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To date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical control region haplotypes. The goal of this study has been to identify polymorphic coding region sites for development in a Pyrosequencing assay that would effectively enhance the discriminatory power of mitochondrial DNA analysis. With this goal in mind, five duplexes have been successfully developed and tested, utilizing the ten polymorphic sites that had been selected, with most sites being specific to Caucasians. Validation studies were performed to test the durability of the assay. The specificity of the assay to primate and non-primate species was determined to be limited to primate species only. Sample variations, including mixtures, dilutions and environmental exposure, were utilized to assess the sensitivity of the Pyrosequencing method. It was found that a minimum initial DNA input of 10fg was necessary for reliable results. The Pyrosequencing assay was able to detect mixtures at a 1:1 ratio and environmental samples exposed to the elements from up to 1 week for blood and 6 weeks for semen. Samples designed to simulate typical casework materials were analyzed and found to provide for consistent results, including trace fingerprints and digested hair shafts. These validation results provide the conclusion that this assay is suitable for use in forensic casework and demonstrate that the mitochondrial coding region provides a viable alternative to hypervariable region analysis.

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京都大学 / 新制・課程博士 / 博士(農学) / 甲第23246号 / 農博第2453号 / 新制||農||1084(附属図書館) / 学位論文||R3||N5336(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 白井 理, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Beluga whales in Canadian waters are subdivided into at least six genetically distinct stocks maintained by geographic separation and philopatry to estuaries in summer. Belugas in eastern and western Hudson Bay have previously been shown to be compose genetically distinct populations using mitochondrial restriction analysis. It is not known whether these stocks are further subdivided on the basis of specific estuarine use. Mitochondrial DNA control region sequences were used to investigate variation among belugas sampled at several sites along eastern Hudson Bay, Hudson Strait and Ungava Bay. 320 bp were sequenced, including the highly variable 5' region of control region, in 126 belugas. 17 variable sites and 17 haplotypes, which clustered into 2 related groups, were detected among the whales sequenced. Haplotypes of group A were found mostly in eastern Hudson Bay sites, while B group haplotypes were predominant in northern populations. Significant differences in frequencies of haplotype groups were found between eastern Hudson Bay and Southern Hudson Strait/Ungava Bay populations, indicating they are genetically distinct populations. Haplotype distribution patterns also suggested possible differences between belugas using different estuaries along eastern Hudson Bay. The presence of both groups in each population indicated some exchange of individuals between populations, and/or between eastern and western Hudson Bay. Multilocus DNA fingerprinting was used to investigate the extent of gene flow between eastern and western Hudson Bay belugas via interbreeding on common wintering grounds in Hudson Strait. Belugas from St. Lawrence estuary and the Mackenzie Delta were also analyzed to measure their genetic relatedness to Hudson Bay whales as well as for purposes of comparison to earlier fingerprinting analyses. While results supported lower genetic diversity within the St. Lawrence population, the range of bandsharing within and between populations was otherwise low (0.09 -0.17 for Jeffreys 33.15 and 0.12-0.22 for Jeffreys 33.6). Mantel tests showed differences among St. Lawrence, Hudson Bay, and Mackenzie Delta populations, but not within Hudson Bay. The conflicting nature of the data did not allow conclusions regarding gene flow. Therefore, DNA fingerprinting was not considered to have provided sufficient resolution in addressing this issue. / Thesis / Master of Science (MS)

Oxidative stress (OS) has been associated with aging and age-related diseases in humans, as well as with the decline in economic trait performance in poultry and other domesticated animals. However, the potential effects of OS on the poultry immune system are not well understood. In addition, the impact of bird genetic variation on redox balance remains to be elucidated. Thus, the central hypothesis of this dissertation is: The bird's adaptive immunocompetence is impacted by their OS level, which is not only influenced by environmental factors, but also related to genetic phenotype of either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). In the first phase of this study, White Leghorn chickens were provided ethanol at different concentrations in drinking water to induce OS. Biomarkers including malondialdehyde (MDA), glutathione (GSH), and plasma uric acid (PUA) were measured to assess OS before and after ethanol treatment. The adaptive immune response during an OS event was measured by plasma IgG and IgM levels, major lymphoid organ weights, CD4+/CD8+ cell ratio, and histopathological analysis of the immune organs. Results showed that when OS was induced by 10% ethanol, chicken adaptive immune responses decreased; however, when birds were exposed to 2% ethanol, there was an enhancement in antioxidant defense and immune response; These results would suggest a negative correlation between OS level and chicken adaptive immune response. In the second phase of the study, subsets