Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.

The majority of the vertebrate genome sequence is not coding for proteins. In recent years, the evolution of this noncoding fraction of the genome has gained interest. These studies have been greatly facilitated by the availability of full genome sequences. The aim of this thesis is to study evolution of the noncoding vertebrate genome through bioinformatic analysis of large-scale genomic datasets. In a first analysis we addressed the use of conservation of sequence between highly diverged genomes to infer function. We provided evidence for a turnover of the patterns of negative selection. Hence, measures of constraint based on comparisons of diverged genomes might underestimate the functional proportion of the genome. In the following analyses we focused on length variation as found in small-scale insertion and deletion (indel) polymorphisms and microsatellites. For indels in chicken, replication slippage is a likely mutation mechanism, as a large proportion of the indels are parts of tandem-duplicates. Using a set of microsatellite polymorphisms in chicken, where we avoid ascertainment bias, we showed that polymorphism is positively correlated with microsatellite length and AT-content. Furthermore, interruptions in the microsatellite sequence decrease the levels of polymorphism. We also analysed the association between microsatellite polymorphism and recombination in the human genome. Here we found increased levels of microsatellite polymorphism in human recombination hotspots and also similar increases in the frequencies of single nucleotide polymorphisms (SNPs) and indels. This points towards natural selection shaping the levels of variation. Alternatively, recombination is mutagenic for all three kinds of polymorphisms. Finally, I present the program ILAPlot. It is a tool for visualisation, exploration and data extraction based on BLAST. Our combined results highlight the intricate connections between evolutionary phenomena. It also emphasises the importance of length variability in genome evolution, as well as the gradual difference between indels and microsatellites.

Genetic variation within and among populations is a result of past and ongoing processes. Among the most important of such processes are dispersal, habitat fragmentation and selection. This thesis use neutral genetic variation as a tool to investigate these processes in three bird species. In the Siberian jay, the timing of dispersal is dependent on social dominance among siblings. Mark-recapture data, radio-tracking and genetic variation was used to investigate whether timing of dispersal had an effect on dispersal distance. The results show that early dispersing individuals also disperse longer. In the same species, genetic correlation between neighbours was used to find areas with high production of philopatric individuals, which could be indicative of high habitat quality. Great snipe populations in northern Europe have a breeding range divided into two regions. A QST-FST approach was applied to study variation in selection between regions. Differentiation between the regions in neutral molecular markers was low, indicating high gene flow, or short time available for neutral divergence. Morphological divergence between the regions was high, and QST > FST, which indicates divergent selection. Thus, neutral genetic markers can be misleading in identifying evolutionary significant units, and the QST-FST approach might be valuable to identify targets for conservation. Rock ptarmigan, or its ancestors, originated in Beringia, and spread throughout the Holarctic region. Their distribution has subsequently been affected by glaciations, most likely leading to withdrawals and re-colonisations. Neutral genetic variation among five populations around the northern Atlantic was investigated. There was strong genetic structure among the populations, and evidence that Scandinavian rock ptarmigan has been isolated from other populations for considerable time. Rock ptarmigan in Svalbard showed slightly lower genetic variation than others, and comparisons with other studies suggested an eastern colonisation route to Svalbard.

Abstract Gaining accurate population information is crucial for the conservation and management of species. The National Monitoring Program for Large Carnivores monitors the Swedish lynx population (species Lynx lynx) by surveying family groups, non-invasive sampling and genetic analysis. Ten microsatellite regions were used as genetic markers to retrieve unique individual genotypes, through polymerase chain reactions (PCR) with specific primer-pairs and capillary-electrophoresis. Complete genotypes were matched using an internal database. The aim of this degree project was to show how monitoring of lynx through genetic analysis is carried out at the Department of Evolutionary Biology at Uppsala University, and to evaluate how effective these methods are and how they might be improved. Even though most of the methods used were fairly robust and reproducible, non-invasive sampling and microsatellite analysis posed some problems regarding DNA quality and quantity, and increased the risks of certain genotyping errors. These risks might be worth taking though, as genetic analysis, in combination with field observations, gives a more comprehensive picture of the Swedish lynx population.

