Abstract . A draft of the whole human genomic sequence has been completed and published in 2001. Researches on structural genomics, functional genomics, proteomics, evolutionary and medical sciences have just begun since. The existence of ninety percent repetitive, non-coding sequences in the human genome and in other mammalian species as well, indicating some potential but unaware functions of these repetitive regions within the mammalian genomes remained to be discovered. Among all features in the genomic sequences, microsatellite has been characterized as one type of short tandem repeats (STRs), abundant in the human genome, which potentially plays some important roles in biological processes, such as makers for molecular biology, the enhancers for transcription, and protein-binding sites. Changes on microsatellite copy numbers also involve in the cause of a couple of diseases have been reported. The sequence of human chromosome 20 has been finished in Dec. 2001, which provided us a valuable resource to study about gene density and the distribution of repetitive sequences such as microsatellties as well. To build a more detail chromosome 20 microsatellite map, including di-, tri- and tetra-nucleotide microsatellites, we therefore designed a sequence-based, molecular markers discovery system by bioinformatics approaches. The results indicate that these repetitive sequences distribute across the chromosome 20 evenly. We further analyzed the GC content of the human chromosome 20. A comparison of GC content, microsatellite distribution and gene density of each contig shows some correlation among them. The slippage rates of di-nucleotide microsatellite were predicted by the formula basing on Markov Chain. Some interesting results were found in this thesis. The same approaches were applied on human chromosome 14, which was being completed sequencing in Feb. 2003. The results implied that our strategies might be useful in the advanced genetic studied or medicine researches.

Using four AFLP markers and seven polymorphic microsatellite loci, we examined the genetic structure of the rare and endangered Spiranthes parksii Correll (Orchidaceae). Spiranthes parksii is not distinguishable from sympatric S. cernua (L.) Rich based on these data, though low levels of polymorphisms exist within both. These low levels of genetic diversity are likely a result of high levels of agamospermic reproduction through adventitious embryony. These results suggest that both S. parksii, as well as the sympatric, open flower form of S. cernua, are products of the more widely distributed S. cernua complex. Further, another local form of S. cernua, distinguished by its tendency to produce closed flowers is genetically distinct from both S. parksii, as well as the openflower form of S. cernua, as shown by AFLPs and microsatellite loci. This is the first known set of microsatellite primers developed specifically for use in Spiranthes. The application of these markers may be used to address other unresolved relationships among species of Spiranthes, many of which are also endangered or have populations in decline.

Friedreich ataxia (FRDA) is the most common inherited ataxia disorder, caused by a GAA repeat expansion mutation within the first intron of the FXN gene. The subsequent deficiency of frataxin protein leads to neurological disability, increased risk of diabetes mellitus, cardiomyopathy and premature death. The exact FRDA disease mechanism is not yet clear, despite some understanding of epigenetic, transcriptional and DNA repair system effects that lead to frataxin reduction. Previous studies have shown that mismatch repair (MMR) genes can affect other trinucleotide repeat disorders by destabilisation of the repeats. Furthermore, it has been proposed that frataxin deficiency might lead to cell malignancy by an as yet undefined mode of action. Therefore, the principle aim of this thesis was to use human and genetically altered mouse cells and tissues to understand the effects of MMR proteins on GAA repeat instability and FXN transcription, and also to identify potential changes in MMR transcription that might cause malignancy in FXN-defective human cells. Firstly, by using FXN and MMR genetically altered mice, MMR proteins were shown to be involved in both intergenerational and somatic GAA repeat instability, although their effects in the two systems were different. Thus, Msh2 or Msh3 were both found to protect against intergenerational transmission of GAA contractions, while loss of Msh2 or Msh3 reduced somatic GAA repeat expansions and increased levels of FXN transcription in brain and cerebellum tissues. Loss of Msh6 induced both intergenerational GAA repeat expansions and contractions, while the frequency of somatic GAA repeat expansions was reduced. Curiously, the level of FXN transcription was also reduced in Msh6-deficient brain and cerebellum tissues. On the other hand, Pms2 was found to protect against both intergenerational and somatic GAA repeat expansions, with loss of Pms2 causing increased GAA repeat expansions and decreased levels of FXN transcription in brain and cerebellum tissues. Finally, loss of Mlh1 led to a reduced frequency of both intergenerational and somatic GAA repeat expansions, but the level of FXN transcription was also reduced in brain and cerebellum tissues. Furthermore, upregulation of MMR mRNA expression was detected in human FRDA fibroblast cells, but downregulation was seen in FRDA cerebellum tissues, suggesting tissue-dependent control of FXN and MMR expression. In summary, these studies indicate that the MMR system can affect GAA repeat expansion instability and FXN transcription through different mechanisms of action. Furthermore, frataxin deficiency can also affect the levels of MMR mRNA expression in a tissue-dependent manner. These findings will assist future investigations aimed at identifying novel FRDA therapies.

