O presente trabalho teve por objetivo identificar fragmentos homólogos a genes de resistência em Brassica oleracea e Zea mays, por meio da amplificação por PCR, utilizando oligonucleotídeos homólogos a regiões conservadas de genes de resistência de plantas. Em B. oleracea, os oligonucleotídeos foram desenhados com base na seqüência de um gene homólogo ao RPS2 de Arabidopsis thaliana descrito em B. oleracea. Um fragmento de 2,5 Kb foi amplificado em duas linhagens. Os fragmentos amplificados apresentaram polimorfismo de comprimento entre as linhagens, gerando um marcador molecular. Este marcador foi utilizado em uma população F2 segregante para resistência a Xanthomonas campestris pv. campestris oriunda do cruzamento entre as linhagens BI-16 e Lc201. O marcador, no entanto, não apresentou-se ligado a nenhum gene de resistência a este patógeno. Análise da expressão por meio de RT-PCR detectou a expressão do fragmento homólogo nas linhagens resistente e suscetível de B. oleracea com e sem inoculação, indicando que o gene é expresso constitutivamente. Em Z. mays, oligonucleotídeos sintetizados com base em seqüências de milho homólogas a genes de resistência, denominadas Pics, e a ESTs de milho, também homólogos a genes de resistência, foram utilizados para amplificação em linhagens resistente e suscetível a Exserohilum turcicum, Colletotrichum graminicola e Phaeosphaeria maydis. Um par de oligonucleotídeos amplificou um fragmento polimórfico entre as linhagens resistente e suscetível a E. turcicum. Este foi utilizado em uma população segregante, mas também não observou-se ligação com o gene Ht de resistência a E. turcicum. Nas demais linhagens, os fragmentos foram monomórficos. Os oligonucleotídeos baseados em ESTs amplificaram fragmentos em todas as linhagens parentais. Esses fragmentos foram digeridos com enzimas de restrição, mas não apresentaram polimorfismo entre nenhuma das linhagens. Os resultados indicaram que a estratégia de utilização de seqüências conservadas é eficiente para amplificação de genes homólogos. O polimorfismo entre estes homólogos pode ser usado como marcador molecular para detecção de genes de interesse. Todavia, nem sempre estes marcadores estão ligados a esses genes. / The aim of this work was to identify homologs of resistance genes in Brassica oleracea and Zea mays by PCR amplification using primers based on conserved domains of plant resistance genes. In B. oleracea, the primers were based on the sequence of a homolog of the Arabidopsis thaliana RPS2 gene previously described in B. oleracea. A 2.5 Kb fragment was amplified on two lines. These fragments showed length polymorphisms between lines, based on which a molecular marker was developed. This marker was used in a F2 population, derived from the crossing between the inbred lines BI-16 and Lc201, and which segregates to resistance to Xanthomonas campestris pv. campestris. The marker, however, was not linked to any Xcc resistance gene. Expression analyses by RT-PCR detected the expression of these homologs on both resistant and susceptible lines with and without inoculation, indicating that the gene is constitutively expressed. In Z. mays, primers based on resistance gene homologs sequences, named Pics, and on maize ESTs homologous to disease resistance genes, were used to amplify genomic fragments on resistant and susceptible lines to Exserohilum turcicum, Colletotrichum graminicola and Phaeosphaeria maydis. A set of primers amplified a polymorphic fragment between lines resistant and susceptible to E. turcicum. This fragment was used in a segregating population, but no linkage was detected between this marker and the E. turcicum resistance gene Ht. Among the other lines, the fragments were not polymorphic. The primers based on ESTs amplified fragments on all parental lines. These fragments were digested with restriction enzymes but did not reveal any polymorphism between lines. The results indicated that the strategy of using conserved sequences is efficient to amplify disease resistance gene homologs. The polymorphism among these homologs may be used as a molecular marker, but these markers are not always linked to disease resistance genes.

