Paralelamente ao modelo Arabidopsis thaliana, o tomateiro (Lycopersicon esculentum) tem sido crescentemente utilizados em abordagens genéticas de questões fisiológicas. Uma das principais vantagens de Arabidopsis como “planta de laboratório” tem sido seu pequeno porte e ciclo de vida curto. Contudo, a cultivar Micro-Tom (MT) de tomateiro possui tamanho muito reduzido (8 cm) e pode produzir até 5 gerações por ano. Mutantes fotomorfogenéticos em tomateiro deficientes na síntese do cromóforo do fitocromo (au), mutantes deficientes na síntese das apoproteínas PHYA e PHYB1 (fri e tri, respectivamente) e mutantes superexpressando o fitocromo (hp, atv e Ip) constituem-se em um modelo para estudos da fotomorfogênese. No que se refere à capacidade de regeneração in vitro como uma resposta fotomorfogenética, poucos trabalhos têm sido realizados. O presente trabalho teve como objetivo transferir as mutações au, fri, tri, hp, Ip e atv, bem como o locus de regeneração (Rg1) da cultivar MsK, para a cultivar Micro-Tom. As linhagens obtidas foram utilizadas para verificar o efeito da fotomorfogênese na competência para regeneração in vitro. Para tanto, foram realizados tratamentos com luz branca, vermelho (V) e vermelho-extremo (VE) em explantes radiculares, caulinares e foliares do genótipo micro-MsK em meio MS mais 5mM de BAP e tratamentos com luz branca em explantes radiculares, caulinares e foliares de micro-mutantes fotomorfogenéticos também em meio MS mais 5mM de BAP. Para todos os tratamentos utilizou-se a cultivar MT como controle. Sob V, as raízes de micro-MsK apresentaram-se diferenciadas, enquanto sob VE não ocorreu diferenciação. O maior número de gemas formadas tanto para caule quanto para folhas de micro-MsK ocorreu sob V, enquanto sob VE foi observado um decréscimo na formação de gemas. A partir destes resultados sugere-se que a forma ativa do fitocromo, induzida pelo V, interage com o Rg1 na aquisição de competência para regeneração. Nos tratamentos com luz branca, raízes de micro-MsK e de mutantes micro-hp, micro-atv e micro-Ip apresentaram-se diferenciadas, enquanto não houve diferenciação para o mutante micro-au ou para o controle MT. O número de gemas formadas alcançou maiores valores para folhas de micro-hp e micro-Ip e a para caules de micro-atv. Apenas um número muito reduzido de gemas foi formado a partir de folhas de micro-au. Com base na alta competência para regeneração de micro-MsK e de mutantes que superexpressam o fitocromo, sugere-se que o fitocromo promove, em uma via de sinalização, a indução de fatores de regeneração (Rg1). Alternativamente, o locus Rg1 poderia promover a alta capacidade regenerativa tornando os explantes mais competentes ao efeito da superexpressão do fitocromo, o qual poderia induzir outros fatores de regeneração. / Parallel to Arabidopsis thaliana model, the tomato (Lycopersicon esculentum) has been increasingly used as a genetic approach to address physiological questions. One of the main advantages of Arabidopsis as a “laboratory plant” has been its small size and short life cycle. However, the tomato cultivar Micro-Tom (MT) possesses reduced size (8 cm) and can produce up to 5 generations per year. Tomato photomorphogenic mutants deficient for the synthesis of phytochrome chromophore (au) or the apoprotein PHYA and PHYB1 (fri and tri, respectively), as well as mutants superexpressing phytochrome (hp, atv and Ip) consist on a model to study photomorphogenesis. Concerning the in vitro regeneration capacity as a photomorphogenic response, fewer works have been carried through. The current work aimed at transfering the mutations au, fri, tri, hp, Ip and atv, as well as the regeneration locus (Rg1) of cv MsK to the cv Micro-Tom (MT). The genotypes obtained were used to verify the effect of photomorphogenesis on the competence for in vitro regeneration. Root, stem and leaf explants from MT and Micro-MsK were incubated in MS plus 5mM BAP under white, red (R) and far-red (FR) light. Root, stem and leaf explants from MT and photomorphogenic micro-mutants were incubated in MS plus 5mM BAP under white light. Under R, roots of micro-MsK were presented differentiation, while under FR the differentiation did not occur. Under R, stem explants from micro-MsK formed more shoots than did leaf explants, while under FR was observed a decrease in shoot formation for all types of explants. These results suggest that the active form of phytochrome, induced by R, interacts with the Rg1 in the acquisition of competence for regeneration. In the treatments with white light, roots of micro-MsK and of mutants micro-hp, micro-atv and micro-Ip presented differentiation, while no differentiation was observed for the mutant micron-au or control MT. The number of shoots formed reached the highest values for leaf explants of micro-hp and micro-Ip and for stem explants of micron -atv. Only a low number of shoots was formed from micro-au leaf explants. On the basis of the high competence for regeneration of micro-MsK and mutants that super express phytochrome, it is suggested that the phytochrome promotes, in a signaling pathway, the induction of regeneration factors ( Rg1 ). Alternatively, the Rg1 locus may turn the explant most competent to respond to phytochrome, which could induces others regeneration factors.

