[Truncated abstract] Narrow-leaf lupin (Lupinus angustifolius L.) was first recorded as having been introduced into Germany during the mid-19th century for use as green manuring and as fodder crops. However, it was not until post World-War I that there was any serious attempt to domesticate the species. Since that time several key domestication genes have been incorporated to enable the species to be grown as a crop over a range of climates, harvested as a bulk commodity and, the seed used for both animal and human consumption. However, the recent domestication of this species has seen a rather limited use of wild germplasm largely as a result of the difficulty in retaining these key domestication genes. To make the task of retaining these genes manageable, it was decided to resort to molecular technology. A mapping population of F8 derived recombinant inbred lines (RILs) has previously been established by the Department of Agriculture and Food, Western Australia, from a cross between a domesticated breeding line 83A:476 and a wild type P27255 in narrow-leaf lupin. The parents together with 89 RILs (of a population of 115) were subjected to DNA fingerprinting using microsatelliteanchored fragment length polymorphism (MFLP) to rapidly generate DNA markers for construction of a linkage map. Five hundred and twenty two unique markers of which 21% were co-dominant, were generated and mapped. Phenotypic data for the domestication traits: mollis (soft seeds), leucospermus (white flower and seed colour); Lentus (reduced pod-shattering), iucundis (low alkaloid), Ku (early flowering) and moustache pattern on seed coats; were included. Three to 7 molecular markers were identified within 5 cM of each of these domestication genes. The anthracnose resistance gene Lanr1 was also mapped. Linkage groups were constructed using MapManager version QTXb20, resulting in 21 linkage groups consisting of 8 or more markers. ... Five pairs of QTLs were found to be involved in epistasis, 2 of these having an effect on early vigour and another 3 influencing the time to opening of the first florets. Variation explained for each trait ranged from 28% for seed size, to 88% for days to flowering. We showed that it was possible to use this data to predict genotypes of superior progeny for these traits under Mediterranean conditions. QTL regions were compared on a second published linkage map and regions of conserved synteny with the model legume Medicago truncatula high-lighted. The work presented in this thesis demonstrates the importance of tight linkage between markers and genes of interest. It is especially important when dealing with genetically diverse material as found in the wild. One of the main problems faced by molecular scientists is the phenomenon known as linkage disequilibrium in marker populations caused by either small population size or 4 insufficient opportunity for recombination. This frequently results in the development of markers with little or no application outside of the population in which it was developed. Although the relatively small size of the population used in this study exposes it to such constraints, in this case excellent and valuable results were achieved in developing useful markers to at least 3 of the domestication traits within a relatively short time period of less then 4 years.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-11-04T19:45:06Z No. of bitstreams: 2 Dissertação - Edivaldo Barbosa de Almeida Júnior - 2012.pdf: 2762643 bytes, checksum: 0890998c45a55f15544debf5f715e8c4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-11-04T19:46:09Z (GMT) No. of bitstreams: 2 Dissertação - Edivaldo Barbosa de Almeida Júnior - 2012.pdf: 2762643 bytes, checksum: 0890998c45a55f15544debf5f715e8c4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-04T19:46:09Z (GMT). No. of bitstreams: 2 Dissertação - Edivaldo Barbosa de Almeida Júnior - 2012.pdf: 2762643 bytes, checksum: 0890998c45a55f15544debf5f715e8c4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-03-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The cagaiteira (Eugenia dysenterica DC)., is a native species of Cerrado. The plant is known for fruit production, which are used in natura or processed in several ways. It also provides food for the local fauna and, therefore, it conservation is important for maintenance for the communities. In order to maintain the productivity potential of the species, we should invest on plant breeding programs. To support these programs and help the species conservation, it is important to characterize the genetic variability available to breeders, both in germplasm collections and natural populations. This could also help to recommend priority areas to collect and conserve the germplasm. Neutral molecular markers have been used to