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Die Allele (*R1, *R2, *S1 und *S2) des A. gambiae complement-like thioester-containing Protein 1 (TEP1) bestimmen die Fitness der Mücken, welches die männlichen Fertilität und den Resistenzgrad der Mücke gegen Pathogene wie Bakterien und Malaria-Parasiten. Dieser Kompromiss zwischen Reproduktion und Immunnität hat Auswirkungen auf die Größe der Mückenpopulationen und die Rate der Malariaübertragung. Wie die genetische Diversität von TEP1 die genetische Struktur natürlicher Vektorpopulationen beeinflusst, ist noch unklar. Die Zielsetzung dieser Doktorarbeit waren: i) die biogeographische Kartographierung der TEP1 Allele und Genotypen in lokalen Malariavektorpopulationen in Mali, Burkina Faso, Kamerun, und Kenia, und ii) die Bemessung des Einflusses von TEP1 Polymorphismen auf die Entwicklung humaner P. falciparum Parasiten in der Mücke. Die Analysen der TEP1 Polymorphismen zeigten, dass die natürliche Selektion auf Exone, sowie Introne wirkt, was auf eine starke funktionale Beschränkung an diesem Lokus hindeutet. Außerdem zeigen unsere Daten die strukturierte Erhaltung natürlicher genetischer Variation im TEP1 Lokus, in welchem die Allele und Genotypen spezifische evolutionäre Wege verfolgen. Diese Ergebnisse weisen auf die Existenz von arten- und habitatspezifischen Selektionsdrücken hin, die auf den TEP1 Lokus wirken. Resultate haben gezeigt, dass TEP1*S1 und *S2 Mücken gleichermassen empfänglich für Plasmodium-Infektionen sind. Insgesamt tragen die Resultate der biogeographischen Kartographierung des TEP1 Lokus und der Züchtungs- und Infektionsexperimente zu einem besseren Verständnis über den Einfluss der verschiedenen Vektorarten und lokale Umwelteinflüsse auf die Vektorpopulationen und Malariaübertragung bei. Des weiteren kann die hier beschriebene hochdurchsatz-genotypisierungs Methode, zur Studie lokaler A. gambiae Mückenpopulationen, in der Feldforschungsarbeit eingesetzt werden. Dieser neue Ansatz wird die epidemiologisch relevante Überwachung und Vorhersage dynamischer Prozesse in lokalen Malariavektorpopulationen unterstützen, welche die Entwicklung neuer Strategien der Vektorkontrolle ermöglichen könnten. / The alleles (*R1, *R2, *S1 and *S2) and genotypes of A. gambiae complement-like thioester-containing protein 1 (TEP1) determine the fitness in male fertility and the degree of mosquito resistance to pathogens such as bacteria and malaria parasites. This trade-off between the reproduction and the immunity impacts directly on mosquito population abundance and malaria transmission respectively. How TEP1 genetic diversity influences the genetic structure of natural vector populations and development of human malaria parasites is unclear. The aims of this thesis were to: i) map distribution of TEP1 alleles and genotypes in local malaria vector populations in Mali, Burkina Faso, Cameroon and Kenya, and ii) assess the impact of TEP1 polymorphism on development of human P. falciparum parasites in mosquitoes. Analyses of TEP1 polymorphism revealed that natural selection acts in concert on both exons and introns, suggesting strong functional constrains acting at this locus. Moreover, our data demonstrate a structured maintenance of natural TEP1 genetic variation, where the alleles and the genotypes follow distinct evolutionary paths. These findings suggest the existence of species- and habitat-specific selection patterns that act on TEP1 locus. Results revealed that the TEP1*S1 and *S2 mosquitoes are equally susceptible to Plasmodium infections. Collectively, results of my thesis on the biogeographic TEP1 mapping, and on the breeding and infection experiments contribute to a better understanding of how the vector species and local environmental factors, shape vector population structures and malaria transmission. Furthermore, the high throughput TEP1 genotyping approach reported here could be used for field studies of local A. gambiae mosquito populations. This new approach will benefit surveilance and prediction of dynamics in local malaria vector populations that may have epidemiological significance, and therefore inform the development of novel vector control measures.

