The darkbarbel catfish (Peltoebagrus vachelli) is a small bodied benthic fish that inhabits the Yangtze River in China and is commercially valued as food. The objectives of this project were to develop the first set of microsatellite primers specific to P. vachelli and use them to examine the levels of genetic variability and population structure of three populations collected at three sites (Yibin, Luzhou, and Song Ji) along an unfragmented stretch of the upper Yangtze River. Microsatellite primers were designed from an enriched library of genomic DNA and a total of eight primer pairs were optimized to produce reliable polymorphic PCR amplicons on a LI-COR 4200 IR2 platform. A high level of variability was detected amongst the isolated loci with the number of alleles per locus ranging from 14 – 29 and mean observed and expected heterozygosity values of 0.84 and 0.90 respectively across the entire sample set. Overall levels of genetic diversity were high within the three populations with mean observed and expected heterozygosities ranging from 0.823 – 0.869 and 0.864 – 0.921 respectively. Little evidence of genetic structure was detected (global FST = 0.0065, p < 0.05) within the sampled region and pairwise tests of differentiation were not significant (all FST p > 0.05). These results imply historically large populations of P. vachelli in the upper portion of the Yangtze River that, in the absence of artificial barriers, are part of a single panmictic unit. As plans have been put forth for construction of hydroelectric installations within the sampled region, this study provides a baseline estimate of the levels of genetic variation present in P. vachelli within the remaining undammed stretch of the Upper Yangtze River which will serve as a foundation for future analyses on the effects of river fragmentation within this region. Additionally the isolation and characterization of the microsatellites will provide useful molecular markers for a variety of other applications in this and closely related species such as parentage analysis, determination of stocks, and maintenance of genetic variation in stocking practices. / Thesis (Master, Biology) -- Queen's University, 2012-06-29 18:00:25.509

No description available.

The aims of this study were: (i) to determine the association between p53, bcl-2, pRb, p21, cyclin A and p-glycoprotein immunoexpression and prognosis, and (ii) to determine the frequency of loss of heterozygosity and microsatellite instability at 11 p, 16q and mismatch repair gene loci and their association with prognosis, in nephroblastomas in South African children. There were 138 cases (111 of whom received preoperative chemotherapy) in the immunohistochemical study and, 70 cases (48 with preoperative chemotherapy) in the microsatellite study. The following monoclonal antibodies were used after heat induced epitope retrieval; p53, bcl-2, pRb, p21, cyclin A and p-glycoprotein. Six polymorphic microsatellite markers were selected from the 11p region, 5 from the 16q region and 6 from the loci of known mismatch repair genes. Automated fluorescent DNA technology was used in the analysis. The results of the immunohistochemical and microsatellite studies were correlated with patient age, gender, preoperative chemotherapy, SlOP histological classification, SlOP histological risk group, clinicopathological stage, patient outcome and survival using X2 , Fisher's exact test, Cox regression model and Kaplan-Meier estimates. The majority of patients presented with advanced disease. Anaplastic tumours and high-risk histology were associated with high disease stage. Mortality was directly related to increasing stage and histological risk group. Multivariate analysis showed that clinicopathological stage was the only factor significantly associated with survival (p<0.001) (hr=5.6, 95%CI: 2.1-14.9). High expression of p53 was more frequent in anaplastic tumours suggesting that p53 mutations are common events in this tumour type (p<0.001). Despite the strong association with tumour histology, there was no association with stage. Although p53 expression was found to be a predictor of survival in the univariate analysis this was not retained in the multivariate analysis. Tumours treated with preoperative chemotherapy showed higher bcl-2 immunoreactivity (p=0.027 but lower levels of pRb (p=0.040) and cyclin A expression (p<0.001). All anaplastic tumours showed high expression of pRb compared to the other histological types (p=0.003). Expression of xxii pRb was significantly associated with survival in the univariate analysis but not in the multivariate analysis. High cyclin A expression was associated with high risk histology (p<0.001). Cyclin A expression was found to be a significant predictor of survival in both the univariate (hr=1.7; 95%CI 1.2-2.4; p=0.002) and multivariate analyses (hr=1.7; 95%CI1.1-2.7; p=0.032). Although tumours with high risk histology were more likely to express high levels of p-glycoprotein, this did not reach significance. LOH at 11 p was seen in 64.7% of 68 informative cases. LOH at 11 p13 was more frequent than LOH at 11p15. LOH for both 11p13 and 11p15 was found in 39.7% of all tumours. MSI at 11 p was seen in 22.1 % of informative cases. The majority showed MSI for one marker only. LOH 16q was seen in 66.7% of 66 informative cases. MSI at 16q was seen in 16.7% of cases. LOH for 016S496 and 016S520 appear to be related to tumour histology and risk group. The most frequent locus for LOH was 16q21-22, which is known to harbour important genes, such as, E2F4 and E-cadherin. LOH for MMR markers was seen in 43.5% of 69 informative cases. MSI was seen in 11.6% of tumours. In the multivariate analysis there was no significant correlation between LOH at any of the loci studied and survival. There were no tumours with high frequency MSI. Low frequency MSI was of no clinicopathological significance. The following conclusions are made: (i) p53 mutations determined by high p53 expression is a frequent finding in anaplastic tumours, (ii) Bcl-2 may play a role in the chemoresistance of nephroblastomas, (iii) Rb gene alterations are not important in the development of nephroblastoma and anaplasia, (iv) Cyclin A expression is an independent predictor of survival, (v) p-glycoprotein may be responsible for the chemoresistance in a proportion of nephroblastomas, (vi) MSI is a rare occurrence in nephroblastoma and does not play a role in the development of nephroblastoma, (vii) LOH at 11 p and 16q are frequent findings in nephroblastomas, (viii) LOH for the specific 16q markers (016S496 and 016S520) may have an important prognostic role in nephroblastoma. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2008.