evaluate the distribution of genetic variability in natural populations. The genetic structure of populations is the result of historical interaction between genetic drift, mutation, and gene flow. To detect the influence of adaptive processes in the genetic differentiation of populations we used 𝑄𝑆𝑇 index. The comparison of 𝑄𝑆𝑇 to the 𝐹𝑆𝑇, for neutral loci, provides values to test hypotheses about the role of natural selection. Therefore, the aim of this study was to characterize the germplasm collection of the cagaiteira from the EA/UFG. We used quantitative traits and microsatellite markers to make inferences about the role of natural selection in the differentiation of the cagaiteira subpopulations of Goiás, Southeast Brazil. Data collected from the quantitative traits were: plant height (AB), height of the first bifurcation (AB), the stem circumference (CC) and mean diameter of the crown projection (DC), leaf length (CL), leaf width (LL), leaf format (FF) and footstalk length (CP). Molecular data were obtained by amplification of eight microsatellite loci. We estimated the following quantitative genetic parameters: heritability and genetic variation coefficient, and the molecular parameters: gene diversity and allelic richness. We compared the probability distributions of the genetic structure parameters for both, quantitative and molecular data (𝑄𝑆𝑇 vs. 𝐹𝑆𝑇). From the quantitative genetic parameters we found modest responses to selection for the traits: AP, CC and DC; and significant responses for CL, LL, FF and CP. It was observed that the samples collected in natural populations are well represented in the germplasm collection, supported by molecular gene diversity. The traits AP, DC and DC are under convergent natural selection, and the traits CL, LL, FF and CP are under divergent natural selection into the cagaiteira subpopulations of Southeast Goiás. / A cagaiteira (Eugenia dysenterica DC.) é uma das espécies nativas do Cerrado que se destaca pela produção de frutos, que são utilizados in natura ou processados de várias formas. Os frutos também fornecem alimento para a fauna local e, portanto, a sua conservação é importante para manutenção das comunidades. A utilização do potencial produtivo da espécie, no entanto, depende de programas de domesticação que visem o incremento da produção. Para subsidiar esses programas e visando também a conservação é necessária a caracterização da variabilidade genética disponível para o melhorista, nas coleções de germoplasma e nas populações naturais, com o objetivo de recomendar áreas prioritárias para coleta ou conservação in situ do germoplasma. A distribuição da variabilidade genética nas populações naturais tem sido avaliada por meio de marcadores moleculares neutros. Nesse caso, a estrutura genética das populações é o resultado da interação histórica entre a deriva genética e a mutação, com o fluxo gênico. Para detectar a influência de processos adaptativos na diferenciação genética das populações tem sido utilizado um índice, o 𝑄𝑆𝑇. Quando a estimativa do 𝑄𝑆𝑇 é comparada com a do 𝐹𝑆𝑇 , para locos neutros, é possível testar hipóteses quanto à atuação da seleção natural. Assim, o objetivo do trabalho foi caracterizar a coleção de germoplasma de cagaiteira da EA/UFG, a partir de caracteres quantitativos e com marcadores microssatélites, para fazer inferências quanto à atuação da seleção natural na diferenciação das subpopulações de cagaiteira do sudeste do Estado de Goiás, Brasil. Foram coletados dados referentes aos caracteres: altura da planta (AP), altura da primeira bifurcação (AB), circunferência do caule (CC), diâmetro médio de projeção da copa (DC), comprimento do limbo foliar (CL), largura do limbo (LL), formato das folhas (FF) e comprimento do pecíolo (CP). Os dados moleculares foram obtidos pela amplificação de oito locos microssatélites. Foram estimados os parâmetros genéticos quantitativos: herdabilidade e coeficiente de variação genética; e moleculares: diversidade genética e riqueza alélica. Foram realizados os testes de comparação entre as distribuições de probabilidade dos parâmetros de estrutura genética para dados quantitativos e moleculares (𝑄𝑆𝑇 vs 𝐹𝑆𝑇), através de 1000 bootstrap. A partir dos parâmetros genéticos quantitativos é possível esperar respostas modestas para a seleção dos caracteres: AP, CC e DC e respostas expressivas para: CL, LL, FF e CP. Foi observado que a coleção de germoplasma de cagaiteira da EA/UFG representa bem as coletas realizadas nas populações naturais da espécie, com base na diversidade genética estimada a partir de marcadores microssatélites. Os caracteres: AP, CC e DC estão sob seleção natural convergente e as variáveis das folhas: CL, LL, FF e CP estão sob seleção divergente nas subpopulações de cagaiteira do sudeste do Estado de Goiás.

A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...) / At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role.

A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...) / At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role.

A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...) / At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role.

Made available in DSpace on 2015-03-26T13:42:19Z (GMT). No. of bitstreams: 1 texto completo.pdf: 525415 bytes, checksum: 988d62e0043ef473ad492ea9ce941239 (MD5) Previous issue date: 2006-09-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / In the genetic mapping in outbred populations not always it is possible to determine the linkage phase of the alleles. Thus, heterozygous individuals are discarded from these analyses due to the lack of information, once it is not possible, through their genotype, to distinguish the origin of their parental alleles. In this way, the main objective of this work was to propose the endosperm genotyping as a strategy to identify the allelic origin of those heterozygotes individuals. Initially, fragments from the endosperm representing 10, 25 and 50% of the corn seeds weight were extracted and the seeds were submitted to the germination test. The results suggest that the elimination of up to 50% of the endosperm did not affected the seed germination. The methodology of semiquantitative PCR was optimized to differentiate doses of the alleles in the mixtures of DNA derived from leaves of two maize inbred lines (L3 and L1113- 01). It was represented different allelic proportions observed in the endosperm of their reciprocal crosses, based on the maximum amount of endosperm that could be used for DNA extraction. SSR markers were generated by semiquantitative PCR technique and the amplified fragments were evaluated in both agarose gels treated with ethidium bromide and poliacrylamide gels using fluorescently labeled primers. Gel resolution using agarose did not allow the differentiation of the mixtures of parental DNAs. However, through the regression analysis and comparison of the band intensity corresponding to the same allele in the different mixtures, the initial concentration of each one of the alleles could be inferred. The requirement of an allelic pattern limited the use of this technique to QTL analysis in populations where at least one of the genitors is known. Although the resolution of poliacrylamide gels using fluorescent markers was more efficient in the endosperm genotyping, once it was allowed to differentiate the maternal origin of reciprocal hybrids seed´s. So, the strategy of endosperm genotyping using fluorescent SSR primer amplified by semiquantitative PCR allowed the determination of allelic origin in the heterozygous offspring derived from outbred populations, including these individuals in the QTL detection, and consequently, increasing the precision of this analysis. / No mapeamento genético e detecção de QTLs em populações exogâmicas nem sempre é possível a determinação da fase de ligação dos alelos. Assim, indivíduos heterozigotos são descartados dessas análises por serem não informativos, uma vez que não é possível, por meio do seu genótipo, distinguir a origem de seus alelos em relação aos dois genitores. Dessa forma, o objetivo principal deste trabalho foi propor a genotipagem do endosperma para identificar a origem alélica dos indivíduos heterozigotos. Inicialmente, fragmentos do endosperma representando 10, 25 e 50% do peso das sementes de milho foram retirados, sendo as sementes submetidas ao teste de germinação. Observou-se que a remoção de até 50% do endosperma não afetou a taxa de germinação das sementes. A metodologia de PCR semiquantitativo foi otimizada para diferenciar doses dos alelos nas misturas de DNA foliar de duas linhagens de milho (L3 e L1113-01), representando as diferentes proporções alélicas observadas no tecido endospermático dos seus cruzamentos recíprocos, tendo como base a quantidade máxima de endosperma que podia ser utilizada na extração do DNA. Marcadores SSR foram gerados pela técnica de PCR semiquantitativo, e os fragmentos amplificados foram avaliados tanto em gel de agarose tratado com brometo de etídio quanto em gel de poliacrilamida, usando-se primers fluorescentes. A resolução do gel de agarose não possibilitou a diferenciação das misturas dos DNAs parentais. No entanto, por meio da análise de regressão e da comparação da intensidade da banda correspondente a um mesmo alelo nas diferentes misturas, pôde-se inferir a concentração inicial de cada um dos alelos. A necessidade de um padrão de alelos limitou o uso dessa técnica nas análises de QTLs em populações nas quais pelo menos um dos genitores é conhecido. Já a resolução do gel de poliacrilamida utilizando marcadores fluorescentes foi mais eficiente na genotipagem de endospermas, uma vez que possibilitou a diferenciação da origem materna das sementes dos híbridos recíprocos. Assim, a estratégia de genotipagem do endosperma utilizando primers SSR fluorescentes amplificados pela técnica de PCR semiquantitativo possibilitou a determinação da origem dos alelos dos descendentes heterozigotos derivados de populações exogâmicas, permitindo a inclusão destes na detecção de QTLs e, conseqüentemente, aumentando a precisão das análises.

