Submitted by (edna.saturno@ufrpe.br) on 2017-02-13T14:39:22Z No. of bitstreams: 1 Hozane Leite Dantas.pdf: 728024 bytes, checksum: e578bad7a4f79d4fe9d1abd9c3ccc3d9 (MD5) / Made available in DSpace on 2017-02-13T14:39:22Z (GMT). No. of bitstreams: 1 Hozane Leite Dantas.pdf: 728024 bytes, checksum: e578bad7a4f79d4fe9d1abd9c3ccc3d9 (MD5) Previous issue date: 2010-02-09 / The São Francisco River is 2700 km long, draining areas of the States of Minas Gerais, Goias, Bahia, Pernambuco, Alagoas, and Sergipe, as well as part of the Federal District. Before the construction of Paulo Afonso Hydroeletric Power Plant, waterfalls of Paulo Afonso (north-eastern Brazil) and its respective lakes already constituted a natural obstacle to fish migration. Nowadays, the construction of hydroelectric dams between medium and submedium São Francisco River associated flooding along rivers, pollution and the introduction of exotic species can alter ecosystems and pose serious threats to native aquatic species, especially those that perform migration for reproduction. One of the most threatened fish migratory species is the Pseudoplatystoma corruscans, a catfish species, locally named surubim. The surubim is the first predator of São Francisco River and it is considered the most appreciated freshwater fish on the north-eastern Brazil. The conservation of freshwater fish species has traditionally been done thought restocking programs. Genetic variability is essential to maintain the capability of restocked fish to adapt to a changing environment. Therefore, if long term persistence of species is the goal that conservation biology is intending to achieve, genetic variability is of the utmost importance to preserve. One approach that have been successfully used in uncovering cryptic population structure in freshwater is microsatellite markers. Samples from three populations were analyzed for six different microsatellite markers. PCR products were separated by agarose gel electrophoresis and stained with silver nitrate. The results showed a wide diversity of alleles for all populations, where a total of 47 alleles were found. The expected heterozygosities showed an average of 0.7498 for the population of Remanso, 0.7142 for the population of Três Marias and 0.7372 for the population of Bom Jesus da Lapa. The average observed heterozygosity were 0.7170, 0.6643 and 0.7132 for the populations of Remanso, Três Marias and Bom Jesus da Lapa respectively. All populations showed loci in deviation from Hardy-Weinberg, but locus Pcor2 was the only one who showed deviation in all populations. The average values of FIS was 0.0348 in Remanso, 0.0583 in Três Marias and 0.0160 in Bom Jesus da Lapa. The fixation index FST obtained was 0.0246, indicating a small degree of differentiation between the populations studied. The universe of alleles shared among populations associated with the low value of FST indicates that this is a panmitic population. These results indicated that breeders can be São Francisco River basin, saving time in restocking programs in terms of growing fish. Hence, this study can now be used as a parameter for the construction of the founder stock to be used in restocking programs or in evaluating the genetic structure of existing stocks founders, as well as monitoring offspring from these programs. / O rio São Francisco apresenta 2700 km de extensão, percorre cinco estados brasileiros e é conhecido como “rio da integração nacional”. Construção de hidrelétricas associadas a outros fatores comprometem os peixes nesse ambiente, principalmente aqueles que efetuam piracema. Dentre esses se destaca o Pseudoplatystoma corruscans, conhecido como surubim ou pintado em algumas regiões, que vêm sofrendo com as problemáticas existentes nesta bacia comprometendo seriamente seus estoques naturais, especialmente na região do Submédio São Francisco. Diante disto se faz necessário investimentos em programas de propagação artificial para a recuperação deste recurso que mantenham a diversidade genética local. O presente trabalho objetivou avaliar a estrutura genética de populações selvagens de surubim do rio São Francisco, através da utilização de marcadores moleculares de microssatélite. Amostras de três populações foram analisadas para seis diferentes marcadores de microssatélite. Os produtos de PCR foram separados por eletroforese em gel de poliacrilamida e corados com nitrato de prata. Os resultados mostraram uma ampla