of chickens were selected based on their high (H)- or low (L)-OS to assess for variations in their genetic phenotypes. Using MDA levels, 36 chickens were chosen to scan a 2734-bp region of mtDNA, but no definitive SNP was detected. In another experiment, 40 chickens were conversely selected according to three biomarkers for OS. Although no variation was found at eight SNP loci tested across the mitochondrial genome, mtDNA damage measured by 8-hydroxy-2′-deoxy-guanosine was shown to increase with time, and at higher levels in the high OS birds (p < 0.05). Thses results suggest that long-term high OS levels in chickens may increase the somatic mutation of their mtDNA. In the final phase of this dissertation, the effect of nDNA on OS, measured via a genome-wide association study was performed with 18 H and 18 L chickens using the latest chicken 60k SNP microarray for genotyping. Among 56,483 SNPs successfully genotyped, 13 SNPs across five independent loci were associated with OS at significance level of p ≤ 0.001, and another 144 SNPs were also associated with OS (p ≤ 0.01). These results indicate new loci and related genes for their genetic influence upon redox balance. In general, experiments carried out on White Leghorn chickens here have shown that adaptive immune response is tightly related to changes of OS. Further, genetic variance in nDNA is associated with the risk of high OS or the ability to better resist it. / Ph. D.

The Nile tilapia Oreochromis niloticus is native to Africa and middle East, and is an important source of nutrition for many in sub-Saharan Africa. Understanding the genetic diversity within and differentiation among wild populations can help identify O. niloticus populations that are imperiled and require directed management, especially because of increasing threats to the species' long-term persistence in the wild, including habitat destruction, overfishing, climate change, and hybridization with farmed populations. Knowledge of the genetic variation among wild populations also can contribute to foundation and selection of genetically diverse populations for aquaculture. I assessed the genetic variation among tilapia populations using fin-clips collected between December 2014 and July 2017 from 14 farmed sources, mostly originating from cage farms on the Volta Lake, and 13 wild sources from nine river basins in Ghana. I also conducted a laboratory growth experiment in Ghana with two wild populations to evaluate the tolerance of different genotypes to high temperatures, to inform their development for aquaculture in West Africa. I found that pure O. niloticus populations persist in the wild but some have been extensively introgressed with the closely related species, O. aureus, which has not previously been documented in Ghana. Additionally, some wild populations appear to have recently declined significantly in numbers, likely due to overfishing and habitat modification, the latter primarily as a result of illegal alluvial mining ongoing in Ghana. Analysis of the farmed populations revealed that at least two farms were growing the unapproved genetically improved farmed tilapia (GIFT) and related strains, and that escaped individuals are admixed into some wild populations. The results of my laboratory experiment showed that O. niloticus populations occurring in northern Ghana already may be adapted to warmer temperatures and could be developed and used purposefully in aquaculture, taking advantage of their adaptation. To protect remnant pure O. niloticus populations in the wild, timely conservation decisions should be made and implemented. Protecting wild O. niloticus populations also would ensure that pure germplasms are available to develop aquaculture stocks from native populations. / Ph. D. / The Nile tilapia Oreochromis niloticus is an important food source for many people in Africa. However, many wild populations may be at risk of population decline and extinction because of increasing human activities such as overfishing and farming of non-native strains. Understanding the genetic differences among wild populations and comparing them with farmed strains can inform protection of wild populations and also help develop aquaculture strains using native populations as genetic resources. I assessed the genetic differences among tilapia populations using fin-clips I collected between December 2014 and July 2017 from 14 farmed sources, mostly originating from cage farms on the Volta Lake, and 13 wild sources from nine river basins in Ghana. I also conducted a laboratory study with two wild populations to test their tolerance to high water temperature. My research showed that pure O. niloticus populations still occur in Ghanaian rivers, but some have reproduced widely with a similar species, O. aureus, which is not known to occur in Ghanaian rivers. I also found that some wild populations may have reduced population sizes because of overfishing or because their environments have been impacted by illegal mining occurring in almost all Ghanaian rivers. My results indicated that at least two farms were growing the genetically improved farmed tilapia (GIFT) and related varieties, some of which have escaped the farms and mixed with wild populations. The results of my laboratory experiment showed that O. niloticus populations occurring in northern Ghana may be adapted to warmer water temperatures and could be selectively bred and used in aquaculture. The information generated from my research should help in making timely conservation decisions, which should help protect the remnant pure O. niloticus populations in the wild and contribute to developing aquaculture responsibly.

