Im Rahmen der Dissertation wurde ein Verfahren zur Quantifizierung monosomer Zellpopulationen innerhalb eines disomen Normalgewebes auf Basis der Pyrosequenzierung von Einzelbasenmutationen etabliert und hinsichtlich seiner Genauigkeit untersucht. Dabei liegt ein besonderer Schwerpunkt auf der Entwicklung eines Verfahrens zur Festlegung von Grenzwerten für die Detektion monosomer Population sowie für genetisch heterogene Subpopulationen. Zur Bestimmung der Genauigkeit wurden Mischreihen von DNA zweier Genotypen angefertigt und das Allelverhältnis durch Pyrosequenzierung gemessen. Diese Ergebnisse wurden genutzt, um Grenzwerte für die Detektion von LOH3-positiven Zellen im UMM estzulegen. In diesen Vorversuchen konnte die Anwendbarkeit der Analysemethode für Proben aus UMM sowohl aus Enukleations wie auch aus Feinnadelaspirationspräparaten demonstriert werden. Es wurde dann in einem weiteren Schritt analysiert, wie viele differente Loci für eine korrekte Diskriminierung zweier Genotypen analysiert werden müssen. Hier wurde gezeigt, dass zum einen die Anzahl der untersuchten SNP aber auch das gemessene Allelverhältnis maßgeblichen Einfluss auf die Genauigkeit der Analyse haben. Basierend auf diesen Daten wurde ein Verfahren entwickelt, das aus der gewünschten Genauigkeit eine Berechnung des Umfangs eines zu etablierenden SNP-Panels ermöglichte.

Doctor of Philosophy / Department of Grain Science and Industry / Dirk E. Maier / Increased mandates for the production of transportation fuels from renewable resources have thrust the conversion of lignocellulosic biomass, e.g., energy crops and agricultural residues, to ethanol into commercial production. The conversion of biomass to ethanol has been implemented; transportation and storage logistics are still obstacles to overcome by industry. Limited harvest windows throughout the year necessitate extended periods of biomass storage to maintain a consistent, year-round supply to the biorefinery. Sorghum biomass stored with no coverage (NN), covered with tarp (NT), wrapped in plastic (PN) and covered with a tarp and wrapped in plastic (PT) for six months was analyzed for changes in biomass components—cellulose, hemicellulose and lignin, cellulose and hemicellulose degrading enzymes, and conversion to ethanol yields. Treatment NN had increased enzyme activity, and reduced cellulose content and ethanol yields; while biomass covered maintained enzyme activity, cellulose content and ethanol yields. Sequencing of the Large SubUnit (LSU) region and the internal transcribed spacer (ITS) regions of ribosomal RNA gene gave consistent results of fungal community dynamics in biomass stored as previously described. Fungal community richness and diversity increased, while evenness decreased in uncovered biomass during storage. Covered and uncovered storage treatments and over time were found to exhibit distinctly different fungal communities. In contrast, bacterial communities were found to be unresponsive to storage treatments and durations. Cladosporium, Alternaria and Cryptococcus were found to be the most abundant in the stored biomass. Covering of biomass strongly limits the arrival and establishment of new fungal propagules in stored biomass, reducing biomass degradation by these often pathogenic, saprobic or endophytic communities. Overall, covering of biomass during storage is essential for optimal substrate retention for downstream processing into ethanol. In addition, storage and transportation logistics of three real-world scenarios were evaluated for the conversion of wheat straw, corn stover and sorghum stalks residues to ethanol at a biorefinery located in Southwest Kansas. Economic evaluation revealed that transport and storage of residues at satellite storage facilities was most economical for farmers and would create opportunity for the operation of profitable facilities that would supply the local biorefinery on demand throughout the year.

