Soybean (Glycine max (L.) Merr.) cyst nematode (SCN) (Heterodera glycines Ichinohe), an obligate sedentary endoparasite, is the most economically destructive pathogen in soybean production and causes over $1 billion in annual losses in the United States. Planting resistant cultivars is the primary management method to control SCN for the long-term purpose, but the nature of genetic resistance is little known. The Rhg1 (Resistance to H. glycines) locus on chromosome 18 is found as a major quantitative trait locus (QTL) that contributes resistance to SCN. The chemical mutagen ethylmethane sulfonate (EMS) can be utilized to induce genetic mutations in soybean populations, which screened by an efficient reverse genetic strategy known as Targeting Induced Local Lesions IN Genomes (TILLING) for functional gene analyses. The objective of this study was to analyze the function of SNAP gene (Glyma18g02590) at rhg1 allele from `Forrest' (`Peking'-derived SCN resistant cultivar) using TILLING. Soybean cultivar `Forrest' seeds were mutagenized with EMS and grown to generate M1 plants. M1 plants were self-pollinated to produce approximately 3000 M2 plants. Genomic DNAs were extracted from young leaves of individual M2 plants and quantified to normalize concentration of DNAs. The DNA samples were then pooled eight-fold in 96-well plates for mutations screening by TILLING. Moreover, 12 phenotypic traits including chlorophyll deficiency, leaf shape, branch architecture, seed color, seed weight, fatty acid phenotype were identified in the mutagenized population, analyzed and archived in this study.

Soybean [Glycine max (L.) Merr.] is a valuable leguminous crop providing protein and oil for food, feed, and industry. Carbohydrates comprise 30-35% of the soybean seed dry weight. The market for soybean-based products is increasing significantly with the increase of consumer awareness about the benefits of plant-based products. The soybean sugar contains different molecules, including sucrose and polysaccharides: raffinose and stachyose. Sucrose is desirable whereas high contents of raffinose and stachyose are undesirable because they are not digested in humans and monogastric animals due to the lack of α-galactosidases. This causes a reduced gastrointestinal performance. The improvement of the soybean seed sugar profile could considerably improve the soybean seed taste and value. In this dissertation, we aim to improve soybean seed sugar content and composition in soybean seeds using genetic and genomic approaches. In the current study, we used QTL (Quantitative Trait Loci) mapping to identify genomic regions associated with soybean seed sugar content. Then we identified soybean candidate genes that are involved in the sugar biosynthesis pathway. Additionally, we produced mutagenized soybean populations and used TILLING (Targeting Induced Local Lesions IN Genomes) by sequencing approach to validate the function of the candidate genes. 26 QTL controlling seed sugar contents were identified and mapped on 16 soybean chromosomes, among them five QTL regions were found in both locations, Illinois and North Carolina, in this study on chrs. 2, 5, 13, 17, and 20. Furthermore, novel QTL regions were identified including qSUC-4, qSUC-8, and qSUC-11 mapped on chrs. 4, 10, and 18 respectively, that were identified for the first time in this study. Additionally, a total of 57 candidate genes were identified, among them 16 genes located within 10 MB of the identified QTL. Amongst these genes a cluster of four involved in the sugars’ pathway was collocated within 6 MB with two QTL that were detected in this study on chromosome17. Furthermore, five candidate genes InDels between Forrest and Williams 82 sequences, including Glyma.09G073600, Glyma.08G143500, Glyma.17G111400, Glyma.05G003900, and Glyma.09G016600 have SNPs. These SNPs could potentially explain the difference in sugar content between Forrest and Williams 82 cultivars. The soybean sucrose synthase and invertase candidate genes were characterized through a comprehensive analysis of phylogenetic tree, synteny analysis, gene structure, and conserved domains variations. Additionally, TILLING (Targeting Induced Local Lesions IN Genomes) by Target Sequencing (TbyS) technology was used to identify mutants with improved sugar content. The results have shown that many mutations have been identified in soybean sucrose synthase and invertase genes that resulted in high sucrose content including the sucrose synthase mutants SL446 (R582W) and F1115 (G249E) on Glyma.02G240400 that have a sucrose content of 9.5% and 9.1%, respectively. Remarkably, two invertase mutants including F1291 (G632E) on Glyma.20G177200 and F1384 (D184V) on Glyma.10G145600 have shown an increased sucrose content and reduced raffinose and stachyose contents without altering the agronomic performance of the soybean plant. The obtained soybean mutants with enhanced sugar content can be useful in breeding programs to improve the soybean sugar content and composition.