Les sources hydrothermales océaniques profondes renferment des écosystèmes extrêmes, situés dans la zone abyssale des Océans. Dans ces environnements dépourvus de lumière, la production primaire est réalisée par la chimiosynthèse microbienne. Ces milieux sont colonisés par des espèces animales, dont la plupart vivent en associations plus ou moins fortes avec des micro-organismes. La crevette Rimicaris exoculata est une espèce hydrothermale endémique des sites de la Ride-Médio-Atlantique (MAR), qui domine la plupart des sites qu’elle colonise. Ce crustacé a pour particularité de posséder deux communautés symbiotiques : une située dans son céphalothorax hypertrophié et une inféodée à son tractus digestif. Tout d’abord, ce travail de thèse s’est concentré sur l’étude de la communication bactérienne (Quorum Sensing ou QS) au sein des communautés ectosymbiotiques de R. exoculata au cours de son cycle de mue et de vie. Ensuite, ce travail s’est focalisé sur l’identification d’un peptide antimicrobien (PAM), puis à rechercher sa fonction dans l'immunité et le contrôle des symbiotes chez Rimicaris exoculata. Ce travail a permis, d’une part, de confirmer la présence de deux gènes du QS (luxS et luxR) dans les communautés ectosymbiotiques de R. exoculata sur quatre sites hydrothermaux : Rainbow, TAG, Snake Pit et Logatchev. Ces gènes étant plus divergents que ceux de l'ARNr 16S, leur utilisation comme marqueurs génétiques biogéographiques pour retracer l'origine des individus est discuté. Ce travail a permis, d’autre part, d’identifier pour la première fois un PAM (sus nommé Re-crustin), chez un arthropode hydrothermal. Les données suggèrent une participation de ce PAM dans le contrôle de l’ectosymbiose. L’ensemble de ces travaux apporte de nouvelles hypothèses sur l’interaction entre les épibiontes du céphalothorax et la crevette Rimicaris exoculata. / Deprived of light, the deep-sea hydrothermal vents are extremes ecosystems sustained by micobial chemosynthesis. These environments are colonized by animal species living in close relationships with these chemoautotrophic micro-organisms, eating them or establishing long term interactions with them, may they be trophic or not only. The shrimp Rimicaris exoculata is an endemic hydrothermal species of the Mid-Atlantic Ridge (MAR) sites. This crustacean represents the predominant macrofauna of some sites of the MAR. It lives in symbiotic association with two distinct microbial communities qualified as ectosymbiosis. One is located in its gill chamber and one in its gut. First, this work focused on the study of bacterial communication (Quorum Sensing or QS) within the ectosymbiontic communities during the molting and life cycles of R. exoculata. Then, we focused on an antimicrobial peptide (AMP) identification and search for its function in R. exoculata immunity and in controlling symbionts. Two QS genes (luxS and luxR) were identified in the R. exoculata ectosymbiontic community at different shrimp molt stages and life stages at the Rainbow, TAG, Snake Pit and Logatchev vent sites.As these genes are more divergent than that of 16S rRNA, they could be then used as biogeographical genetic markers tools to trace back the origin of individuals to a location or between locations along its life cycle. This work reports also the first description of an AMP in an extremophile arthropod (namely Recrustin). Data suggest a participation of this AMP in the control of the ectosymbiosis in Rimicarisexoculata. All this work provides new hypotheses wich are discussed in the manuscript, dealing with the interaction between symbionts and Rimicaris exoculata.

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En aquest treball es pretén utilitzar la informació que deixa la recombinació al nostres genomes per fer inferències sobre la història evolutiva recent de les poblacions humanes. Per fer-ho, s’ha desenvolupat un mètode novedós, anomenat IRiS, que permet la detecció de recombinacions antigues específiques en un conjunt de seqüències. Hem validat extensivament IRiS i l'hem sotmès a diferents escenaris per tal d’avaluar-ne l’ eficàcia. Un cop els events de recombinació són detectats, es poden utilitzar com a marcadors genètics per estudiar els patrons de diversitat de les poblacions humanes. Finalment, hem aplicat aquesta innovadora aproximació a un conjunt de poblacions humanes del Vell Món, que varen ser genotipades específicament amb aquesta finalitat, aportant nous coneixements en la història evolutiva recent dels humans / The aim of this work is to use the information left by recombination in our genomes to make inferences on the recent evolutionary history of human populations. For that, a novel method called IRiS has been developed that allows detecting specific past recombination events in a set of extant sequences. IRiS is extensively validated and studied in whole set of different scenarios in order to assess its performance. Once recombination events are detected, they can be used as genetic markers to study the recombinational diversity patterns of human populations. We apply this innovative approach to a whole set of different human populations within the Old World that were specifically genotyped for this end and we provide new insights in the recent human evolutionary history of our species.

