La pourriture grise, causée par le champignon Botrytis cinerea, est l'une des principales maladies aériennes fongiques sur diverses cultures d’importance agronomique. La diversité génétique de B. cinerea est très forte et la capacité rapide d’adaptation de ce champignon à une pression sélective est également avérée. Ce champignon est ainsi capable de développer des résistances à une grande variété de composés fongicides de synthèse ou d'origine naturelle. Des méthodes alternatives de lutte ont de ce fait été développées ces dernières années : divers agents de lutte biologique (ALB) présentant différents modes d’actions ont été identifiés et pour certains d’entre eux commercialisés pour contrôler B. cinerea. Cependant la durabilité de la lutte biologique est un domaine encore très peu étudié. La perte d'efficacité d'un ALB pourrait résulter de la préexistence d’isolats moins sensibles de pathogènes dans les populations naturelles et/ou de la capacité de l’agent pathogène à produire, sous une pression de sélection continue exercée par l’ALB, des mutants ayant une sensibilité réduite. L'objectif global de la présente étude est d'évaluer le risque potentiel de perte d'efficacité de la lutte biologique vis-à-vis de B. cinerea. Dans cette étude, les efforts ont été concentrés sur la pyrrolnitrine, un antibiotique produit par divers ALBs, dont certains sont efficaces contre B. cinerea. Les objectifs spécifiques de l'étude étaient (i) d’évaluer la diversité de la sensibilité à la pyrrolnitrine au sein de la population naturelle de B. cinerea, (ii) d'estimer le risque de perte d'efficacité des ALBs produisant la pyrrolnitrine due à la pression de sélection exercée par la pyrrolnitrine et (iii) d'étudier le mécanisme de résistance à la pyrrolnitrine chez B. cinerea. Parmi 204 isolats de B. cinerea, une gamme importante de sensibilité à la pyrrolnitrine a été observée, avec un facteur de résistance de 8,4 entre l’isolat le plus sensible et l'isolat le moins sensible. La production de 20 générations successives pour 4 isolats de B. cinerea, sur des doses croissantes de pyrrolnitrine, a abouti au développement de mutants avec des niveaux élevés de résistance à l'antibiotique, et à une réduction in vitro de la sensibilité à la bactérie productrice de pyrrolnitrine Pseudomonas chlororaphis PhZ24. La comparaison entre les mutants résistants à la pyrrolnitrine et leurs parents sensibles pour la croissance mycélienne, la sporulation et l'agressivité sur plantes a révélé que la résistance à la pyrrolnitrine est associée à un fort coût adaptatif. Des observations cytohistologiques sur tomates ont confirmé que l’isolat sensible à la pyrrolnitrine attaque le pétiole rapidement et envahit la tige, alors que le mutant résistant à la pyrrolnitrine ne s'étend pas au-delà du pétiole. De plus, ce dernier mutant forme un mycélium anormal et des cellules ressemblant à des chlamydospores. Les résultats ont d'autre part révélé que les mutants de B. cinerea résistants à la pyrrolnitrine sont résistants au fongicide iprodione, suggérant ainsi qu'une pression exercée par la pyrrolnitrine sur le champignon conduit à une résistance au fongicide. Réciproquement, la production de générations successives sur iprodione conduit à une résistance à l'antibiotique. Afin d'étudier les déterminants moléculaires de la résistance de B. cinerea à la pyrrolnitrine, le gène histidine kinase Bos1, impliqué entre autres dans la résistance aux fongicides chez B. cinerea a été séquencé chez les souches sensibles et les mutants résistants. La comparaison des séquences a mis en évidence des mutations ponctuelles différentes chez les mutants de B. cinerea obtenus sur la pyrrolnitrine et ceux obtenus sur l'iprodione. De plus, les résistances à la pyrrolnitrine et à l'iprodione ne sont pas systématiquement associées à une mutation ponctuelle dans le gène Bos1. Enfin, aucune modification n'a été détectée dans la taille des allèles de neuf locus microsatellites quelle que soit la pression sélective exercée et quelle que soit le phénotype du mutant produit. Cette étude montre qu'un champignon pathogène des plantes est capable de développer progressivement une moindre sensibilité à un agent de lutte biologique mais que cette moindre sensibilité est associée à une forte perte de fitness / Gray mould, caused by Botrytis cinerea, is a severe disease on a wide range of crops. Disease control generally relies on chemicals, although biological control strategies have been intensively studied over the last decades. This pathogen can withstand a wide variety of fungitoxic compounds including fungicides and natural molecules. This capacity to adapt to different stress might, potentially, compromise the durability of biological control methods. The global purpose of that work was to estimate the potential of B. cinerea to overcome the efficacy of biological control agents. Knowledge