeira ,,x11\" em quatro épocas do ano e acompanhar o desenvolvimento do meristema apical em gemas axilares de plantas adultas, em relação às plantas de ,,Valência\". Também foi avaliado o perfil de expressão dos genes envolvidos na indução e no florescimento em plantas adultas e juvenis das duas laranjeiras. Plantas adultas de ,,x11\" enxertadas em ,,Cravo\" e ,,Swingle\" foram podadas no outono, inverno, primavera e verão e, em seguida, realizou-se a caracterização do florescimento em ramos novos e a determinação da viabilidade e germinação in vitro de grãos de pólen. O acompanhamento morfo-anatômico do meristema apical foi realizado em quatro estádios das brotações axilares das duas laranjeiras após a poda de outono. A expressão dos genes integradores das vias de indução (FT, SOC1 e LFY), genes repressores (FLC, SVP e TFL1) e os genes de identidade do meristema floral (AP1, BAM e WUS) foram analisados por RT-PCR em três estádios de desenvolvimento das brotações de plantas adultas e juvenis. A caracterização fenotípica do florescimento em ,,x11\" demonstrou que as podas de primavera e de outono induziram a formação de ramos com flores terminais, sendo que no outono ocorreu a formação de ramos vegetativos. A poda de inverno resultou em ramos multiflorais e a poda de verão flores abortadas. O número de dias até a formação de botões florais variou entre 5 e 20 dias após a poda, com ramos medindo entre 18 a 24 cm e número médio de folhas variando entre 9 e 12. A viabilidade e germinação in vitro de grãos de pólen foram maiores após a poda de inverno. Pelas análises morfo-anatômicas foi possível observar a diferenciação de botão floral em laranjeira ,,x11\" quando as brotações apresentavam 13 mm de comprimento. A análise de transcritos das plantas adultas de ,,x11\" no estádio 1 indicou maiores níveis de expressão nos genes FT e TFL1 enquanto os genes BAM e LFY foram reprimidos em relação às plantas de ,,Valência\". No estádio 2, os genes FT, LFY e BAM apresentaram um maior número de transcritos, porém o gene TFL1 teve um baixo número de transcritos quando comparado com plantas de ,,Valência\". No estádio 3, uma elevada expressão relativa foi observada no gene LFY nas plantas adultas de ,,x11\" em relação à ,,Valência\". As plantas juvenis de ,,x11\" nos três estádios não apresentaram grandes alterações de expressão dos nove genes em relação às plantas juvenis de ,,Valência\". A exceção foi para o gene BAM, que apresentou maiores expressões nos estádios 1 e 3, mas no estádio 2, sofreu repressão em relação a \"Valência\". Palavras-chave: Citrus sinensis (L.) Osbeck. Florescimento precoce. Indução. Diferenciação floral. Genes do florescimento / The ,,x11\" plant is a spontaneous mutant of sweet orange, with seedlings flowering from the first or second year of the growing and adult plants can flower several times a year. These features make this mutant into an excellent material for functional genomics studies related to flowering and fruiting. The present work aimed to characterize the flowering of ,,x11\" sweet orange in four seasons and to follow the development of the apical meristem in axillary buds of adult plants, compared to ,,Valencia\" sweet orange. In addition, the expression profile of genes involved in the induction and flowering was evaluated in adult and juvenile plants of the both sweet oranges. Adult plants of ,,x11\" grafted on \'Rangpur\' and \'Swingle\' were pruned in fall, winter, spring and summer, and then, the characterization of the flowering in new branches and the viability and in vitro germination of pollen grains were evaluated. The following morpho-anatomical apical meristem was carried out in four stages of the axillary sprouts in both sweet oranges after fall pruning. Gene expression of floral pathway integrators (FT, SOC1, and LFY), repressor genes (FLC, TFL1 and SVP) and the genes of floral meristem identity (AP1, BAM and WUS) were analyzed by RT-PCR in three stages development of sprouting from juvenile and adult plants. Phenotypic characterization of flowering in ,,x11\" showed that the spring and fall pruning induced the formation of terminal branches with flowers, and the fall pruning also presented vegetative branches. The winter pruning resulted in multifloral branches and the summer pruning produced aborted flowers. The number of days up to the arising of flower buds ranged between 5 and 20 days after pruning, with branches measuring between 18 and 24 cm and number of leaves between 9 and 12. The