Viticulture is a multibillion-dollar market based on grapes, one of the most valuable fruits in agriculture, but it is also characterized by the highest uses of fungicides per hectare for disease management. In fact, despite the availability of genetic resources resistant to the most diffuse fungal pathogens, those cover less than a fifth of the worldwide vine area. The remaining 80% is represented mainly by susceptible Vitis vinifera L. varieties. Instead, re-sistance traits are known to derive from other Vitis species, due to their coevolution with the pathogens, and their exploitation in breeding is a valuable strategy to reduce pesticide treat-ments. Black rot (BR) is a destructive disease caused by the ascomycete, Phyllosticta ampeli-cida, whose telomorphic form was known as Guignardia bidwellii. In the European continental area in the last two decades its pressure increased due to the concomitant decreased usage of chemicals and the occurrence of mild-rainy summer because of the climate change. This work was established to fight the spread of BR by means of the introduction of BR resistance in susceptible genetic background. The study consisted of (i) the reconstruction of BR resistant donor pedigree based on the collection of historical phenotypic data, (ii) the eval-uation of parental lines and selections of the breeding program of the Edmund Mach Founda-tion (Italy), (iii) the microscopic inspection of disease progression, and (iv) QTL analysis of the segregating population (N=146) ‘Merzling’ (hybrid, resistant) × ‘Teroldego’ (V. vinifera, suscep-tible). The outcomes revealed a large unexplored pool of disease resistance donors of 148 va-rieties belonging to 14 different species, that permitted to evaluate and identify five new promising genotypes readily exploited for breeding. Concurrently, a new inoculation method based on spore production from fresh infected leaves was developed. Ex vivo (detached leaves) inoculation did not reveal significant differences among the assessed genotypes and highlight-ed the tendency of spores to accumulate and germinate near leaf hairs, while disease progres-sion did not occur. The screening of the segregating population, both under greenhouse and field conditions, allowed the discrimination on chromosome 14 between two distinct QTLs as-sociated with leaf/shoot and bunch resistance. The first QTL confirmed the Resistance to Gui-gnardia bidwellii (Rgb)1 locus previously identified by three studies and the high-density link-age mapping allowed to reduce it from 2.4 to 0.7 Mb along the PN40024.v4 reference genome. The region resulted enriched in genes belonging to phloem dynamics and mitochondrial pro-ton transfer. The second QTL associated with bunch resistance was designated Rgb3. Located at 9 cM (6 Mb) upstream Rgb1, it was characterized by a cluster of Germin-like protein 3 genes and lipid transfer and localization, notably known to be linked to broad spectrum disease re-sistance and stress response. No resistance (R)-genes have been annotated in the region under-lying the QTL in the PN40024 reference genome. In conclusion, this work provided new insights for grapevine breeding programs by the identification of previously unknown BR resistance donors, the development of protocols and good practices towards large-scale resistance screening as well as the discovery of a novel QTL associated with bunch resistance. Marker validation in different genetic backgrounds is ongoing for its routinary implementation in marker-assisted breeding and further studies are planned to dissect BR resistance mechanism by the sequencing of the genomic regions, the study of the Germin-like 3 gene cluster and the cell wall characterization.

No description available.

No description available.