diversidade de alelos para todas as populações sendo encontrados 47 alelos no total. As heterozigosidades esperadas apresentaram média de 0,7498 para a população de Remanso, 0,7142 para a população de Três Marias e 0,7372 para a população de Bom Jesus da Lapa. As médias da heterozigosidade observada foram 0,7170, 0,6643 e 0,7132 para as populações de Remanso, Três Marias e Bom Jesus da Lapa respectivamente. Todas as populações apresentaram loci em desvio do Equilíbrio de Hardy-Weinberg. Os valores médios de FIS encontrados foram 0,0348 para Remanso, 0,0583 para Três Marias e 0,0160 para Bom Jesus da Lapa. O índice de fixação FST obtido foi de 0,0246, indicando um pequeno grau de diferenciação entre as populações estudadas. O universo de alelos compartilhados entre as populações estudadas associados ao baixo valor do FST indicam que se trata de uma população panmítica. Com esse resultado, reprodutores poderiam ser capturados ao longo de toda a extensão do São Francisco reduzindo o tempo de manejo em programas de repovoamento. Este trabalho caracterizou as populações selvagens quanto a sua variabilidade genética, podendo agora ser utilizado como paramêtro para a construção do estoque fundador que será utilizado em programas de repovoamento ou na validação de estoques fundadores já existentes, bem como no monitoramento das progênies desses advindos desses programas.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-16T16:50:10Z No. of bitstreams: 1 Miguel Arcanjo dos Santos Neto.pdf: 268842 bytes, checksum: ffc97fe7f597d810577265479d03c8e2 (MD5) / Made available in DSpace on 2017-02-16T16:50:10Z (GMT). No. of bitstreams: 1 Miguel Arcanjo dos Santos Neto.pdf: 268842 bytes, checksum: ffc97fe7f597d810577265479d03c8e2 (MD5) Previous issue date: 2008-07-29 / The surubim, Pseudoplatystoma corruscans, is one of the most important native fish species in South American hydrographic basins, especially in São Francisco River, where it is considered a top chain predator. Different aspects have contributed to the decline of this species populations, especially at the submedium São Francisco, thus raising the necessity of a restocking program to recover the resource. A basic principle in such programs is that a large number of unrelated wild fish should ideally be used as founders, when possible or, alternatively, a small number of unrelated wild fish with few genetic relatedness could be used. Therefore, the genetic structure of the founder stock constitutes an essencial information to the success of such programs. This project aimed to evaluate the genetic structure of the founder stock of the restocking program of surubim maintained by Chesf in Paulo Afonso city, through the use of microsatellite molecular markers. DNA of 80 breeders were genotyped for 5 different microsatellite markers. PCR products were separated by polyacrylamide gel electrophoresis stained with silver nitrate. Statistical analysis were carried out using GENEPOP software, in which parameters such as number of alleles (A), observed (Ho) and expected heterozygosities (He), Hardy-Weinberg deviation, linkage disequilibrium and inbreeding coefficient (FIS) were calculated. GenAlEx 6.1 was used to calculate the number of effective alleles and relatedness coefficient (rxy). Results showed that the number of alleles ranged between from 6 to 18 and the number of effective alleles, from 3.28 to 9.25. Average observed and expected heterozygosities were 0.63 and 0.84, respectively. All loci, except Pcor5, showed deviation from Hardy-Weinberg equilibrium (P<0,001). Average inbreeding coefficient (FIS) for the 5 loci was 0.24. The analysis of linkage disequilibrium, when corrected by Bonferroni, were significant for loci Pcor21 and Pcor10 (P<0.05). The average relatedness coefficient (rxy) was -0.008 for a total number of 2926 possible pairwise combinations. A great genetic diversity, expressed in number of alleles and in a low value of average relatedness, was obtained despite a deficit of heterozygotes was observed in this stock. Such deficit seems to reflect the strategy adopted in the composition of this stock, in which individuals caught at the medium and lower São Francisco River, separated by two dams, were mixed together. It is feasible to conclude that this founder stock retained genetic diversity comparable to those found in the wild stocks and could be used in a restocking program. / Pseudoplatystoma corruscans (surubim) é