Microarray technology is a promising approach for the simultaneous analysis of multiple single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variation. In this thesis enzyme-assisted microarray-based methods were developed to improve the accuracy and genotype discrimination power of the current methods for SNP genotyping. The improved technology was applied for analysing recessively inherited disease mutations, for Y-chromosomal SNPs in a population study, for an evolutionary analysis of SNPs in flycatchers and for multiplexed quantitative determination of SNP-allele frequencies in pooled DNA samples. A robust attachment chemistry for immobilising oligonucleotides on glass surface was established, based on an evaluation of eight covalent coupling methods. A four-colour fluorescence detection strategy, which enabled a multiplexed quantitative analysis for as little as 2% of a minority allele frequency in pooled samples was generated. Twenty-five Y-chromosomal SNPs were screened in a collection of 300 samples from five Finno-Ugric-speaking populations using minisequencing on microarrays. In these populations six distinct haplotypes were defined by the six SNPs that were polymorphic. Data from five microsatellite markers was combined with the SNP data, revealing shared Y-chromosomal haplotypes between the Finns and the Saami, indicating, in accordance with earlier data, at least two founding Y-chromosomal lineages in these populations. Database screening and subsequent validation of 125 potential SNPs in the highly repetitive type 1 interferon genes and genes coding for proteins in the interferon-related regulatory pathways revealed 25 informative SNPs in the Finnish and Swedish populations. These SNPs were included in a panel for microarray based genotyping that should find a variety of applications in genetic studies due to the important immunoregulatory functions of the IFN family. The significance of sex-chromosome evolution on speciation was investigated in two naturally hybridising flycatcher species (N=459) by analysing a panel of 20 SNPs using minisequencing on microarrays. A strong selection against gene flow across the species boundary of sex-linked genes was observed, as well as a sex-chromosomal influence on male plumage characteristics that have previously been shown to reinforce isolation in these birds. The results suggest a major role for sex-chromosome-mediated isolation of the two flycatcher species.

Genomic selection consists of using predicted effects of genetic markers to predict breeding values and/or genotypic values of genotyped individuals. With this approach, selection can be carried based only on those predicted breeding values, reducing the need for further phenotypic evaluations. This represents a great advance in terms of cost and effectiveness of selection in breeding programs of all kinds of crops. In the first chapter of this work, we explore one of the ways genomic selection can be used to increase efficiency when breeding clonally propagated crops for multiple traits. Using stochastic simulations, we show that an economic selection index should be preferred over independent culling. Our results show that the use of genomic selection may render the cost-efficiency benefit of independent culling obsolete when all early generation individuals are genotyped and accurate prediction of all traits becomes available simultaneously. Despite the potential benefits of selecting based on predicted breeding values, for some clonally propagated species the complexity of their genomes limits the implementation of genomic selection in breeding programs. Since including allele dosage information has been shown to improve performance of genomic selection models in autotetraploid species, our objective in the second chapter of this work was to assess the accuracy of genome-wide prediction in the highly complex polyploid sugarcane when incorporating allele dosage information. In this chapter, we expanded GBLUP genomic selection models developed for autotetraploids to include higher levels of ploidy. Two types of model were used, one with additive effects only and one with additive and digenic dominance effects. We observed a modest improvement in the performance of the prediction model when ploidy and allele dosage estimates were included, indicating that this is a possible way of improving genomic selection in sugarcane. The results obtained in both studies can assist researchers and breeders of clonally propagated crops, opening new research opportunities and indicating the most efficient ways to implement genomic selection. / A seleção genômica consiste no uso de efeitos preditos de marcadores genéticos para predizer os valores genéticos e/ou genotípicos de indivíduos genotipados. Desta forma, a seleção de genótipos superiores pode ser feita baseada apenas em valores genéticos preditos, reduzindo a necessidade de avaliações fenotípicas