on the potential development of resistance to biological control agents can help to devise or improve resistance management strategies. In this work, efforts have been focused on the antibiotic pyrrolnitrin produced by various bacteria described as potential biological control agents against B. cinerea. The specific objectives of the study were (i) to evaluate the diversity in susceptibility to pyrrolnitrin among natural population of B. cinerea, (ii) to estimate the risk of loss of efficacy of pyrrolnitrinproducing biological control agent due to selection pressure exerted by pyrrolnitrin and (iii) to study the mechanism of resistance to pyrrolnitrin in B. cinerea. An important range of sensitivity to pyrrolnitrin with an 8.4-fold difference in EC50 values between the most sensitive and the least sensitive isolates was observed within the 204 isolates tested. The production of 20 generations, for 4 isolates of B. cinerea, on increasing doses of pyrrolnitrin, resulted in the development of mutants of B. cinerea with high levels of resistance to the antibiotic and a reduced sensitivity in vitro to the pyrrolnitrin-producing Pseudomonas chlororaphis PhZ24. Comparison of the pyrrolnitrin-resistant mutants and their sensitive parent isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin has resulted in a high fitness cost. Additional cytohistological investigations revealed that while the sensitive isolate spread throughout the petiole and rapidly invaded the stem via the abscission zone, the pyrrolnitrinresistant mutant failed to extend beyond petiole to invade the stem. Moreover, the pyrrolnitrin-resistant mutant formed abnormal mycelium and chlamydospore-like cells. The comparison of resistance to pyrrolnitrin and to the iprodione fungicide in B. cinerea revealed that fungicide pressure exerted on the fungus is able to build-up resistance to pyrrolnitrin. Comparison of sequences of the osmosensing class III histidine kinase encoding gene bos1 revealed different mutations in pyrrolnitrin- and iprodione-resistant mutants. However, resistance to pyrrolnitrin and to iprodione was not systematically associated with a point mutation in the Bos1 gene. Finally, no changes were observed in the allele size at nine microsatellite loci whatever the four selective pressure endured by the fungus despite their phenotypic changes. This study provides evidence that a fungal plant pathogen is able to gradually build-up resistance to an antibiotic produced by a biocontrol agent

Le retrait des nématicides rend urgent la mise au point de méthodes alternatives de lutte contre les nématodes parasites des cultures et la création de variétés résistantes est une voie prometteuse. En vignoble, le nématode Xiphinema index a un impact économique élevé en transmettant le Grapevine fanleaf virus (GFLV), principal virus du court-noué de la vigne et première virose de la vigne à l’échelle mondiale. Des porte-greffe résistants vis-à-vis du vecteur X. index basés sur la source de résistance muscadine (Muscadinia rotundifolia) sont en cours de sélection chez la vigne afin de stopper ou retarder l’infection. Sur cette culture, une étude antérieure avait montré que ce nématode parthénogénétique méiotique est aussi capable de se reproduire (rarement) de façon sexuée. Un travail préliminaire de phylogéographie avait permis de révéler les groupes prédominants de diversité et de sélectionner des populations représentatives pour la création de lignées monofemelles. La durabilité de la résistance doit prendre en compte la diversité du nématode. Dans ce contexte, la thèse a d’abord complété et approfondi l’approche phylogéographique en utilisant une très large gamme d’échantillons originaires de l’aire mondiale de répartition de la vigne. Nos résultats permettent de proposer des hypothèses fortes afin de localiser l’aire native du nématode X. index au Moyen-Orient et de retracer ses itinéraires de dissémination à partir de l’Antiquité. IIs illustrent également le lien étroit depuis cette époque entre la dissémination du nématode et celle de la vigne domestiquée par l’homme. La deuxième partie de la thèse a évalué la durabilité de la résistance de matériel porte-greffe issu de la muscadine en serre (nématodes non virulifères sur plants entre 3 et 6 ans) et en vignoble (nématodes virulifères sur plants âgés de 16 ans). En serre, des accessions résistantes F1 ou BC1, préalablement obtenues à partir d’in vitro ou de boutures ligneuses, ont été inoculées avec un mélange de 4 lignées représentatives, chaque lignée étant traçable avec des marqueurs microsatellites. Nous avons montré que les nématodes issus de plants obtenus par multiplication in vitro surmontent progressivement la résistance tandis que le matériel issu de boutures exprime une résistance durable. La multiplication progressive des nématodes sur le matériel résistant uniquement dans