Walleye (Sander vitreus) is an economically valuable freshwater fish throughout North America. In Alberta, pressure from sport fishing and commercial fishing make effective management and protection of this species crucial to its sustainability. Walleye from 12 Alberta lakes were genetically characterized using 15 microsatellite markers. Each lake contained a genetically distinct walleye subpopulation within a larger population of the river basin in which the lake was situated. Differentiation between subpopulations varied (ST=0.05 to 0.29). Patterns of genetic divergence aligned closely with the current hydro-geographical landscape, except where stocking events have occurred. Vicariance and natal philopatry are likely mechanisms maintaining the current genetic structure. The markers detected sufficient genetic variation between most subpopulations to assign an individual fish to a subpopulation of origin. The utility of genetic assignment was illustrated for stocking assessment and forensic enforcement. These genetic data will help to inform management decisions, monitor population status and enforce harvest restrictions for Alberta walleye. / Systematics and Evolution

The Baird’s pocket gopher (Geomys breviceps) is a solitary, fossorial rodent found throughout areas of Texas, Arkansas, Oklahoma, and Louisiana. Research focusing on the population genetics of pocket gophers and other species with limited vagility and isolated populations is lacking. Through the use of mitochondrial and microsatellite data, a series of population genetic analyses were completed to better understand the population structure and gene flow among a series of G. breviceps localities. Pocket gophers were captured from five localities in the Brazos Valley and used in this study. Due to the lack of microsatellite loci available for G. breviceps, 10 loci were created for use in this study. Overall estimates from the population genetic analyses showed high levels of gene flow amongst nearby localities with decreasing levels as distance between localities increased. Findings suggest that 2-3 localities located within 2 km of each other function as one genetic cluster thus showing 3-4 total genetic clusters total in this study. Results also suggest that the Baird’s pocket gopher is capable of moving at least 2 km, but further analyses should be completed to better understand dispersal distance.