um peixe encontrado nas principais bacias hidrográficas sul-americanas, sendo considerado o primeiro predador da bacia do Rio São Francisco. Diversos fatores vêm comprometendo seriamente as populações de surubim no São Francisco, especialmente no submédio, despertando a necessidade de se investir em programas de propagação artificial que recuperem este recurso. Uma premissa básica de tais programas é utilizar um grande número de indivíduos selvagens como estoque fundador, quando possível, ou alternativamente, um número menor de indivíduos selvagens, mas com pouca relação genética de parentesco. A estrutura genética do plantel de fundadores constitui, portanto, uma informação essencial para o sucesso de um programa de repovoamento. O presente trabalho objetivou avaliar a estrutura genética do plantel de fundadores do programa de repovoamento do surubim, a ser desenvolvido pela Chesf em Paulo Afonso, através da utilização de marcadores moleculares de microssatélite. DNA de 80 reprodutores foram analisados para 5 diferentes marcadores de microssatélite. Os produtos de PCR foram separados por eletroforese em gel de poliacrilamida corados com nitrato de prata. As análises estatísticas foram realizadas utilizando-se o programa computacional GENEPOP, onde parâmetros como número de alelos (A), heterozigosidades observada (Ho) e esperada (He), desvio do equilíbrio de Hardy-Weinberg, desequilíbrio de ligação e coeficiente de consangüinidade (FIS) foram calculados. O programa GenAlEx 6.1 foi utilizado para calcular o número de alelos efetivos e o estimador de relação genética (rxy). Os resultados mostraram que o número de alelos variou de 6 a 18 e o de alelos efetivos (Ae), de 3,28 a 9,25. As heterozigosidades médias observadas e esperadas encontradas foram de 0,63 e 0,84, respectivamente. Todos os loci, a exceção do Pcor5, mostraram desvio do equilíbrio de Hardy-Weinberg (P<0,001). O coeficiente de consangüinidade (FIS) médio calculado para os 5 loci foi de 0,24. As análises de desequilíbrio de ligação, quando corrigidas pelo método de Bonferroni, mostraram-se significativas apenas para os loci Pcor21 e Pcor10 (P<0,05). O valor médio do coeficiente de relação genética (rxy) foi de -0,008 para um total de 2926 combinações de possíveis casais. Uma grande diversidade alélica caracterizou o estoque fundador e, muito embora um déficit de heterozigotos tenha sido registrado, uma baixa relação de parentesco foi encontrada para o plantel em questão. Tal déficit parece refletir a estratégia de composição deste plantel em que indivíduos, oriundos do médio e do baixo São Francisco, separados por duas barragens, foram unidos. É possível concluir que o plantel fundador manteve uma diversidade alélica comparável àquela encontrada em estoques selvagens e poderá ser usado em um programa de repovoamento.

Submitted by DANILLA CRISTINA LEMOS SOUZA null (danillacristina@ig.com.br) on 2017-03-23T12:18:23Z No. of bitstreams: 1 TESE - Danilla Souza com ficha.pdf: 2474993 bytes, checksum: 4fede4ee91d0e6d92b69f3cdf397643c (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-03-23T14:55:55Z (GMT) No. of bitstreams: 1 souza_dcl_dr_bot.pdf: 2474993 bytes, checksum: 4fede4ee91d0e6d92b69f3cdf397643c (MD5) / Made available in DSpace on 2017-03-23T14:55:55Z (GMT). No. of bitstreams: 1 souza_dcl_dr_bot.pdf: 2474993 bytes, checksum: 4fede4ee91d0e6d92b69f3cdf397643c (MD5) Previous issue date: 2017-02-16 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Myracrodruon urundeuva (Anacardiaceae) é uma espécie arbórea de ampla distribuição geográfica. Por possuir madeira de reconhecido valor econômico, associada à alta taxa de fragmentação dos seus habitats, encontra-se ameaçada de extinção. Com o intuito de manter a variabilidade genética existente das populações fragmentadas e reduzir perdas na base genética da espécie, sementes de polinização aberta de M. urundeuva foram coletadas em diferentes regiões, no Brasil, para formação do banco de conservação ex situ da Faculdade de Engenharia de Ilha Solteira, em Selvíria-MS. Esse trabalho foi realizado com o objetivo de caracterizar o sistema de reprodução e estimar parâmetros genéticos populacionais em progênies de M. urundeuva, procedentes de diferentes biomas brasileiros, por meio de caracteres quantitativos de crescimento e marcadores moleculares do tipo microssatélites. Os caracteres altura, diâmetro a altura do peito (DAP), diâmetro médio de copa e sobrevivência (SOB) foram utilizados para avaliar cinco populações: Aquidauana-MS, Selvíria-MS, Itarumã-GO, Paulo de Faria-SP e Seridó-RN. Os valores altos a medianos observados para SOB (96,4% - 70,8%) indicam boa adaptação das populações de M. urundeuva e potencial para uso em reflorestamentos. As estimativas do coeficiente de variação genética oscilaram de 3% a 24,6%, em nível de indivíduo, e de 1,5% a 12,3%, entre progênies, com os maiores valores obtidos para o DAP da população de Seridó. As herdabilidades individuais variaram de moderada (0,41) a baixa (0,01) para os caracteres apresentados. Empregando-se o valor genético dos indivíduos, o DAP foi utilizado para estimar ganhos genéticos na seleção de indivíduos dentro de progênies, sendo a população de Itarumã a mais indicada ao melhoramento genético, no referido ano de avaliação destas populações (população de Itarumã aos 11 anos de idade). Para análise molecular, 20 locos microssatélites foram desenvolvidos, a partir do sequenciamento de nova geração (MiSeq Sequencing System, Illumina), e seis desses foram utilizados para caracterizar as populações de Aquidauana, Itarumã, Paulo de Faria e Seridó (1.224 indivíduos). O número total de alelos (215), o número de diferentes alelos (67) e os valores de heterozigosidades observados demonstraram que estas populações ainda conservam índices de diversidade genética consideravelmente altos. Foi detectada alta diferenciação genética entre as populações (33%), relacionada, em partes, à distância que as separam; e foi confirmado que a espécie dioica M. urundeuva reproduz-se por cruzamento. Estes cruzamentos não são aleatórios nas populações, com aumento do grau de parentesco nas populações que sofreram forte pressão antrópica. Os coeficientes de coancestria dentro de progênies foram superiores ao esperado em progênies de meios-irmãos (0,125), portanto, estas progênies são constituídas também de irmãos-completos. Visando à utilização em reflorestamentos, estratégias de coleta de sementes devem ser adotadas, de modo a evitar a obtenção de sementes advindas de cruzamentos correlacionados ou endogâmicos, estabelecendo-se o valor mínimo de coleta em 55 árvores matrizes, em cada população, de modo a garantir um tamanho efetivo de população de 150. As informações obtidas com este estudo são úteis para fins de conservação e melhoramento genético da espécie, como também para seleção de matrizes potenciais para recuperação ambiental. / Myracrodruon urundeuva (Anacardiaceae) is a tree species with a wide geographic distribution. Because it has wood of recognized economic value, associated with the high rate of fragmentation of its habitats, it is threatened with extinction. In order to maintain the existing genetic variability of the fragmented populations and reduce losses in the genetic base of the species, M. urundeuva seeds were collected in different regions in Brazil for the formation of the ex situ conservation bank of the Faculty of Engineering of Ilha Solteira (FEIS/UNESP), in Selvíria-MS. This work was conducted with the aims to characterize the mating system and estimate population genetic parameters in M. urundeuva offsprings from different Brazilian biomes, using quantitative growth traits and molecular markers of the microsatellite type. The height, diameter at breast height (DBH), mean crown diameter and survival were used to evaluate five populations: Aquidauana-MS, Selvíria-MS, Itarumã-GO, Paulo de Faria -SP and Seridó-RN. The values high to median observed for survival (96.4% - 70.8%) indicate good adaptation of populations of M. urundeuva and potential for use in reforestation. Estimates of the coefficient of genetic variation ranged from 3% to 24.6%, at the individual level, and from 1.5% to 12.3%, among progenies, with the highest values obtained for the DBH of the Seridó population. The individual heritabilities ranged from moderate (0.41) to low (0.01) for the characters. Using the genetic value of the individuals, DBH was used to estimate genetic gains in the selection of individuals within progenies, and the Itarumã population being the most indicated for breeding, in the said year of evaluation of these populations (Itarumã population at 11 years old). For molecular analysis, 20 microsatellite loci were developed from the new generation sequencing (MiSeq Sequencing System, Illumina), and six of these were used to characterize the populations of Aquidauana, Itarumã, Paulo de Faria and