Fusarium head blight (FHB), a fungal disease caused principally by Fusarium graminearum, and barley yellow dwarf (BYD) caused by BYD luteoviruses are two serious fungal and viral diseases of wheat resulting in high economic losses annually. Wuhan, a Chinese wheat cultivar resistant to FHB, and Maringa, a Brazilian cultivar tolerant to BYDV were inter-crossed and crossed with Roblin, a Canada western red spring wheat susceptible to both FHB and BYDV, to determine the genetic basis of resistance/tolerance and to combine the two traits. Four hundred ninety nine F1-derived doubled haploid (DH) lines were generated from reciprocal crosses using corn pollen-mediated DH technology. The DH lines and the parents were evaluated for disease symptoms, reduction in height and spike mass for BYD and for disease incidence, disease severity and Fusarium-damaged kernels for FHB in field and controlled environments. A subset (20/150) of the best performing DH lines from Wuhan/Maringa populations for both BYD and FHB were further evaluated. Plants were point inoculated with F. graminearum in greenhouse experiments, and macroconidial spray inoculations and spread of corn inoculum were used in field environments to evaluate FHB. BYDV inoculations were performed by placing ten to fifteen viruliferous aphids (Rhopalosiphum padi infected with BYDV-PAV isolate 9301PAV), at the one to two leaf stage for both greenhouse and field trials. The studies showed that both FHB and BYDV are quantitatively inherited. Transgressive segregants were observed and the broad sense heritability was high (0.90 to 0.97) for all traits evaluated. Results from independent testing of diseases on Wuhan/ Maringa populations showed fourteen DH lines were as, or more resistant than Wuhan for FHB and Maringa for BYDV tolerance and have combined both BYDV tolerance and FHB resistance. Identifying such lines facilitates the pyramiding of independent genes to obtain adequate levels of enduring resistance. A further experiment was conducted on the 14 lines by inoculating them with BYDV and F. graminearum successively on the same plant. Six out of 14 selected DH lines demonstrated high resistance to FHB and tolerance to BYDV. These six lines can be used in FHB/BYDV resistance/tolerance breeding programs.

More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.

Resumo: Estudos de associação baseados em desequilíbrio de ligação (DL) são importantes ferramentas para construção de mapas de ligação e utilização em programas de melhoramento assistidos por marcadores. Em geral, são utilizadas populações segregantes ou linhagens isogênicas para composição destes programas. Com o objetivo de comparar o desequilíbrio de ligação em linhagens duplo-haplóides (DH) e linhagens obtidas por meio de autofecundação, duzentas e quarenta e cinco linhagens - cento e setenta e cinco convencionais e setenta DHs - foram submetidas à análise de marcadores do tipo Single Nucleotide Polymorphism (SNP), utilizando-se mil duzentos e trinta e quatro marcadores distribuídos pelos dez cromossomos. O resultado da regressão entre as distâncias mostrou um R2 de 0,6546 para linhagens duplo-haplóides e uma equação de tendência logarítmica, sendo y= -0,024ln(x) + 0,159. Para as linhagens convencionais, o R2 foi de 0,5727, com a equação que explica a tendência, sendo y = -0,008ln(x) + 0,0659. Os dados de DL foram analisados individualmente, por cromossomo; e, assim como na análise conjunta, individualmente, todos os cromossomos tiveram o mesmo comportamento quando se comparam o DL de linhagens DH e os convencionais, sendo que os valores de DL nas linhagens DH foram em geral mais altos que nas convencionais. Os resultados indicam que, para a obtenção de linhagens DH, a recombinação ocorre em blocos maiores quando comparado com as linhagens convencionais. / Abstract: Association studies based on linkage disequilibrium (LD) are important tools for linkage maps construction and to use in marker-assisted breeding programs. Typically, segregating populations or isogenic lines are used to compose them. With objective of compare the linkage disequilibrium on double haploid lines (DH) and lines obtained by self pollination (Conventional), two hundred and forty five inbred lines, where one hundred seventy five conventional and seventy DHs where submitted to the analysis of Single Nucleotide Polymorphism (SNP) molecular markers, using one thousand two hundred and thirty four markers random distributed over the ten chromosomes. The regression output among the distances showed R2 of 0.6546 for double haploids lines and one logarithmic trend equation, being y = -0.024ln(x) + 0.159 For conventional lines, R2 was 0.5727, with an equation that explains the trend, being y = -0.008ln(x) + 0.0659. LD data were analyzed by chromosomes individually and as such in the joint analysis, all individual chromosomes showed the same patternr when comparing the LD of DH lines versus conventional lines, being the LD of DH lines generally higher that the conventional lines. Results show that to obtain DH lines the recombination occurs in larger blocks when compared against the conventional lines. / Orientador: José Roberto Moro / Coorientador: Heyder Diniz Silva / Banca: Ricardo Machado da Silva / Banca: Herberte Pereira da Silva / Banca: Antonio Carlos Silva / Banca: Sandra Helena Unêda Trevisoli / Doutor