Studying species at the molecular level can provide insights into how ecological and biological processes interrelate resulting in the diversity we see today. This information can be applied to conserve species at risk of extinction, or to better manage genetic diversity in species of economic importance. Species that inhabit freshwater riverine systems commonly exhibit population structures that are related to their relative dispersal capability, contemporary stream structure and/or historical stream structure. This thesis examined the populations genetic structure of wild and cultured stocks of the commercially farmed freshwater crayfish, C. quadricarinatus (von Martens), using genetic markers characterized by different modes of inheritance. C. quadricarinatus is distributed naturally in riverine systems in northern Australia, and southern Paupa New Guinea (PNG) and inhabits a variety of freshwater ecosystems ranging from ephemeral to permanent. Life history characteristics of C. quadricarinatus suggest a high level of genetic structuring among wild stocks might exist. However, seasonal flooding coupled with low topography across its distribution in northern Australia may promote sufficient gene flow among rivers to produce genetic homogeneity. Historical gene flow may also influence modern genetic structure as many distinct riverine catchments that C. quadricarinatus inhabits, were once connected at times of lower sea level. Insight into genetic relationships among C. quadricarinatus populations will allow for better management practices of wild populations in the future. The study investigated phylogenetic relationships among C. quadricarinatus representing 17 discrete natural drainages across the natural range in Australia and PNG, using 16s and COI gene sequences. Sequence analysis of both genes resolved two distinct genealogical lineages in Australia and three in PNG. The two divergent Australian lineages concur with original taxonomic descriptions of Reik (1969) based on external morphological differences. The three C. quadricarinatus populations sampled in PNG were all genetically distinct from each other, with one exhibiting a close association with an Australia lineage. The immense physical barriers (rugged mountain ranges) to gene flow in PNG will almost certainly have reduced dispersal capabilities for C. quadricarinatus. During times of lowered sea levels in the past, Australia and southern PNG were a single landmass with terrestrial and freshwater organisms theoretically able to disperse over associated land and via freshwater connections. The close genetic relationship between PNG and Australian C. quadricarinatus support a recent freshwater connection and hence gene flow between northern Australia and PNG C. quadricarinatus populations. Genetic differentiation among some C. quadricarinatus lineages exhibit as much genetic divergence at 16s RNA sequences as taxonomically recognised sub-species in the Cherax genus. Since C. quadricarinatus was originally described as different species based on external morphological differences (Reik, 1969), it is recommended that the taxonomy of C. quadricarinatus in Australia and PNG be re-evaluated. C. quadricarinatus specific microsatellite markers were developed for this study. Five variable loci were employed to investigate the extent of contemporary gene flow among fourteen C. quadricarinatus wild river populations in northern Australia. High FST and genetic distance estimates observed among pair wise comparisons of C. quadricarinatus populations are consistent with limited or no gene flow occurring among drainages. Speculation that C. quadricarinatus may disperse between adjacent or nearby drainages at times of flood, either across floodplains, or via flood plumes therefore seems highly unlikely among the populations examined in the current study. No significant correlation was observed between geographic distance and genetic distance among C. quadricarinatus populations here. C. quadricarinatus populations most closely resemble an island-like model, where gene flow is independent of geographic distance among populations and where genetic divergence occurs to a greater or lesser extent as a result of genetic drift within otherwise isolated populations. A significant number of C. quadricarinatus populations showed deviations from expected Hardy-Weinberg equilibrium (HWE). Samples sizes may not have been sufficiently large to reflect a true representation of genotypic proportions present in the sampled populations due to the highly variable nature of microsatellite loci. Deviations from HWE equilibrium, however, can also result from null alleles. Null allele estimates suggested a large proportion of null alleles were present in the C. quadricarinatus populations analysed. This may be a result of C. quadricarinatus populations confined to discrete drainages experiencing independent evolution, resulting in mutations in primer binding sites. The growing economic potential of C. quadricarinatus culture, both domestically and internationally, prompted expanding the current study to examine genetic diversity levels in commercial C. quadricarinatus stocks. The study employed five microsatellite markers to quantify genetic diversity in four Australian and three C. quadricarinatus culture stocks from overseas. Many C. quadricarinatus culture stocks also showed deviations from HWE expectations. This was not a surprising result given that the wild populations also deviated and domestication can also influence HWE. Relatively high levels of genetic diversity were observed. This probably results from intentional mixing of discrete river strains for production of the first commercial stock. Genetic differentiation estimates among culture stocks and assignment tests indicated that overseas culture stocks are most likely derived from the first commercial culture stock developed in Australia and then disseminated widely (the Hutchings stock). Robin Hutchings was a known supplier of live C. quadricarinatus to many international culture initiatives. Assignment of culture stocks back to their wild origins indicated that all C. quadricarinatus culture stocks sampled possess alleles that originate from the Flinders River (proportions ranged from 33-94%). Domestication of C. quadricarinatus to date has not resulted in significant reductions in levels of genetic diversity (heterozygosity or alleles richness) when compared to wild populations sampled in this study. Comparing culture stocks to wild populations to gauge their 'genetic health' may not be a suitable scale for evaluating genetic diversity in culture stocks. Wild populations are essentially evolving independently, are subjected to harsh seasonal environmental fluctuations resulting in periodic population crashes (genetic bottlenecks), with little or no recruitment from neighbouring drainages (gene flow). This study does however indicate that there is a large amount of genetic diversity distributed among wild populations that has yet to be exploited in culture. Genetic diversity in wild populations provides a resource for future stock improvement programs for C. quadricarinatus culture and thus requires careful conservation and appropriate management.