Seridó (1,224 individuals).The total number of alleles (215), the number of different alleles (67) and the observed heterozygosity values demonstrated that these populations still retain considerably high genetic diversity indices. High genetic differentiation was detected among populations (33%), related, in part, the distance separating them; and it was observed that the species dioica M. urundeuva is reproduced by mating. These matings are not random in populations, with increased degree of relationship in populations with strong anthropic pressure. The coancestry coefficients within offspring were higher than expected in half-sibs offsprings (0.125), so these offsprings are also composed of full-sibs. Aiming at the use in reforestation, seed collection strategies should be adopted, in order to avoid obtaining seeds produced from related or inbred matings, establishing the minimum collection amount in 55 seed trees, in each population, so to ensure an effective population size of 150. The informations obtained from this study are useful for the purpose of conservation and breeding of the species, but also for selection of potential matrices for environmental recovery. / FAPESP: 2013/24503-1

Statistical inference from molecular population genetic data is currently a very active area of research for two main reasons. First, in the past two decades an enormous amount of molecular genetic data have been produced and the amount of data is expected to grow even more in the future. Second, drawing inferences about complex population genetics problems, for example understanding the demographic and genetic factors that shaped modern populations, poses a serious statistical challenge. Amongst the many different kinds of genetic data that have appeared in the past two decades, the highly polymorphic microsatellites have played an important role. Microsatellites revolutionized the population genetics of natural populations, and were the initial tool for linkage mapping in humans and other model organisms. Despite their important role, and extensive use, the evolutionary dynamics of microsatellites are still not fully understood, and their statistical methods are often underdeveloped and do not adequately model microsatellite evolution. In this thesis, I address some aspects of this problem by assessing the performance of existing statistical tools, and developing some new ones. My work encompasses a range of statistical methods from simple hypothesis testing to more recent, complex computational statistical tools. This thesis consists of four main topics. First, I review the statistical methods that have been developed for microsatellites in population genetics applications. I review the different models of the microsatellite mutation process, and ask which models are the most supported by data, and how models were incorporated into statistical methods. I also present estimates of mutation parameters for several species based on published data. Second, I evaluate the performance of estimators of genetic relatedness using real data from five vertebrate populations. I demonstrate that the overall performance of marker-based pairwise relatedness estimators mainly depends on the population relatedness composition and may only be improved by the marker data quality within the limits of the population relatedness composition. Third, I investigate the different null hypotheses that may be used to test for independence between loci. Using simulations I show that testing for statistical independence (i.e. zero linkage disequilibrium, LD) is difficult to interpret in most cases, and instead a null hypothesis should be tested, which accounts for the “background LD” due to finite population size. I investigate the utility of a novel approximate testing procedure to circumvent this problem, and illustrate its use on a real data set from red deer. Fourth, I explore the utility of Approximate Bayesian Computation, inference based on summary statistics, to estimate demographic parameters from admixed populations. Assuming a simple demographic model, I show that the choice of summary statistics greatly influences the quality of the estimation, and that different parameters are better estimated with different summary statistics. Most importantly, I show how the estimation of most admixture parameters can be considerably improved via the use of linkage disequilibrium statistics from microsatellite data.