This research explores the capacity for functional photoreception in larvae of the mountain pine beetle (Dendroctonus ponderosae), an extremely important forest pest insect that is well adapted for development beneath the bark of pine trees. Phototaxis tests, gene expression analysis and development experiments were integrated to assess mountain pine beetle larvae for light sensitivity. When presented with a phototaxis choice test, larvae preferred dark over light microhabitats, revealing that larvae sense and respond behaviourally to light. Long wavelength opsin transcription was identified in all life stages, including eggs and larvae, suggesting that D. ponderosae possesses extraretinal photosensitive capabilities across its life cycle. The long wavelength opsin could function in phototaxis or the development phenology of immature beetles, while the ultraviolet opsin, only found to be expressed in pupae and adults, is likely to function in dispersal via the compound eyes. Results from two development experiments reveal an effect of photoperiod treatment on beetle development rate when reared from the egg stage, but not when reared from mature larvae, indicating that a critical photosensitive life stage(s) must occur in D. ponderosae prior to the third larval instar. An effect of photoperiod on adult emergence rates, however, appears to be independent of larval rearing conditions. The discovery of opsin expression and negative phototaxis in eyeless mountain pine beetle larvae, in addition to an effect of photoperiod on immature development and adult emergence rates, suggest that light and photoperiodism likely function in survival and life cycle coordination in this species. / Graduate / 2018-10-17

Abstract Adaptation to local environment is important for all organisms to guarantee survival and to maximize reproduction. Populations of the same species may live in environments that differ markedly. Due to differential selection pressures this can lead to population differentiation, which can be studied both at the phenotypic and at the gene level. The growing season cued by long days is typically short in the north, whereas southern populations have long growing seasons and are adapted to short days. Seasonal fluctuations in temperature also differ between northern and southern environments. Daylength and temperature regulate the timing of flowering in plants. Environmental regulation of flowering and its genetic basis has been extensively studied in the annual model species Arabidopsis thaliana. The perennial growth and flowering habit has been studied especially in trees, but studies on herbaceous plants species have been lacking. In this thesis, I have studied adaptation to growing season length in a perennial herbaceous model species Arabidopsis lyrata. Individuals from populations adapted to northern and southern environments in Europe were grown in same conditions in the growth chambers and in the field. Differentiation between the populations was studied by observing their flowering phenotypes and by studying the expression of genes that are candidates for governing the phenotypic differentiation. The main result in the thesis was that adaptation to short growing season in north can be seen as