In this study, die-off patterns of helminth ova, fecal coliforms, and Salmonella spp. in biosolids were documented using three small-scale sand drying beds located in a greenhouse. Treatments involved tilling the biosolids with differing frequencies. The results indicate that the inactivation rate for helminth ova was 0.88, 0.55, and 0.22 eggs/4 g TS day-1 for the intensively-tilled, moderately-tilled, and control beds, respectively. Achievement of Class A criteria was only possible in the intensively-tilled bed by Day 70 of the experiment. Salmonella spp. were inactivated to Class A levels in 9 days for the intensively and moderately-tilled beds. Regrowth of Salmonella spp. occurred thereafter in all beds, but high levels were seen only in the control bed. Fecal coliforms reached Class A criteria late in the experiment. Tilling treatments enhanced the inactivation rate of helminth ova and offer a potentially cost-effective method of pathogen reduction.

In this work the possibilities of plain tessellation design are investigated. The structure of tessellation is extremely complicated therefore their creation without computer is difficult and requires special knowledge. Specialized computer software may be successfully used as an efficient instrument in this design process. This investigation consist of: § The history of tessellation § Types of tessellation § Tessellation of Escher type § Practical applications of tessellation § Computer software for tessellation design In this work the computer program for tessellation design and some experiments are presented. Appendixes contain the examples of created tessellation and program code.

Soybean (Glycine max) is one of the most widely grown crops in the world. Soybeans are used as food and feed products due to their high protein, oil, and carbohydrate seed contents. Soybeans are one of the most important protein sources presenting several benefits for human and animal consumption. Moreover, the carbohydrate content of soybean is important since it contributes to the taste of soyfood such as tofu, soymilk, natto, and livestock. Sucrose is the predominant carbohydrate found in soybeans which is followed by other raffinose family oligosaccharides (RFOs) such as raffinose and stachyose. Higher sucrose contents contribute to improving the digestibility of products. However, higher raffinose and stachyose contents make soymeal indigestible and can cause undesirable flatulence in humans and animals. When these RFOs are consumed by animals they cannot be hydrolyzed in monogastric animals due to the absence of alpha-galactosidase activity in their body. Therefore, it would be desirable to reduce these polysaccharides in seeds for easier consumption, while also investigating the impact of oligosaccharide content changes on seed germination and vigor. TILLING has proven to be effective on different agronomic crops such as soybean, maize, rice, wheat, etc. TILLING mutants carrying mutations at the raffinose and stachyose synthase genes may reduce their activity which may impact sucrose content in the seeds without causing unwanted poor agronomic features. Hence, developing high sucrose lines with reduced RFOs for commercial usage will be useful for developing better cross lines for the future and will be healthier and desirable for human and animal consumption. In the current study, we developed an EMS soybean mutagenized population and then TILLING by sequencing was used to study the function of previously discovered raffinose synthase and stachyose synthase genes in the isolated soybean mutants.

The correct development of multicellular organisms is tightly regulated by intrinsic and extrinsic factors at specific time points. Disturbance on any level of these multiple processes may result in drastic phenotypes or eventually death of the organism. The midbrain-hindbrain boundary (also termed isthmic organizer) is a region of high interest as well in early as also in later development. The isthmic region carries organizer identity by the expression and subsequent release of FGF8. False patterning events of this region in early developmental stages would therefore display dramatic results over time. As it has been shown that the midbrain-hindbrain boundary (mhb) in the zebrafish is a compartment (or lineage restriction) boundary I tried to understand the underlying molecular mechanism for its correct establishment. In this work I focused both on embryological, molecular and genetic means to characterize involved molecules and mechanisms. In the first part of the thesis I followed in vivo cell transplantation assays, having started with an unbiased one. Cells of either side the mhb were challenged with this boundary by bringing them into direct cell contact with their ectopic counterpart. In a biased approach, cells overexpressing mRNA of specific candidate genes were transplanted and their clonal distribution in host embryos was analyzed. In the second part of the thesis I started interfering with specific candidate genes by transiently knocking down their protein translation. The adhesion molecules of the Eph/ephrin class had been shown to restrict cell mixing and thereby creating compartment boundaries in other tissues, such as the hindbrain, in the zebrafish and other organisms. Additionally, we generated several stable genetic mutant lines in cooperation with the Tilling facility at the Max-Planck-Institute. The only acquired potential null mutant ephrinB2bhu2971 was analyzed and characterized further. I observed that a knock down or knock out of only one of the ephrinB2 ligands does not seem to be sufficient for a loss of compartment boundary formation. The combinatory approach of blocking translation of EphrinB2a in ephrinB2bhu2971 mutants gave very complex and interesting phenotypes, which need to be investigated further.