Species long-term persistence is to a great extent contingent on the ability of populations to mount variable responses to perturbations; the breadths of which are largely dependent on the amount of heritable variation present at the population level. However, populations are not necessarily equivalent in their amount of genetic variation, or in their responses to future environmental conditions, and information about the magnitude and spatial distribution of intraspecific genetic variation is integral to conservation planning, and preserving species evolutionary potential. Using neutral molecular markers, I demonstrate that latitude is an important determinant of the amount and spatial distribution of genetic variation within and among Nearctic fishes. Latitudinal declines observed among species were mirrored by declines within species, and encourage a cautionary approach for interspecific comparisons and inferences of broad spatial patterns of genetic variation when data for individual species are obtained from only a portion of their range. I subsequently examined the magnitude and spatial distribution of microsatellite based variation for 33 spawning runs from across the range of American shad (Alosa sapidissima). Sequential reductions of intraspecific genetic variation with latitude were observed among spawning runs from formerly glaciated regions; consistent with stepwise post-glacial range expansion, and successive population founder events. Canadian populations exhibited temporally stable genetic differentiation characterized by a significant pattern of isolation by distance, and exhibited evidence of metapopulation structure. Although isolation by distance was observed among U.S. spawning runs, population structure was comparatively weak; a possible consequence of increased levels of gene flow (human-mediated or natural). Different spatial patterns of population structure were detected across the species’ range, but are not likely due to alternative reproductive strategies (iteroparity vs. semelparity). Rather, these different patterns probably reflect different management strategies in Canada (no stocking) and the United States (stocking), alternative glacial histories, or combinations thereof. Reciprocal patterns of genetic diversity and differentiation observed across the species’ range suggests that U.S spawning runs contribute more to diversity and less to differentiation than Canadian spawning runs. These results have implications for future shad restoration efforts, and the long-term persistence of the species.

Ancient DNA (aDNA) was isolated from the bones of 290 individuals and four species of extinct New Zealand moa. All sampled bones had been recovered from a small geographic area (~10 km radius) near Waikari in North Canterbury. A total of 217 specimens were 14C-AMS dated, providing a temporal framework for the genetic analyses and an unprecedented opportunity to study extinct megafauna at the population level. Taxon and sex were determined for each individual, using aDNA technology. This revealed a large excess of females (overall ♂:♀ = 1:5.1), and significant compositional differences for the moa assemblages between fossil sites. Balanced sex ratios were observed among juvenile moa, suggesting that a gender-bias developed as the birds matured, probably as a result of higher male mortality. Female territoriality and ecological niche-separation are discussed in this context. Mitochondrial DNA (mtDNA), amplified using a quantitative PCR procedure, provided a measure of DNA preservation in each radiocarbon-dated fossil. This assessment showed that DNA degrades over thousands of years according to an exponential decay model, and the average molecular half-life for the here targeted DNA fragment was estimated to be 521 years. By using high-throughput sequencing, six polymorphic moa microsatellite markers were identified and characterised. These are the first microsatellite primers developed exclusively for extinct taxa. A high-resolution genetic study of the four sympatric moa populations was carried out, combining information from mtDNA, microsatellites, sex-identification, and radiocarbon age. Genetic diversity, past demography, kinship, and other aspects of moa biology were analysed. The populations showed a remarkable extent of genetic stability throughout the 3000-4000 years preceding their extinction, suggesting that they were large and viable before suddenly disappearing. The results represent significant advances in aDNA research and thanks to the high resolution in microsatellite markers, moa have here been studied, almost as if they were still alive.