long daylength requirement for flowering and as fast developmental rate. Critical daylength for flowering likely regulates especially the timing of flowering cessation in the end of the growing season. Flowering time of individuals from northern populations also responded more strongly to cold treatment (representing winter) than that of the southern population. The cold requirement for flowering guarantees that the plants only flower after the winter in the spring with suitable conditions. Expression studies indicated that population differentiation in flowering could be at least partly governed by the expression variation in a few candidate genes. The results in this thesis are valuable for instance for understanding perennial species in general, including tree and crop species, and for predicting how plants response to changing climate. / Tiivistelmä Ympäröiviin oloihin sopeutuminen on tärkeää kaikille organismeille selviytymisen ja jälkeläistuoton kannalta. Saman lajin eri populaatiot saattavat elää ympäristöissä, joiden olosuhteet poikkeavat toisistaan huomattavasti. Tällöin populaatioihin kohdistuvat erilaiset valintapaineet ja populaatiot erilaistuvat. Erilaistuminen havaitaan tarkastelemalla yksilöiden ilmiasuja ja geenejä. Kasvukauden pituus määrittää eteläisten ja pohjoisten kasvuympäristöjen valintapaineita. Pohjoisessa kasvukausi on lyhyt ja sen alkamisesta ja loppumisesta kertoo pitkä päivänpituus. Etelässä on pitkä kasvukausi ja siellä elävät populaatiot ovat sopeutuneet lyhyeen päivänpituuteen. Myös vuodenaikaiset lämpötilavaihtelut eroavat pohjoisten ja eteläisten alueiden välillä. Muutokset päivänpituudessa ja lämpötilassa säätelevät kasveilla kukkimisen ajankohtaa. Kukkimiseen vaikuttavia ympäristötekijöitä ja kukkimista sääteleviä geenejä on tutkittu paljon yksivuotisella mallilajilla lituruoholla (Arabidopsis thaliana). Monivuotisten kasvien kasvun ja kukkimisen säätelyä on tutkittu etenkin puilla, mutta hyvin vähän ruohovartisilla kasveilla. Tässä väitöskirjatyössä tutkin kasvukauden pituuteen sopeutumista monivuotisella ruohovartisella lajilla, idänpitkäpalolla (Arabidopsis lyrata). Pohjoisiin ja eteläisiin ympäristöoloihin sopeutuneiden eurooppalaisten populaatioiden yksilöitä kasvatettiin samanlaisissa olosuhteissa kontrolloiduissa kasvatushuoneissa ja kenttäolosuhteissa. Populaatioiden erilaistumista tarkkailtiin kukkimiseen liittyvissä ominaisuuksissa sekä eroja selittävien kandidaattigeenien ekspressiossa. Tutkimuksen päätulos oli, että pohjoisen populaation lyhyeen kasvukauteen sopeutuminen voidaan havaita pitkän päivän vaatimuksena kukkimiselle ja nopeana kehityksenä. Kenttäolosuhteissa päivänpituus sääteli etenkin kukkimisen lopetusta kasvukauden lopussa. Pitkä kylmäkäsittely nopeutti kukkimista etenkin pohjoisessa populaatiossa. Kukkimista edeltävä kylmävaatimus takaa, etteivät kasvit kuki syksyllä epäsuotuisissa olosuhteissa, vaan vasta keväällä talven jälkeen. Populaatioiden erilaistuminen kukkimisen päivänpituus- ja kylmävaatimuksessa selittyy todennäköisesti ainakin osittain kandidaattigeenien ekspressioeroilla. Tämän väitöskirjatyön tuloksia voidaan soveltaa monivuotisten viljelykasvien jalostuksessa tai ennustettaessa kasvipopulaatioiden sopeutumista ilmastonmuutokseen.