A candidate gene approach was used to identify potential genetic markers associated with wool quality traits including mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVD), prickle factor, curvature, yellowness, brightness, staple strength, staple length, yield, greasy fleece weight (GFW) and clean fleece weight (CFW). Inheritance of potential genetic markers was studied in two half-sib Merino families and assessed for association with the wool quality traits. The sire for one of the half-sib families is referred to as MV144-58-00, and wool measurements from its progeny were taken at 12 (n = 131), 24 (n =128) and 36 (n = 37) months of age. The sire for the second half-sib family is referred to as Stoneyhurst, and wool measurements from its progeny (n = 35) were taken at 12 months of age. Genes that code for the keratin intermediate-filament proteins (KRTs) (KRT1.2, KRT2.10) and the keratin intermediate-filament-associated proteins (KAPs) (KAPl.1, KAPl.3, KAP3.2, KAP6.1, KAP 7, KAP8) were targeted for this investigation, along with the beta 3-adrenergic receptor (ADRB3) gene and microsatellites BfMS and OarFCB193. Polymerase chain reaction (PCR) was used to amplify specific DNA fragments from each locus and PCR- single strand conformational polymorphism (PCR-SSCP) analysis was used to detect polymorphism within the half-sib families for all the loci, except for the KAP1.1 gene, where length polymorphism was detected using agarose gel electrophoresis. Only the loci that were heterozygous for the sire (KAP1.1, KAP1.3, KRT1.2, ADRB3, KAP8) and hence were informative, were genotyped in the progeny. The total number of alleles observed at the KAP1.1, KAP1.3, KRT1.2, KAP8 and the ADRB3 loci were four, ten, six, five and six, respectively. Analysis of each of the informative loci revealed allelic associations with various wool traits. In the MV144-58-00 (genotypes KAP1.1 AB; KAP1.3 BD; KRT1.2 AB; ADRB3 CE) half-sib, inheritance of the KAP1.1 A allele was associated with a higher yield at 24 months of age (P = 0.037). This trend also observed at 36 months of age (P = 0.078). At 12 months of age, the KAP1.1 A allele tended to be associated with increased staple length (P = 0.08). At 36 months of age, the inheritance of the KAP1.1 B allele tended towards being associated with whiter wool (P = 0.080). The MV144-58-00 KAP1.3 D allele tended to be associated with increased yield at 24 and 36 months of age (P = 0.091 and 0.059, respectively), and with lower FDSD at 12 months of age (P = 0.055). The sire KAP1.3 B allele was associated with whiter wool colour at 36 months of age (P = 0.045). The inheritance of the MV144-58-00 KR T1.2 B allele was associated with or tended to be associated with a smaller FDSD (P = 0.040), an increase in staple strength (P = 0.025) and an increase in GFW (P = 0.069) at 12 months of age. At 24 months of age, the KR T1.2 B allele tended to be associated with increased yield (P = 0.057). At 36 months of age, the KRTl.2 A allele was associated with whiter wool (P = 0.019) and tended to be associated with increased crimp within the wool fibre (P = 0.089). In the Stoneyhurst (genotypes KAP1.1 BC; KAP1.3 CJ; KRT1.2 DE; ADRB3 CE) half-sib, inheritance of the KAP1.1 B allele was associated with longer staple length (P = 0.018) and a decrease in wool brightness (P = 0.039). In contrast, KAP1.1 C allele was associated with lowest staple length (P = 0.018) and brighter wool colour (P = 0.039). Associations observed with the inheritance of Stoneyhurst KAP 1.1 alleles were similar to the inheritance ofKAPl.3 alleles. Stoneyhurst KAP1.3 J allele was associated with longer staple length (P = 0.017) and a decrease in wool brightness (P = 0.010). In contrast, KAP1.3 C allele was associated with lowest staple length (P = 0.017) and brighter wool colour (P = 0.010). The Stoneyhurst KRT12 D allele was associated with longer staple length and a decrease in wool brightness (P = 0.033). In contrast, KRT1.2 E allele was associated with lowest staple length (P = 0.033) and brighter wool colour (P = 0.022). Sire alleles at the ADRB3 gene locus were associated with variation in staple strength (P = 0.025) for MV144-58-00's progeny, and with variation in yield (P = 0.023) for Stoneyhurst's progeny. The results obtained in this thesis are consistent with KAP1.1, KAP1.3 and KRT1.2 being clustered on one chromosome because both sires in this study passed on two major KAP1.1-KAP1.3-KRT1.2 haplotypes to their progeny, and the associations with wool traits were very similar for all the three loci. The major sire derived KAP1.1 – KAP1.3 - KRT1.2 haplotypes observed within the MV144-58-00 half-sib were: BBA (frequency of 43.4%; n = 43) and ADB (frequency of 44.4%; n = 44). Other minor haplotypes observed were: ADA (frequency of 4.0%; n = 4); BDA (frequency of 2.0%; n = 2); BBB (frequency of 3.0%; n = 3) and BDB (frequency of 3.0%; n = 3). In the Stoneyhurst half-sib, major sire-derived KAP 1.1 - KAP 1.3 - KR Tl.2 haplotypes observed were CCE (frequency of 53.1 %; n = 17) and BJD (frequency of 40.6%; n = 13). The minor haplotype BJE (frequency of 6.3%; n = 2) was also observed. Statistical analyses within the MVI44-58-00 half-sib showed that KAP1.1 AKAP1.3 D - KRT1.2 B haplotype was associated with increased yield (P = 0.023) and tended towards whiter wool colour (P = 0.059), smaller FDSD (P = 0.081) and stronger