<p>Sex chromosomes are useful in elucidating the evolutionary factors affecting diversity and divergence. In particular, Y chromosome analyses may complement studies using mitochondrial DNA for inferring sex-specific population genetic processes.</p><p>Y chromosome studies have been scarce due to limited access to genetic markers and the dynamic evolution of Y. Conserved Y-specific primers that could amplify a diverse set of mammalian species were developed from comparison of gametologous X and Y sequences. Y-specific sequence, generally more than one kb, was amplified for all 20 species examined.</p><p>Intraspecific diversity on mammalian Y was found to be reduced even when male-biased mutation rate and effective population size were corrected for. A number of factors can cause this low variation on Y of which selection on a haploid chromosome seems most important.</p><p>The field vole (<i>Microtus agrestis</i>), a common and well-studied small mammal in Eurasia, was examined for X and Y variability. Earlier studies on mtDNA had shown that the field vole is separated in two distinct lineages in Europe. The X and Y chromosome sequences confirmed the deep split and suggested that the two lineages of field vole should be reclassified as two separate species.</p><p>Two distinct Y chromosome haplogroups were found in modern European cattle, distributed among breeds according to a north-south gradient. Ancient DNA analysis of European aurochsen showed the northern haplogroup to be the most common, possibly indicating local hybridization between domestic cows and wild aurochs bulls in Europe.</p>

<p>The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling.</p><p>Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors.</p><p>To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval.</p><p>The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC.</p><p>We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals.</p><p>In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.</p>

<p>Management of wolves is a complex issue, and molecular genetics is an important tool in this work. Molecular genetics can provide important information at the species, population and individual level, which can be essential for the development of management programs aiming at the long term survival of wolf populations.</p><p>In this thesis I developed new genetic markers on the canine Y chromosome to estimate the number of founders of the Scandinavian wolf population. This knowledge is important to reconstruct the history of the population and to design the most appropriate conservation strategies. Next, genetic markers with different pattern of inheritance have been used to identify hybrids between wolves and dogs. This allowed us to determine the direction of hybridization and to evaluate its possible impact on the gene pool of a wolf population. Furthermore, I also developed a method for a more reliable identification of the predator responsible of an attack by using saliva remains left on the prey. Since predation on livestock is perhaps the main reason for the negative opinions about the predator, the correct identification of the responsible for an attack (wolf, dog or hybrid) is essential. </p><p>Finally, this thesis has also been focusing on the domestication of dogs. By using Y chromosome markers (paternally inherited), it has been possible to complement previous studies based on mtDNA sequences (maternally inherited) and autosomal markers (inherited from both parents). In this way I have obtained a more complete picture of the domestication process and of the origin of breeds. This has shown that there has been a bias in the contribution of the two sexes in the origin of dog breeds (fewer males then females contributing to each breed) and that the origin of dogs was not marked by extensive backcrosses with male wolves over the entire species range.</p>

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 54-58).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 104-113).

The United States is the second largest producer of oranges and grapefruit. However, the US citrus industry experiences constraints in production due to pests, diseases and environmental concerns. Furthermore, due to the low diversity in current commercial scion cultivars any exotic diseases, if introduced into any of the producing states could be devastating. To maintain the US industry competitiveness it is necessary to improve cold, pest and disease resistance to allow expansion of citrus production areas in the US, and to improve fruit quality characteristics such as sweetness, vitamins and phytochemical contents and seedlessness. Sexual hybridization in most Citrus species is complicated because they are highly apomictic. Polyembryony makes it difficult to create large segregating populations for selection. Somatic hybridization by protoplast fusion circumvents sexual incompatibilities and is a powerful tool in genetic improvement. Symmetric and asymmetric hybdridization (gamma irradiation plus iodoacetamide) via protoplast fusion were performed with the objective of producing somatic hybrids of Citrus paradisi with C. sinensis and C. reticulata with C. sinensis. These hybrids could be used for grapefruit improvement and to create genetic diversity. Furthermore, irradiated Swinglea glutinosa microprotoplasts were fused with ‘Ruby Red’ grapefruit and ‘Mucott’ tangor to assess the possibility of introgression of pieces of S. glutinosa chromosomes into the recipient protoplasts, a possible first step for radiation hybrid mapping. Double-inactivated fusions (irradiation + iodoacetamide) produced tetraploid and aneuploid plants, and hybridity was confirmed by amplified fragment length polymorphism (AFLP) analysis. This is the first report of obtaining rooted Citrus asymmetric hybrid plants, produced by irradiation plus iodoacetamide. AFLP confirmed presence of S. glutinosa into the receptor genomes, showing a possible donor introgression.