viability and in vitro germination of pollen grains were higher after winter pruning. It was observed the differentiation of floral bud in ,,x11\" sweet orange by the morpho-anatomical analysis when the sprouting was 13 mm in length. The analysis of transcripts of the ,,x11\" adult plants in stage 1 showed higher levels of expression in FT and TFL1 genes while the BAM and LFY genes were repressed in relation to ,,Valencia\" plants. In stage 2, FT, LFY and BAM genes had a larger number of transcripts, but the TFL1 gene had a low number of transcripts compared with ,,Valencia\" plants. In stage 3, high expression was observed in LFY gene in ,,x11\" adult plants relation to the ,,Valencia\". Juvenile plants of ,,x11\"in the three stages showed no significant changes of expression of nine genes in relation to juvenile plants of ,,Valencia\". The exception was the BAM gene, which showed higher expression in stages 1 and 3, but in stage 2, had a repression when compared to ,,Valencia\"

A laranjeira ,,x11\" é um mutante espontâneo de laranja doce, com seedlings florescendo a partir do primeiro ou segundo ano de cultivo e plantas adultas podendo florescer em várias épocas num mesmo ano. Estas características tornam este mutante um excelente material para estudos de genômica funcional relacionado ao florescimento e a frutificação. Os objetivos deste trabalho foram caracterizar o florescimento da laranjeira ,,x11\" em quatro épocas do ano e acompanhar o desenvolvimento do meristema apical em gemas axilares de plantas adultas, em relação às plantas de ,,Valência\". Também foi avaliado o perfil de expressão dos genes envolvidos na indução e no florescimento em plantas adultas e juvenis das duas laranjeiras. Plantas adultas de ,,x11\" enxertadas em ,,Cravo\" e ,,Swingle\" foram podadas no outono, inverno, primavera e verão e, em seguida, realizou-se a caracterização do florescimento em ramos novos e a determinação da viabilidade e germinação in vitro de grãos de pólen. O acompanhamento morfo-anatômico do meristema apical foi realizado em quatro estádios das brotações axilares das duas laranjeiras após a poda de outono. A expressão dos genes integradores das vias de indução (FT, SOC1 e LFY), genes repressores (FLC, SVP e TFL1) e os genes de identidade do meristema floral (AP1, BAM e WUS) foram analisados por RT-PCR em três estádios de desenvolvimento das brotações de plantas adultas e juvenis. A caracterização fenotípica do florescimento em ,,x11\" demonstrou que as podas de primavera e de outono induziram a formação de ramos com flores terminais, sendo que no outono ocorreu a formação de ramos vegetativos. A poda de inverno resultou em ramos multiflorais e a poda de verão flores abortadas. O número de dias até a formação de botões florais variou entre 5 e 20 dias após a poda, com ramos medindo entre 18 a 24 cm e número médio de folhas variando entre 9 e 12. A viabilidade e germinação in vitro de grãos de pólen foram maiores após a poda de inverno. Pelas análises morfo-anatômicas foi possível observar a diferenciação de botão floral em laranjeira ,,x11\" quando as brotações apresentavam 13 mm de comprimento. A análise de transcritos das plantas adultas de ,,x11\" no estádio 1 indicou maiores níveis de expressão nos genes FT e TFL1 enquanto os genes BAM e LFY foram reprimidos em relação às plantas de ,,Valência\". No estádio 2, os genes FT, LFY e BAM apresentaram um maior número de transcritos, porém o gene TFL1 teve um baixo número de transcritos quando comparado com plantas de ,,Valência\". No estádio 3, uma elevada expressão relativa foi observada no gene LFY nas plantas adultas de ,,x11\" em relação à ,,Valência\". As plantas juvenis de ,,x11\" nos três estádios não apresentaram grandes alterações de expressão dos nove genes em relação às plantas juvenis de ,,Valência\". A exceção foi para o gene BAM, que apresentou maiores expressões nos estádios 1 e 3, mas no estádio 2, sofreu repressão em relação a \"Valência\". Palavras-chave: Citrus sinensis (L.) Osbeck. Florescimento precoce. Indução. Diferenciação floral. Genes do florescimento / The ,,x11\" plant is a spontaneous mutant of sweet orange, with seedlings flowering from the first or second year of the growing and adult plants can flower several times a year. These features make this mutant into an excellent material for functional genomics studies related to flowering and fruiting. The present work aimed to characterize the flowering of ,,x11\" sweet orange in four seasons and to