staple strength (P = 0.092). In the Stoneyhurst half-sib, the KAP1.1 B - KAP1.3 J - KRT1.2 D haplotype was associated with longer staple length (P = 0.010), while the KAP1.1 C - KAP1.3 C - KRT1.2 E haplotype showed a strong trend with increased wool brightness (P = 0.096). Result from this study indicated that the keratin genes on chromosome 11 are recombining relatively frequently at recombination "hotspots". A high rate of recombination among loci that impact on wool traits would make breeding for consistent wool quality very difficult. The results presented in this thesis suggest that genes coding for the KRTs and KAPs have the potential to impact on wool quality. KAP1.1, KAP1.3 and KRT1.2 could potentially be exploited in gene marker-assisted selection programmes within the wool industry to select for animals with increased staple length, 'increased staple strength, higher yield and brighter wool. This study was however limited to two half-sib families, and further investigation is required.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-03-29T20:09:57Z No. of bitstreams: 2 Dissertação - Eni Liudmiliza Leite Buma - 2017.pdf: 1928297 bytes, checksum: 00dfb2987c7dc920fba208a5861948fe (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-31T10:28:49Z (GMT) No. of bitstreams: 2 Dissertação - Eni Liudmiliza Leite Buma - 2017.pdf: 1928297 bytes, checksum: 00dfb2987c7dc920fba208a5861948fe (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-31T10:28:49Z (GMT). No. of bitstreams: 2 Dissertação - Eni Liudmiliza Leite Buma - 2017.pdf: 1928297 bytes, checksum: 00dfb2987c7dc920fba208a5861948fe (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-07 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Water courses that pass through areas of production, whether agricultural, pasture or housing, are subject to discharge of municipal and industrial sewage systems. These waters can be directed to river basins and redistributed to networks of water treatment and public supply. Normally, surface raw water has high concentrations of total coliforms, thermotolerant coliforms and E. coli. However, the microbiological indicator of fecal contamination, E. coli, does not present specificity, limiting, therefore, the host identification causing fecal pollution in water stream. As an alternative, bacteria of the genus Bacteroides have been suggested as potential alternative indicators of fecal pollution. Bacteroides are strictly anaerobic, exclusive and specific to the human gastrointestinal tract, and are also present in homeothermic animals. Because its inability to withstand aerobic environments, Bacteroides are considered as promising indicators of recent fecal pollution. Their identification in water bodies is usually performed by the presence of the 16S rRNA genetic marker of the order Bacteroidales. The objective of this study was to evaluate the microbiological and physico-chemical quality of the water of the Ribeirão João Leite Basin, responsible to supply 50% of water to the city of Goiânia, as well as the evaluation of the 16S rRNA Bacteroidales marker as an indicator of human and / or animal fecal pollution in waters of this basin. For this purpose, 91 samples of surface freshwater were collected from 13 points located along the Ribeirão João Leite Hydrographic Basin extension. Also, human and animal fecal samples were collected to test the sensitivity and specificity of primers to trace the 16S rRNA Bacteroidales marker. Based on the CONAMA Resolution 357/2005 Class II for fresh water, 5.5% (5/91) of the samples had a turbidity level above > 100 NTU (119-180 NTU), while 33% presented values below <103 CFU / 100 mL for thermotolerant E. coli. The mean values were found to be between 1.24 x 103-5.03 x 104 CFU / 100 mL. The 16S rRNA Bacteroidales ruminant host marker was identified in points with high agricultural and cattle influence, on the other hand, the presence of the 16S rRNA Bacteroidales marker as an indicator of human fecal pollution was not detected in the 5 analyzed points. The results obtained will be able to collaborate with sanitary measures to reduce the level of turbidity and also to identify the source of the fecal contamination in these bodies of water, thus minimizing the risk of dissemination of waterborne diseases. / Cursos de água que atravessam áreas de produção, sejam elas agrícolas, de pastagens ou habitacional, estão sujeitos à captação de sistemas de esgoto municipais e industriais. Essas águas podem ser direcionadas a bacias hidrográficas e redistribuídas a redes de estação de tratamento de água e abastecimento público. Normalmente, águas de mananciais apresentam elevados níveis de concentrações de coliformes totais, coliformes termotolerantes e E. coli. Entretanto, o indicador microbiológico de poluição fecal E. coli, não apresenta especificidade, limitando, portanto, a identificação do hospedeiro causador da poluição fecal em determinada corrente de água. Como alternativa, as bactérias do gênero Bacteroides vêm sendo sugeridas como potenciais indicadores alternativos de poluição fecal. Bacteroides são estritamente anaeróbicas, exclusivas e específicas ao trato gastrointestinal humano, apresentando especificidade a animais homeotérmicos. Por serem incapazes de resistir a ambientes aeróbios são consideradas como promissores indicadores de poluição fecal recente. A