le cas où il est issu d’in vitro écarte a priori l’hypothèse d’une adaptation génétique du nématode. Elle apparaît liée à une architecture différente du système racinaire chez les plants issus de ce type de multiplication, multiplication qui pourrait induire des changements physiologiques discrets mais durables dans les tissus racinaires apicaux à partir desquels les nématodes se nourrissent. Le génotypage des nématodes par microsatellites a permis de détecter un taux bas mais croissant d’individus hybrides entre lignées sur les plants âgés de 4 à 6 ans, ce qui confirme l’aptitude de multiplication sexuée précédemment observée en vignoble. Du fait que l’observation d’individus hybrides apparaît indépendante du type de propagation et du statut de résistance de la plante, nos résultats écartent l’hybridation comme mode d’adaptation du nématode qui serait à même d’expliquer le contournement de la résistance chez les plants issus d’in vitro. En vignoble, après 16 années, les nématodes ont été quasi-impossibles à détecter sur l’accession résistante BC1 qui est également peu affectée par les attaques virales, tandis que des effectifs de nématodes plus élevés ont été retrouvés sur une accession témoin sensible dont les plants sont par contre très majoritairement morts ou en dépérissement. Considérés globalement, nos résultats montrent que la stratégie de résistance basée sur la muscadine apparaît durable. Cette stratégie ciblée sur le nématode vecteur contribuera à réduire significativement l’impact du GFLV transmis par X. index. / The ban of most nematicides renders urgent control alternatives against plant-parasitic nematodes and breeding for resistant plant varieties is promising. In vineyards, the nematode Xiphinema index has a high economical impact by transmitting Grapevine fanleaf virus (GFLV), the main virus of ‘Court-noué’ disease and the first grapevine viral disease worldwide. Resistant rootstocks are being selected in grapevine, using Muscadinia rotundifolia (muscadine) as a resistance source to the vector, in order to arrest or delay GFLV transmission. In this crop, a previous study had shown that this meiotic parthenogenetic nematode is able to reproduce sexually (rarely) in the field. A preliminary phylogenetic work had allowed to reveal the predominant diversity groups and to select representative populations for the creation of single-female lines. Resistance durability is a real challenge that must consider the key information of the nematode diversity. In this context, the PhD project first completed and deepened our phylogeographical approach using an extended geographic coverage of the worldwide nematode distribution. Our results allow proposing strong hypotheses to locate the native area of X. index in the Middle-East and trace its dissemination routes from the Antiquity. They also highlight the close link since this epoch between dissemination of the nematode and domesticated grapevine by man. The second part of the PhD project has then evaluated the durability of muscadine-derived rootstock material in greenhouse (non viruliferous nematodes on plants aged 3 to 6 years) and field (viruliferous nematodes on plants aged 16 years) conditions. In the greenhouse, F1 and BC1 resistant accessions, previously obtained from both in vitro and hardwood-cutting propagation, were inoculated with 4 mixed representative X. index lines, traceable each with microsatellite markers. We showed that nematodes from plants obtained from in vitro progressively overcame the resistance while the material obtained from cuttings displayed a durable resistance. Nematode progressive multiplication in resistant accessions obtained only from in vitro removes a priori the hypothesis of a nematode genetic adaptation and appears linked to a different architecture of the root system in this propagation type. This type may have induced discrete but durable physiological changes in apical root tissues from where nematodes feed. Nematode microsatellite genotyping allowed detecting a low but increasing rate of hybrid individuals from 4 to 6 years, which confirms data from the vineyard. As the hybrid occurrence appears independent from the propagation type and the resistance status of the plant, our data discard hybridization as the mode of adaptation of the nematode underlying resistance breakdown from in vitro plants. In field conditions, after 16 years, nematodes were almost undetectable on the resistant BC1 accession, also almost unaffected by the viral attacks, while higher numbers were detected on a susceptible control accession, whose plants were by contrast in high majority dead or poorly vigorous. Taken all together, our results show that the muscadine-derived resistance strategy appears durable. This strategy focused on vector control will significantly contribute to reduce the impact of GFLV transmitted by X. index.