O milho é uma espécie alógama cujo produto comercial são os híbridos, os quais originam-se do cruzamento de duas linhagens endogâmicas. Uma forma para obtenção de tais linhagens é por meio das técnicas de indução de haploidia e posterior obtenção dos duplo-haploides, permitindo até 100% de homozigose. Essas técnicas retornam resultados importantes no melhoramento de milho. Uma variável de interesse importante, obtida a partir dessas técnicas é a taxa de indução de haploidia, a qual trata-se de uma proporção entre o número de sementes haploides e o número total de sementes. O conjunto de dados foi obtido pelo cruzamento da linhagem indutora LI- ESALQ, com cinco genótipos comerciais de milho (2B587PW, 30F53H, BM820, DKB390 e STATUS VIPTERA), em duas gerações F1 e F2, por meio de um delineamento em blocos ao acaso, na área experimental do Departamento de Genética da ESALQ/USP. A teoria dos modelos lineares generalizados (MLGs) possibilita mais opções para a distribuição da variável resposta, exigindo somente que a mesma pertença à família exponencial sob a forma canônica. Tal classe de distribuições pode ser ainda expandida para modelos que permitem efeitos aleatórios no preditor linear, caracterizando a classe dos modelos lineares generalizados mistos (MLGMs). O objetivo deste trabalho foi analisar a taxa de indução de haploidia em milho tropical, utilizando um modelo binomial misto, com efeito aleatório em nível de indivíduo. O método de estimação foi o de máxima verossimilhança. Com base em tal modelagem, verificou-se que o genótipo 30F53H, destacou-se em relação aos demais quanto à eficiência da taxa de indução de haploidia. Todas as análises foram implementadas no software R. / The maize is an allogeneic species whose commercial product are the hybrids, which are gerated by the crossing of two endogenous lines. An alternative to obtain these lines is using the haploid induction techniques and subsequent doubled haploid production, that allows up to 100% homozygous. Artificial production of doubled haploids is important in plant breeding. An important variable, that results from these techniques, is the haploid induction rate, which is a proportion between the number of haploid seeds and the total number of seeds. The data set was obtained by crossing the inductive line LI-ESALQ, with five commercial genotypes of corn (2B587PW, 30F53H, BM820, DKB390 and STATUS VIPTERA), in two generations F1 e F2, in a randomized block design, in the experimental area of Department of Genetics, ESALQ/USP. The generalized linear models (GLMs) allow more options for the variable response distribution, requiring only that it belongs to the exponential family in canonical form. The GLM class can be expanded to models that allow random effects in the linear predictor, the mixed generalized linear models (MGLM) class. This work aimed to analyze the haploid induction rate in the tropical maize. The binomial mixed model, that included random effects in individual level, was proposed. The maximum likelihood method was used to estimate the parameters. The result revealed that the genotype 30F53H stands out in relation to the others regarding the efficiency in the haploid induction rate. All the analyzes were implemented in the software R.