Turtles have long life spans, overlapping generations and promiscuous mating systems. Thus, they are an ideal system with which to investigate the application of conservation genetics methods and assumptions to long-lived organisms. Turtles are also one of the most threatened groups of vertebrates and conservation genetics studies are essential to effective recovery of turtle species. This thesis has two main objectives: 1) to evaluate some common population genetics assumptions with respect to turtles and other long-lived organisms, and 2) to collect important information on the population genetics of threatened turtles in Ontario, which can be used to inform species recovery. In Chapters Two and Three, I describe the development of novel microsatellite markers for the snapping turtle and spiny softshell. In Chapter Four I demonstrate significant genetic structure in populations of the endangered spotted turtle in Ontario, and find that “bottleneck tests” may fail to detect recent population declines in small turtle populations. I also show that spotted turtles do not show the typical correlation between population size and genetic diversity. In Chapter Five I use microsatellite markers developed in Chapter Two and document population structure in the widespread snapping turtle for the first time. I compare these results with results from Chapter Four to test the traditionally accepted hypothesis that genetic diversity is reduced in small, isolated populations compared to large, connected populations. As in Chapter Four, my results suggest that the usual patterns of genetic structure and loss of diversity may not apply to turtles. In Chapter Six I conduct a conservation genetics study of the endangered Blanding’s turtle. Finally, in Chapter Seven I combine results from spotted, snapping and Blanding’s turtles to test whether vagility predicts population structure, genetic diversity and significant barriers to gene flow in three species sampled across a single landscape. Analyses reveal minimal congruence in barriers to gene flow and the three species show unexpected and contrasting patterns of diversity across the landscape. Discordant patterns among species highlight areas for further research and shed light on possible cryptic behaviour, and I discuss potential further directions for research in the Summary.

There are around 20.000 genes in the human genome all of which could potentially be expressed. However, it is obvious that not all of them can be active at the same time. Thus, there is a need for coordination achieved through the regulation of transcription. Transcriptional regulation is a crucial multi-component process involving genetic and epigenetic factors, which determine when and how genes are expressed. The aim of this thesis was to study two of these components, the transcription factors and the DNA sequence elements with which they interact. In papers I and II, we tried to characterize the regulatory role of repeated elements in the regulatory sequences of nitric oxide synthase 2 gene. We found that this type of repeat is able to adopt non B-DNA conformations in vitro and that it binds nuclear factors, in addition to RNA polymerase II. Therefore it is probable that these types of repeats can participate in the regulation of genes. In papers III-V, we intended to analyze the genome-wide binding sites for six transcription factors involved in fatty acid and cholesterol metabolism and the sites of an epigenetic mark in a human liver cell line. For this, we applied the chromatin immunoprecipitation (ChIP) method together with detection on microarrays (ChIP-chip) or by detection with the new generation massively parallel sequencers (ChIP-seq). We found that all of these transcription factors are involved in other liver-specific processes than metabolism, for example cell proliferation. We were also able to define two sets of transcription factors depending on the position of their binding relative to gene promoters. Finally, we demonstrated that the patterns of the epigenetic mark reflect the structure and transcriptional activity of the promoters. In conclusion, this thesis presents experiments, which moves our view from genetics to genomics, from in vitro to in vivo, and from low resolution to high resolution analysis of transcriptional regulation.

<p><p>Understanding the ecological and evolutionary responses of plant species to shifts in climate (and other rapid environmental perturbations) will require an improved knowledge of interactions between ecological and evolutionary processes as mediated by reproduction and gene flow. This dissertation research examines the processes of seed dispersal, intra- and inter-specific gene flow, and reproductive success in two red oak populations in North Carolina; the variation in these processes from site to site; and their influence on genetic structure, population dynamics, and migration potential.</p> <p><p>Using genetic and ecological data collected from two large long-term study sites, I develop a hierarchical Bayesian model to identify the parents of sampled seedlings and characterize the scale of effective seed and pollen dispersal. I examine differences in scale of dispersal between the Appalachian and Piedmont sites in light of the spatial genetic structure and ecological differences of the two sites. I then use the pedigree and dispersal estimates derived from these analyses to examine variation in reproductive success and to test hypotheses about the causes and consequences of such variation. Using parentage estimates and measures of genetic differentiation between species, I study the likely extent of hybridization in these mixed-species secondary forests. Finally, using the SLIP stand simulator, I explore the implications of new genetic dispersal estimates for migration potential in oaks.</p> <p><p>I find that effective seed dispersal distances are longer than estimated using seed trap data. While at the Piedmont site the large number of seedling found >100 m from their mother trees suggests that animal dispersers play a vital role, at the Appalachian site seedling distributions conform more closely to the original gravity-created pattern of seed density. Individual trees vary widely in their reproductive success. Seedling production was found to be positively associated with annual seed production, but exhibited hump-shaped or reversing relationships with age (suggesting the effect of senescence) and growth rate (suggesting tradeoffs in allocation). Germination fraction was negatively associated with fecundity, suggesting that density-dependent mortality may be acting on the high concentrations of seeds near highly fecund adults. Due to overlapping generations and variation in individual reproductive success, effective population size is estimated to be less than half the size that numbers of "adult" individuals would suggest, with consequences for the relative strength of drift and selection. Hybridization may boost effective population size somewhat; my analyses suggest that inter-specific gene flow is common at both study sites. Finally, simulations show that dispersal has a relatively stronger effect on migration rate and population growth than fecundity or size at maturity, and that genetic estimates of seed dispersal can yield significantly higher rates of migration and/or population persistence than seed-trap based estimates under both competitive and non-competitive conditions.</p> / Dissertation