Made available in DSpace on 2015-03-26T13:42:35Z (GMT). No. of bitstreams: 1 texto completo.pdf: 566480 bytes, checksum: ec7963fd634f205e34b49003fe8588c0 (MD5) Previous issue date: 2007-03-06 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The genomic era came to transform science as a whole. The intention of this work was to evaluate different significance levels on the identification of the molecular markers, related with its quantitative characteristics of low, medium and high heritability (h²), aiming genetic improvement. A comparison between the significance levels of 1%, 5%, 10% and 20% was accomplished through a computer system of genetic simulation (Genesys), used for the simulation of three genomes, where each was constituted by only one low, medium or high h² characteristic, respectively. First from each of these genomes, was simulated a base population of 1000 animals (500 males and 500 females). Next, it was obtained an initial population for each one of the base population, totalizing three initial populations of 1000 animals each (10 males, 10 female/males and 10 son/female/males). After obtaining this population, was initiated the evaluation process of the four significance levels considered, using the selection assisted by markers (MAS), where the markers were identified by a linear regression analysis, admitting a minimum significance level for it. A total of four selection processes based on the initial population, according to the significance level adopted, was conducted through 20 consecutive generation, repeating each one 10 times to minimize the genetic oscillation effects. The analysis of the four significance level s differences was performed individually for each of the three characters, through a medium phenotype gain, looking forward to, besides checking the existence of gain distinction, if this difference will also appear simultaneously in the low, medium and high heritability characteristics. To complement and better explain the phenotype results obtained, other genetic parameters were considered, such as the endogamy coefficient, the number of markers used in the selection, the number of set alleles, the favorable and adverse allele setting and the selection s limit. The results obtained, indicated superiority in the significance level of higher magnitude ( 10% and 20%) related to the lower values ones (1% and 5%), and the phenotypic gains obtained after the assisted selection by the molecular marker, besides its benefits on the genetic parameter (lower endogamy coefficient; higher number of markers used, lower markers setting, lower percentage of adverse alleles set, and lower selection limits) for all three characteristics, although in an more expressive way for the low heritability character, and less for the high h². That way, even if the lower levels (1% and 5%) present a higher precision on the markers detection - QTLs ( quantitative trait loci), they are not satisfying enough related to the genetic benefits, resulting in a lower phenotypic and genotypic gain, making the choice of a certain statistic significance acceptable according to its aim and to the interdisciplinary research in question. It is notorious the relevance of statistics association on the identification of markers related to the QTLs and, consequently on