Arsenic (As) is a problem with rice production systems throughout the world as high As concentrations are reported in rice grains originating from several parts of the world. This characteristic is mainly due to the flooded conditions utilized in rice culture. We hypothesized that the soluble As concentrations in the rice rhizosphere can be decreased by growing rice more aerobically through intermittent flooding. Intermittent water management practices might also change microbial populations in the rice rhizosphere that might potentially impact As chemistry and bioavailability. Two field-scale experiments were conducted over two years to study the impact of intermittent and continuous flooding on As speciation and microbial populations in the rice rhizosphere. As levels and speciation in the rhizosphere soil, root-plaque and pore-water were determined using a high performance liquid chromatography-inductively coupled plasmamass spectroscopy (HPLC-ICP-MS). The microbial populations were assessed from the rhizosphere soil and root-plaque samples using quantitative polymerase chain reaction (qPCR) and 16S rRNA sequencing. Pore-water and root-plaque total-As concentrations significantly decreased in the intermittent compared to the continuous flood plots. Inorganic arsenite (iAsIII) was predominant in pore-water and inorganic arsenate (iAsV) in root-plaque and soil. Rootplaque sequestered significantly higher levels of As (almost tenfold higher) than the adjacent rhizosphere soil. Grain As concentrations also decreased by 35 to 45 percent in the intermittent compared to the continuously flooded plots. Organic As species, monomethyl and dimethyl arsenate were detected in the rhizosphere with relative increases and decreases among the treatments. Bacteria were the predominant group (91 to 94 percent and 48 to 78 percent of total community in root-plaque and rhizosphere soils, respectively). Archaea were also a major component of rhizosphere soil with their populations being higher under continuous flooding. The relative abundance of iron-reducing bacteria was around 3 to 6 percent of the total community in root-plaque and around 6 to 6 percent in soil, with significantly lower abundance in the intermittent compared to the continuously flooded plots. Results of these studies demonstrated that intermittent flooding could be a potential management option to reduce grain As in rice cultivated on fields with moderate to high As concentrations.

Acid mine drainage (AMD) is an environmental problem of a global scale. Passive remediation strategies utilizing the metabolism of sulfate-reducing bacteria have emerged as promising options for the mitigation of impacted AMD sites. In order to test the effect of varying complex and simple carbon sources on AMD remediation efficiency, pilot-scale bioreactors were constructed and exposed to AMD in situ over a ten-month period. Geochemical analyses suggested that the efficiency of AMD remediation depended more on the seasonal weather patterns of Southern Illinois, USA than the substrate composition of each bioreactor. Enrichment cultures targeting sulfate-reducing organisms yielded several isolates most closely related to members of the genera Desulfovibrio and Clostridium. Microbial community analysis was performed using fluorescent in situ hybridization, 16S rRNA gene targeted pyrosequencing, and quantitative polymerase chain reaction (qPCR). Results suggested that the depth from which samples were taken as well as the substrate composition impacted the microbial communities within each bioreactor. Over the course of the experiment the community changed from one similar to that of a bovine rumen to one more adapted to the acidic nature and high metal content of AMD. Community abundance based on 16S rRNA gene and dsrB gene copy number suggested an overall decrease in the bacterial population over the course of the study.

Background: Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with EGFR mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with KRAS mutations do not benefit of such treatment. Methods: This study investigates the frequency of EGFR and KRAS mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes. Results: Five EGFR mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. KRAS mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations. Conclusions: This study confirms previous observations regarding the frequency of EGFR and KRAS mutations in non-small cell lung cancer. Mutations in EGFR and KRAS were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.

We developed a set of hypervariable microsatellite markers for the Pacific red snapper (Lutjanus peru), an economically important marine fish for small-scale fisheries in the west coast of Mexico. We performed shotgun genome sequencing with the 454 XL titanium chemistry and used bioinformatic tools to search for perfect microsatellite loci. We selected 66 primer pairs that were synthesized and genotyped in an ABI PRISM 3730XL DNA sequencer in 32 individuals from the Gulf of California. We estimated levels of genetic diversity, deviations from linkage and Hardy-Weinberg equilibrium, estimated the frequency of null alleles and the probability of individual identity for the new markers. We reanalyzed 16 loci in 16 individuals to estimate genotyping error rates. Eighteen loci failed to amplify, 16 loci were discarded due to unspecific amplifications and 32 loci (14 tetranucleotide and 18 dinucleotide) were successfully scored. The average number of alleles per locus was 21 (+/- 6.87, SD) and ranged from 8 to 34. The average observed and expected heterozygosities were 0.787 (+/- 0.144 SD, range 0.250-0.935) and 0.909 (+/- 0.122 SD, range 0.381-0.965), respectively. No significant linkage was detected. Eight loci showed deviations from Hardy-Weinberg equilibrium, and from these, four loci showed moderate null allele frequencies (0.104-0.220). The probability of individual identity for the new loci was 1.46(-62). Genotyping error rates averaged 9.58%. The new markers will be useful to investigate patterns of larval dispersal, metapopulation dynamics, fine-scale genetic structure and diversity aimed to inform the implementation of spatially explicit fisheries management strategies in the Gulf of California.