sua identificação em corpos de água é geralmente realizada pela presença do marcador genético 16S rRNA da ordem Bacteroidales. Este estudo teve como objetivos avaliar a qualidade microbiológica e físico-química da água bruta superficial da Bacia Hidrográfica do Ribeirão João Leite, responsável por 50% de abastecimento da cidade de Goiânia, e também, a avaliação do marcador 16S rRNA Bacteroidales como indicador da fonte de poluição fecal humana e/ou animal em águas desta bacia. Para tal, foram coletadas 91 amostras de água bruta superficial de 13 pontos localizados ao longo da extensão da Bacia Hidrográfica do Ribeirão João Leite, assim como, amostras de matéria fecal humana e animal de modo a testar a sensibilidade e especificidade de oligonucleotídeos iniciadores para o rastreamento do marcador 16S rRNA Bacteroidales. Baseado na Resolução CONAMA 357/2005 Classe II para águas doces, 5,5% (5/91) das amostras apresentaram nível de turbidez acima de >100 NTU (119-180 NTU), enquanto que 33% apresentaram valores inferiores a <103 CFU/100 mL para E. coli termotolerante, sendo os valores médios encontrados entre 1,24 x 103–5,03 x 104 CFU/100 mL. O marcador 16S rRNA Bacteroidales hospedeiro bovino (ruminante) foi identificado em água bruta superficial dos pontos coletados com alta influência agropecuária, em contrapartida, dos 5 pontos analisados não foi detectado a presença do marcador 16S rRNA Bacteroidales como indicador de poluição fecal humana. Os resultados obtidos poderão colaborar com medidas sanitárias que visam a redução do nível da turbidez e na identificação da origem da contaminação microbiológica fecal nesses corpos d’água minimizando, desta forma, o risco de disseminação das doenças de veiculação hídrica

Dentre os diversos aspectos geralmente observados na caracterização genética de populações naturais, a avaliação do grau de estruturação da variabilidade genética entre e dentro dos indivíduos e a obtenção de estimativas de parâmetros genéticos indicadores do sistema reprodutivo da espécie assumem grande importância. Os parâmetros de maior interesse neste caso são o índice de fixação intrapopulacional (f) e a taxa de fecundação cruzada (t). Pelo uso de simulações computacionais, este trabalho demonstra o caráter dinâmico do índice de fixação intrapopulacional em diferentes locos ao longo das gerações em decorrência do caráter finito da população e de variação nas taxas médias de fecundação cruzada entre gerações. Sugere-se que este caráter dinâmico representa uma explicação para a elevada variação, comumente reportada na literatura, das estimativas de f obtidas com locos diferentes avaliados em uma mesma população. Utilizando a abordagem Bayesiana, um modelo hierárquico de análise é proposto para a estimação de f, incorporando as informações obtidas de múltiplos locos não ligados, levando-se em conta a condicionalidade do processo de estimação ao polimorfismo dos locos utilizados. O modelo proposto incorpora o caráter dinâmico de f para diferentes locos e permite a estimação do número efetivo de indivíduos reprodutivamente ativos em uma população. Propõe-se ainda um modelo Bayesiano para a estimação da taxa de fecundação cruzada com base na informação de múltiplos locos, admitindo-se a possibilidade de ocorrência de apomixia. Os modelos propostos são avaliados por simulação e exemplos de aplicação a dados reais de marcadores moleculares codominantes são discutidos. Os resultados obtidos demonstram a aplicabilidade das metodologias propostas e o elevado potencial de aplicação da estatística Bayesiana em estudos de genética de populações. / Among the various aspects generally considered in the genetic characterization of natural populations of plant species, the evaluation of the degree of genetic structure within and among individuals and the estimation of parameters related to the species mating system are of great importance. In general, considerable effort is focused on the estimation of the intrapopulation fixation index (f) and the outcrossing rate (t). Using computer simulated data, the dynamic nature of f for different loci along generations is illustrated. The dynamic nature of f is shown to result from the finite condition of populations and from the variation in the mean values of the outcrossing rates among generations. It is suggested that this dynamic behavior explains the inconsistency, commonly reported in the literature, of f estimates obtained for different loci in a given population. Using a Bayesian approach, we propose a hierarchical model for the estimation of f, incorporating information obtained from different unlinked loci and considering the conditionality of the estimation process to genetic polymorphism. The proposed model incorporates the dynamic nature of f values for different loci and allows the estimation of the effective number of reproductively active individuals in a given population. Using a similar approach, a Bayesian model is also proposed for estimating the outcrossing rate using multiple loci information and incorporating the possibility of apomixis. The models proposed are evaluated by computer simulations and examples using real data from codominant molecular markers are presented. Results obtained illustrate the applicability of the proposed methods and reveal the great potential of use of Bayesian statistics in population genetic studies.