Doctor of Philosophy / Department of Biology / Philip S. Gipson / Understanding factors that influence genetic variability, demographic vital rates, and resource selection is important for conservation and management of wildlife populations. I examined factors influencing microsatellite variability, demographic vital rates, and habitat use for a reintroduced elk (Cervus elaphus) population at Fort Riley, Kansas based on data collected from 2003 – 2007. Levels of allelic richness, observed heterozygosity, and expected heterozygosity for the Fort Riley population were intermediate to other North American elk populations. Genetic variability in restored North American elk populations was not well explained by founding population size, number of founding populations, or number of years since the last translocation. I examined the influence of demographic vital rates on the rate of population change to test the hypothesis that variability in calf survival has a greater influence on rates of population change than adult survival. Survival for prime-age adult elk had the highest stage-specific elasticity value, but life-stage simulation analysis indicated that variation in calf survival had the highest correlation with variation in population growth rate. These results suggest that calf survival varies temporally and is the vital rate most directly related to variation in population growth rate for this population. I assessed the relative influence of risk-related and resource-related factors on elk habitat selection by comparing predictor variables included in top resource selection function models at the landscape and home range scales. All predictor variables, with the exception of fall and spring prescribed burns, were included in top models across seasons at both spatial scales. Elk selected low elevation areas, gentle slopes, edge habitat, and areas close to streams at both spatial scales. At the landscape scale, elk generally avoided roads and preferred areas on or near Fort Riley. At both spatial scales, elk used riparian woodlands more frequently than grasslands and selected for agricultural crops when seasonally available. These findings do not support the idea that risk-related factors are the primary determinant of elk habitat use at the landscape scale as has been found for ungulates in areas with natural predators.

Increasing human pressure combined with sea level rise and increased storminess is threatening coastal ecosystems around the world. Among these ecosystems, saltmarshes are particularly endangered due to their position in temperate areas with low wave action where human density is often high (e.g. estuaries). Around the UK, centuries of land reclamation have led to a substantial decrease of the area of saltmarsh. Over the past decades, restoration schemes have been implemented in numerous coastal locations in an attempt to counteract this loss. Such schemes involve allowing sea water to inundate a previously embanked area and letting the vegetation develop naturally, thereby reverting to saltmarsh through natural colonisation. However, surveys of restored areas that have looked at the recovery of plant species diversity or functional characteristics often show that restored saltmarshes do not reach the state of a natural saltmarsh ecosystem. While there is much data at the species level, recovery of plant intra-specific diversity (genetic diversity) has not been assessed in restored saltmarsh although this component of biodiversity is receiving increasing attention for its effect on ecosystem function. This thesis represents the first attempt to (1) characterize the nation-wide genetic structure of two important north-west European saltmarsh plant species, the common saltmarsh grass (Puccinellia maritima) and the sea arrowgrass (Triglochin maritima) and (2) compare levels of genetic diversity and structure between restored and natural ecosystems. Microsatellite molecular markers were developed for both species. Using innovative methods to analyse the genetic data obtained for these two polyploid species, this thesis highlights that genetic diversity at the national scale is organised regionally for both species, although gene-flow is still restricted between populations within the same region. Gene-flow between populations is determined by different processes depending on the species. While coastal processes mainly influence gene dispersal in P. maritima, overland routes of dispersal are involved for T. maritima. These differences are believed to be due to differences in dispersal ecology between the two species. Although gene-flow exists between distant saltmarshes, the genetic analysis of P. maritima and T. maritima colonists arriving on restored sites highlighted their local origin and reaffirmed that it is preferable to restore saltmarsh where a nearby natural saltmarsh can act as a source of colonists. A multiple paired-site comparison identified similar genetic diversity between restored and natural saltmarshes indicating that restoration of local genetic diversity is rapid for both species. A single site comparison at Skinflats in the Forth estuary compared fine-scale spatial genetic structure between the restored and natural saltmarsh. Interestingly, no structure was detected for T. maritima either in restored or natural saltmarsh. In contrast, a strong genetic structure organised along the elevation gradient was observed in the natural saltmarsh for P. maritima but was absent in the restored saltmarsh. The origin of this structure is not clear but could be due to restricted gene-flow between individuals from different elevations due to strong post-zygotic selection, as suggested in previous work. In any case, this lack of structure in the restored saltmarsh indicates that genetic recovery is incomplete in this respect for P. maritima. This thesis introduces the growing field of restoration genetics to saltmarsh ecology and identifies the principal population genetic trends in two of the species dominating the vegetation of north-west European saltmarshes community. The information given here will be useful for restoration practitioners and provides a strong foundation for future work characterizing the importance of genetic diversity for saltmarsh function.