The correct development of multicellular organisms is tightly regulated by intrinsic and extrinsic factors at specific time points. Disturbance on any level of these multiple processes may result in drastic phenotypes or eventually death of the organism. The midbrain-hindbrain boundary (also termed isthmic organizer) is a region of high interest as well in early as also in later development. The isthmic region carries organizer identity by the expression and subsequent release of FGF8. False patterning events of this region in early developmental stages would therefore display dramatic results over time. As it has been shown that the midbrain-hindbrain boundary (mhb) in the zebrafish is a compartment (or lineage restriction) boundary I tried to understand the underlying molecular mechanism for its correct establishment. In this work I focused both on embryological, molecular and genetic means to characterize involved molecules and mechanisms. In the first part of the thesis I followed in vivo cell transplantation assays, having started with an unbiased one. Cells of either side the mhb were challenged with this boundary by bringing them into direct cell contact with their ectopic counterpart. In a biased approach, cells overexpressing mRNA of specific candidate genes were transplanted and their clonal distribution in host embryos was analyzed. In the second part of the thesis I started interfering with specific candidate genes by transiently knocking down their protein translation. The adhesion molecules of the Eph/ephrin class had been shown to restrict cell mixing and thereby creating compartment boundaries in other tissues, such as the hindbrain, in the zebrafish and other organisms. Additionally, we generated several stable genetic mutant lines in cooperation with the Tilling facility at the Max-Planck-Institute. The only acquired potential null mutant ephrinB2bhu2971 was analyzed and characterized further. I observed that a knock down or knock out of only one of the ephrinB2 ligands does not seem to be sufficient for a loss of compartment boundary formation. The combinatory approach of blocking translation of EphrinB2a in ephrinB2bhu2971 mutants gave very complex and interesting phenotypes, which need to be investigated further.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Die Identifizierung des albostrians Gens erfolgte mittels Karten-basiertem Klonieren. Begonnen wurde mit der Kartierung in zwei kleinen F2-Kartierungspopulationen, MM4205 und BM4205, die zur Lokalisierung des Genes auf dem langen Arm von Gerstenchromosom 7H führte. Durch Kartierung mit hoher Auflösung in Verbindung mit extensiver Markersättigung konnte der betreffende DNA-Bereich schrittweise von anfangs 14,29 cM auf schließlich 0,06 cM eingeschränkt werden, wobei insgesamt 1344 F2-Pflanzen analysiert wurden. Zwischen den nächsten flankierenden genetischen Markern konnte in einem Bereich von 46 Kbp ein einzelnes Gen identifiziert werden. Durch Sequenzvergleich des abgeleiteten Genprodukts mit Einträgen in Datenbanken konnte das Protein der CMF-Genfamilie putativer Transkritionsregulatoren mit DNA-bindenden oder Protein-Protein-Wechselwirkungs-Eigenschaften zugeordnet werden. Eine erste Bestätigung der Identität des Kandidatengens mit dem albostrians-Gen konnte durch Analyse einer EMS-induzierten TILLING-Population (abgeleitet von der Gerstensorte ‚Barke’) erreicht werden. Unter den 42 gefundenen induzierten Mutationen gab es eine Mutation, die zu einem vorzeitigen Stopcodon und damit nach der Translation potenziell zu einem verkürztem Protein führt. Die Nachkommenschaft dieser heterozygoten Mutante spaltete in grüne und albino Pflanzen auf. Der albino-Phänotyp war perfekt mit dem homozygoten Status der nonsense-Mutation in den untersuchten 245 M4-Nachkommen von fünf heterozygoten M3-Pflanzen der Mutantenfamilie verbunden. Nach transienter Transformation von Gerstenblatt-Epidermiszellen mittels biolistischem Cobombardement von ALBOSTRIANS::GFP-Fusionsprotein mit dem mCherry-markierten Organellenmarker pt-rk-CD3-999 konnte die Lokalisation des ALBOSTRIANS-Proteins in den Plastiden und im Kern beobachtet werden. / Map-based cloning was employed for identification of the albostrians gene. Starting with mapping in two small F2 mapping populations, MM4205 and BM4205, the locus could be assigned to the long arm of barley chromosome 7H. High-resolution genetic mapping in conjunction with extensive marker saturation allowed to reduce the genetic target interval iteratively from initially 14.29 cM to finally 0.06 cM by analyzing a total of 1344 F2 plants. A single gene could be identified in a physical distance of 46 Kbp between the closest flanking genetic markers. Functional annotation of the deduced protein revealed it to represent a member of the CMF gene family of putative transcriptional regulators comprising DNA binding or protein-protein interaction properties. The identified candidate gene was first confirmed by screening an EMS-induced TILLING population derived from barley cv. ‘Barke’. Among the 42 identified induced mutations a single mutation introduced a premature stop codon potentially resulting in a shorter protein upon translation. Progeny of this heterozygous mutant segregated for green and albino plants. The albino phenotype was perfectly linked with the homozygous state of the stop codon mutation in 245 M4 offspring of five heterozygous M3 plants of the mutant family. Transient transformation by biolistic co-bombardment of barley epidermal cells with an ALBOSTRIANS::GFP fusion protein and an mCherry labelled organelle marker pt-rk-CD3-999 revealed the ALBOSTRIANS protein is targeting to plastids and nucleus.