O presente trabalho teve por objetivo identificar fragmentos homólogos a genes de resistência em Brassica oleracea e Zea mays, por meio da amplificação por PCR, utilizando oligonucleotídeos homólogos a regiões conservadas de genes de resistência de plantas. Em B. oleracea, os oligonucleotídeos foram desenhados com base na seqüência de um gene homólogo ao RPS2 de Arabidopsis thaliana descrito em B. oleracea. Um fragmento de 2,5 Kb foi amplificado em duas linhagens. Os fragmentos amplificados apresentaram polimorfismo de comprimento entre as linhagens, gerando um marcador molecular. Este marcador foi utilizado em uma população F2 segregante para resistência a Xanthomonas campestris pv. campestris oriunda do cruzamento entre as linhagens BI-16 e Lc201. O marcador, no entanto, não apresentou-se ligado a nenhum gene de resistência a este patógeno. Análise da expressão por meio de RT-PCR detectou a expressão do fragmento homólogo nas linhagens resistente e suscetível de B. oleracea com e sem inoculação, indicando que o gene é expresso constitutivamente. Em Z. mays, oligonucleotídeos sintetizados com base em seqüências de milho homólogas a genes de resistência, denominadas Pics, e a ESTs de milho, também homólogos a genes de resistência, foram utilizados para amplificação em linhagens resistente e suscetível a Exserohilum turcicum, Colletotrichum graminicola e Phaeosphaeria maydis. Um par de oligonucleotídeos amplificou um fragmento polimórfico entre as linhagens resistente e suscetível a E. turcicum. Este foi utilizado em uma população segregante, mas também não observou-se ligação com o gene Ht de resistência a E. turcicum. Nas demais linhagens, os fragmentos foram monomórficos. Os oligonucleotídeos baseados em ESTs amplificaram fragmentos em todas as linhagens parentais. Esses fragmentos foram digeridos com enzimas de restrição, mas não apresentaram polimorfismo entre nenhuma das linhagens. Os resultados indicaram que a estratégia de utilização de seqüências conservadas é eficiente para amplificação de genes homólogos. O polimorfismo entre estes homólogos pode ser usado como marcador molecular para detecção de genes de interesse. Todavia, nem sempre estes marcadores estão ligados a esses genes. / The aim of this work was to identify homologs of resistance genes in Brassica oleracea and Zea mays by PCR amplification using primers based on conserved domains of plant resistance genes. In B. oleracea, the primers were based on the sequence of a homolog of the Arabidopsis thaliana RPS2 gene previously described in B. oleracea. A 2.5 Kb fragment was amplified on two lines. These fragments showed length polymorphisms between lines, based on which a molecular marker was developed. This marker was used in a F2 population, derived from the crossing between the inbred lines BI-16 and Lc201, and which segregates to resistance to Xanthomonas campestris pv. campestris. The marker, however, was not linked to any Xcc resistance gene. Expression analyses by RT-PCR detected the expression of these homologs on both resistant and susceptible lines with and without inoculation, indicating that the gene is constitutively expressed. In Z. mays, primers based on resistance gene homologs sequences, named Pics, and on maize ESTs homologous to disease resistance genes, were used to amplify genomic fragments on resistant and susceptible lines to Exserohilum turcicum, Colletotrichum graminicola and Phaeosphaeria maydis. A set of primers amplified a polymorphic fragment between lines resistant and susceptible to E. turcicum. This fragment was used in a segregating population, but no linkage was detected between this marker and the E. turcicum resistance gene Ht. Among the other lines, the fragments were not polymorphic. The primers based on ESTs amplified fragments on all parental lines. These fragments were digested with restriction enzymes but did not reveal any polymorphism between lines. The results indicated that the strategy of using conserved sequences is efficient to amplify disease resistance gene homologs. The polymorphism among these homologs may be used as a molecular marker, but these markers are not always linked to disease resistance genes.