The Upper Tennessee River drainage of Virginia and Tennessee, Green River drainage of Kentucky, and Cumberland River drainage of Kentucky and Tennessee are known for their freshwater mussel species diversity. These river systems harbor dense populations of freshwater mussels that have significant impacts on surrounding ecosystems; however, due to their sedentary lifestyles, freshwater mussels are particularly susceptible to many biotic and abiotic stressors. Managers strive to preserve the fragile ecosystems that include freshwater mussels and, hence, study the life-history strategies of endangered and common freshwater mussel species. The goals of this project were to inform management decision-making based on whether Leaunio ortmanni is endemic to the Green River drainage and a species distinct from Leaunio vanuxemensis based on molecular identification, estimation of genetic diversity, and morphometric analysis, and to screen for cryptic biodiversity of populations of the Cambarunio iris species complex in the Upper Tennessee, Cumberland, and Green River drainages. I used the mitochondrial DNA (mtDNA) gene from the first subunit of the NADH dehydrogenase (ND1) as a genetic marker for species-level assessment of L. ortmanni and L. vanuxemensis from the Green and Cumberland River drainages. Additional mtDNA sequences of L. ortmanni and L. vanuxemensis were added to increase sample sizes and coverage of historical distribution. A Bayesian phylogenetic analysis of mtDNA sequences did not result in monophyletic lineages for either species based on the ND1 marker. Haplotype networks of mtDNA sequences demonstrated that haplotype sharing is occurring between the two focal taxa, and also among additional taxa, all of which previously belonged to the genus Villosa. A total of eight nuclear DNA microsatellites were successfully genotyped for the two focal taxa. The nuclear DNA microsatellites showed a strong phylogeographic signal between L. ortmanni of the Green River drainage and L. vanuxemensis of the Cumberland River drainage. An assignment-test based analysis in program STRUCTURE and a phylogenetic tree constructed using Nei's D genetic distance indicated well-differentiated populations across the two drainages. Additionally, the nuclear DNA microsatellite analysis showed a recent loss of genetic diversity across all populations, including when populations were pooled together at the sub-basin level. Further delineation of the focal taxa was assessed using geometric morphometrics and decision tree and random forest analyses. Decision tree and random forest analyses identified periostracum color, nacre color, overall shape, and sex to be distinguishing factors for field identification of L. ortmanni and L. vanuxemensis. Geometric morphometrics comparing species, shape, and drainage showed clear differentiation in shell shape between L. ortmanni and L. vanuxemensis. This study was able to delineate these two taxa, showing that L. ortmanni and L. vanuxemensis are separate species, and that L. ortmanni warrants listing under the Endangered Species Act. Management actions should focus on broodstock collection and propagation strategies to increase genetic diversity within established populations. Additionally, propagation and augmentation should look to reintroduce populations of L. ortmanni into its historical geographic range in unoccupied sections of the mainstem Green River. In addition, I assessed genetic diversity and differentiation again using ND1 and eight nuclear DNA microsatellite loci, and morphological differences among different shell forms of C. iris in the Upper Tennessee, Cumberland, and Green river basins and of the sister species C. taeniatus in the Cumberland, and Green river basins. Additional mitochondrial DNA sequences of C. iris and C. taeniatus were added to increase sample sizes and coverage of historical distribution. Mitochondrial DNA analysis demonstrated haplotype sharing between taxa, but with many unique haplotypes occurring in the upper Tennessee River basin. Nuclear DNA microsatellite loci revealed low levels of genetic diversity within