El melón (Cucumis melo) y el calabacín (Cucurbita pepo) son especies cucurbitáceas de gran importancia económica a nivel nacional y mundial. Para optimizar su producción se requiere de la obtención de nuevas variedades mejor adaptadas a los sistemas de cultivo, más resistentes frente a nuevas enfermedades o plagas y con mejores características organolépticas, que respondan a las cada vez mayores exigencias del mercado. La mejora debe realizarse de una forma eficiente y competitiva, apoyándose en los crecientes conocimientos genéticos en estas dos especies y en los últimos avances biotecnológicos. El desarrollo de herramientas genómicas con el fin de impulsar la mejora de estos cultivos es el principal objetivo de la presente Tesis doctoral. El desarrollo de marcadores moleculares es esencial para la construcción de mapas genéticos, para la realización de una selección más eficiente, para el análisis y cartografía de QTLs (Quantitative trait loci) y para el desarrollo de líneas de premejora, además de ser una herramienta fundamental para el análisis de la biodiversidad. En esta Tesis se han desarrollo y/o validado marcadores de alta calidad, de tipo microsatélite (Simple Sequence Repeats, SSRs) y SNPs (Single Nucleotide Polymorphisms), para estas dos especies. La generación de información de secuencia, necesaria para el desarrollo de este tipo de marcadores, ha cambiado en el transcurso de los trabajos presentados en la Tesis, habiéndose abordado finalmente la secuenciación del transcriptoma de melón mediante técnicas de secuenciación de alto rendimiento (NGS, Next generation sequencing). La obtención de grandes colecciones de SSRs y SNPs en ambas especies, resultado del ensamblaje de ESTs (Expressed Sequence Tags) procedentes de secuencias Sanger previamente disponibles y de las nuevas secuencias obtenidas por secuenciación masiva, ha supuesto un gran avance para estas especies no modelo, permitiendo la construcción de mapas más densos en melón y del primer mapa ba / Esteras Gómez, C. (2012). Desarrollo y aplicación de herramientas genómicas para la mejora de especies cucurbitáceas por calidad y resistencia a enfermedades [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/17046

The aim of the current thesis was the analysis of the genetics of sex determination of farmed fish with sexual dimorphism, using Next Generation Sequencing. Three different species of farmed fish with sex-determining systems of varying complexity were studied. Both full-sibs and more distantly related specimens of Atlantic halibut (Hippoglossus hippoglossus), Nile tilapia (Oreochromis niloticus) and European sea bass (Dicentrarchus labrax) were used for this study. Application of Restriction-site Associated DNA sequencing (RAD-seq) and double digest Restriction-site Associated DNA sequencing (ddRAD-seq), two related techniques based on next generation sequencing, allowed the identification of thousands of Single Nucleotide Polymorphisms (SNPs; > 3,000) for each of the above species. The first SNP-based genetic maps for the above species were constructed during the current study. The first evidence concerning the location of the sex-determining region of Atlantic halibut is provided in this study. In the case of Nile tilapia both novel sex-determining regions and fine mapping of the major sex-determining region are presented. In the study of European sea bass evidence concerning the absence of a major sex-determining gene was provided. Indications of putative sex-determining regions in this species are also provided. The results of the current thesis help to broaden current knowledge concerning sex determination in three important farmed fish. In addition the results of the current thesis have practical applications as well, towards the production of mono-sex stocks of those species for the aquaculture industry.