In the interest of understanding the genetic basis of adaption to environment, we developed F2 lines from an F1 interspecific cross between the rare serpentine endemic, Caulanthus amplexicaulis var. barbarae and the non-serpentine Caulanthus amplexicaulis var. amplexicaulis. Using genomic DNA from Caulanthus amplexicaulis var. barbarae, we developed a suite of microsatellite markers. In addition, we developed gene specific markers for genes known in Arabidopsis to be ecologically important. Our suite of markers was used to genotype 186 F2 plants, the basis for our F2 linkage map. In order to further resolve evolutionary relationships among related taxa, we constructed a molecular phylogeny for 52 taxa within the related genera Caulanthus, Guillenia, Sibaropsis, Streptanthella, and Streptanthus, using the sequences from the ribosomal ITS region and two chloroplast regions. To create a useful system to enable comparative genomics within the related taxa of the ecologically and morphologically diverse Streptanthoid Complex, we demonstrated that our molecular tools are portable across a large group of ecologically significant taxa. To use the significant genomic resources available in Arabidopsis, we constructed a collinear comparative map of Caulanthus and the model plant Arabidopsis thaliana based on ancestral linkage blocks with the Brassicaceae family. This comparative map acted as a guide for candidate gene selection in the mapping of sepal color. We identified a region of MYB transcription factors in an orthologous region of Arabidopsis. Sequence data from Caulanthus amplexicaulis var. barbarae and Caulanthus amplexicaulis var. amplexicaulis in this MYB region showed significant sequence divergence between the two taxa. To determine the genetic basis for the tolerance of high concentrations of magnesium in Caulanthus amplexicaulis var. barbarae, we phenotyped multiple individuals from 88 F2:3 families under two nutrient treatments, differing in the ratio of calcium to magnesium. Through QTL analysis, using our F2 linkage map as a framework for the analysis, we identified one major effect QTL on Caulanthus Linkage Group 8 and another QTL on Caulanthus Linkage Group 3. We identified candidate genes for the QTLs using our collinear comparative map to Arabidopsis.

We analyzed a total of 760 worker bees, two samples per colony, 390 colonies in 26 provinces in Turkey to determine and compare the genetic variation of Turkish honey bee (Apis mellifera L.) populations using 10 primers for RAPD and 6 microsatellite loci. Mean gene diversity levels ranged from 0.035 (Sanliurfa) to 0.175 (Antalya) for RAPD and 0.449 (Mugla) to 0.739 (Artvin) for microsatellite markers. Private band patterns and alleles, pairwise FST values support that the Anatolian honey bees belong to C lineage except for Hatay and Sanliurfa populations illustrated from previous findings of mitochondrial DNA studies. Genetic differentiation (GST) from RAPD data ranged from 0.060 (Bilecik and Mugla) to 0.395 (G&ouml / k&ccedil / eada and Sanliurfa). The genetic diversity (FST) for microsatellites ranged from -0.068 (G&ouml / k&ccedil / eada and &amp / #272 / zmir) to 0.347 (Konya and Mugla). The results of the present research are in agreement to that of previous study in Turkish honey bee populations which used different microsatellite loci. That is the genetic variation was the highest in African, the lowest in European and intermediate in the Mediterranean honey bee populations. The data presented here indicate that in spite of extensive migratory beekeeping, there is still a large genetic differentiation among honey bee populations. These results should be considered in establishment of conservation plans particularly in moving of colonies between regions. The most importantly introduction of bees with foreign origin and distribution queen bees from one center to all over the country which will homogenize the gene pool of the populations should be prevented

This study reexamines the nuclear microsatellite analysis by Rooney et al. (1999a) of Bering-Chukchi-Beaufort Seas bowhead whales (Balaena mysticetus) to determine if this population underwent a genetic bottleneck as a result of 19th and early 20th Century commercial whaling. This investigation used more accurate laboratory techniques to score alleles, had a larger sample size that was divided into two groups (mainland Alaska and St. Lawrence Island (SLI)), and used a moderately different set of microsatellite loci which are more variable and thus, more informative. The results corroborate the findings of Rooney et al. (1999a) for mainland Alaska showing no evidence of a genetic bottleneck. However, the SLI data analyses provide conflicting conclusions. The Wilcoxon test is significant for a heterozygote excess (p = 0.042) suggesting that a genetic bottleneck has occurred. This is not substantiated by the exact tests of each locus or the table-wide sign test. There is a possibility that a bottleneck has occurred, but due to the small sample size this is not a definitive conclusion and warrants reanalysis with a larger sample size.