the optimization of genomic improvement-programs. / A era genômica veio para transformar a ciência como um todo. Objetivou-se com esse trabalho avaliar diferentes níveis de significância na identificação de marcadores moleculares relacionados com características quantitativas de baixa, média e alta herdabilidade (h2) de interesse no melhoramento genético. Uma comparação entre os níveis de significância de 1%, 5%, 10% e 20% foi realizada por meio de um sistema computacional de simulação genética (Genesys), utilizado para a simulação de três genomas, onde cada qual era constituído de uma única característica de baixa, média ou alta h2, respectivamente. A partir de cada um dos genomas, simulou-se uma população base de 1000 animais (500 machos e 500 fêmeas). Em seguida, foi obtida uma população inicial para cada uma das populações base, totalizando três populações iniciais com 1000 animais cada (10 machos, 10 fêmeas/macho e 10 filhos/fêmea/macho). Após a obtenção desta população, iniciou-se o processo de avaliação dos quatro níveis de significância considerados, utilizando a seleção assistida por marcadores (MAS), onde os marcadores eram identificados por uma análise de regressão linear admitindo-se um nível mínimo de significância para a análise. Um total de quatro processos de seleção a partir da população inicial, de acordo com a significância adotada, foi conduzido por 20 gerações consecutivas, repetindo-se cada qual por 10 vezes para minimizar os efeitos da oscilação genética. A análise da diferença dos quatro níveis de significância foi realizada individualmente para cada um dos três caracteres através do ganho fenotípico médio, buscando, além de verificar a existência de distinção nos ganhos, se esta diferença também se apresenta simultaneamente nas características de baixa, média e alta herdabilidade. Para complementar e melhor esclarecer os resultados fenotípicos obtidos, outros parâmetros genéticos foram considerados, tais como o coeficiente de endogamia, o número de marcadores usados na seleção, o número de marcadores fixados, a fixação de alelos favoráveis e desfavoráveis e o limite da seleção. Os resultados observados indicaram superioridade dos níveis de significância de maior magnitude (10% e 20%) em relação aos de menores valores (1% e 5%), quanto aos ganhos fenotípicos obtidos após a seleção assistida por marcadores moleculares além de benefícios nos parâmetros genéticos (menor coeficiente endogâmico; maior número de marcadores utilizados; menor fixação de marcadores; menor porcentagem de alelos desfavoráveis fixados; e menores limites de seleção) para todas as três características, embora de forma mais expressiva para o caráter de baixa herdabilidade e menos para o de alta h2. Assim, mesmo que os níveis menores (1% e 5%) apresentem uma maior precisão na detecção de marcadores - QTLs (quantitative trait loci), eles deixam a desejar no que diz respeito às melhorias genéticas, resultando em ganhos fenotípicos e genéticos inferiores, fazendo com que a escolha de uma determinada significância estatística seja aceitável de acordo com o objetivo e a interdisciplinaridade da pesquisa em questão. É notório a relevância de associações estatísticas na identificação de marcadores ligados a QTLs e, consequentemente, na otimização de programas de melhoramento genômico.