Conventional plastics have been used for decades in a diverse range of applications, however, many are resistant to degradation, leading to environmental pollution and their manufacture is dependent on non-renewable fossil fuels. Therefore, there has been an increasing need for eco-friendly biodegradable materials from renewable resources. Poly(lactic acid) (PLA) is a compostable polyester with a hydrolysable backbone that is susceptible to biodegradation and produced from renewable feedstocks. PLA has mechanical qualities comparable to non-biodegradable plastics, and currently is commercialized as food-packaging polymer for short shelf-life products. However, while PLA hydrolysis at elevated temperatures proceeds abiotically, ultimately releasing lactic acid and short chain oligomers, the role of microorganisms is unclear. Since PLA short-shelf life products are disposed after use, understanding the role of microorganisms and the effect of degradation on microbial populations in the environment is important. Therefore, the aims of this research was to (a) determine the relative importance of biotic and abiotic factors on PLA degradation; (b) to isolate putative fungal PLA degraders from the surface of PLA when buried in compost or soil and to test their ability to degrade PLA; (c) to assess the impact of PLA degradation on fungal communities when entering compost systems. The roles of abiotic and biotic factors in the degradation of high molecular weight PLA were investigated by comparing degradation rates in compost, soil and sterile water at temperatures of 25°, 37°, 45°, 50° and 55°C. Tensile strength loss and molecular weight decline of PLA in microorganism-rich compost and soil were greater than chemical hydrolysis in sterile water at elevated temperatures (above 45°C) indicating microorganisms can directly enhance PLA degradation. Since extensive fungal growth was observed on the surface of PLA when buried in compost and soil, putative fungal PLA degraders were isolated from PLA surface and their community profile on PLA surface was compared with the compost and soil community with a molecular method, terminal restriction fragment polymorphism (TRFLP). Among the identified fungi, Thermomyces lanuginosus was the dominant isolate recovered and shown to enhance PLA degradation in compost at 50°C. The fungal community profile on PLA surface was different than the fungal profile in compost and soil suggesting enrichment for PLA degraders on the surface of PLA. In order to determine the impact of PLA degradation on the fungal compost community, two different high molecular weight PLA sources, films and granules were buried in compost at 10%, 25% and 50% (w/w) concentration for 4 months at 25°C and 50°C and the community profile analysed by TRFLP and pyrosequencing. TRFLP revealed that when PLA did not degrade, the fungal community shifted back toward the initial compost community profile, however, when PLA degraded to its monomers releasing lactic acid at 50°C at a concentration of 50% (w/w) it changed the fungal community profile and decreased the fungal diversity. Pyrosequencing revealed that the presence of PLA enriched for Thermomyces in the compost population over time.