Resumo: O oídio em soja, trata-se de uma doença praticamente presente em todos os paises produtores. Os marcadores moleculares microssatélites ou SSR (Simple Sequence Repeat) têm sido amplamente utilizados no processo de seleção assistida de genótipos de soja. Assim, o objetivo deste estudo foi obter marcadores microssatélites próximos ao gene de resistência ao oídio em soja. O estudo foi realizado em duas populações F2, oriundas de cruzamentos entre parentais contrastantes quanto à resistência ao oídio. Para o estudo, foram selecionados marcadores microssatélites a uma distância de até 42 cM ao redor do gene Rmd (resistência ao oídio). Utilizou-se o método de BSA (Bulked Segregant Analysis) na avaliação dos marcadores, para a comparação com a análise fenotípica das populações. Na análise foram utilizados dez iniciadores SSRs para as duas populações, sendo identificados quatro marcadores polimórficos para o cruzamento 1 (MGBR95-20937 x IAC-Foscarin 31) e três para o cruzamento 2 (MGBR 46/Conquista x EMBRAPA 48). Pela análise de Qui-quadrado da avaliação fenotípica, confirmou-se à segregação esperada (3:1) de um gene dominante condicionando a resistência. Os marcadores polimórficos também segregaram conforme o esperado (1:2:1) já que possuem natureza codominante. Para as populações 1 e 2, os melhores resultados foram obtidos com os marcadores Sat_366 e Sat_393, respectivamente, localizando-se a 9,41 e 12,45 cM de distância do gene, sendo considerados promissores na seleção assistida para resistência ao oidio em soja. / Abstract: Powdery mildew in soybeans, it is a disease present in virtually all producing countries. The molecular markers microsatellites or SSR (Simple Sequence Repeat) have been widely used in the assisted selection of soybean genotypes. The objective of this study was to obtain microsatellites markers near the gene for resistance to powdery mildew in soybeans. The study was conducted in two populations F2, from crosses between contrasting parents about the resistance to powdery mildew. For the study, were selected microsatellites markers at a distance of 42 cM around the gene Rmd (resistance to powdery mildew). It was used the method of BSA (Bulked Segregant Analysis) in the evaluation of markers, for comparison with the phenotypic analysis of populations. In the analysis were used in ten initiators SSRs for the two populations, and identified four polymorphic markers for the crossing 1 (MGBR95-20937 x-IAC Foscarin 31) and three for the crossing 2 (EMBRAPA MGBR 46/Conquista x 48). For the analysis of chi-square of the phenotypic evaluation, it is confirmed the segregation expected (3:1) of a dominant gene conditioning the resistance. The polymorphic markers also segregation as expected (1:2:1) that have already codominante nature. For the populations 1 and 2, the best results were obtained with the markers Sat_366 and Sat_393, respectively, finding itself to 9.41 and 12.45 cM distance of the gene and are considered promising in assisted selection for resistance to soybean in powdery mildew. / Orientador: Antonio Orlando Di Mauro / Coorientadora: Sandra Helena Unêda Trevisoli / Banca: Eduardo Antonio Gavioli / Banca: João Carlos de Oliveira / Mestre

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T19:32:32Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-KlaudiaTenório_dissert.pdf: 4615109 bytes, checksum: fd3f355eef0c71a4e121182d43b7657b (MD5) / Made available in DSpace on 2017-04-06T19:32:32Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-KlaudiaTenório_dissert.pdf: 4615109 bytes, checksum: fd3f355eef0c71a4e121182d43b7657b (MD5) Previous issue date: 2012 / O camarão de água doce Macrobrachium carcinus (LINAEUS, 1758) pode ser encontrado em regiões tropicais e subtropicais. No Brasil, está distribuído desde o Pará até Rio Grande do Sul, em rios que deságuam no oceano Atlântico. Esta espécie tem sofrido com a sobrepesca, a poluição ambiental, a destruição dos ambientes naturais, barramento dos rios e riachos, os quais impedem o acesso ao mar impossibilitando o desenvolvimento dos estágios larvais do animal, além da introdução de espécies exóticas, como o M. rosenbergii. Por essas razões, o Ministério do Meio Ambiente (MMA) inseriu o M. carcinus na lista oficial das espécies ameaçadas de extinção em vários estados do Nordeste. Visando fornecer informações para as iniciativas de repovoamento, este estudo teve como objetivo avaliar a diversidade e estrutura genética, bem como aspectos da ecologia desta espécie em quatro estados do Nordeste (Ceará, Pernambuco, Sergipe/Alagoas e Bahia), utilizando seis marcadores de microssatélites, dados biométricos e análises de conteúdo estomacal. Um total de 143 animais foi coletado, sendo de 32 a 40 animais para cada um dos rios avaliados. Após as análises, o número de alelos variou de dois a 18 alelos, com heterozigosidades médias esperadas e observadas de 0,6256 e 0,5985 respectivamente. Três dos seis loci estão em desequilíbrio de Hardy-Weinberg e apresentam alelos nulos em todas as populações. O valor total de FST foi de 0, indicando ausência de estruturação genética. Os estudos biológicos mostram que o pico reprodutivo mais provável para esta espécie ocorra em fevereiro. A análise estomacal revelou predominância de itens de origem vegetal. Estes resultados sugerem que um único programa de repovoamento poderia fornecer pós-larvas a todos os rios da região Nordeste, diminuindo os custos de operação e garantido que rios distantes de laboratórios de multiplicação também sejam beneficiados. / The Macrobrachium carcinus (LINAEUS, 1758) can be found in tropical and subtropical regions. In Brazil, it is distributed from Pará to the Rio Grande do Sul, in rivers that empty into the Atlantic Ocean. This specie has suffered with overfishing, pollution, destruction of natural environments, rivers and streams dams that block the access to the sea, what inhibits the development of larval stages of the animal, and the introduction of exotic species such as M. rosenbergii. Because these reasons, the Brazilian Ministry of Environment (MMA) has inserted the M. carcinus in the official list of endangered species in several Northeastern states like Piauí, Cear, Pernambuco, Alagoas, Sergipe and Bahia. In order to provide information to restocking initiatives, the aim of this study was to assess the genetic diversity and structure, as well as aspects of ecology of this species in four regions of the Brazilian Northeast (Ceará, Pernambuco, Sergipe/Alagoas and Bahia), by the use of six microsatellite markers, biometric data and stomach content analysis. A total of 143 animals was collected, with 32-40 animals for each of the rivers evaluated. After the analyses, the number of alleles ranged from two to 18 alleles, with expected average heterozygosities from 0,6256 and observed average heterozygosities from 0,5985. Three of the six loci are in Hardy-Weinberg disequilibrium and present null alleles in all populations. The total value of FST was 0, what indicates no genetic structuration. Biological studies show that reproductive peak for this species most likely to occur in February. The stomach analysis revealed predominance of plant items.These results suggest that a single restocking program could provide post-larvae to all rivers of Brazilian Northeast, which reduce operating costs and ensure the benefit of the rivers that are distant from multiplication laboratories also.