Sex chromosomes are useful in elucidating the evolutionary factors affecting diversity and divergence. In particular, Y chromosome analyses may complement studies using mitochondrial DNA for inferring sex-specific population genetic processes. Y chromosome studies have been scarce due to limited access to genetic markers and the dynamic evolution of Y. Conserved Y-specific primers that could amplify a diverse set of mammalian species were developed from comparison of gametologous X and Y sequences. Y-specific sequence, generally more than one kb, was amplified for all 20 species examined. Intraspecific diversity on mammalian Y was found to be reduced even when male-biased mutation rate and effective population size were corrected for. A number of factors can cause this low variation on Y of which selection on a haploid chromosome seems most important. The field vole (Microtus agrestis), a common and well-studied small mammal in Eurasia, was examined for X and Y variability. Earlier studies on mtDNA had shown that the field vole is separated in two distinct lineages in Europe. The X and Y chromosome sequences confirmed the deep split and suggested that the two lineages of field vole should be reclassified as two separate species. Two distinct Y chromosome haplogroups were found in modern European cattle, distributed among breeds according to a north-south gradient. Ancient DNA analysis of European aurochsen showed the northern haplogroup to be the most common, possibly indicating local hybridization between domestic cows and wild aurochs bulls in Europe.

The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling. Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors. To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval. The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC. We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals. In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.

Management of wolves is a complex issue, and molecular genetics is an important tool in this work. Molecular genetics can provide important information at the species, population and individual level, which can be essential for the development of management programs aiming at the long term survival of wolf populations. In this thesis I developed new genetic markers on the canine Y chromosome to estimate the number of founders of the Scandinavian wolf population. This knowledge is important to reconstruct the history of the population and to design the most appropriate conservation strategies. Next, genetic markers with different pattern of inheritance have been used to identify hybrids between wolves and dogs. This allowed us to determine the direction of hybridization and to evaluate its possible impact on the gene pool of a wolf population. Furthermore, I also developed a method for a more reliable identification of the predator responsible of an attack by using saliva remains left on the prey. Since predation on livestock is perhaps the main reason for the negative opinions about the predator, the correct identification of the responsible for an attack (wolf, dog or hybrid) is essential. Finally, this thesis has also been focusing on the domestication of dogs. By using Y chromosome markers (paternally inherited), it has been possible to complement previous studies based on mtDNA sequences (maternally inherited) and autosomal markers (inherited from both parents). In this way I have obtained a more complete picture of the domestication process and of the origin of breeds. This has shown that there has been a bias in the contribution of the two sexes in the origin of dog breeds (fewer males then females contributing to each breed) and that the origin of dogs was not marked by extensive backcrosses with male wolves over the entire species range.

DNA as forensic evidence is valuable in criminal investigations. Implementation of new, sensitive and fast technologies is an important part of forensic genetic research. This thesis aims to evaluate new sensitive methods to apply in forensic DNA analysis including analysis of old skeletal remains. In Paper I and II, two novel systems for analysis of STRs, based on the Pyrosequencing technology, are presented. In Paper I, Y chromosomal STRs are analysed. Markers on the male specific Y chromosome are especially useful in analysis of DNA mixtures. In Paper II, ten autosomal STRs are genotyped. The systems are based on sequencing of STR loci instead of size determination of STR fragments as in routine analysis. This provides a higher resolution since sequence variants within the repeats can be detected. Determination of alleles is based on a termination recognition base. This is the base in the template strand that is excluded from the dispensation order in the sequencing of the complementary strand and therefore terminates the reaction. Furthermore, skeletal remains are often difficult to analyse, due to damaging effects from the surrounding environment on the DNA and the high risk of exogenous contamination. Analysis of mitochondrial DNA is useful on degraded samples and in Paper III, mtDNA analysis of 700 years old skeletal remains is performed to investigate a maternal relationship. The quantity and quality of DNA are essential in forensic genetics. In Paper IV the efficiency of DNA isolation is investigated. Soaking skeletal remains in bleach is efficient for decontamination but result in a lower DNA yield, especially on pulverised skull samples. In conclusion, this thesis presents novel sequencing systems for accurate and fast analysis of STR loci that can be useful in evaluation of new loci and database assembly as well as the utility of mtDNA in forensic genetics.