Une distribution nationale de moustiquaires imprégnées d'insecticide à longue durée d'action à destination des populations vulnérables du Niger a été effectuée fin 2005. Déjà montrée lors d'études pilotes à l'échelle du village, l'efficacité de cet outil dans le contrôle du paludisme restait à évaluer à l'occasion de vastes programmes opérationnels qui se multiplient en Afrique. Peu d'études des populations de vecteurs ont été publiées dans ce cadre. Nous avons mis en place un suivi entomologique au niveau de sites sentinelles répartis dans la zone Sahélienne du Niger, ayant couvert trois saisons de transmission, dont une avant intervention considérée comme période contrôle. Les paramètres entomologiques de la transmission ont été déterminés pour An. gambiae s.l., et la distribution spatiale des deux principaux vecteurs, An. gambiae et An. arabiensis, a été précisée. Le suivi temporel a mis en évidence une baisse globale du niveau de transmission de P. falciparum, probablement entrainée par la forte hausse d'utilisation de moustiquaires imprégnées. Cependant la hausse de la résistance des populations aux pyréthrinoïdes semble avoir été rapidement amorcée faisant craindre à moyen terme une perte d'efficacité de cet outil central des stratégies de lutte contre le paludisme. L'étude de la structure génétique des populations d'An. gambiae et d'An. arabiensis à l'aide de marqueurs microsatellites a montré une homogénéité génétique dans l'espace, entre les villages, même séparés par plusieurs centaines de kilomètres, ainsi que dans le temps, entre la saison de transmission 2005 contrôle et la saison 2006 après distribution. Ces résultats ont suggéré qu'au cours de la première année d'intervention, la couverture en moustiquaires imprégnées atteinte n'a pas eu d'effet de masse suffisant pour entrainer une baisse de la diversité génétique ou une modification des fréquences alléliques des populations. La faible différenciation spatiale observée pourrait être expliquée par des échanges de gènes importants à l'intérieur de la zone d'étude, hypothèse appuyée par l'expansion rapide de la mutation kdr dans l'ensemble des sites où An. gambiae est présent. L'évaluation rigoureuse de tels programmes de contrôle permettra d'améliorer les outils de contrôle et par exemple de préserver l'efficacité des pyréthrinoïdes, seule classe d'insecticides actuellement disponible pour l'imprégnation des moustiquaires.

Les microsatellites, séquences répétées en tandem de période une à six paires de bases, sont des entités génomiques présentes dans tous les organismes qu'ils soient animaux, végétaux ou microbiens. Ils présentent un cycle de vie caractérisé par trois phases principales : une apparition et une maturation, une dynamique à l'état mature, puis une dégénérescence. Nous nous intéressons dans cette thèse à la première phase, l'apparition des microsatellites. <br />Pour traiter ces questions, nous nous sommes basés sur l'analyse de la séquence du génome humain. L'une des lacunes de ce type d'analyse est qu'il faut d'abord extraire les microsatellites du génome, et qu'il existe plusieurs algorithmes de nature et fonctionnement différents. La première partie de cette thèse se concentre donc sur la comparaison de quelques-uns des principaux algorithmes de recherche de répétitions en tandem, et dresse un portait des différentes qualités et limitations de chacun des algorithmes.<br />Deux possibilités majeures sont détaillées, l'apparition par l'intermédiaire d'éléments transposables (ETs), et l'apparition spontanée à partir d'une séquence quelconque. Dans le premier cas, l'étude est focalisée sur le rôle des queues polyA des séquences Alu chez les primates. La question de l'apparition à partir d'une séquence quelconque cherche à établir l'impact de trois mécanismes mutationnels différents sur la création et le développement primordial des microsatellites : la mutation ponctuelle, le glissement de polymérase et la micro-duplication adjacente de quelques nucléotides. Un modèle général d'apparition des microsatellites est aussi proposé, suggérant une dynamique d'apparition plus complexe que ce qui était précédemment supposé.