subsequentes. Isto representa um grande avanço em termos de custos e eficiência da seleção em programas de melhoramento de todos os tipos de culturas. No primeiro capítulo deste trabalho, nós exploramos uma das maneiras com que a seleção genômica pode ser utilizada para aumentar a eficiência no melhoramento simultâneo para múltiplos caráteres em espécies de propagação vegetativa. Utilizando simulações estocásticas, nós mostramos que um índice de seleção econômico deve ser utilizado no lugar da eliminação independente (independent culling). Os resultados mostram que o uso da seleção genômica pode tornar o custo-benefício da eliminação independente obsoleto se indivíduos em gerações iniciais forem genotipados e predições acuradas para todos os caráteres estiverem disponíveis desde o início. Apesar dos potenciais benefícios de realizar a seleção com base em valores genéticos preditos, para algumas espécies de propagação vegetativa a complexidade de seus genomas é um fator limitante para a efetiva implementação da seleção genômica em programas de melhoramento. Considerando que incluir a informação de dosagem alélica melhorou a performance de modelos de seleção genômica em espécies autotetraploides, nosso objetivo no segundo capítulo deste trabalho foi avaliar a acurácia da predição genômica com informação de dosagem alélica em cana-de-açúcar, que é uma complexa espécie poliploide. Neste capítulo, nós expandimos modelos GBLUP de seleção genômica desenvolvidos para autotetraploides para incluir níveis mais altos de ploidia. Dois modelos foram utilizados, um modelo com somente efeitos aditivos e um modelo com efeitos aditivos e efeitos de dominância digênica. Nós observamos uma modesta melhora na performance do modelo preditivo quando estimativas de ploidia e dosagem alélica foram incluídas, indicando que esta é uma possível maneira de aprimorar a seleção genômica em cana-de-açúcar. Os resultados obtidos nos dois estudos podem auxiliar pesquisadores e melhoristas de espécies de propagação vegetativa, abrindo portas para novas pesquisas e indicando as maneiras mais eficientes para implementação da seleção genômica.

Polimorfismos do tipo indel são os mais abundantes depois dos SNPs, representando 3,6 milhões das variantes caracterizadas pelo projeto 1000 Genomes. Com uma distribuição que pode ser estimada em mais de um indel a cada 1000 pb, são facilmente encontrados em regiões de interesse. A baixa taxa de mutação e a possibilidade de desenhar primers alelo-específicos são as principais características dos indels que os diferenciam de STRs. O uso de primers aleloespecíficos na detecção e dosagem de misturas de DNA apresenta maior sensibilidade e acurácia que as técnicas usualmente empregadas. Aqui foram descritos, para 10 lócus indel, pares de primers flanqueadores e alelo-específicos para ambos os alelos (inserção e deleção) e foi realizado o estudo populacional em 160 indivíduos. A determinação de fenótipos e avaliação de especificidade dos primers, dos quais 28 foram específicos, foi realizada por PCR convencional seguida de PAGE. As análises populacionais e forenses mostraram que esses lócus apresentam alta variabilidade (heterozigose de 30-50%) e consequentemente, alta informatividade. Os valores de PIC, PE e PD variaram de 0,2763 a 0,3750; 0,1381 a 0,1875 e 0,4978 a 0,6250 respectivamente. Os valores cumulativos de PCE e PCD foram respectivamente 0,8508 e 0,9999. Assim, esse conjunto de indels é indicado para serem testados na detecção e quantificação de misturas de DNA a partir da amplificação alelo-específica. / Indels polymorphisms are the most abundant after SNPs, representing 3.6 million of the variants characterized by the 1000 Genomes project. With a distribution that can be estimated at more than one indel per 1000 bp, they are easily found in regions of interest. The low mutation rate and the possibility of designing allele-specific primers are the main characteristics of the indels that differentiate them from STRs. The use of allele-specific primers in the detection and dosage of DNA mixtures is more sensitive and accurate than regularly employed techniques. Here, for 10 indel loci, pairs of flanking primers and allele-specific primers, for both alleles (insertion and deletion), were described and a population study was performed on 160 individuals. Determining phenotypes and evaluation of primers specificity, of which 28 were specific, was performed by conventional PCR followed by PAGE. In population and forensic analysis, these loci showed high variability (heterozygosis of 30-50%) and consequently high informativeness. The values of PIC, PE and PD ranged from 0.2763 to 0.3750, 0.1381 to 0.1875 and 0.49978 to 0.6250 respectively. Combined values of PCE and PCD were respectively 0.8508 and 0.9999. Thus, this set of indels is indicated to be tested for detection and quantification of DNA mixtures using the allele-specific amplification method.