follow the development of the apical meristem in axillary buds of adult plants, compared to ,,Valencia\" sweet orange. In addition, the expression profile of genes involved in the induction and flowering was evaluated in adult and juvenile plants of the both sweet oranges. Adult plants of ,,x11\" grafted on \'Rangpur\' and \'Swingle\' were pruned in fall, winter, spring and summer, and then, the characterization of the flowering in new branches and the viability and in vitro germination of pollen grains were evaluated. The following morpho-anatomical apical meristem was carried out in four stages of the axillary sprouts in both sweet oranges after fall pruning. Gene expression of floral pathway integrators (FT, SOC1, and LFY), repressor genes (FLC, TFL1 and SVP) and the genes of floral meristem identity (AP1, BAM and WUS) were analyzed by RT-PCR in three stages development of sprouting from juvenile and adult plants. Phenotypic characterization of flowering in ,,x11\" showed that the spring and fall pruning induced the formation of terminal branches with flowers, and the fall pruning also presented vegetative branches. The winter pruning resulted in multifloral branches and the summer pruning produced aborted flowers. The number of days up to the arising of flower buds ranged between 5 and 20 days after pruning, with branches measuring between 18 and 24 cm and number of leaves between 9 and 12. The viability and in vitro germination of pollen grains were higher after winter pruning. It was observed the differentiation of floral bud in ,,x11\" sweet orange by the morpho-anatomical analysis when the sprouting was 13 mm in length. The analysis of transcripts of the ,,x11\" adult plants in stage 1 showed higher levels of expression in FT and TFL1 genes while the BAM and LFY genes were repressed in relation to ,,Valencia\" plants. In stage 2, FT, LFY and BAM genes had a larger number of transcripts, but the TFL1 gene had a low number of transcripts compared with ,,Valencia\" plants. In stage 3, high expression was observed in LFY gene in ,,x11\" adult plants relation to the ,,Valencia\". Juvenile plants of ,,x11\"in the three stages showed no significant changes of expression of nine genes in relation to juvenile plants of ,,Valencia\". The exception was the BAM gene, which showed higher expression in stages 1 and 3, but in stage 2, had a repression when compared to ,,Valencia\"

La domestication du mil (Pennisetum glaucum), dans le Sahel, a engendré une large gamme de variétés, très diversifiées pour de nombreuses caractéristiques agronomiques. En particulier, la diversité de la durée du cycle des variétés locales de mil est une composante essentielle des stratégies mises en œuvre par les agriculteurs pour faire face aux fluctuations des précipitations et assurer une certaine stabilité de la production. Au cours des dernières décennies, des évolutions dans les pratiques agricoles ont été observées, en réponse à des changements écologiques et sociaux. Une des conséquences de ces évolutions pourrait être l’existence de flux de gènes entre variétés à cycle court et variétés à cycle long du fait de l’émergence de situations de parapatrie entre ces deux types de variétés, naguère isolées. Par ailleurs, l’existence de recouvrement des périodes des floraisons de ces deux types variétaux a déjà été préalablement observée. Une telle situation amène donc à s’interroger sur la dynamique évolutive passée et actuelle de la diversité de la longueur du cycle du mil dans le Sahel. Dans la première partie de ma thèse, j’ai évalué les possibilités d’occurrence de flux de gènes entre variétés précoces et tardives de mil dans le Sud-ouest du Niger, en utilisant une approche comparative entre situations contrastées pour la distribution spatiale de ces deux types de variétés. J’ai réalisé : 1) une étude des périodes de floraison de deux variétés de mil (précoce (Haïni Kiré) : 75 à 95 jours entre le semis et la maturité et tardive (Somno) : 105 à 125 jours de durée de cycle) dans plusieurs champs paysans, et dans deux villages. 