Abstract Local adaptation is important evolutionary process leading to adaptive population differentiation. Currently, examining its genetic basis is a major goal of evolutionary and ecological genetics. In my thesis I studied local adaptation and its genetic basis in populations of a perennial outcrossing model plant Arabidopsis lyrata by combining common garden experiments at the native field sites and in controlled conditions with quantitative trait locus mapping. Estimates of fitness in the field – both at the level of multiple components as well as hierarchical total fitness – showed that populations of A. lyrata were locally adapted. The studied populations were also phenotypically differentiated in ecologically relevant traits. Different components of fitness were important for the advantage of the locals depending on the environment. Local alleles were associated with high fitness in the field, suggesting that differing directional selection pressures have been involved in phenotypic differentiation. Mostly different genomic regions governed local adaptation in different environments, but the results also suggested that some of these regions could involve rarely documented fitness tradeoffs (antagonistic pleiotropy). Loci governing flowering time differentiation differed between the studied environments, highlighting the need to conduct experiments both in the wild and in controlled conditions. In contrast to most existing studies, F2 hybrids in general had surprisingly high fitness at one study site, largely due to beneficial dominance effects at loci governing survival in that environment. In addition to nuclear genes, cytoplasmic genomes also were found to have a role in local adaptation. / Tiivistelmä Luonnonvalinta saa aikaan paikallista sopeutumista ja adaptiivista erilaistumista. Paikallisen sopeutumisen perinnöllisen taustan selvittäminen on tällä hetkellä yksi tärkeimpiä evolutiivisen ja ekologisen genetiikan tavoitteita. Tässä väitöskirjatyössä tutkin paikallista sopeutumista ja sen geneettistä taustaa monivuotisella, ristipölytteisellä mallikasvilla, idänpitkäpalolla (Arabidopsis lyrata). Käytin työssäni geenikartoitusta kasveilla joita kasvatettiin yhdenmukaisissa oloissa sekä populaatioiden luontaisilla kasvupaikoilla että kontrolloiduissa olosuhteissa. Kenttäolosuhteissa arvioitu kelpoisuus osoitti idänpitkäpalkopopulaatioiden olevan paikallisesti sopeutuneita sekä yksittäisten kelpoisuuteen vaikuttavien ominaisuuksien että hierarkkisen kokonaiskelpoisuuden tasolla. Tutkitut populaatiot olivat myös erilaistuneita ekologisesti tärkeissä ominaisuuksissa. Kelpoisuuteen vaikuttavat ominaisuudet myös poikkesivat ympäristöjen välillä. Paikalliset alleelit olivat yhteydessä korkeaan kelpoisuuteen luonnossa, minkä perusteella voitiin päätellä erisuuntaisen luonnonvalinnan vaikuttaneen populaatioden erilaistumiseen. Kromosomiston eri alueet olivat tärkeitä sopeutumisessa eri ympäristöihin, mutta myös joidenkin samojen genomin alueiden havaittiin mahdollisesti vaikuttavan vastakkaisesti kelpoisuuteen eri ympäristöissä. Myös kukkimisajan erilaistumiseen vaikuttavat genomin alueet poikkesivat eri ympäristöjen välillä erityisesti verrattaessa kenttäkokeita kasvatushuone- ja kasvihuonekokeisiin. Toisin kuin useimmissa tutkimuksissa on havaittu, F2-sukupolven jälkeläistön kelpoisuus oli yllättävän korkea yhdessä kenttäkoeympäristössä. Tähän vaikuttivat kelpoisuuden kannalta suotuisat dominoivat geenivaikutukset, jotka paransivat kasvien selviytymistä kyseisessä ympäristössä. Tumassa sijaitsevien geenien lisäksi myös soluelimien perimällä havaittiin olevan yhteys paikalliseen sopeutumiseen.

Knowledge of the genetic variation of crop collections is essential for their efficient use in plant breeding programs. The Ethiopian Highland Maize Germplasm Collection Mission was launched throughout the highlands of Ethiopia in 1998 and 287 traditional maize accessions were collected from farmers’ fields. To date, no information was available on the morphological and genetic diversity in this important collection. Various molecular marker techniques and quantitative genetics approaches were applied to accurately unravel the extent of phenotypic and genetic diversity, to study patterns of morphological and molecular variation and to determine association of molecular markers with quantitative trait variation, with the view of designing a sound breeding program and management strategy for maize in the highlands of Ethiopia. The morphological study confirmed that traditional Ethiopian highland maize accessions contain large amounts of variation for agro-morphological traits. The broad trait diversity observed among the accessions suggested ample opportunities for the genetic improvement of the crop through selection directly from the accessions and/ or the development of inbred lines for a future hybrid program. Selection practices followed by local farmers are mostly consistent within agroecology and gave rise to morphologically distinct maize accessions in different agroecologies. This underscores the importance of considering farmers’ knowledge of diversity in the collection and evaluation of local accessions. The results of amplified fragment