O gênero Mazama é composto por cinco espécies no Brasil. São animais de visualização dificultada por causa de comportamentos evasivos, o que torna as capturas e os estudos comportamentais quase impossíveis. Assim, o uso de metodologias não invasivas para estudos ecológicos e genéticos destas espécies se torna necessário. A análise do DNA fecal está dentro das técnicas mais promissoras para esse fim. Este estudo objetivou genotipar amostras fecais, de uma população de veado-mateiro (Mazama americana) para a obtenção de informações genéticas e ecológicas. Para tanto, foram coletadas, com auxílio de um cão farejador, georreferenciadas e estocadas em etanol absoluto, 52 amostras fecais de cervídeos. A coleta realizou-se em um fragmento (21o20S 47o17W) de 600 ha de floresta estacional semidecidual. Dessas amostras coletadas, 31% (n=16) foram classificadas como frescas e 69% (n=36) como não frescas. O DNA foi extraído em torno de 30 dias após a coleta, usando o kit comercial QIAamp® DNA Stool Mini Kit, seguindo o protocolo do fabricante. Das 52 amostras, 45 foram identificadas por PCR/RFLP como pertencentes a M. americana e as demais apresentaram problemas de amplificação e digestão, permanecendo sem identificação. Amplificaram-se por PCR cinco locos microssatélites, e o sucesso de amplificação, visualizado em gel de agarose, variou com o tamanho dos locos e com a classe das amostras. O sucesso de amplificação foi de 65% das amostras da categoria fresca e 35% das amostras da categoria não fresca. Encontrou-se uma correlação negativa (R= -0,82) entre o tamanho dos fragmentos dos locos de microssatélites e o sucesso de amplificação. Foi possível identificar o sexo do animal em 43,7% das amostras fecais, pela amplificação do gene da amelogenina. Os locos microssatélites amplificados foram analisados em sequenciador automático. Os eletroferogramas gerados pelo seqüenciador impossibilitaram a genotipagem da maioria dos locos e amostras, tornando inviável qualquer análise genética e ecológica com confiabilidade. Fica evidente a dificuldade de se trabalhar com a metodologia do DNA fecal para a identificação individual e sexagem de amostras obtidas de Cervídeos florestais em vida livre. Algumas melhorias metodológicas (coleta de amostras fecais frescas, seleção de iniciadores para locos menores e quantificação do DNA extraído por PCR em tempo real) são sugeridas para o aumento nos índices de sucesso na genotipgem em estudos futuros. / Mazama genus is composed by five species in Brazil. All of them are difficult to observe due to their evasive behaviors, what makes the captures and behavioral studies almost impossible. Thus, the use of non invasive methodologies is necessary to study the ecology and genetics of these species. The fecal DNA analysis is one of the most promising techniques for this purpose. This study aimed to genotype a Mazama americana population faecal samples for obtaining genetics and ecological information. For this, 52 deer faecal samples were collected in a 600ha seasonal semideciduos forest fragment (21o20S 47o17W), with the help of a detection dog, stored in ethanol and georeferenced. Of these samples 31% (n=16) was classified as fresh and 69% (n=36) as not fresh. About thirty days after the collection the DNA was extracted using the QIAamp® DNA Stool Mini Kit following the manufacturers instructions. From the 52 samples collected and extracted, 45 were identified by PCR/RFLP as M. americana and the others showed amplification and digestion problems, remaining without identification. Five microsatellite loci were amplified by PCR and the amplification success, visualized in agarose gel, varied with the loco size and age class. The amplifications success occurred in 65% of the fresh samples and in 35% of the non-fresh samples and a negative correlation (R= -0.82) was found between amplification success and loci sizes. It was possible to identify the animal sex in 43% of the samples by the amelogenin gene. The microsatellite loci amplifications were analyzed in an automatic sequencer. The majority of the samples and loci were impossible to genotype because of the quality of the elestroferograms, what made impossible any reliable genetic and ecological analysis. It is evident the difficulty to work with the faecal DNA methodology using field collected forests deer samples for individual and sexual identifications. Some methodological improvements (collect fresh samples, select primers for shorter loci and quantify the extracted DNA by real time PCR) are suggested to increase the genotyping success indexes in future studies