In the present study, the ease of rearing, short generation time and molecular research tools available for the zebrafish model (Danio rerio, Hamilton) were exploited to investigate transcriptional regulation in relation to feeding, photoperiod and experimental selection. Chapter 2 describes transcriptional regulation in fast skeletal muscle following fasting and a single satiating meal of bloodworms. Changes in transcript abundance were investigated in relation to the food content in the gut. Using qPCR, the transcription patterns of 16 genes comprising the insulin-like growth factor (IGF) system were characterized, and differential regulation between some of the paralogues was recorded. For example, feeding was associated with upregulation of igf1a and igf2b at 3 and 6h after the single-meal was offered, respectively, whereas igf1b was not detected in skeletal muscle. On the other hand, fasting triggered the upregulation of the igf1 receptors and igfbp1a/b, the only binding proteins whose transcription was responsive to a single-satiating meal. In addition to the investigation of the IGF-axis, an agnostic approach was used to discover other genes involved in transcriptional response to nutritional status, by employing a whole-genome microarray containing 44K probes. This resulted in the discovery of 147 genes in skeletal muscle that were differentially expressed between fasting and satiation. Ubiquitin-ligases involved in proteasome-mediated protein degradation, and antiproliferative and pro-apoptotic genes were among the genes upregulated during fasting, whereas satiation resulted in an upregulation of genes involved in protein synthesis and folding, and a gene highly correlated with growth in mice and fish, the enzyme ornithine decarboxylase 1. Zebrafish exhibit circadian rhythms of breeding, locomotor activity and feeding that are controlled by molecular clock mechanisms in central and peripheral organs. In chapter 3 the transcription of 17 known clock genes was investigated in skeletal muscle in relation to the photoperiod and food content in the gut. The hypothesis that myogenic regulatory factors and components of the IGF-pathway were clock-controlled was also tested. Positive (clock1 and bmal1 paralogues) and negative oscillators (cry1a and per genes) showed a strong circadian pattern in skeletal muscle in anti-phase with each other. MyoD was not clock-controlled in zebrafish in contrast to findings in mice, whereas myf6 showed a circadian pattern of expression in phase with clock and bmal. Similarly, the expression of two IGF binding proteins (igfbp3 and 5b) was circadian and in phase with the positive oscillators clock and bmal. It was also found that some paralogues responded differently to photoperiod. For example, clock1a was 3-fold more responsive than clock1b. Cry1b did not show a circadian pattern of expression. These patterns of expression provide evidence that the molecular clock mechanisms in skeletal muscle are synchronized with the molecular clock in central pacemaker organs such as eyes and the pineal gland. Using the short generation time of zebrafish the effects of selective breeding for body size at age were investigated and are described in chapter 4. Three rounds of artificial selection for small (S-lineage) and large body size (L-lineage) resulted in zebrafish populations whose average standard length were, respectively, 2% lower and 10% higher than an unselected control lineage (U-lineage). Fish from the L-lineage showed an increased egg production and bigger egg size with more yolk, possibly contributing to the larger body size observed in the early larval stage (6dpf) of fish from this lineage. Fish from S- and L-lineage exposed to fasting and refeeding showed very similar feed intake, providing evidence that experimental selection did not cause significant changes in appetite control. Investigation of the expression of the IGF-axis and nutritionally-response in skeletal muscle after fasting and refeeding revealed that the pattern of expression was not different between the selected lineages, but that a differential responsiveness was observed in a limited number of genes, providing evidence that experimental selection might have changed the way fish allocate the energy acquired through feeding. For example, a constitutive higher expression of igf1a was recorded in skeletal muscle of fish from the L-lineage whereas igfbp1a/b transcripts were higher in muscle of fish from the S-lineage. These findings demonstrate the rapid changes in growth and transcriptional response in skeletal muscle of zebrafish after only three rounds of selection. Furthermore, it provides evidences that differences in growth during embryonic and larval stages might be related to higher levels of energy deposited during oogenesis, whereas differences in adult fish were better explained by changes in energy allocation instead of energy acquisition. In chapter 5 the main findings made during this study and their impact on the literature are discussed.