All-male fry are preferred to prevent uncontrolled reproduction before harvest in intensive Nile tilapia (Oreochromis niloticus) aquaculture. Males also grow faster than females. An alternative approach to direct hormonal masculinisation of tilapia fry is to produce fry that are genetically male. However, sex determination system in tilapia is fairly complex. Recent developments have resulted in a linkage map and genetic markers that can be used to analyse the sex determination system. To analyse the genetic sex determination mechanism and to develop marker-assisted selection in the Stirling Nile tilapia population, a fully inbred line of clonal females (XX) was verified using test crosses and DNA markers (mostly microsatellites) to use as a standard reference line in sex determination studies. A series of crosses were performed involving this line of females and a range of males. Three groups of crosses were selected (each group consisted of three families) from progeny sex ratio distributions, and designated as type ‘A’ (normal XY males x clonal XX females), type ‘B’ (putative YY males x clonal XX females) and type ‘C’ (unknown groups of males x clonal XX females), for sex linkage study. For type ‘A’, inheritance of DNA markers and phenotypic sex was investigated using screened markers from tilapia linkage group 1 (LG1) to confirm the LG1-associated pattern of inheritance of phenotypic sex and the structure of LG1. Screened markers from LG1, LG3 and LG23 were used to investigate the association of markers with sex in families of type ‘B’ and ‘C’. In addition, a genome-wide scan of markers from the other 21 LGs was performed to investigate any association between markers and sex, in only families of cross type ‘B’. LG1 associated pattern of inheritance of phenotypic sex was confirmed by genotype and QTL analyses in families of cross type ‘A’. Analyses of genotypes in families of type ‘B’ and ‘C’ showed strong association with LG1 markers but no association with LG3 and LG23 markers. Genome wide scan of markers from all other LGs did not show any significant association between any markers and the sex. The allelic inheritance of two tightly linked LG1 markers (UNH995 and UNH104) in families of type ‘B’ and ‘C’ identified polymorphism in the sex determining locus: one of the alleles was associated mostly with male offspring whereas another allele was associated with both progeny (mostly males in type ‘B’ families, and approximately equal numbers in type ‘C’ families). This knowledge was used to identify and separate supermales (‘YY’ males) that should sire higher proportions of male progeny, reared to become sexually mature for use as broodstock. Two of them were crossed with XX females (one clonal and one outbred) to observe the phenotypic expression of the strongest male-associated allele in progeny sex. The observations of 98% male (99 males out of 101 progeny) and 100% male (N=75) from these two crosses respectively, suggest that a marker-assisted selection (MAS) programme for genetically male Nile tilapia production could be practical. This study also suggests that the departures from the sex ratios predicted using a “simple” XX/XY model (i.e., YY x XX should give all-male progeny) were strongly associated with the XX/XY system, due to multiple alleles, rather than being associated with loci in other LGs (e.g., LG3, LG23). This study also tentatively names the allele(s) giving intermediate sex ratios as “ambivalent” and emphasizes that the presence and actions of such allele(s) at the same sex-determining locus could explain departures from predicted sex ratios observed in some earlier studies in Nile tilapia.

I examined the population genetic structure of three known subspecies of Glaucomys sabrinus from Appalachia, Washington State, and two previously unexamined populations from Mount Rogers National Recreation Area (MRNRA) in Southwestern Virginia. Mean FST (0.107) and an AMOVA (P G. sabrinus subspecies populations in the southern Appalachians are genetically differentiated. Glaucomys sabrinus at MRNRA were less inbred than expected. Gene flow, a consensus tree based on Nei's genetic distance, elevated heterozygosity and morphometric data suggest that the MRNRA G. sabrinus population is an intergrade of the two recognized Appalachian subspecies, G. s. fuscus and G. s. coloratus. I compared inbreeding and the level of parasite infestation in the two MRNRA populations of G. sabrinus and found that Whitetop Mountain (150 ha habitat) was more inbred than the population on Mount Rogers (400 ha habitat, P Strongyloides robustus were greater in the more fragmented Whitetop Mountain population, although the difference was not statistically significant (P= 0.278). A Mantel comparison of genetic diversity and parasite infestation among individuals did show a highly significant negative correlation (P G. sabrinus form a unique insular population with high genetic diversity that is nonetheless susceptible to increased inbreeding, and elevated parasitism caused by fragmentation. MRNRA G. sabrinus should retain endangered species status.

