Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Hybrid development has become an essential component in many major crop species due to the exploitation of heterosis. However, parental combinations that exhibit high heterotic gains can be difficult to obtain if no prior genetic architecture is known. The current research focuses on heterotic gene pool development using genetic distance and multivariate cluster analysis. Seventy-nine inbred accessions were grouped using three different methods including; 1) phenotypic classification based on 20 qualitative and quantitative traits; 2) Sequence related amplified polymorphisms (SRAP) using 29 forward and reverse primer combinations; 3) genotyping-by-sequencing (GBS) using 80,005 single nucleotide polymorphisms. Both genotypic methods (SRAP and GBS) were compared to each other, and hierarchical clustering produced similar results with 68% homology between the two methods. Heterotic cluster accuracy was investigated between the two genotypic methods through hybrid performance. Forty-four hybrid combinations along with parental cultivars were grown at one location in Winnipeg, MB with three replicates in a randomized complete block design. Hybrid yield and heterosis were regressed against the genetic distance from each genotypic method. GBS was a better predictor of hybrids with high yield (R = 0.47, p <.01); however, SRAP was a better predictor for mid-parent and high-parent heterosis at R = 0.53 and R = 0.61, respectively. This research provides experimental evidence that SRAP and GBS heterotic pool definitions have utility in the prediction of high heterotic parental combinations / May 2016

Canola (Brassica napus) oil has immense economic value due to its use as food. The production of biodiesel has led to the recent increase in the demand for canola oil, and further reinforced the need to improve the oil content and composition of canola seeds. The content and quality of canola oil is dependent on the process of fatty acid biosynthesis and accumulation of triacylglycerols (TAGs) during seed development. The biochemical pathways leading to the synthesis of fatty acids and TAGs are thoroughly understood, and many of the genes encoding the metabolic enzymes involved in this pathway have been isolated and functionally characterized. Despite comprehensive understanding of fatty acid and TAG biosynthesis, alternate factors and genes involved in the modification of seed oil content and composition are being investigated. This has initiated a new sector of research focusing on a series of transcription factors: LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3) and WRINKLED1 (WRI1), which play a key role during embryo and seed development. Previous studies in Arabidopsis have demonstrated that the noted transcription factors influence the synthesis and accumulation of oil during seed development. These findings have prompted the design of two studies with the objective of further characterizing the role of these genes. In the first study, a homolog of Arabidopsis LEC1 was isolated from Brassica napus (Bn) microspore derived embryos. BnLEC1 contained the same distinct features identified in LEC1 genes from other plant species. BnLEC1 was inserted into an expression vector, which was subsequently used to transform Arabidopsis plants. The transgenic lines were characterized by differences in seed oil composition, and one line showed a reduction in the number of seeds counted per silique. Expression analysis revealed that the transgene was not expressed in the transgenic lines. To account for the discrepant findings, the possibility of endogenous gene silencing mechanisms suppressing transgene expression has been discussed. As an extension of this research, a second study assessed the differential expression of LEC1, LEC2, FUS3 and WRI1 in double haploid Brassica napus lines characterized by different levels of seed oil content. The expression level of the noted genes was determined at 7, 14, 21 and 28 days after pollination. Numerous changes in the transcript level were observed, but the trends were not consistent among high and low oil content lines.

The objective of this research was to determine the relationship between seedling root length and seed yield in spring canola. Field and growth room experiments were conducted using the same eight genotypes. In the field experiment, root length and root area had a strong positive relationship to seed yield with R2 values at the 1-2 leaf stage of 0.90 and 0.93, respectively. Shoot dry weight had a strong positive relationship to seed yield at the cotyledon stage, with an R2 of 0.99. In the growth room, root length was compared to short- and long-term seed yield. The R2 values when root length was compared to short- and long-term seed yield were 0.95 and 0.96, respectively.

The meristem genes SHOOT MERISTEMLESS (STM), CLAVATA1 (CLV1) and ZWILLE (ZLL) are essential for the formation and maintenance of the shoot apical meristem (SAM). As an extension of that work, this thesis examines the function of these genes during seed oil accumulation and microspore-derived embryogenesis. Using a transgenic approach it is demonstrated that only the over-expression of BnSTM increases the percentage of total seed oil and a reduction of glucosinolate (GLS) levels achieved by transcriptional regualtion. The over-expression of BnSTM also affected in vitro embryogenesis by increasing the number and quality of microspore-derived embryos (MDEs) in contrast to the MDEs down-regulating BnSTM. The MDEs with ectopic expression of BnSTM were found to regulate embryonic SAM by altering cytokinin synthesis, catabolism, perception and signaling. Taken together, these findings provide evidence for a novel function of BnSTM in promoting desirable changes in seed oil and GLS levels and enhancing in vitro embryogenesis.

Canola (Brassica napus) oil has immense economic value due to its use as food. The production of biodiesel has led to the recent increase in the demand for canola oil, and further reinforced the need to improve the oil content and composition of canola seeds. The content and quality of canola oil is dependent on the process of fatty acid biosynthesis and accumulation of triacylglycerols (TAGs) during seed development. The biochemical pathways leading to the synthesis of fatty acids and TAGs are thoroughly understood, and many of the genes encoding the metabolic enzymes involved in this pathway have been isolated and functionally characterized. Despite comprehensive understanding of fatty acid and TAG biosynthesis, alternate factors and genes involved in the modification of seed oil content and composition are being investigated. This has initiated a new sector of research focusing on a series of transcription factors: LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3) and WRINKLED1 (WRI1), which play a key role during embryo and seed development. Previous studies in Arabidopsis have demonstrated that the noted transcription factors influence the synthesis and accumulation of oil during seed development. These findings have prompted the design of two studies with the objective of further characterizing the role of these genes. In the first study, a homolog of Arabidopsis LEC1 was isolated from Brassica napus (Bn) microspore derived embryos. BnLEC1 contained the same distinct features identified in LEC1 genes from other plant species. BnLEC1 was inserted into an expression vector, which was subsequently used to transform Arabidopsis plants. The transgenic lines were characterized by differences in seed oil composition, and one line showed a reduction in the number of seeds counted per silique. Expression analysis revealed that the transgene was not expressed in the transgenic lines. To account for the discrepant findings, the possibility of endogenous gene silencing mechanisms suppressing transgene expression has been discussed. As an extension of this research, a second study assessed the differential expression of LEC1, LEC2, FUS3 and WRI1 in double haploid Brassica napus lines characterized by different levels of seed oil content. The expression level of the noted genes was determined at 7, 14, 21 and 28 days after pollination. Numerous changes in the transcript level were observed, but the trends were not consistent among high and low oil content lines.

The objective of this research was to determine the relationship between seedling root length and seed yield in spring canola. Field and growth room experiments were conducted using the same eight genotypes. In the field experiment, root length and root area had a strong positive relationship to seed yield with R2 values at the 1-2 leaf stage of 0.90 and 0.93, respectively. Shoot dry weight had a strong positive relationship to seed yield at the cotyledon stage, with an R2 of 0.99. In the growth room, root length was compared to short- and long-term seed yield. The R2 values when root length was compared to short- and long-term seed yield were 0.95 and 0.96, respectively.

The meristem genes SHOOT MERISTEMLESS (STM), CLAVATA1 (CLV1) and ZWILLE (ZLL) are essential for the formation and maintenance of the shoot apical meristem (SAM). As an extension of that work, this thesis examines the function of these genes during seed oil accumulation and microspore-derived embryogenesis. Using a transgenic approach it is demonstrated that only the over-expression of BnSTM increases the percentage of total seed oil and a reduction of glucosinolate (GLS) levels achieved by transcriptional regualtion. The over-expression of BnSTM also affected in vitro embryogenesis by increasing the number and quality of microspore-derived embryos (MDEs) in contrast to the MDEs down-regulating BnSTM. The MDEs with ectopic expression of BnSTM were found to regulate embryonic SAM by altering cytokinin synthesis, catabolism, perception and signaling. Taken together, these findings provide evidence for a novel function of BnSTM in promoting desirable changes in seed oil and GLS levels and enhancing in vitro embryogenesis.

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.

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.