length polymorphism (AFLP) and microsatellite or simple sequence repeat (SSR) marker analyses showed that bulking leaf samples from 15 individual plants per out-bred accession is an effective means of producing representative profiles of individual plants, thereby reducing the cost of DNA extraction and subsequent marker analysis of open-pollinated varieties. Cluster analyses based on AFLP and SSR data showed that most of the accessions collected from the Northern agroecology were genetically distinct from the Western and Southern accessions suggesting that differentiation for adaptive traits for drought conditions may have occurred in the Northern accessions. However, there was very little genetic differentiation between the Western and Southern accessions suggesting gene flow between the two agroecologies and recent introduction of similar improved varieties in these agroecoogies . In both marker systems, high mean genetic diversity was observed among the traditional Ethiopian highland maize accessions. This is possibly due to (i) the continuous introduction of maize from abroad by different organizations; (ii) genetic variation generated through farmers management practices; and (iii) the presence of different environmental conditions in the highlands of Ethiopia to which local landraces may have been adapted. The correlation between the morphological dissimilarity matrix and the matrices of genetic dissimilarity based on SSR and AFLP markers were 0.43 and 0.39, respectively (p = 0.001 in both cases). The correlation between SSR and AFLP dissimilarity matrices was 0.67 (p = 0.001). These significant correlations indicate that the three independent sets of data likely reflect the same pattern of genetic diversity, and validate the use of the data to calculate the different diversity statistics for Ethiopian highland maize accessions. From this study, three groups of maize accessions with distinctive genetic profiles and morphological traits were identified that will be useful for future collection, conservation and breeding programs of maize for the highlands of Ethiopia. A pilot association study using SSR markers and quantitative trait variation indicated that molecular markers could be useful to identify genetic factors controlling earliness, tallness, grain yield and associated traits, which could be exploited by various breeding schemes. The analytical tools outlined in this dissertation can be a useful tool in managing genetic variation of open-pollinated crops and will aid in the conservation of unique genetic diversity. Production stability and global food security are linked to the conservation and exploitation of worldwide genetic resources and this research attempts to add to that body of knowledge. Copyright 2005, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Beyene, YA 2005, Genetic analysis of traditional Ethiopian Highland Maize (Zea Mays l.) using molecular markers and morphological traits : implication for breeding and conservation, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02212006-112610 / > / Thesis (PhD (Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

<p>Domestication has led to genetic changes that affect quantitative traits in farm animals. Both candidate gene analysis using association tests and genome scans based on linkage analysis have been performed to understand the molecular basis underlying quantitative genetic variation in horses, pigs and chickens. To test a possible association of polymorphisms in the <i>PRKAG3</i> gene, previously found to be associated with excess glycogen content in pig skeletal muscle, with quantitative traits in the horse, the major coding part of the equine <i>PRKAG3</i> sequence was identified. Bioinformatic characterization of the equine <i>PRKAG3</i> gene was conducted. A single nucleotide polymorphism (SNP) causing a missense mutation (Pro258Leu) was found. Screening this SNP showed that the Leu258 allele was more frequent in breeds with heavy muscularity. To assess previously reported associations between polymorphisms in the <i>MC4R</i> gene and obesity-related traits further, we conducted linkage analysis between the <i>MC4R</i> locus and fatness-related traits using a Wild BoarxLarge White intercross. No significant association between segregation at the <i>MC4R</i> locus and fatness was detected in this pedigree. A genome scan of quantitative trait loci (QTLs) has been performed in an intercross between chicken lines divergently selected for growth. Divergent parental lines have been established by selecting for high and low 56-day body weight for over 40 generations. The selection has led to approximately a 9-fold difference in 56-day body weight between lines and resulted in correlated responses for a number of traits including appetite, immune response, body composition and metabolic traits. Phenotypic data on growth and other correlated traits were collected from more than 800 F2 individuals. Genome scans using 145 markers on 26 linkage groups have identified QTLs affecting growth and correlated responses to selection for 56-day body weight. No major QTL explaining a large portion of phenotypic variation in growth was revealed in this study. </p>