Abstract I used individual-based capture-mark-recapture data and genetic markers to gain understanding of the demographic and genetic processes operating in small and declining populations of two different species, the willow tit Parus montanus and the Siberian flying squirrel Pteromys volans. Both species have declined in Finland and the flying squirrel has been considered to be vulnerable. The willow tit study was conducted in northern Finland, near city of Oulu. The population size in the studied area has on average been stable during the past decade. Adult survival in the willow tit was high and fairly stable and was positively correlated with recruitment. Adult survival has been the most influential vital rate to the population growth rate. Local recruitment and immigration have high variation, inducing variation in the population growth rate. Female willow tits use extrapair copulations to maximise offspring heterozygosity. Heterozygous individuals are supposedly of higher quality than homozygous ones. I found weak negative association between individual homozygosity and recruitment probability. The flying squirrel populations have declined during the past ten years. Furthermore, adult survival has declined in one of the populations, most likely due to habitat loss and fragmentation that decrease the adult survival and limit dispersal. The flying squirrel populations were studied in western Finland. The flying squirrel densities in the studied areas are the highest in Finland and therefore these areas have been regarded as favourable for the flying squirrel. My results question this view. Microsatellite analyses strengthen the view of populations doing poorly, because the heterozygosities in all the populations and particularly in the most isolated one were rather low. High FST values indicate low dispersal even between adjacent populations. Following work should investigate the spatial variation in individual performance and the dispersal processes in these populations. For the flying squirrel it is vital to determine the size and quality of the patches that can support flying squirrels and the ones that apparently can not. Present estimates of survival and genetic diversity can be used to reconstruct a meaningful PVA and projections for these populations.

Abstract The evolution of microsatellites was studied within and between the pine species. Sequences showed that microsatellites do not necessarily mutate in a stepwise fashion and that size homoplasy is common due to flanking sequence and repeat area changes within and between the species. Thus, some assumptions of statistical methods based on changes in repeat numbers may not hold. Sequences from cross-species amplifications revealed evidence of duplications of microsatellite loci in pines. On two independent occasions, the repeat area of the microsatellite had undergone a rapid expansion during the last 10-25 million of years. Microsatellite markers were used together with other molecular markers (allozymes, RFLPs, RAPDs, rDNA RFLPs) and an adaptive trait (date of bud set) to study patterns of genetic variation in Scots pine (Pinus sylvestris) in Finland. All molecular markers showed high level of within population variation, while differentiation among populations was low (FST = 0.02). Of the total variation in bud set, 36.4 % was found among the populations which experience a steep climatic gradient. Thus, the markers applied were poor predictors of population differentiation of the quantitative trait studied The distribution of genetic variation was studied in five natural populations of radiata pine (Pinus radiata), species which has gone through bottlenecks in the past. Null allele frequencies were estimated and used in later analyses. Microsatellites showed high level of variability within populations (He = 0.68-0.77). Allele length distributions and average number of alleles per locus showed some traces of bottlenecks. Instead, comparison of observed genetic diversities and expected diversities suggested post-bottleneck expansion of populations. Genetic differentiation (FST and RST) among populations was over 10 %, reflecting situation in the isolated radiata pine populations. Using microsatellites and a newly developed Bayesian method, individual inbreeding coefficients were estimated in five populations of radiata pine. Most individuals were outbred while some were selfed. Presumably, in ancestral radiata pine populations the recessive deleterious alleles have been eliminated after bottlenecks and the mating system has changed as a consequence.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22480号 / 農博第2384号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5260(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 吉村 剛, 教授 井鷺 裕司, 講師 YANG Chin-Cheng / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM