Brassica napus (canola) é a terceira cultura oleaginosa mais produzida no mundo, fornecendo cerca de 13% de toda a oferta mundial de óleo vegetal. Durante o desenvolvimento de sementes, B. napus acumula compostos de reservas em estádios e tecidos específicos. A maioria destes compostos de reservas são lípidos (30-40%) e proteínas (17-26%), sendo quase que exclusivamente armazenados nos cotilédones do embrião maduro. Os microRNAs (miRNAs) desempenham um papel essencial em diversos aspectos do desenvolvimento de sementes. A função destes pequenos RNAs (sRNAs) endógenos não-codificantes é regular a expressão gênica, principalmente através da clivagem e a inibição de tradução de mRNA alvo. Estudos recentes têm contribuído para a identificação de miRNAs em sementes de B. napus, mas a expressão temporal e as funções regulatórias dos miRNAs em sementes de B. napus, especialmente durante a maturação, são desconhecidas. Para entender os padrões de expressão temporal dos miRNAs durante o desenvolvimento de sementes por RT-qPCR, este trabalho se concentra primeiramente na determinação de genes de referência para serem utilizados como normalizadores em estudos de RT-qPCR e no perfil de expressão dos miRNAs durante o desenvolvimento de sementes. A estabilidade da expressão de 16 mRNA e 43 miRNAs de B. napus foi avaliada em amostras de folha, tecidos florais e diferentes estágios de desenvolvimento de sementes. Nossas análises demonstraram que os miRNAs apresentam uma maior estabilidade de expressão do que genes que codificam proteínas, e a combinação miR156-7, miR11-1 e miR408-1 foi a mais apropriada para ser utilizada como normalizadores em estudos de expressão gênica por RT-qPCR. O perfil de expressão de 40 miRNAs foi avaliado ao longo do desenvolvimento de sementes. A maioria dos miRNAs teve sua expressão induzida durante a maturação de sementes, enquanto um pequeno número foi preferencialmente expressos em estágios iniciais do desenvolvimento das sementes. Um miRNA inédito, denominado miR03-1, apresentou uma expressão diferencial entre os estágios precoces e tardios do desenvolvimento. A expressão do miR03-1 é fortemente induzida 21 dias após o florescimento. A partir de uma abordagem computacional para predição de possíveis genes-alvo de miRNAs de B. napus foi possível identificar um total de 481 alvos putativos para 104 sequências maduras de miRNAs. Todos os alvos preditos estão diretamente relacionados com o metabolismo lipídico, dentre estes fatores de transcrição e enzimas-chave do metabolismo lipídico. O estudo da regulação destes genes por miRNAs em diferentes estágios do desenvolvimento de semente contribuirá para o entendimento dos mecanismos moleculares envolvidos no metabolismo lipidico. / Brassica napus (canola) is the third largest oilseed crop in the world, providing approximately 13% of the world's supply of vegetable oil. During seed development, B. napus build up storage reserves in specific stages and tissues. The vast majority of these reserves are made up of lipids (30–40%) and proteins (17–26%) that are almost exclusively stored in the cotyledons of the maturing embryo. MicroRNAs (miRNAs) play essential roles in various aspects of seed development, including embryo development and the timing of seeds maturation. The function of these endogenous small non-coding RNAs (sRNAs) is to regulate gene expression, mainly through cleavage and translation inhibition of target. Several recent studies have contributed to the identification of miRNAs in seeds from B. napus, but temporal expression and regulatory functions of miRNAs in seeds of B. napus, especially during seed maturation, are unknown. To understand the temporal expression patterns of miRNAs during seed development by RT-qPCR, this work focuses primarily on the determination of appropriate reference genes for use in RT-qPCR studies and in the expression profile of miRNAs during seed development. The expression stability of 16 previously reported reference genes and 43 B. napus miRNAs have been evaluated in leaf and flower tissues, as wel in different seed development stages. Our analyses showed that miRNAs presented higher expression stability than protein-coding genes and the combination of miR156-7, miR11-1 and miR408-1 was appropriate as normalizers in studies of gene expression by RT-qPCR. In addition, the expression profile of 40 miRNAs was studied throughout seed development. The majority of miRNAs increased their expression during seed maturation, while a small part of the miRNAs was preferentially expressed at early seed developmental stages. A new miRNA named miR03-1 showed differential expression between the early and late stages of seed development and is strongly induced 21 days after flowering. In parallel, a computational approach was carried out to predict candidate target genes for B. napus miRNAs. A total of 481 putative targets were predicted for 104 sequences of mature miRNAs. All predicted targets are directly related to the lipid metabolism, such as transcription factors and key enzymes of lipid metabolism. The study of regulation of these genes by miRNAs in different stages of seed development will contribute to understanding the molecular mechanisms involved in lipid metabolism.