Master of Science / Genetics Interdepartmental Program / Bikram S. Gill / TILLING (Targeting Induced Local Lesions IN Genomes) is a high-throughput, reverse genetics strategy for scanning mutagenized populations for point mutations in loci of interest. Originally, TILLING was used to investigate gene function in Arabidopsis and has since been similarly applied for gene functional analysis in other organisms. TILLING also allows the generation of novel genetic variation in specific genotypes and, thus, has been implemented as a tool for crop improvement. Ethyl methanesulfonate (EMS) is a widely used mutagen to induce point mutations in most TILLING protocols. M1 plants are then self-pollinated and M2 seed harvested. A single seed is grown from each M2 progeny and tissue taken for DNA isolation. M3 seed is cataloged. DNA is pooled to increase the efficiency and aid in mutation detection. Polymerase chain reaction (PCR) is used to amplify a locus of interest using the M2 DNA pools as a template. The PCR products are digested with an endonuclease that cleaves mismatched, mutant DNA, and the digested products are visualized. The pools for which PCR products are positive for a mutation are deconvoluted to determine which individual plant of the pool was responsible for the mutation. DNA from the positive individual is sequenced to determine the type of mutation (missense, nonsense, synonymous). Individuals with mutations that are more likely to disrupt gene function (nonsense and certain missense) are studied further by growing the corresponding M3 generation. In bread wheat, Triticum aestivum, TILLING is complicated by polyploidy: genes that have homoeologs require that the functionality of each be studied. If functional homoeologs are present for all three genomes, mutants must be identified for each homoeolog, followed by successive intercrossing to produce a triple mutant plant. As a model for wheat genetics, we propose TILLING in diploid wheat. EMS mutant populations were created in diploid wheat (Triticum monococcum ssp. monococcum) and the hexaploid bread wheat cultivar ‘Jagger’. The diploid and hexaploid wheat populations were screened for mutations at the waxy locus, GBSS1, as a validation of our population and for comparative analysis of mutation rates in 2x and 6x wheat. For diploid wheat, GBSSI was screened in 716 M2 plants, and one mutant was found for 1.9 Mb screened. 3 For hexaploid wheat, GBSSI was screened in 518 M2 plants, and 30 mutants were identified within a total of 657 Kb screened, giving a mutation frequency of one mutation per 22 Kb. The reasons for this vast difference in mutation frequency between diploid and hexaploid wheat are discussed. The diploid wheat population was further examined by screening for mutations within four lignin biosynthesis candidate genes, for a total of 2 Mb screened. A single mutant was discovered for both of the lignin genes PAL6 and HCT, giving a mutation frequency of one mutation per 1 Mb screened.