populations of C. iris within the Upper Tennessee River basin, but high levels of divergence from C. iris and C. taeniatus of the Green and Cumberland River basins. The nuclear DNA analysis showed high admixture within and among sampled populations of C. iris throughout the Upper Tennessee River Basin with minimal geographic structuring among sub-basins. Further, phenotypic diversity was assessed using geometric morphometrics and decision tree and random forest analysis. Decision-tree and random forest analysis identified maximum height from the umbo to the ventral margin, periostracum color, shell width, and ray coverage to be defining characteristics for field identification of the focal taxa. Geometric morphometrics showed high overlap of shell shape for the focal taxa regardless of locality. While cryptic biodiversity was not detected in the Upper Tennessee River Basin, on a larger geographic level, cryptic biodiversity was detected using the combination of the mtDNA, nuclear DNA, and morphological data, which showed that C. taeniatus and C. iris from the Green River drainage were distinct from populations of C. iris in the upper Tennessee River basin. / Master of Science / Worldwide, freshwater mussel species diversity is greatest in North America; however, both abundance and diversity have declined in Canada, the United States, Mexico and the countries of Central America. Among rivers of North America, the Ohio River and its large tributaries, which include the Green, Cumberland, and Tennessee River drainages, have been noted for their high levels of biodiversity of freshwater mussels. Freshwater mussels contribute many services to freshwater ecosystems, including nutrient recycling and storage, structural habitat, substrate and food web modification. Dense populations of freshwater mussels have significant impacts on the surrounding ecosystem; however, due to their sedentary lifestyles, freshwater mussels are particularly susceptible to many biotic and abiotic stressors. Examples of stressors include agricultural runoff, temperature fluctuations, and dams which can alter stream conditions into more lake-like conditions and therefore affect the distributions of host fish populations. These stressors put mussels at risk for extirpation, which in turn, reduces the ecological biodiversity of these river drainages. Like other freshwater mussel species, the rainbow mussel (Cambarunio iris) of the Upper Tennessee River drainage, Mountain Creekshell (Leaunio vanuxemensis) of the Cumberland and Tennessee River drainages, and Kentucky Creekshell (Leaunio ortmanni) of the Green River drainage are in danger of decline due to anthropogenic changes in biotic and abiotic factors. Habitat degradation and loss have been of particular concern to managers. Life-history strategies for these three species are still widely unknown, and due to the overlap in their distributions, both phylogenetic and morphological analyses are needed to distinguish between species and populations to determine the best approach for management and conservation. The lack of understanding of taxonomic relationships in combination with morphologically similar characters poses a threat to conservation of these three species. The phylogenetic species concept is defined as an irreducible group whose members are descended from a common ancestor and who all possess a combination of certain defining, or derived traits, whereas the biological species concept is defined as groups or populations that are reproductively isolated from each other, meaning individuals from different groups cannot breed with one another. The results of my study have led to a better understanding of the phylogenetic relationships and species status of individuals collected from different localities of L. ortmanni and L. vanuxemensis and C. iris collected from the Green and Cumberland River drainages and the Upper Tennessee River drainage. In addition, morphological analyses were conducted to identify which traits are best for external identification of these three look-alike species, so they can be more reliably identified in the field.