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sn-Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation of sn-1 position of sn-glycerol-3-phosphate to produce lysophosphatidic acid and Coenzyme A. GPATs are involved in several lipid synthetic pathways and play important physiological roles in plant development. The present doctoral thesis includes three related studies, which aim to molecularly and functionally characterize several plant GPAT genes and the encoded enzymes. The first study characterized three endoplasmic reticulum-bound GPAT4s encoded by three homologous GPAT4 genes of Brassica napus (oilseed rape), focusing primarily on their functional divergence and physiological roles in plant development and lipid biosynthesis. The three homologous GPAT4 genes exhibited different expression patterns and altered epigenetic features. Phenotypic rescue of a gpat4 gpat8 Arabidopsis double mutant and analysis of the gpat4 RNAi B. napus lines suggested physiological roles for the GPAT4s in cuticle formation of the rosette leaves, early flower development, pollen development and storage lipid biosynthesis. The second study investigated stable internal reference genes for gene expression studies in B. napus. This project identified four reliable reference genes to be used in gene expression analysis of BnGPAT4 homologues in both vegetative tissues and developing seeds. The third study focused on molecular cloning and biochemical characterization of a soluble plastidial GPAT isolated from a chilling-tolerant plant, western wallflower (Erysimum asperum). A truncated form of recombinant EaGPAT, with the putative transit peptide deleted, was functionally expressed in yeast. A series of enzymatic assays were performed in order to determine the optimum in vitro reaction conditions for the recombinant EaGPAT. The recombinant EaGPAT was further assayed with different acyl-CoAs and exhibited a substrate preference for 18 carbon unsaturated acyl-CoAs. With this substrate preference, the EaGPAT could potentially be used as a biotechnological tool for improving plant chilling-tolerance or increasing unsaturated fatty acid content of seed oil. Overall, the present doctoral studies revealed the functional divergence and important physiological roles of the GPAT4s in B. napus, and biochemically characterized a plastidial GPAT from E. asperum. The knowledge obtained from these studies provides new insights into the role of GPAT in plants and will be useful for further development of biotechnological approaches to modify seed oil biosynthesis in oleaginous crops. / Plant Science

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Brassica napus L., which is commonly known as canola, is the largest sources of edible oil in Canada. The remaining plant material, such as the stem, remains unused for any immediate application and is returned to the soil for decomposition. An investigation has been conducted to extract, characterize and modify the fibre materials from B. napus stems for textile and apparel applications. In order to find the optimum retting conditions for retting time, four different retting parameters were evaluated including, retting temperature, material liquor ratio, water exchange and the reuse of retted water. It was discovered that the virgin-retted fibres from Brassica plants exhibit most of the required textile properties including dye absorbency, strength, and thermal behaviour. However, the virgin-retted fibres do not exhibit the required spinning (yarn transformation) properties (softness, flexibility and individual fibre entity). In order to modify the Brassica fibres for spinnability, three treatment methods were applied: 1) alkali, acid and softener treatment; 2) pectinase enzyme treatment; and 3) enhanced enzyme treatment. According to Method 5 of the American Association of Textile Chemists and Colorists (AATCC), Brassica fibers obtained from treatments 2 and 3 showed similar spinning properties, and demonstrated superior spinning properties to Brassica fibres obtained from treatment one. To determine the variability of the cultivars upon textile and spinning properties, seeds from twenty different Brassica cultivars consisting of three different species, B. napus, B. juncea L. and B. rapa L., were collected, planted, and harvested upon reaching physiological maturity. The virgin water-retted fibre samples were then treated with pectinase enzyme, and different spinning properties (stiffness, softness, individual fibre entity) and textile properties (fibre decomposition temperature, tenacity and dye absorbency) of enzyme-treated samples were evaluated. The current research suggests that producing fibers from canola stubble and stems could be an additional income source for canola growers. / October 2016