Microsatellites are short (1-6bp long) highly polymorphic tandem repeats, found in all genomes analyzed so far. Popular genetic markers for many applications including population genetics, pedigree analysis, genetic mapping and linkage analysis, some microsatellites also can cause a variety of human neurodegenerative diseases and may act as agents of adaptive evolution through the regulation of gene expression. As a consequence of these diverse uses and functions, the mutational and evolutionary dynamics of microsatellite sequences have gained much attention in recent years. Mostly, the focus of studies investigating microsatellite evolution has been to develop more refined evolutionary models for estimating parameters such as genetic distance or linkage disequilibrium. However, there is an incentive in using our understanding of the evolutionary processes that affect these sequences to examine the functional implications of microsatellite evolution. What has emerged from nearly two decades of study are highly complex mutational dynamics, with mutation rates varying across species, loci and alleles, and a multitude of potential influences on these rates, most of which are not yet fully understood. The increasing availability of whole genome sequences has immensely extended the scope for studying microsatellite evolution. For example, where once it was common to examine single loci, it is now possible to examine microsatellites using genome wide approaches. In the first part of my dissertation I discuss approaches and issues associated with detecting microsatellites in genomic data. In Chapter 2 I undertook a meta-analysis of studies investigating the distribution of microsatellites in yeast and showed that studies comparing the distribution of microsatellites in genomic data can be fraught due to the application of different definitions for microsatellites by different investigators. In particular, I found that variation in how investigators choose the repeat unit size of a microsatellite, handle imperfections in the array and especially the choice of minimum array length used, leads to a large divergence in results and can distort the conclusions drawn from such studies, particularly where inter-specific comparisons are being made. In a review of the currently available suite of bioinformatics tools (Chapter 3), I further showed that this bias extends beyond a solely theoretical controversy into a methodological issue because most software tools not only incorporate different definitions for the key parameters used to define microsatellites, but also employ different strategies to search and filter for microsatellites in genomic data. In this chapter I provide an overview of the available tools and a practical guide to help other researchers choose the appropriate tool for their research purpose. In the second part of my thesis, I use the analytical framework developed from the previous chapters to explore the biological significance of microsatellites exploiting the well annotated genome of the model organism Saccharomyces cerevisiae (baker’s yeast). Several studies in different organisms have indicated spatial associations between microsatellites and individual genomic features, such as transposable elements, recombinational hotspots, GC-content or local substitution rate. In Chapter 4, I summarized these studies and tested some of the underlying hypotheses on microsatellite distribution in the yeast genome using Generalized Linear Models (GLM) and wavelet transformation. I found that microsatellite type and distribution within the genome is strongly governed by local sequence composition and negative selection in coding regions, and that microsatellite frequency is inversely correlated with SNP density reflecting the stabilizing effect point mutations have on microsatellites. Microsatellites may also be markers for recent genome modifications, due to their depletion in regions nearby LTR transposons, and elements of potential structural importance, since I found associations with features such as meiotic double strand breaks, regulatory sites and nucleosomes. Microsatellites are subject to local genomic influences, particularly on small (1-2kb) scales. Although, these local scale influences might not be as dominant as other factors on a genome-wide scale they are certainly of importance with respect to individual loci. Analysis of locus conservation across 40 related yeast strains (Chapter 5) showed no bias in the type of microsatellites conserved, only a negative influence of coding sequences, which supports again the idea that microsatellites evolve neutrally. Polymorphism was rare, and despite a positive correlation with array length, there was no relationship with either genomic fraction or repeat size. However, the analysis also revealed a non-random distribution of microsatellites in genes of functionally distinct groups. For example, conserved microsatellites (similar to general microsatellites in yeast) are mostly found in genes associated with the regulation of biological and cellular processes. Polymorphic loci show further an association with the organization and biogenesis of cellular components, morphogenesis, development of anatomical structures and pheromone response, which, is absent for monomorphic loci. Whether this distribution is an indication of functionality or simply neutral mutation (e.g. genetic hitch-hiking) is debatable since most conserved microsatellites, particularly variable loci, are located within genes that show low selective constraints. Overall, microsatellites appear as neutrally evolving sequences, but owing to the sheer number of loci within a single genome, individual loci may well acquire some functionality. More work is definitely needed in this area, particularly experimental studies, such as reporter-gene expression assays, to confirm phenotypic effects.

Thesis(M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniversitet, 2007. / Härtill 5 uppsatser + 3 appendix.

Title from first page of PDF document. Includes bibliographical references (p. 24-26).