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.

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.

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.

Il ricino (Ricinus communis L.) è una tra le dieci principali colture oleaginose a livello mondiale. La ricina, eliminata nei processi industriali per la produzione di olio di ricino, costituisce un rischio sia per lo sfruttamento della pianta come biomassa per la generazione di biocarburanti vegetali di seconda generazione, sia per la possibilità di ottenere scorie tossiche dalla lavorazione del materiale. L’utilizzo di tecniche di mutagenesi chimica, associata ad AFLP e metodiche di sequenziamento bersaglio specifiche (TILLING) hanno dimostrato la possibilità di ottenere e riconoscere, in tempi relativamente brevi e in modo specifico, mutazioni nel genoma d’interesse. A partire da una popolazione monovarietale di ricino, sono stati messi a punto diversi trattamenti (basati su EMS ed MNU), al fine di ottenere una popolazione mutagenizzata chimicamente al cui interno ricercare piante prive, o con un contenuto ridotto, di ricina nel seme e di conseguenza caratterizzate da un maggior valore agronomico ed economico. In seguito all’autoimpollinazione delle piante sopravvissute, analisi AFLP e di sequenziamento sulle generazioni successive hanno mostrato come le piante tendano ad accumulare variazioni nel loro genoma rispetto alla generazione precedente. Dall’analisi di sequenza di circa 1 Mb del gene per la ricina nella popolazione mutagenizzata non è emerso nessun cambiamento nella sequenza nucleotidica, in accordo con i risultati di altri studi in cui si è visto come la frequenza di mutagenesi possa variare con la specie considerata. / Castor (Ricinus communis L.) is one of the ten major oil crops worldwide. Ricin, eliminated in industrial processes for the production of castor oil, constitutes a risk to both the exploitation of the plant as a biomass to generate second-generation biofuels, both for getting toxic waste from the processing of the material. The use of chemical mutagenesis techniques, AFLP and methods associated with specific target sequences (TILLING) have demonstrated the ability to obtain and recognize, in a relatively short time, and specifically, mutations in the genome of interest. Starting from a population of castor-variety, have been developed different treatments (based on EMS and MNU), in order to obtain a chemically mutagenized population of plants with a reduced content of ricin in the seed and therefore characterized by a greater agronomic and economic value. After self-pollination of the survived plants, AFLP analysis and sequencing showed how plants tend to accumulate changes in their genome than the previous generation. A sequence analysis of about 1 Mb of the gene for ricin in mutagenized populations has revealed no change in the nucleotide sequence, in agreement with results of other studies in which we have seen that the frequency of mutation may vary with the species under consideration.

Quinoa has been utilized for millennia in the Andes region of South America as a nutritious and hardy food crop. In recent years interest in quinoa has grown as need increases for an alternative to traditional cereal crops that can tolerate marginal environments while offering superior nutrition. Growers outside the Andes have experienced several complications adopting quinoa, including undesirable secondary metabolites, poor yield, lodging, and height inconsistency. Unfortunately, access to native ecotypes for crop improvement is limited, and desirable traits are difficult to introduce into available quinoa cultivars because of its allotetraploid genome and tendency to self-pollinate. A genome-wide survey of induced mutations in 244 sequenced M2 families was created from a bank of EMS-treated quinoa seeds and assembled into a library of mutant lineages with predicted variants and their effects on genes to assist in identifying agronomically valuable mutations in target genes as a supplement to crop improvement efforts. Using this library, eight families containing mutations in genes associated with reduced height "GAI1, GA20OX, GID1, and L " were identified. Several individuals exhibited a shorter than average phenotype; however, because each family contains thousands of EMS-induced mutations, the causative mutation of the reduced height phenotype in each family could not be definitively identified. In one family, absence of the GAI1 mutant allele, but the presence of a mutant CKX3 allele, provided a correlation between a mutation and the short phenotype. Genotyping each generation would be required for a targeted mutant allele to be tracked through selection.