Phoma stem canker (aka Blackleg) caused by the fungal pathogen Leptosphaeria maculans is a major disease affecting Canola (Brassica napus L.). This study examined 674 L. maculans isolates collected in 2010 and 2011 from western Canada at ten avirulence gene loci. Overall, certain alleles were more prevalent with AvrLm6 and AvrLm7 present in >85% of isolates and AvrLm3, AvrLm9, and AvrLepR2 present in <10% of isolates. This study also examined the genetic diversity of Leptosphaeria maculans populations around the world. Blackleg disease is found in most countries where Brassica spp. are cultivated and there are indications that L. maculans is an expanding species displacing the less aggressive Leptosphaeria biglobosa. Twenty two microsatellite primers were used to screen 96 isolates from 8 countries. A phylogenetic tree to assess the evolutionary relationship between regions was generated and the results indicated that genetic diversity was correlated with geographic location. / October 2015

Quantitative trait loci (QTL) analysis for Sclerotinia stem rot resistance was carried out in five doubled haploid (DH) populations of Brassica napus. Sclerotinia stem rot is caused by the necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary. Sclerotinia stem rot has worldwide occurrence and causes significant yield losses in many crop species. Several screening methods have been recommended in the literature to evaluate plant resistance to Sclerotinia stem rot. Four controlled environment based screening methods: 1) excised leaf assay, 2) cotyledon assay, 3) mycelial stem inoculation technique and 4) petiole inoculation technique compared for their ability to differentiate between plant susceptibility/resistance, their reliability and suitability for large scale screening using eight B. napus cultivars/lines of varying reaction to S. sclerotiorum. The petiole inoculation technique and the mycelium stem inoculation technique were identified as reliable methods in this study. Previously developed, five B. napus DH populations (H1, H2, H3, DH179 and DH180) segregating for resistance to Sclerotinia stem rot were used in this study. The petiole inoculation technique was used to evaluate resistance to Sclerotinia stem rot. Data on days to wilting was recorded for a two week period. Twelve plants per line were screened in each evaluation and each population was evaluated three times. Two to three day-old mycelial cultures of S. sclerotiorum isolate Canada 77 was used. QTL analyses were carried out using a LOD threshold value of 2.5 on each individual replicate and on the average of all the replicates. In the H1 population, the number of QTL detected ranged from four to six in each analysis. In the H2 population, there were three to six QTL in each analysis. There were two to six QTL in each analysis of the H3 population. In the DH179 population, the number of QTL detected ranged from three to five in each analysis. In DH180 population, the number of QTL identified varied from three to six in each analysis. A number of common QTL were found between the replicates of each population. Five common QTL were identified between these populations. The markers linked to these QTL are now available for marker assisted selection.

Quantitative trait loci (QTL) analysis for Sclerotinia stem rot resistance was carried out in five doubled haploid (DH) populations of Brassica napus. Sclerotinia stem rot is caused by the necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary. Sclerotinia stem rot has worldwide occurrence and causes significant yield losses in many crop species. Several screening methods have been recommended in the literature to evaluate plant resistance to Sclerotinia stem rot. Four controlled environment based screening methods: 1) excised leaf assay, 2) cotyledon assay, 3) mycelial stem inoculation technique and 4) petiole inoculation technique compared for their ability to differentiate between plant susceptibility/resistance, their reliability and suitability for large scale screening using eight B. napus cultivars/lines of varying reaction to S. sclerotiorum. The petiole inoculation technique and the mycelium stem inoculation technique were identified as reliable methods in this study. Previously developed, five B. napus DH populations (H1, H2, H3, DH179 and DH180) segregating for resistance to Sclerotinia stem rot were used in this study. The petiole inoculation technique was used to evaluate resistance to Sclerotinia stem rot. Data on days to wilting was recorded for a two week period. Twelve plants per line were screened in each evaluation and each population was evaluated three times. Two to three day-old mycelial cultures of S. sclerotiorum isolate Canada 77 was used. QTL analyses were carried out using a LOD threshold value of 2.5 on each individual replicate and on the average of all the replicates. In the H1 population, the number of QTL detected ranged from four to six in each analysis. In the H2 population, there were three to six QTL in each analysis. There were two to six QTL in each analysis of the H3 population. In the DH179 population, the number of QTL detected ranged from three to five in each analysis. In DH180 population, the number of QTL identified varied from three to six in each analysis. A number of common QTL were found between the replicates of each population. Five common QTL were identified between these populations. The markers linked to these QTL are now available for marker assisted selection.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2001. / Härtill 7 uppsatser.