La résilience des agricultures subsahariennes face aux perturbations environnementales et socio-économiques repose en partie sur le maintien des diversités spécifique, variétale et génétique présentes au sein des agrosystèmes, mais aussi sur la préservation du droit pour les cultivateurs de reproduire et d'échanger librement les semences et les savoirs relatifs aux variétés. Pourtant, peu de données sont réellement disponibles sur la circulation effective des semences et les mécanismes modulant les flux de gènes et les introgressions génétiques entre variétés cultivées dans les agrosystèmes sahéliens. Chez le mil en particulier, il n’existe que des données très parcellaires sur les relations entre la diversité de la plante d’une part et la diversité socio-culturelle des agriculteurs d'autre part. Dans la première partie je me suis intéressé aux interactions entre l’organisation en groupes socio-culturels des agriculteurs et la structure génétique des populations de mil dans le bassin du lac Tchad. Les analyses de la diversité génétique suggèrent l’existence d’une barrière sociale à la diffusion des gènes sur l’ensemble de cette région. Néanmoins ces barrières ne sont pas suffisantes pour empêcher les introgressions génétiques entre populations cultivées par des agriculteurs appartenant à des groupes ethnolinguistiques différents. Dans le deuxième chapitre, je me suis intéressé au rôle des processus d’adaptation locale sur la circulation des gènes. Les résultats m’ont conduit à proposer l’hypothèse selon laquelle les flux de gènes entre types nommés précoces et tardifs sont plus importants dans les régions du nord où la pluviométrie est faible. Enfin dans le troisième chapitre j’ai quantifié la diversité biochimique des grains de mil de plusieurs variétés de mil, dont les usages culinaires varient selon les groupes ethnolinguistiques ou dont les qualités gustatives sont appréciées différemment. J’ai utilisé pour cela une approche combinée de protéomique et de métabolique. Parmi les 1072 spots protéiques quantifiés seulement 7 permettent de distinguer les types nommés photopériodiques des types nommés non ou peu photopériodiques. Les données de métabolomique suggèrent la présence de champignons, qui pourraient être des endophytes, dans certains des échantillons analysés. / The resilience of sub-Saharan farming systems to environmental and socioeconomic disturbances is partly based on the maintenance of agro-biodiversity, but also on preserving the right for farmers to reproduce and freely exchange seeds and related knowledge. However, few data are actually available on effective seed flow and on mechanisms modulating gene flow and genetic introgression between landraces grown in Sahelian farming systems. For pearl millet especially, there are only very few data on the relationship between diversity of this crop on the one hand and the socio-cultural diversity of farmers on the other hand. In the first part of my thesis, I was interested in assessing a potential relationship between ethnolinguistic diversity and population genetic structure of pearl millet in the Lake Chad Basin. Analysis of molecular polymorphisms suggests the existence of social barrier to seed flow among ethnolinguistic groups. However, these barriers are not sufficient to prevent genetic introgression between pearl millet populations cultivated by farmers belonging to different ethno-linguistic groups. In the second chapter, I focused on the role of local adaptation on gene flow. The results led me to propose the hypothesis that gene flow between early and late landraces are higher in the northern regions where rainfall is weak. Finally in the third part, I have quantified the biochemical compound of pearl millet seeds belonging to different varieties that show different uses or culinary preferences among farmers belonging to different ethnolingiuistic groups. I used a combined approach of proteomics and metabolics. Among the 1072 protein spots quantified only 7 distinguish the very photoperiodic non photoperiodic landrace. Metabolomics data suggest the presence of fungi, possibly endophytes, in some of the samples analyzed.

Cotton has been a world-wide economic staple in textiles and oil production. There has been a concerted effort for cotton improvement to increase yield and quality to compete with non-natural man-made fibers. Unfortunately, cultivated cotton has limited genetic diversity; therefore finding new marketable traits within cultivated cotton has reached a plateau. To alleviate this problem, traditional breeding programs have been attempting to incorporate practical traits from wild relatives into cultivated lines. This incorporation has presented a new problem: uncultivated cotton hampered by photoperiodism. Traditionally, due to differing floral times, wild and cultivated cotton species were unable to be bred together in many commercial production areas world-wide. This worldwide breeding problem has inhibited new trait incorporation. Before favorable traits from undomesticated cotton could be integrated into cultivated elite lines using marker-assisted selection breeding, the markers associated with photoperiod independence needed to be discovered. In order to increase information about this debilitating trait, we set out to identify informative markers associated with photoperiodism. This study was segmented into four areas. First, we reviewed the history of cotton to highlight current problems in production. Next, we explored cotton’s floral development through a study of floral transition candidate genes. The third area was an in-depth analysis of Phytochrome C (previously linked to photoperiod independence in other crops). In the final area of study, we used Genotype-By-Sequencing (GBS), in a segregating population, was used to determine photoperiod independence associated with single nucleotide polymorphisms (SNPs). In short, this research reported SNP differences in thirty-eight candidate gene homologs within the flowering time network, including photoreceptors, light dependent transcripts, circadian clock regulators, and floral integrators. Also, our research linked other discrete SNP differences, in addition to those contained within candidate genes, to photoperiodicity within cotton. In conclusion, the SNP markers that our study found may be used in future marker assisted selection (MAS) breeding schemas to incorporate desirable traits into elite lines without the introgression of photoperiod sensitivity.