Linhagens endogâmicas (F@1,0) são usualmente utilizadas para a produção de híbridos de milho. Devido a elevada depressão por endogamia no milho, as linhagens endogâmicas apresentam baixa produtividade, encarecendo o custo das sementes de híbridos simples e tornando-os inacessíveis para grande parte dos agricultores dos países em desenvolvimento. Uma alternativa para contornar o problema seria utilizar linhagens parcialmente endogâmicas (0,0 < F < 1,0), selecionadas para capacidade de combinação e uniformidade. Relatos de literatura mostram que (a) híbridos simples de linhagens S3 (F=0,875) devem apresentar performances superiores as de híbridos triplos e duplos de linhagens endogâmicas; (b) a correlação genética entre híbridos simples de linhagens S3 e de linhagens endogâmicas (F@1,0) é elevada (r=0,94); e (c) a produtividade de linhagens S3 é em média 20% superior a de linhagens endogâmicas. Entretanto, a maior dificuldade em se produzir híbridos simples de linhagens parcialmente endogâmicas refere-se à manutenção destas, por apresentarem variabilidade genética. Devido a isto, o objetivo deste estudo foi avaliar a viabilidade de se produzir e utilizar híbridos simples de linhagens parcialmente endogâmicas S3. Para isso, oito linhagens S3 da população BR-105 e dez linhagens da população BR-106, as quais estão alocadas em grupos heteróticos distintos, selecionadas para capacidade de combinação e uniformidade, originais e mantidas por intercruzamentos e seleção moderada por cinco gerações, foram utilizadas. Durante as gerações de manutenção, pelo menos 50 plantas foram usadas. Estas linhagens e cruzamentos destas com dois testadores de grupos heteróticos diferentes, foram avaliados em quatro ambientes no ano agrícola de 1999/2000. Além disso, as linhagens originais e mantidas foram genotipadas utilizando o marcador molecular AFLP para estimar a similaridade genética entre elas. Os resultados obtidos mostraram que, excetuando-se uma linhagem da população BR-105 em que provavelmente ocorreu contaminação, apenas dois caracteres nas linhagens per se e apenas um caráter nos cruzamentos apresentaram alterações positivas e significativas, de treze caracteres avaliados. Entretanto estas alterações são muito pequenas para serem detectadas visualmente. Os resultados das similaridades genéticas entre as linhagens originais e mantidas, mostraram valores elevados, sendo que o limite superior do intervalo de confiança para a maioria das linhagens atingiu o valor 1,0, indicando que a manutenção das linhagens da forma como foi conduzida as suas integridades genéticas foram mantidas. Estes resultados permitiram concluir que seria viável a utilização de linhagens parcialmente endogâmicas S3 para a produção comercial de híbridos simples de milho. / Inbred lines (F@1.0) are usually used for the production of maize single-crosses. Because of the high inbreeding depression in maize, the inbred lines are lower yielding, which causes the seed prices to be costly and then inaccessible for most of the farmers in the developing countries. One way to circumvent the problem would be the use of partly inbred lines (0.0< F<1.0) selected for combining ability and for uniformity within the lines. Reported results have shown that: (a) theoretically, single-crosses from S3 lines (F=0.875) are expected to have superior performance than that of three-way and double-crosses; (b) the genetic correlation of single-crosses from S3 lines and from their inbred lines (F@1.0) counterparts is fairly high (r=0.94); and (c) S3 lines are on the average 20% higher yielding than highly inbred lines. However, the main difficulty in the production of single-cross from partly inbred lines is the maintenance of their genetic integrity because of the variability within them. Therefore, the ob-jective of this research was to study the feasibility of the development and the production of single-crosses from S3 lines. The genetic material included eight original S3 lines from the BR-105 population, and ten original S3 lines from the BR-106 population, selected for combining ability and for uniformity within them, and their counterparts maintained by sib-mating and mild selection for five generations. During the generations of maintenance at least 50 plants per line were used. The populations BR-105 and BR-106 have been assigned to distinct heterotic groups. The original, the maintained lines and their crosses with testers from different heterotic groups were evaluated in four environments in the growing season of 1999/2000. Also, the S3 lines were genotyped with the AFLP molecular marker in order to estimate the genetic similar-ity between the original and their maintained counterparts. The results showed that out of the 13 traits evaluated only two traits in the lines per se, and only one trait in the crosses changed significantly from the original lines to the maintained counterparts. However those changes are too low to be visually detected. The estimates of the genetic similarities between the original and their maintained counterparts S3 lines were high, and the upper bound of the confidence interval for most of the lines reach the limit value, i.e., 1.0. These results showed that the ap-proach uses for the maintenance of the S3 lines was effective and thus the genetic integrity of the lines were maintained. The results of this research could allow one to expect that would be feasible the use of partly inbred S3 lines for the commercial production of maize single-crosses.