Magister Scientiae - MSc / Doubled Haploid (DH) Technology is an important tool for plant breeding and biotechnological applications as it accelerates the breeding cycle of plants by shortening the time required to attain homozygosity. Anther culture has become one of the most frequent and well-established methods for the induction of haploid embryogenesis and regeneration in barley. Anther culture is easily reproduced and workable for a wide range of genotypes. The aim of this study was to determine the optimal pre-treatment for barley anther culture. Three pre-treatments, 0.3 M Mannitol, 0.7 M Mannitol and a cold treatment with a moist cloth (CMC), were studied. The results suggest that CMC is the optimal pre-treatment to use for green plant regeneration. Anthers treated with CMC showed a higher response percentage than that of 0.7 M Mannitol and 0.3 M Mannitol. CMC also induced a significantly higher callus formation and green plant regeneration frequencies than 0.7 M Mannitol and 0.3 M Mannitol. Further research has to be conducted to further optimize green plant yields per treatment as well as reduce the number of albinos regenerated through barley anther culture.

The development of mouse (Mus musculus) haploid parthenogenetic embryos does not reach the same level as normal embryos. The aim of this diploma thesis was to find out whether haploid parthenogenetic embryos of mice differ in the nucleocytoplasmic ratio. The volume of the nucleus increases with ploidity. The nucleocytoplasmic ratios of haploid embryos do not significantly change between the two-cell and four-cell stage (p = 0.052), there is a significant difference (p < 0.001) for diploid and tetraploid embryos. Non-standard nucleocytoplasmic ratio could be related to the problematic development. Understanding the regulation of preimplantational development of parthenogenetic embryos will increase the efficiency of haploid embryonic stem cell derivation.

A hybrid between a highly regenerative diploid clone (BARD 1-3) of Solanum phureja and haploid inducer IVP 101 was transformed with Agrobacterium tumefaciens strain 4404 containing plasmid pHB2892 with genes for green florescent protein (GFP) and kanamycin resistance. Hemizygous primary transformants (To) were produced from three leaf discs: 17 diploid plants from one leaf disc, three and nine tetraploids from the other two leaf discs. GFP expression was observed qualitatively under fluorescence microscopes and quantitatively with a GFP meter. Anther culture of tetraploids produced 29 plants, none with high levels of GFP. Segregation ratios for tetraploid T1 seedlings fit models for single duplex insertions (35 transgenic: 1 non) or double simplex insertions (15 transgenic: 1 non). Diploid T1 seedlings segregated for deleterious traits: dwarfed size and curled leaves, as well as the GFP transgene. Similar segregation patterns in diploid families implied that all diploids may have been from the same transformation event. The cumulative segregation showed the dwarfed and curled plants fit a single recessive gene ratio (3 normal: 1 mutant), and GFP fit a double-copy insertion ratio (15 transgenic: 1 non). There was substantial GFP silencing evidenced by the loss of expression in plants that had originally been selected for high GFP. However, six selections were found to be free of deleterious traits, consistently high expressers of GFP, and producers of stainable pollen with less 2n than IVP 101. / Master of Science

