Most human traits are influenced by a combination of genetic and environmental effects. Heritability expresses the proportion of trait variance that can be explained by genetic factors, and the 1980s heralded the beginning of studies that aimed to pinpoint genetic loci that contribute to trait variation, also known as quantitative trait loci (QTLs). Subsequently, the availability of cheap, high-resolution genotyping chips ushered in the era of genome-wide association studies (GWAS). These genetic studies have discovered many associations between single-nucleotide polymorphisms (SNPs) and complex traits, but these associations do not explain the genetic component of these traits entirely. This is known as the ‘missing heritability’ problem. Within this thesis, 40 medically-relevant human complex traits are studied in order to identify new QTLs. These traits include eye biometric traits, blood biochemical traits and anthropometric traits measured in approximately 28,000 individuals belonging to family-based samples from the general Scottish population (the Generation Scotland study) or from population isolates from Croatian (Korčula, Vis) or Scottish (Shetland, Orkney) islands. These individuals had been genotyped using commercially-available arrays, and unobserved genotypes were imputed using the Haplotype Reference Consortium (HRC) dataset. In parallel to standard GWAS, these traits are analysed using two other statistical genetics approaches: variance component linkage analysis and regional heritability (RH) mapping. Each study is analysed separately, in order to detect study-specific genetic effects that may not generalise across populations. At the same time, because most traits are available in several studies, this also enables meta-analysis, which boosts the power of discovery and can reveal cross-study genetic effects. These methods are a priori complementary to each other, exploiting different aspects of human genetic variation, such as the segregation of variants within families (identity by descent, IBD), or the presence of the same variant throughout the general population (identity by state, IBS). The strengths and weaknesses of these methods are systematically assessed by applying them to real and simulated datasets.

Chicken is a major protein source and intensively selected for economically important traits by humans. As such, this generated a huge range of phenotypes that representing a diverse spectrum of genetic variation. Understanding the functional basis of the genetic variants that underlie these traits, however, remains a formidable endeavour particularly for complex traits. Nonetheless, molecular phenotyping of an organism from sequenced data is doable with the advances in bioinformatics analysis and unparalleled surveys of genome wide genetic variants. This provides the opportunity to gain insights into the genome architecture and assists in identifying chromosomal regions underlying selection through a “sequences to consequences” approach. Combining a whole genome re-sequencing (WGS) approach with the knowledge of selection history, this thesis aimed to study the chromosomal regions and genetic variants underlying traits of interest in various selected chicken populations. To achieve this, genetic (quantitative and population genetics), genomic and bioinformatics approaches were employed and integrated to investigate the genome wide selection signatures in a number of different lines of chicken selected for different complex traits. This includes analysing: (i) divergently selected broilers for fatness traits (Chapter 2), (ii) a closed population of layer chickens (Chapter 3), (iii) selection signatures unique to broiler or layer chickens (Chapter 4) and (iv) selection signatures in colony stimulating factor 1 (CSF1) associated with gene expression differences in broiler and layer populations (Chapter 5). Candidate genes and nucleotides underlying potential selection regions were identified, and attempts were made to further elucidate the potential interplay between genes and the biological pathways involved in regulating traits in these selected chicken lines. Incorporating integrative approaches, variants within selection signatures were annotated to provide further evidence of their functional consequences. Overall, non-coding regions were enriched in selection signatures implied that causative variants may have regulatory roles. Capitalising on the millions of genetic variants discovered from WGS, chromosomal regions subject to selection were detected using a number of population genetics statistics. In broiler chicken lines divergently selected for very low-density plasma lipoprotein (VLDL) (Chapter 2), incorporating signatures of selection helped to improve the resolution of previously mapped quantitative traits loci (QTL) intervals. This research demonstrated that the integration of the analysis of selection signatures with functional annotation of genetic variants enabled refinement and characterisation of the QTL for fatness traits. In a closed population of brown leghorn layers (Chapter 3), evidence of selection signatures was found through Tajima’s D analysis. The analysis unravelled selection signatures encoding genes involved in numerous pathways and genes having key roles such as in behaviour, including feather pecking. Combining population differentiation statistic (FST) and Tajima’s D, a number of regions subject to divergent selection between broilers and white egg layers were identified (Chapter 4). Selection signatures were found to harbour mutations involved in cellular and tissue development, including genes having important roles in growth, fatness, egg shell strength and muscle development. These regions and the overlapping genes thereby may be potentially contributing to the different phenotypic variations observed between broilers and layers. In Chapter 5, a revised gene model for colony stimulating factor 1 (CSF1) showing complex pattern of alternate transcripts was predicted from transcriptome analysis of RNA isolated from 21 different tissues. In parallel, selection signatures analysis with the FST statistic, identified selection signatures that differentiate broilers to white egg layers (3 regions) or brown egg layers to white egg layers (4 regions). All these selection signatures were located within non-coding regions, indicating potential divergent selection of CSF1 within regulatory regions. Overall, the results presented in this thesis using the “sequences to consequences” approach, link several genomic regions and genes to phenotypic variation in domesticated chicken lines. The work reported here serves as a foundation for further study to decipher the relationship between “genotype and phenotype” and its functional consequences due to selection.

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.