Les zones arides couvrent environ un tiers de la surface terrestre de la planète. Des études visant à comprendre la dispersion microbienne dans les déserts ont été réalisées. En effet, les communautés microbiennes du sable des déserts peuvent jouer un rôle important dans la stabilité des sols. Le pyroséquençage pour les ARNr 16S à partir de l’ADN total extrait des sols des échantillons de sable peut donner des renseignements clés sur la structure des communautés bactériennes qui les composent. Dans cette étude, la diversité et la structure des communautés bactériennes de la surface du sol des déserts des l'États de l'Utah et du Maine ont été mises en évidence. Nous avons mise en œuvre une procédure permettant l'analyse des séquences de l’ADNr 16S en combinant des outils préexistants dédiés à la métagénomique. Ainsi, des corrélations entre certains facteurs environnementaux et la diversité bactérienne dans les deux déserts, ont pu être établis.Le désert du Maine situé dans le nord-est Etats-Unis est une étendue de boue glaciaire, entourée par une forêt de pins. Le sol de ce désert possède les caractéristiques d’on sable avec de très faibles capacités de rétention d'eau, d’une rétention des éléments nutritifs, ainsi qu’une valeur de pH relativement faible (pH 5,09). Les échantillons provenant de ce site présentent donc des propriétés particulièrement intéressantes à étudier en lieu avec la diversité bactérienne. Deux échantillons de sable de la surface du désert du Maine ont été obtenus, et le pyroséquençage des gènes d'ADNr 16S obtenus après amplification par PCR à partir de l'ADN total extrait a été utilisé pour évaluer la diversité bactérienne, la structure de la communauté bactérienne et l'abondance relative des principaux taxons. Nous avons observé que les échantillons de sol provenant du désert du Maine présentent une diversité bactérienne singulière, avec une prédominance de Proteobacteria et Actinobacteria. Les bactéries du genre le plus abondant, Acidiphilium, représentent 12,5% du total des séquences d'ADNr 16S. Au total, 1 394 OTU ont été comptabilisées. En comparant les résultats de notre population bactérienne avec des études portant sur des sols avec caractéristiques similaires, nous avons constaté que les échantillons du Maine contiennent une faible diversité du phylum Acidobacteria que les sols acides des certains forêts, et moins de Firmicutes ainsi que plus de Proteobacteria que les sols des déserts oligotrophes.Le Désert de l'Utah présente des caractéristiques géographiques qui ressemblent à Mars. En effet il est caractérisé par la présence de collines de couleur rouge et de sols constitués de grès. Les sites d'échantillonnage couvrent le Gobblin Valley State Park et autour, notamment sur le plateau du Colorado. Avec des approches similaires à ceux utilisés pour le désert du Maine, des corrélations entre facteurs environnementaux (paramètres physico-chimiques) et diversité de structure des communautés bactériennes obtenus, ont été étudiés. Les phylums prédominants sont les Proteobacteria, Actinobacteria, Bacteroidetes et Gemmatimonadetes. Les genres les plus abondants dans nos échantillons sont Cesiribacter, Lysobacter, Adhaeribacter, Microvirga et Pontibacter. Mais de façon notable, il semble que l'abondance relative des Alphaproteobacteria et des Gemmatimonadetes est significativement corrélée aux certains facteurs environnementaux des sols, par exemple de pH et des concentration des matières organiques. / Aridity is the dominant climatic factor over approximately 30% of the land surface of the world. Research concerning microbial populations in two U.S. deserts has been performed to determine the diversity of these bacteria. Pyrosequencing-based profiling of 16S rRNA amplicons from surface soils of sand samples can provide key insights into the structure of bacterial communities and their diversity. In this study, we demonstrated the bacterial diversity and community structures of surface soil in the Corolado Plateau in the Utah State and the Desert of Maine using pyrosequencing of 16S rRNA amplicons. We built our pipeline for the analysis of 16S rRNA pyrosequencing data by combining several existing tools of metagenomics. We also examined correlations between certain environmental factors and bacterial diversity in the two deserts.The Desert of Maine is a tract of glacial silt, surrounded by a pine forest, in the state of Maine located in the northeastern USA. The soil of the Desert of Maine has a sandy texture with poor water holding abilities, nutrient retention capabilities and a relatively low pH value (pH 5.09). Samples from this site thus present an interesting place to examine the bacterial diversity in mineral sandy loam soils with an acidic pH and low concentrations of organic materials. Two surface sand samples from the Desert of Maine were obtained, and pyrosequencing of PCR amplified 16S rDNA genes from total extracted DNA was used to assess bacterial diversity, community structure and the relative abundance of major bacterial taxa. We found that the soil samples from the Desert of Maine showed high levels of bacterial diversity, with a predominance of members belonging to the Proteobacteria and Actinobacteria phyla. Bacteria from the most abundant genus, Acidiphilium, represent 12.5% of the total 16S rDNA sequences. In total, 1394 OTUs were observed in the two samples, with the number of common OTUs observed in both samples being 668. By comparing our bacterial population results with studies on related soil environments, we found that the samples contained less Acidobacteria than soils from acid soil forests, and less Firmicutes plus more Proteobacteria than soils from oligotrophic deserts.Deserts in Utah has geographic features that resemble Mars, characterized by red-colored hills, soils and sandstones. Our sample sites cover the Goblin Valley State Park and nearby regions on the Colorado Plateau. We also examined physicochemical parameters of soil from the sample sites to investigate correlations between bacterial community structure and environmental drivers. The predominant phyla of the samples represent members of the Proteobacteria, Actinobacteria, Bacteroidetes, and Gemmatimonadetes. The most abundant genera in our samples are Cesiribacter, Lysobacter, Adhaeribacter, Microvirga and Pontibacter. We found that the relative abundance of Alphaproteobacteria and Gemmatimonadetes are significantly correlated to some environmental factors of soils, such as pH and concentration of organic matters.