Resumo: O Brasil é o país com o maior rebanho de búfalo de rio, Bubalus bubalis, no continente americano e também o maior produtor deste animal fora do continente asiático. A produção de leite e derivados vem aumentando devido à potencialidade dessa espécie em produzir leite com baixos custos e elevado rendimento industrial. Apesar das características economicamente importantes inerentes ao búfalo, as pesquisas científicas são limitadas em muitos países onde o búfalo é economicamente importante e como conseqüência a pesquisa genômica encontrase defasada quando comparado com outras espécies de interesse econômico. Marcadores moleculares são essenciais para avaliar informações qualitativas e quantitativas da diversidade molecular com o objetivo de aperfeiçoar a utilização e conservação da variabilidade genética e o relacionamento entre vários rebanhos. Microssatélites diferem dos outros tipos de seqüências de DNA por seu extenso polimorfismo dentro e entre populações, sendo considerados excelentes tipos de marcadores moleculares. O presente estudo teve como objetivos construir bibliotecas genômicas parciais enriquecidas com microssatélites, isolar e caracterizar os microssatélites obtidos, além de determinar as condições de amplificação por PCR para alguns dos locos isolados da biblioteca. Foram desenvolvidas 6 bibliotecas genômicas parciais - (CA)15 , (CT)15 , (AGG)8 , (GATA)8 , (GAAA)8, (AAAAC)8. O processo de clonagem gerou um total de 1.824 clones recombinantes, sendo que 954 foram seqüenciados para identificação de microssatélites. Cento e treze microssatélites foram encontrados. Desses, foram identificados 96 com unidade de repetição dinucleotídica (84,95%), 10 repetições trinucleotídicas (8,85%), 6 repetições tetranucleotídicas (5,3%) e 1 repetição pentanucleotídica (0,89%). As seqüências de microssatélites... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Brazil is the largest river buffalo (Bubalus bubalis) breeding center outside the Asian continent, the origin of the domestic buffalo. All buffalo breeds have a strong milk/meat attributes. Their extensive use in agriculture world wide, and especially in developing countries, begs for genetic resources to evaluate and improve traits important to local and regional economies. Among the different types of DNA markers, microsatellites are useful for studying genetic variability within and between populations due its high heterozygosity. The goal of this study was to construct partial genomic libraries enriched with repeated sequences to isolate and characterize microsatellites for river buffalo. The cloning process generated a total of 1824 recombinant clones, from which 954 were sequenced for the microsatellites search. One hundred and thirteen new microsatellites were isolated, containing the following type of repeats: dinucleotide repeats (96 sequences - 84.95%), trinucleotide repeats (10 sequences - 8.85%), tetranucleotide repeats (6 sequences - 5.3%) and pentanucleotide repeats (1 sequence - 0.89%). The new microsatellites were structurally categorized into 3 categories: pure repeats (90 sequences - 79.64%), pure interrupted (21 sequences - 18.59%) and compound interrupted repeats (2 sequence - 1.77%). PCR primer pairs were designed for ten microsatellites, from which four had the PCR conditions optimized. The microsatellites isolated in this study will be used to evaluate the genetic variability of Brazilian populations of river buffalo and to the gene mapping program established for this specie. / Orientador: Maria Elisabete Jorge Amaral / Coorientador: Artur Luiz da Costa da Silva / Banca: Vera Fernanda Martins Hossepian de Lima / Banca: Sandra Regina de Carvalho Marchesin / Mestre

A doença vassoura-de-bruxa do cacaueiro (Theobroma cacao), causada pelo basidiomiceto Crinipellis perniciosa, levou a total destruição da lavoura sul-baiana, previamente cultivada principalmente com variedades altamente suscetíveis, tornando o Brasil, um país tipicamente exportador de cacau em importador em poucos anos. A perda da resistência do genótipo ?Scavina 6?, única fonte de resistência reconhecida contra C. perniciosa, está associada à variabilidade genética do patógeno. Diante disto, o objetivo deste estudo foi caracterizar a diversidade genética de isolados de C. perniciosa derivados de tecido infectado de Theobroma cacao (vassoura vede), originalmente coletados na Amazônia (Amazonas, Pará e Rondônia), uma