Chez les animaux, la conformation unique du noyau du spermatozoïde dont la chromatine est organisée avec des protéines chromosomiques spécifiques telles que les protamines le rend totalement inactif. Le remodelage de la chromatine paternelle à la fécondation par des activités d'origine maternelle sont donc des processus essentiels à la formation d'un embryon diploïde, dont les mécanismes restent très mal connus. Lors de ma thèse j'ai essayé de mieux comprendre ces processus par l'étude, chez la drosophile, d'un mutant létal embryonnaire à effet maternel : maternal haploid (mh). Ce mutant affecte l'incorporation des chromosomes paternels à la première division zygotique menant à la formation d'embryons haploïdes gynogénétiques. L'identification du gène de mh comme CG9203 m'ont permis de caractériser sa fonction. Dans les œufs mh, les chromosomes paternels se condensent anormalement et ne parviennent pas à se diviser correctement lors de la première mitose de l'embryon. Récemment, des études sur son orthologue humain, appelé Spartan/DVC1, ont montré qu'il était impliqué dans la synthèse translésionnelle (TLS), un mécanisme de tolérance aux dommages d'ADN. J'ai pu démontrer que dans les cellules somatiques, la fonction de Spartan dans le TLS est conservée chez la drosophile. Cependant, la fonction maternelle de MH ne relève pas du TLS canonique, mais permet de maintenir l'intégrité de l'ADN paternel avant la réplication. Ensemble, mes travaux soulignent la singularité du pronoyau mâle et la complexité que présente le maintien de son intégrité à la fécondation / In animals, sexual reproduction requires the union between two distinct parental gametes: the spermatozoon and the oocyte. The unique nuclear conformation of the sperm, in which the chromatin is organized with sperm-specific chromosomal protein like protamines, abolishes its activity. The paternal chromatin remodeling and the maintenance of its integrity at fertilization by maternal activities are therefore essential processes for zygote formation. However, although their mechanisms are crucial, they remain poorly understood. During my thesis, I tried to better understand the processes involved during de novo paternal chromatin assembly in Drosophila through the study of a maternal embryonic lethal mutation: maternal haploid (mh). The mutant affects the incorporation of paternal chromosomes during the first zygotic division, leading to the development of gynogenetic haploid embryos. The identification of the mh gene as CG9203, and the generation of the null allele mh2 allowed me to characterize its function. In eggs led by mh mutant females, paternal chromosomes abnormally condense and fail to divide leading to the formation of chromatin bridges at the first embryonic division. Recently, its human ortholog Spartan/DVC1, has been described to be involved in translesion synthesis (TLS), a DNA damage tolerance pathway that ensures replication fork progression. Combining genetic and cytological approaches, I demonstrated that the Spartan function in TLS is conserved in Drosophila. However, I discovered that the critical function of MH during the first embryonic division, was not consistent with a canonical TLS. Alternatively, it is specifically required to maintain paternal integrity and to allow its proper replication at the first cycle. The mh phenotype characterization, led me to compare it with others phenotypes induced by the knock-down of replication factors and to study parental chromosome condensation in the zygote. Surprisingly, one of the proteins allowing the establishment of the pre-replication complex is dispensable for the proper paternal chromosome segregation contrarily to the maternal counterpart. Altogether, these works highlight the difference that exists between the two parental pronuclei and the complexity of maintaining their integrity at fertilization

Sexually reproducing eukaryotes are typically going through a biphasic life cycle with a diploid and a haploid phase. Unlike in plants where selection on haploid pollen genotypes is well established, the possibility of selection occurring in animal sperm is currently not known. One of the main reasons for this lack of knowledge is the general assumption that due to the shortness and the apparent absence of gene expression in haploid sperm, selection during that phase is unlikely to occur. The aim of this thesis was to fill this gap and address some of the main fundamental questions. I investigated the interaction between sperm phenotype and offspring phenotype with a focus on the trans-generational effects of (i) selection on the haploid sperm genotype, (ii) sperm ageing and (iii) sperm-mediated epigenetic effects. For one, we performed several experimental studies to investigate how selection on the sperm phenotype affects offspring performance in two externally fertilizing fishes, Atlantic salmon and zebrafish. We found that in Atlantic salmon, sperm of intermediate post-activation longevity sire offspring that hatch earlier. In zebrafish, longer living sperm sire more viable offspring with a higher fitness than their short-lived sibling sperm. We explored the mechanisms of these trans-generational effects and found that neither intrinsic post-ejaculation sperm ageing (Atlantic salmon and zebrafish) nor pre-ejaculation sperm ageing (zebrafish) affect offspring performance. However, we identified genetic differences between sperm pools that were obtained by selecting different phenotypes within ejaculates of zebrafish males. These results suggest a genetic basis for intra-ejaculate sperm phenotype variation and show that there is potential for haploid selection in sperm. In a separate experiment, we explored the role of sexual selection in shaping sperm-mediated epigenetic effects, and found that short-time changes in male-male competition affect offspring hatching time and survival. In conclusion, this thesis provides evidence that sperm phenotype affects offspring phenotype, and that sperm phenotype is affected by both epigenetic changes influenced by the male environment and differences in the haploid genome of sperm.