Objectifs: Plusieurs polymorphismes nucléaires localisés sur les gènes des récepteurs nicotiniques cholinergiques CHRN sont associés au tabagisme. Cependant, peu d’études ont examiné l’association entre les polymorphismes sur les gènes CHRN et l’étourdissement. Les polymorphismes et les symptômes subjectifs sont peu être lié à la dépendance à la nicotine et à l’étourdissement ressenti lors de l’initiation. Le but de cette étude est d’étudier l’association entre 61 polymorphismes sur huit gènes CHRN (CHRNA3 CHRNA4 CHRNA5, CHRNA6, CHRNA7, CHRNB2, CHRNB3, CHRNB4) et l’étourdissement ressenti lors de l’initiation. Méthodes: Les données provenant d'une étude de cohorte longitudinale composée de 1293 étudiants, ont été analysées selon un devis d'étude gène-candidat. Les données ont été collectées par le biais de questionnaires auto-reportés aux troix mois, durant 5 ans. L’ADN provenent de la salive ou du sang a été génotypé pour 61 polymosphismes localisés sur les gènes CHRN ont été génotypés, à l'aide d'une stratégie de couverture maximale du gène. L'équation d'analyse est une régression logistique, incluant un ajustement sur l’âge, le sexe et l’origine ethnique. Résultats: Trois SNPs sur le gène CHRNA6 (rs7812298, rs2304297, rs7828365) sont associés à notre phénotype (OR (95% CI)= 0.54 (0.36, 0.81), 0.59 (0.40, 0.86) and 0.58 (0.36, 0.95, respectivement),. Trois autres polymorphismes (rs3743077 (CHRNA3), rs755204 (CHRNA4), rs7178176 (CHRNA7)) sont également associés à phénotype (OR (95% CI)=1.40 (1.02, 1.90), 1.85 (1.05, 3.27) and 1.51 (1.06, 2.15), respectivement) Conclusion: Plusieurs SNPs localisés sur les gènes CHRN sont associés à l'étourdissement, un phénotype de l'initiation qui est peut-être associé à la dépendance à la nicotine. / Background: Numerous single nucleotide polymorphisms (SNPs) in multiple nicotinic receptor genes (CHRN) are associated with smoking. However few studies have examined the association between CHRN SNPs and subjective responses to smoking which may relate to sustained smoking, such as dizziness at first inhalation. The objective of this study was to investigate the association between 61 SNPs in eight CHRN genes (CHRNA3 CHRNA4 CHRNA5, CHRNA6, CHRNA7, CHRNB2, CHRNB3, CHRNB4) and dizziness at first inhalation. Methods: Data were available in a longitudinal cohort investigation of 1293 students 12-13 years old at baseline. Students completed self-report questionnaires at-school every 3 months for 5 years during secondary school, and a mailed self-report questionnaire three years later. DNA extracted from blood or saliva was genotyped for 61 CHRN SNPs selected using a gene tagging approach. Associations were modeled using logistic regression controlling for sex, race and age at first cigarette. Results: The minor alleles of three SNPs in CHRNA6 (rs7812298, rs2304297, rs7828365) were associated a decreased probability of dizziness (OR (95% CI)=0.54(0.36, 0.81), 0.59(0.40,0.86) and 0.58(0.36,0.95, respectively), while one SNP in each of three other genes (rs3743077 (CHRNA3), rs755204 (CHRNA4), rs7178176 (CHRNA7)) was associated with an increased probability of dizziness (OR(95% CI)=1.40 (1.02,1.90), 1.85(1.05,3.27) and 1.51(1.06,2.15), respectively). Conclusion: Thus, several SNPs located in CHRN genes are associated with dizziness at first inhalation, a smoking initiation phenotype that may relate to sustained smoking.

En 1975, Wilson et King ont proposé que l'évolution opère non seulement via des changements affectant la structure des protéines, mais aussi via des mutations qui modifient la régulation génétique. L'étude des éléments régulateurs de l'expression génétique a un rôle important dans la compréhension de l'expression de différentes maladies et de la réponse thérapeutique. Nous avons développé un algorithme bio- informatique qui nous permet rapidement de trouver des sites de régulation génétique à travers tout le génome et pour une grande quantité de gènes. Notre approche consiste à trouver des sites polymorphes (SNPs) qui sont en déséquilibre de liaison avec le débalancement allélique (AI) afin de cartographier la région régulatrice et le site responsable. Notre méthode est avantageuse par rapport à d'autres méthodes, car elle n'a pas besoin des données « phasées». De plus, les données de débalancement allélique ne sont pas affectées par des facteurs externes étant donné qu'ils sont mesurés dans la même cellule. Nous avons démontré que notre approche est fiable et qu'elle peut détecter des sites loin du gène. De plus, il peut être appliqué à des données de génotypage sans avoir besoin de les « phaser » . / Wilson and King (1975) proposed that evolution frequently operates through mutations affecting genetic regulation. Likewise, it is expected that genetic variation responsible for inter-individual differences will be due to variation in regulatory sites. Identifying such sites is thus important in the genetic and medical research. We have developed a new bioinformatics algorithm to find genome-wide regulatory sites for a big number of genes. Individuals carrying different alleles at a regulatory site will exhibit allelic imbalance(AI) due to differential expression of the two copies the same locus. Our approach consists of searching polymorphic sites (SNPs) in linkage disequilibrium with AI in order to map regulatory regions. We have detected many SNPs associated to the regulation of different genes pointed in previous studies. We have also found regulatory regions far from the transcription start site (TSS). The major advantage of this method is that phased data is not needed. In addition, AI data has the benefit of not being affected by external factors since it is measured in the same cell. The results show that our approach is reliable and it can detect sites far from the gene.