La construction d’animaux transgéniques est une technique clef qui a permis l’étude de nombreux aspects de la biologie du nématode Caenorhabditis elegans. Les animaux transgéniques peuvent être construits soit en injectant l’ADN exogène dans les gonades syncitiales de l’hermaphrodite adulte, soit en bombardant une population de vers avec des microbilles enrobées d’ADN. Dans les deux cas, l’utilisation de marqueurs génétiques est indispensable pour l’identification des individus transgéniques et la maintenance des lignées. Nous avons développé un vecteur d’expression pour les nématodes contenant le gène de résistance à la néomycine (neo), qui fonctionne comme marqueur génétique. Le gène neo confère la résistance au G-418, un antibiotique qui inhibe la synthèse de protéines chez les eucaryotes et qui est létal pour les nématodes sauvages. Nous avons montré que le marqueur neo est un marqueur génétique très puissant qui permet l’identification rapide des animaux transgéniques et qui permet l’enrichissement des populations transgéniques en présence de l’antibiotique, facilitant ainsi la maintenance des lignées. Ce système ne nécessite aucun contexte génétique particulier pour fonctionner et est donc compatible avec des lignées receveuses mutantes, ainsi que des lignées transgéniques ayant été transformées avec d’autres marqueurs génétiques. De plus, le gène neo est sous le contrôle du promoteur du gène de C. elegans rps-27, codant pour une protéine ribosomale dont la séquence est hautement conservée entre les nématodes. Nous avons utilisé ce gène comme marqueur génétique pour la transgénèse de l’espèce Caenorhabditis briggsae, ce qui suggère que le système neo pourrait aussi être utilisé pour d’autres espèces de la famille Caenorhabditis. Finalement, nous avons aussi montré que le système neo peut être utilisé dans le contexte des techniques d’ingénierie génétique basées sur le transposon Mos1. En conclusion, la sélection en présence de G-418 offre des nouvelles possibilités d’expériences pour la transgénèse de C. elegans et d’autres espèces proches. Les avantages du système neo devraient ainsi contribuer à développer des techniques de transgénèse du ver plus flexibles et efficaces. / The generation of transgenic animals has been instrumental to study many biological aspects of Caenorhabditis elegans biology. Transgenic animals can be obtained by either microinjection of the exogenous DNA into the syncitial gonad of the hermaphrodite or by bombardment of a population of worms with DNA coated microparticles. Both techniques rely on the use of genetic markers to facilitate the recovery of transformed animals and the maintenance of transgenic lines. We developed a nematode expression vector carrying the neomycin resistance gene (neo) as a selection marker. This gene confers resistance to G-418, an antibiotic that normally inhibits protein synthesis in eukaryotes and is lethal for wild-type nematodes. We showed that the neo marker is a potent tool that allows a clear-cut selection of transgenic animals and hands-off maintenance of non-integrated populations on G-418 plates. This system does not imply any prerequisite on the original genotype of the recipient strain and can therefore be used on mutants lines as well as transgenic strains obtained with common markers. Moreover, we placed the neo gene under the control of the C. elegans rps-27 promoter, a highly conserved ribosomal protein throughout the nematode phylogeny. We were able to provide resistance to Caenorhabditis briggsae using this vector; this likely indicates that neo can be used in any species from the Caenorhabditis family. Finally, we demonstrated that this powerful selection system can be used in the context of Mos1 transposon excision-repair methods. Therefore, the neo system offers a wide range of new possibilities for transgenesis both in C. elegans and in other related species. We therefore believe that the benefits of the neo system should contribute to the development of more flexible and efficient techniques for nematode transgenesis.