<p>&nbsp / </p> <p align="left">The domesticated apple (<i><font face="TimesNewRomanPS-ItalicMT">Malus </font><font face="TimesNewRomanPSMT">x </font><i><font face="TimesNewRomanPS-ItalicMT">domestica </font><font face="TimesNewRomanPSMT">Borkh.), belonging to the </font><i><font face="TimesNewRomanPS-ItalicMT">Malus </font><font face="TimesNewRomanPSMT">genus of the Rosaceae family, is one of the edible pomaceous fruits. Since it is one of the important commercial fruit crops worldwide, the quality of the fruit is crucial to breeders and farmers as it ultimately determines acceptance of a cultivar for consumption. Fruit quality is also a critical determinant factor that is used to estimate the potential of apples to have a long shelf life. The introduction of marker-assisted selection (MAS) has allowed hastening of traditional breeding and selection of high-quality apple cultivars. The availability of genetic linkage maps, constructed by positioning molecular markers throughout the apple genome, enables the detection and analysis of major genes and quantitative trait loci (QTLs) contributing to the quality traits of a given genotype.&nbsp / herefore, the primary aim of this study was to construct a genetic linkage map of the &lsquo / Golden Delicious&rsquo / x &lsquo / Dietrich&rsquo / population for the identification of QTLs associated with fruit quality traits and then to examine the apple fruit pulp proteome with a specific focus on fruit firmness. In this regard, genomic DNA was extracted from leaves of the &lsquo / Golden Delicious&rsquo / x Dietrich&rsquo / population and used in megaplex PCR reactions. The PCR products were analysed prior to scoring of alleles. Polymorphic markers were then used to construct genetic linkage maps. The genetic linkage maps constructed in this study comprise of 167 simple sequence repeats (SSR) markers, 33 of these were newly developed markers. The 17 linkage groups of apple were constructed and aligned to existing apple genetic maps. The maps span 1,437.8 cM and 1,491.5 cM for &lsquo / Golden Delicious&rsquo / and &lsquo / Dietrich&rsquo / , respectively.</font></i></i></i></p>

Genes are the foundation of evolution and biodiversity. The genetic structure of natural populations needs to be understood to maintain exploited resources rationally. This thesis focuses on genetic variability and methods to determine spatial and temporal genetic heterogeneities. Intense human exploitation generates particular challenges to conserve genetic diversity of fishes since it has genetic effects. My research concerns one of our most valuable fish species: the Atlantic herring (Clupea harengus). I analyzed Atlantic herring samples from the North and Baltic Seas. The objectives were to determine: 1) spatial genetic structure, 2) whether allozymes and microsatellites provide similar descriptions of the differentiation pattern, or 3) if they are influenced by selection, 4) factors affecting statistical power when testing for genetic differentiation, and 5) the temporal stability of the genetic structure. The results show: 1) very low levels of spatial genetic differentiation in Atlantic herring; a major component is a difference between the Baltic and North Seas, 2) a concordant pattern with allozymes and microsatellites, 3) that selection influences a microsatellite locus, which can be a low salinity adaptation, 4) that statistical power is substantial for frequently used sample sizes and markers; the difference in power between organelle and nuclear loci is partly dependent on the populations’ stage of divergence, and 5) no changes in amount of genetic variation or spatial genetic structure over a 24-year period; the selection pattern in one microsatellite locus remained. The notion that the large population sizes make herring unlikely to lose genetic diversity may be disputed. I found small local effective population sizes, and the evidence of selection hints of a distinct evolutionary lineage in the Baltic. When Atlantic herring is managed as very large units, there can be detrimental genetic effects if certain population segments are excessively harvested.

Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase. / QC 20100415

Intensive aquaculture of Atlantic cod is fast developing in both Northern Europe and Canada. The last six years have seen major improvements in the larval rearing protocols and husbandry techniques for this species. Although breeding programmes are currently being developed by both governmental and private institutions in the main cod producing countries (i.e. Norway, Iceland and Canada), most hatcheries still rely on the mass spawning of their own broodstock. Mass spawning tanks are complex systems where fish are left to spawn naturally and fertilised eggs are collected with the overflowing water, with little or no control over the matings of the animals. Few published studies in other commercial marine species (i.e. turbot and sole) have attempted to analyse the output from such systems using microsatellite markers and several parentage analysis software programs. A review of these publications exposed a lack of consistency in the methods used to analyse such complex datasets. This problem was addressed by carrying out a detailed comparison of two analytical principals (i.e. assignment by strict exclusion and assignment by probabilities) and four parentage software programmes (i.e. FAP, VITASSIGN, CERVUS and PAPA), using the DNA profiles, at 5 loci, from 300 cod fry issued from the mass spawning of a large hatchery cod broodstock tank (consisting of 99 fish). This study revealed large discrepancies in the allocation outcomes between exclusion-based and probability-based assignments caused by the important rate of typing errors present in the dataset. Out of the four softwares tested, FAP (Taggart, 2007) was the most appropriate to use for handling such a dataset. It combined the most conservative method of assignment with the most informative output for the results displayed. In an attempt to study the breeding dynamics in a cod commercial hatchery, parental contributions to five groups of 300 fry (from five single days of spawning and from two commercial mass spawning cod tanks) were analysed, based on the genotyping data from eight loci. The parentage results from the exclusion-based analyses revealed that, on a single day, at least 25 to 30% of the total breeding population contributed to fertilised eggs that resulted in viable offspring at 50 and 83 days post-hatch. Family representations were highly skewed - with the marked dominance of a few males - and effective breeding populations were consistently low (approx. 5% of the total breeding population). Parental contribution to a group of 960 codlings - produced following intensive commercial practices (i.e. including successive size gradings and mixing of batches) and belonging to a single graded group - was also analysed, based on the genotyping data from eleven loci. The effective breeding population size of the juvenile batch (c. 14% of the total broodstock population) was two to three times greater than the effective size observed on a single day of mass spawning. The per-generation rate of inbreeding was however relatively high, for this batch alone, at 2.5%. Based on these results, suggestions were made to manage hatchery cod broodstock populations and implement genetic selection. Early maturation of farmed cod in sea cages (at two or three years old) is a major concern for ongrowers. Understanding the mechanism(s) behind sex determination in cod would probably help the development of a method to control sexual maturation. In an attempt to elucidate sex determination in cod, a protocol to induce gynogenesis was developed. Gynogenetic fish were successfully produced by irradiating cod milt with UV and applying a cold shock (at -6oC) to newly fertilised eggs. However, due to poor survival during larval rearing, only one gynogenetic fish survived long enough to be sexed; not enough to conclude anything on the sex determination mechanism(s) in cod.

Parasitism in plants is often accompanied by a suite of morphological and physiological changes resulting in a condition known as the ‘parasitic reduction syndrome’. With changes including extreme vegetative reduction, frequently beyond any resemblance to its photosynthetic relatives, accompanied by significant losses of genes linked to photosynthesis, the study of parasitic plants can be challenging. Conopholis (Orobanchaceae) is a small holoparasitic genus distributed across eastern and southwestern North America and Central America. This genus has never been the subject of a molecular phylogenetic or morphometric analyses. In addition, very little is known of the relationships among populations and of their post-glacial history. To investigate the species limits and phylogenetic relationships in Conopholis, we conducted a comprehensive molecular phylogenetic study of the genus as well as a fine-scale morphometric study. Based on plastid and nuclear sequences, Conopholis was found to contain three distinct and well-supported lineages which have varying degrees of overlap with previously proposed taxa. The clustering and ordination analyses of the morphometric study corroborated the molecular data, demonstrating the morphological differentiation between the three lineages detected within Conopholis. A taxonomic re-alignment is proposed for the genus that recognizes three species, C. americana, C. panamensis, and C. alpina. To address genetic diversity and phylogeographic structure of C. americana in eastern North America, microsatellite markers were developed and characterized for the first time in this species. Using these newly generated markers along with sequences from the plastid genome, the persistence of a minimum of two glacial refugia at the last glacial maximum were inferred, one in Florida and southern Alabama and another in the Appalachian Mountains near the southern tip of Blue Ridge Mountains. The diversity seen across the southern Appalachian Mountains supports the hypothesis that populations derived from the southern and northern refugia come together in this area.