INTRODUCTION Des réponses thérapeutiques variables aux glucocorticoïdes (GCs) sont observées parmi les patients atteints de la leucémie lymphoblastique aiguë (LLA). Les protéines Bax et Bim ont déjà montré un rôle important dans l’apoptose des cellules leucémiques. L’expression de Bax était plus basse chez les patients leucémiques résistants au médicament, de même une sensibilité diminuée aux GCs a été associée avec une expression réduite de Bim. La différence dans l’expression pourrait être due à des polymorphismes présents dans ces gènes et donc être associés avec la résistance aux GCs. MÉTHODE Dix-huit polymorphismes en régions régulatrices, 2 polymorphismes exoniques et 7 polymorphismes en région 3’UTR de ces gènes ont été analysés chez les témoins (n=50) et ont permis de déterminer un nombre minimal de polymorphismes suffisants pour définir les haplotypes (tagSNPs). Ces 8 polymorphismes ont ensuite été génotypés chez 286 enfants atteints de la LLA et ont été testés pour l’issue de la maladie par l’analyse de survie. RÉSULTATS Une survie sans évènement et une survie sans rechute diminuées ont été observées pour l’haplotype 3 (p=0,03 et p=0,02). Une survie globale diminuée a été associée avec l’homozygotie pour l’allèle exonique T298C>T (p=0,03), de même que pour les haplotypes 1 et 4 (p=0,04 et p=0,02) du gène Bim. CONCLUSION Les polymorphismes ont été associés avec une survie diminuée chez des enfants atteints de LLA. Il reste à tester d’autres polymorphismes présents dans ces deux gènes ainsi qu’à définir leurs fonctions afin de comprendre leurs rôles dans la réponse aux GCs. / INTRODUCTION Variable therapeutic responses to glucocorticoids (GCs) are observed for acute lymphoblastic leukemia (ALL) patients. Proteins Bax and Bim have already shown to play a major role in mediating GC-induced apoptosis in leukemia cells. Bax expression was lower in drug-resistant leukemia samples; likewise lower sensitivity to GC was associated with reduced Bim expression. The difference in the expression can be due to polymorphisms in these genes and therefore associated to GC resistance. METHOD Eighteen polymorphisms in the regulatory region, two exonic polymorphisms and seven polymorphisms in 3’UTR of these genes were analysed in controls (n=50) and have permitted to determine a minimal number of polymorphisms sufficient to define haplotypes (tagSNPs). These 8 single nucleotide polymorphisms (SNPs) were then genotyped in 286 LLA children and were tested for disease outcome by survival analysis. RESULTS A diminished event free survival and a diminished relapse free survival were observed for haplotype 3 (p=0,03 and p=0,02). A diminished overall survival was associated with the exonic T298C>T allele (p=0,03) and with haplotypes 1 and 4 (p=004 and p=0,02) of Bim gene. CONCLUSION Bax and Bim were associated with a diminished survival in LLA children. We still have to test other polymorphisms located in these genes and to define their functions in order to understand their roles in GC response.