O trigo é um alimento básico da humanidade cuja demanda cresce proporcionalmente ao aumento populacional. Existe carência de pesquisas sobre o efeito da nutrição da planta doadora na resposta androgenética. Este trabalho teve por objetivo avaliar a influência de cinco concentrações de nitrogênio e de boro (25%, 50%, 100%, 150% e 200%) a partir da solução de Sarruge modificada, no cultivo de plantas de trigo doadoras de anteras visando à obtenção de estruturas embriogênicas androgenéticas. Para tanto, foram avaliados os componentes do rendimento (número de colmos, diâmetro e comprimento do colmo principal; comprimento, largura e área da folha bandeira; teor de clorofila da folha bandeira, distância entre a folha bandeira e o último nó e comprimento da espiga); o acúmulo de graus dias e duração do ciclo da semeadura até a coleta das espigas para a cultura de anteras; a formação de estruturas embriogênicas androgenéticas e o desenvolvimento dos micrósporos (tétrade, mononucleado central, mononucleado periférico, binucleado e pólen P). Constatou-se que as concentrações de nitrogênio afetaram significativamente os componentes do rendimento, a formação de estruturas embriogênicas androgenéticas e o desenvolvimento dos micrósporos. A duração do ciclo das plantas cultivadas na concentração de 25% de nitrogênio foi menor que nas demais doses, apresentando diferença de até 3,3 dias quando comparada às plantas submetidas a doses mais elevadas. Houve aumento linear na produção de estruturas embriogênicas androgenéticas em função das doses crescentes de nitrogênio utilizadas no cultivo das plantas doadoras das anteras. As doses de nitrogênio interferiram também no estádio de desenvolvimento dos micrósporos, para todas as variáveis estudadas. Essa influência no desenvolvimento dos micrósporos foi efetiva enquanto a folha bandeira menos um encontrava-se no primeiro terço de deslocamento em relação à distância total entre a folha bandeira e o último nó do colmo. As concentrações de boro utilizadas nas soluções de cultivo das plantas de trigo doadoras de anteras não influenciaram nos componentes do rendimento, na formação de estruturas embriogênicas, na duração do ciclo da planta e na maioria das variáveis estudadas com relação ao desenvolvimento dos micrósporos. O desenvolvimento dos micrósporos na espiga seguiu o padrão normal descrito para a espécie, independentemente dos nutrientes estudados. Concluiuse que as concentrações de nitrogênio influenciaram positivamente nos componentes do rendimento (exceto no comprimento da espiga), na formação de estruturas embriogênicas e na distância entre a folha bandeira e o último nó do colmo (marcador morfológico da coleta das espigas para cultura de anteras), enquanto que as concentrações de boro utilizadas no cultivo das plantas doadoras de anteras não apresentaram esse efeito. / Wheat is the staple food of humanity whose demand grows in proportion to the population growth. There is lack of research about the effect of the nutrition of the donor plant in the androgenetic response. This work aim for evaluate the influence of five concentrations of nitrogen and boron (25%, 50%, 100%, 150% and 200%) from the solution of Sarruge modified, in the cultivation of wheat plants donors of anthers aiming the obtainment of androgenetics embryogenic structures. Both for, were evaluated the yield components (number stalks, diameter and length of the main stem; length, width and area of flag leaf; chlorophyll content of flag leaf, distance between flag leaf and the last node and ear length); the accumulation of degree-days and cycle duration of sowing until collection of ears to the anther´s culture; the formation of androgenetics embryogenic structures and the development of microporous (determination of the number of cells in stages: tetrad, central mononucleate, peripheral mononucleate and binucleated and occurrence of pollen P). It was found that the concentrations of nitrogen significantly affected the yield components, the formation of androgenetics embryogenic structures and the development of microspores. The duration of the cycle of the cultivated plants in the concentration of 25% of nitrogen was lower than in other doses, presenting difference of up to 3.3 days when compared to plants subjected to higher doses. There was a linear increase in the production of androgenetics embryogenic structures in function of increasing doses of nitrogen used in cultivation of donor plants of the anthers. The doses of nitrogen interfered also at the stage of development of microspores, for all variables studied. This influence on the development of microspores was effective while the flag leaf less one was in the first third of displacement in relation to the total distance between the flag leaf and the last node of the culm. The concentrations of boron used in solutions of cultivation of wheat donors plants of anthers did not affect the income components, in the formation of embryogenic structures, on the duration of the cycle of the plant and in the majority of the variables studied in relation to the development of the microspores. The development of microspores in spike followed the normal pattern described for the species, regardless of the nutrients studied. It was concluded that the concentrations of nitrogen positively influenced in yield components (except the length of the ear), the formation of embryogenic structures on and the distance between the flag leaf and the last node of the culm (morphological marker of collection of ears to anther culture), while the concentrations of boron used for the cultivation of plants donated anthers did not have this effect.