região de ocorrência endêmica do fungo, usando marcadores moleculares. A identificação de relações genéticas entre isolados amazônicos e da Bahia e a possível existência de isolados geográficos também foram objetos deste trabalho. Primeiramente, a confirmação de identidade dos isolados Amazônicos foi conduzida usando amplificação e digestão da região ITS do rDNA e marcadores teloméricos. Todos os isolados avaliados foram confirmados como C. perniciosa. O primer telomérico TeloC1, previamente apresentado para discriminar o biótipo C, permitiu a separação do biótipo C dos biótipos S e L, porém, revelou variabilidade genética nos isolados de Cametá, PA e Cacaulândia, RO. Usando marcadores telomérico amplificado com o primer TeloA1R e ERIC, uma grande diversidade foi encontrada para os isolados da Região Amazônica em comparação àqueles da Bahia. Dentro dos isolados Amazônicos, a maior diversidade foi detectada para os isolados de Rondônia (Ji-Paraná, Cacaulândia e Ariquemes) e Pará (Cametá), áreas com 11 ocorrência endêmica ou de instalação histórica (mais de 300 anos) do cacaueiro, respectivamente. Isolados coletados em municípios localizados na regiãoTransamazônica, tais como Anapú, Altamira, Brasil Novo, Medicilândia e Uruará apresentaram maior similaridade com aqueles de Santarém e municípios relacionados à Belém, como Cametá, Baião e Mocajuba. Estes resultados sugerem que isolados da região Transamazônica pode ter sido originados de Belém ou Santarém, Pará. Na Bahia, houve a formação de dois grupos de isolados como previamente demonstrado. Os marcadores moleculares Microssatélites, ERIC e teloméricos foram eficientes na detecção da variabilidade genética em C. perniciosa. A diversidade genética observada auxiliará na identificação e escolha de regiões com maior diversidade de isolados para serem usados na seleção para resistência à vassoura-de-bruxa em programas de melhoramento do cacau / Witches broom disease of cacao (Theobroma cacao L.), caused by the basidiomycete Crinipellis perniciosa, devastated the producing region of Southern Bahia, previously cultivated mainly with highly susceptible cultivars, forcing Brazil, a typical exporter country to become a cocoa importer. The loss of resistance of the genotype ?Scavina 6?, the unique source of resistance against C. perniciosa has been associated with pathogen genetic variability. Thus, the objective of this study was to characterize the genetic diversity of C. perniciosa isolated derived from infected tissues of T. cacao (green-brooms), originally collected in the Amazon (Amazonas, Pará and Rondônia states), a region with endemic occurrence of the fungus, using molecular markers. The identification of the genetic relationships among the Amazonian and Bahian isolates, and the possible existence of geographical isolates were also objectives of this work. First, the identification confirmation of the Amazonian isolates was conducted using amplification and digestion of the ITS region of the rDNA and telomeric markers. All isolates evaluated were confirmed as C. perniciosa. The telomeric primer TeloC1, previously shown to discriminate the C biotype, allowed the separation of biotype C from biotypes S and L, but it revealed genetic diversity for isolates from Cametá, PA and Cacaulândia, RO. Using another telomeric marker amplified with TeloA1 primer and ERIC, a large genetic diversity was detected for isolates from the Amazon in comparison to Bahian. Within the Amazonian isolates, more diversity was detected for isolates from Rondônia (Ji-Paraná, Cacaulândia and Ariquemes) and Pará (Cametá), areas with endemic occurrence of wild cacao or historical introduction and cultivation (over 300 years), respectively. Isolates colletected at the Transamazônica roadway, such as from Anapú, Altamira, Brasil Novo, Medicilândia and Uruará presented more similarity with those from Santarém and locales nearer to Belém, such as Cametá, Baião and Mocajuba. These results suggested that isolates from the Transamazônica region might have originated from Belém or Santarém, Pará. In Bahia, there were two groups of isolates as previously demonstrated. Microsatellite, ERIC and telomeric markers were efficient in detecting the genetic variability of C. perniciosa. The genetic diversity observed will help in identifying and choosing regions with more diverse isolates to be used to screen for witches? broom resistance in cacao breeding programs