Objectifs: Plusieurs polymorphismes nucléaires localisés sur les gènes des récepteurs nicotiniques cholinergiques CHRN sont associés au tabagisme. Cependant, peu d’études ont examiné l’association entre les polymorphismes sur les gènes CHRN et l’étourdissement. Les polymorphismes et les symptômes subjectifs sont peu être lié à la dépendance à la nicotine et à l’étourdissement ressenti lors de l’initiation. Le but de cette étude est d’étudier l’association entre 61 polymorphismes sur huit gènes CHRN (CHRNA3 CHRNA4 CHRNA5, CHRNA6, CHRNA7, CHRNB2, CHRNB3, CHRNB4) et l’étourdissement ressenti lors de l’initiation. Méthodes: Les données provenant d'une étude de cohorte longitudinale composée de 1293 étudiants, ont été analysées selon un devis d'étude gène-candidat. Les données ont été collectées par le biais de questionnaires auto-reportés aux troix mois, durant 5 ans. L’ADN provenent de la salive ou du sang a été génotypé pour 61 polymosphismes localisés sur les gènes CHRN ont été génotypés, à l'aide d'une stratégie de couverture maximale du gène. L'équation d'analyse est une régression logistique, incluant un ajustement sur l’âge, le sexe et l’origine ethnique. Résultats: Trois SNPs sur le gène CHRNA6 (rs7812298, rs2304297, rs7828365) sont associés à notre phénotype (OR (95% CI)= 0.54 (0.36, 0.81), 0.59 (0.40, 0.86) and 0.58 (0.36, 0.95, respectivement),. Trois autres polymorphismes (rs3743077 (CHRNA3), rs755204 (CHRNA4), rs7178176 (CHRNA7)) sont également associés à phénotype (OR (95% CI)=1.40 (1.02, 1.90), 1.85 (1.05, 3.27) and 1.51 (1.06, 2.15), respectivement) Conclusion: Plusieurs SNPs localisés sur les gènes CHRN sont associés à l'étourdissement, un phénotype de l'initiation qui est peut-être associé à la dépendance à la nicotine. / Background: Numerous single nucleotide polymorphisms (SNPs) in multiple nicotinic receptor genes (CHRN) are associated with smoking. However few studies have examined the association between CHRN SNPs and subjective responses to smoking which may relate to sustained smoking, such as dizziness at first inhalation. The objective of this study was to investigate the association between 61 SNPs in eight CHRN genes (CHRNA3 CHRNA4 CHRNA5, CHRNA6, CHRNA7, CHRNB2, CHRNB3, CHRNB4) and dizziness at first inhalation. Methods: Data were available in a longitudinal cohort investigation of 1293 students 12-13 years old at baseline. Students completed self-report questionnaires at-school every 3 months for 5 years during secondary school, and a mailed self-report questionnaire three years later. DNA extracted from blood or saliva was genotyped for 61 CHRN SNPs selected using a gene tagging approach. Associations were modeled using logistic regression controlling for sex, race and age at first cigarette. Results: The minor alleles of three SNPs in CHRNA6 (rs7812298, rs2304297, rs7828365) were associated a decreased probability of dizziness (OR (95% CI)=0.54(0.36, 0.81), 0.59(0.40,0.86) and 0.58(0.36,0.95, respectively), while one SNP in each of three other genes (rs3743077 (CHRNA3), rs755204 (CHRNA4), rs7178176 (CHRNA7)) was associated with an increased probability of dizziness (OR(95% CI)=1.40 (1.02,1.90), 1.85(1.05,3.27) and 1.51(1.06,2.15), respectively). Conclusion: Thus, several SNPs located in CHRN genes are associated with dizziness at first inhalation, a smoking initiation phenotype that may relate to sustained smoking.

Background: A large number of human inherited and acquired diseases and phenotypes are caused by mutations in G protein-coupled receptors (GPCR). Genome-wide association studies (GWAS) have shown that variations in the ADGRD1 (GPR133) locus are linked with differences in metabolism, human height and heart frequency. ADGRD1 is a Gs protein-coupled receptor belonging to the class of adhesion GPCRs. Results: Analysis of more than 1000 sequenced human genomes revealed approximately 9000 single nucleotide polymorphisms (SNPs) in the human ADGRD1 as listed in public data bases. Approximately 2.4 % of these SNPs are located in exons resulting in 129 non-synonymous SNPs (nsSNPs) at 119 positions of ADGRD1. However, the functional relevance of those variants is unknown. In-depth characterization of these amino acid changes revealed several nsSNPs (A448D, Q600stop, C632fs [frame shift], A761E, N795K) causing full or partial loss of receptor function, while one nsSNP (F383S) significantly increased basal activity of ADGRD1. Conclusion: Our results show that a broad spectrum of functionally relevant ADGRD1 variants is present in the human population which may cause clinically relevant phenotypes, while being compatible with life when heterozygous.