Penstemon Mitchell (Plantaginaceae) is one of the largest, most diverse plant genera in North America. Their unique diversity, paired with their drought-tolerance and overall hardiness, give Penstemon a vast amount of potential in the landscaping industry—especially in the more arid western United States where they naturally thrive. In order to develop Penstemon lines for more widespread commercial and private landscaping use, we must improve our understanding of the vast genetic diversity of the genus on a molecular level. In this study we utilize genome reduction and barcoding to optimize 454-pyrosequencing in four target species of Penstemon (P. cyananthus, P. davidsonii, P. dissectus and P. fruticosus). Sequencing and assembly produced contigs representing an average of 0.5% of the Penstemon species. From the sequence, SNP information and microsatellite markers were extracted. One hundred and thirty-three interspecific microsatellite markers were discovered, of which 50 met desired primer parameters, and were of high quality with readable bands on 3% Metaphor gels. Of the microsatellite markers, 82% were polymorphic with an average heterozygosity value of 0.51. An average of one SNP in 2,890 bp per species was found within the individual species assemblies and one SNP in 97 bp were found between any two supposed homologous sequences of the four species. An average of 21.5% of the assembled contigs were associated with putative genes involved in cellular components, biological processes, and molecular functions. On average 19.7% of the assembled contigs were identified as repetitive elements of which LTRs, DNA transposons and other unclassified repeats, were discovered. Our study demonstrates the effectiveness of using the GR-RSC technique to selectively reduce the genome size to putative homologous sequence in different species of Penstemon. It has also enabled us the ability to gain greater insights into microsatellite, SNP, putative gene and repetitive element content in the Penstemon genome which provide essential tools for further genetic work including plant breeding and phylogenetics.

Allopolyploids form through the hybridization of two or more diploid genomes. A challenge to reproduction in allopolyploids is that pairing can occur between homologous chromosomes or homeologous chromosomes (i.e.different subgenomes.). Crossover between homeologous chromosomes can result in chromosome rearrangements that lower fertility and overall fitness. Rearrangements can alter the dosage of either entire chromosomes or just parts of chromosomes. Understanding the frequency and extent of rearrangements will help to explain the evolution and genome stabilization of agriculturally important allopolyploid species. Pyrosequencing is a useful tool in the study dosage changes in allopolyploids because it allows quantification of the relative contribution from each progenitor species at any given locus. Here we use pyrosequencing to analyze resynthesized Brassica napus allopolyploids and their progeny. Targets for pyrosequencing were identified using a bioinformatic approach taking advantage of recently-released Brassica genome sequence. SNPs identified through bioinformatics were confirmed through molecular biology. Markers along the A3/C3 homeolog pair were used to identify the occurrence of novel homeologous exchanges during meiosis in the parent plant, and segregation patterns arising from dosage changes in the parent. We identify a higher frequency of homeologous rearrangements at the distal end of the chromosomes. We also observe that the presence of a dosage change in a parent increases the likelihood that the chromosome bearing the dosage change will undergo subsequent rearrangements in neighboring loci.