2) une analyse moléculaire à l’aide de 15 marqueurs microsatellites qui a permis l’estimation des niveaux de différenciation génétique entre populations de mils précoces et tardifs échantillonnés dans 4 villages (incluant les deux villages déjà cités) de la même région.Les résultats ont montré la possibilité effective de flux de pollen et l’existence d’introgressions génétiques entre variétés précoces et tardives. Les mécanismes qui pourraient permettre un maintien sur le long terme d’une différenciation phénologique entre les deux types variétaux malgré l’existence de ces flux de gènes, sont discutés.Dans la deuxième partie, j’ai utilisé une approche « gène candidat » combinée à une démarche de génétique des populations, pour tenter d’identifier des gènes qui auraient pu contribuer à la diversité de la durée de cycle chez le mil. Je me suis focalisé sur trois gènes du contrôle de la transition florale PgHd3a, PgDwarf8 et PgPHYC. Leur implication dans la diversité de la durée de cycle chez plusieurs espèces a déjà été montrée. J’ai estimé les niveaux de différenciation génétique entre les mils domestiques et sauvages, précoces et tardifs pour ces trois gènes J'ai aussi cherché à mettre en évidence, au sein de ces gènes, les empreintes éventuelles d’évènements sélectifs passés. Afin de prendre en compte l’histoire démographique des mils dans les tests de neutralité sélective, j’ai utilisé les données de polymorphisme nucléotidiques de 8 séquences témoins dans le cadre d’une approche Bayésienne.Les résultats obtenus suggèrent fortement que PgHd3a et PgDwarf8 ont été ciblés par la sélection durant la domestication. Cependant, les données ne soutiennent pas l’hypothèse d’un rôle potentiel des trois gènes candidats dans la différenciation de la durée de cycle entre les variétés locales précoces et tardives. L’approc / Domestication of pearl millet (Pennisetum glaucum) in the Sahel of Africa has produced a wide range of diversity in cycle duration of landraces. This diversity allows Sahelian farmers to outface the precipitation fluctuation and to ensure regularity in grain production. Due to ecological and social recent changes, modifications of farmer’s practices could be a factor promoting gene flow between the early and late flowering varieties by increasing the opportunity of neighboring and flowering overlap between them. Such a situation raises questions about the past and current evolutionary dynamics of phenological diversity in this crop.In the first part of my thesis I tried to evaluate the possibility of gene flow between pearl millet varieties in South-West Niger, through a comparative approach among contrasting situations pertaining to the spatial distribution of early and late landraces. Therefore I conducted: 1) a field study where we observed flowering periods, for two types of varieties (early type (Haïni Kiré): 75 to 95 days and late type (Somno): 105 to 125 days of cycle length) in several pearl millet fields, and in two villages 2) a molecular study that allows the assessment of the level of genetic differentiation between late and early flowering populations sampled from four villages (including the two where the field study was conducted) of the same region (Dallol Bosso), using microsatellite markers. I was able to demonstrate the occurrence of pollen flow between the two types of landraces and I also showed evidence of genetic introgression between early and semi-late landraces. Potential mechanisms that would allow for the maintenance of the phenological differentiation between these two varieties and despite the gene flow are discussed.In the second part of this work I used a candidate gene and a population genetics approach, to try to identify genes that may have contributed to the cycle length diversity in pearl millet. I focused on three flowering candidate genes, PgHd3a, PgDwarf8 and PgPHYC which have been shown to be involved in the cycle length genetic diversity in several species, in order to estimate the differentiation between wild and domestic pearl millets and between early and late landraces, on the basis of theses candidate genes. I also tried to track for the fingerprint of eventual past selective events within these candidate genes. To be able to distinguish the effects of selection from the effect of demographic events that occurred during the domestication process, I used 8 neutral STS loci and an Approximate Bayesian Computation approach.My results strongly suggest that PgHd3a and PgDwarf8 were likely targeted by selection during domestication. However, a potential role of any of the three candidate genes in the phenological differentiation between early and late landraces was not supported by our data. The Bayesian approach confirmed the idea, suggested by many authors, that the gene flow from the wild to the domestic genetic pool has contributed significantly to the genetic diversity of the domestic pearl millet.