| 1. |
- Bräutigam, Marcus, 1968, et al.
(författare)
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Development of Swedish winter oat with gene technology and molecular breeding
- 2006
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Ingår i: J. Seed Science. - 0039-6990. ; 116:1-2, s. 12-35
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Tidskriftsartikel (refereegranskat)abstract
- In Sweden, oat (Avena sativa) is only grown as a spring crop. A Swedish winter oat, on the other hand, would give increased yields and would secure oat in Swedish agriculture. During three consecutive winters we performed field trials with oat aiming at identifying potential winter material. More than 300 varieties, originating from breeding programs all over the world, were tested. Plants were rated according to winter survival, vigour and general performance during the following growth season and more than 20 lines were identified that were cold hardier than present commercial oat varieties. In parallel experiments a cDNA library was constructed from cold induced English winter oat (Gerald) and ca 10000 EST sequences were generated. After data mining a UniGene set of 2800 oat genes was obtained. By detailed analysis of microarray data from cold stressed Arabidopsis and by advanced bioinformatics, gene interactions in the complex cold induced signal transduction pathway were deduced. By comparison to the oat UniGene set, several genes potentially involved in the regulation of cold hardiness in oat were identified. An Agrobacterium mediated transformation protocol was developed for one oat genotype. Key regulatory genes in cold acclimation will be introduced to oat by genetic transformation or modified by TILLING. Such genes will be used as molecular markers in intogression of winter hardiness to commercial oat.
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| 2. |
- Chawade, Aakash, 1980, et al.
(författare)
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Development and characterization of an oat TILLING-population and identification of mutations in lignin and beta-glucan biosynthesis genes
- 2010
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Ingår i: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved beta-glucan-, antioxidant-and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda. Results: Here a population of 2600 mutagenised M2 lines, producing 2550 M3 seed lots were obtained. The M2 population was initially evaluated by visual inspection and a number of different phenotypes were seen ranging from dwarfs to giants, early flowering to late flowering, leaf morphology and chlorosis. Phloroglucinol/HCl staining of M3 seeds, obtained from 1824 different M2 lines, revealed a number of potential lignin mutants. These were later confirmed by quantitative analysis. Genomic DNA was prepared from the M2 population and the mutation frequency was determined. The estimated mutation frequency was one mutation per 20 kb by RAPD-PCR fingerprinting, one mutation per 38 kb by MALDI-TOF analysis and one mutation per 22.4 kb by DNA sequencing. Thus, the overall mutation frequency in the population is estimated to be one mutation per 20-40 kb, depending on if the method used addressed the whole genome or specific genes. During the investigation, 6 different mutations in the phenylalanine ammonia-lyase (AsPAL1) gene and 10 different mutations in the cellulose synthase-like (AsCslF6) beta-glucan biosynthesis gene were identified. Conclusion: The oat TILLING population produced in this work carries, on average, hundreds of mutations in every individual gene in the genome. It will therefore be an important resource in the development of oat with specific characters. The population (M5) will be available for academic research via Nordgen http://www.nordgen.org as soon as enough seeds are obtained.
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| 3. |
- Chawade, Aakash, 1980, et al.
(författare)
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Development of a model system to identify differences in spring and winter oat
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Our long-term goal is to develop a Swedish winter oat (Avena sativa). To identify molecular differences that correlate with winter hardiness, a winter oat model comprising of both non-hardy spring lines and winter hardy lines is needed. To achieve this, we selected 294 oat breeding lines, originating from various Russian, German, and American winter oat breeding programs and tested them in the field in south- and western Sweden. By assaying for winter survival and agricultural properties during four consecutive seasons, we identified 14 breeding lines of different origins that not only survived the winter but also were agronomically better than the rest. Laboratory tests including electrolytic leakage, controlled crown freezing assay, expression analysis of the AsVrn1 gene and monitoring of flowering time suggested that the American lines had the highest freezing tolerance, although the German lines performed better in the field. Finally, six lines constituting the two most freezing tolerant lines, two intermediate lines and two spring cultivars were chosen to build a winter oat model system. Metabolic profiling of non-acclimated and cold acclimated leaf tissue samples isolated from the six selected lines revealed differential expression patterns of 245 metabolites including several sugars, amino acids, organic acids and 181 hitherto unknown metabolites. The expression patterns of 107 metabolites showed significant interactions with either a cultivar or a time-point. Further identification, characterisation and validation of these metabolites will lead to an increased understanding of the cold acclimation process in oats. Furthermore, by using the winter oat model system, differential sequencing of crown mRNA populations would lead to identification of various biomarkers to facilitate winter oat breeding. © 2012 Chawade et al.
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| 4. |
- Chawade, Aakash, 1980, et al.
(författare)
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Putative cold acclimation pathways in Arabidopsis thaliana identified by a combined analysis of mRNA co-expression patterns, promoter motifs and transcription factors
- 2007
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Ingår i: BMC GENOMICS. - : Springer Science and Business Media LLC. - 1471-2164. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Background With the advent of microarray technology, it has become feasible to identify virtually all genes in an organism that are induced by developmental or environmental changes. However, relying solely on gene expression data may be of limited value if the aim is to infer the underlying genetic networks. Development of computational methods to combine microarray data with other information sources is therefore necessary. Here we describe one such method. Results By means of our method, previously published Arabidopsis microarray data from cold acclimated plants at six different time points, promoter motif sequence data extracted from ~24,000 Arabidopsis promoters and known transcription factor binding sites were combined to construct a putative genetic regulatory interaction network. The inferred network includes both previously characterised and hitherto un-described regulatory interactions between transcription factor (TF) genes and genes that encode other TFs or other proteins. Part of the obtained transcription factor regulatory network is presented here. More detailed information is available in the additional files. Conclusion The rule-based method described here can be used to infer genetic networks by combining data from microarrays, promoter sequences and known promoter binding sites. This method should in principle be applicable to any biological system. We tested the method on the cold acclimation process in Arabidopsis and could identify a more complex putative genetic regulatory network than previously described. However, it should be noted that information on specific binding sites for individual TFs were in most cases not available. Thus, gene targets for the entire TF gene families were predicted. In addition, the networks were built solely by a bioinformatics approach and experimental verifications will be necessary for their final validation. On the other hand, since our method highlights putative novel interactions, more directed experiments could now be performed.
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| 5. |
- Chawade, Aakash, 1980
(författare)
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Unravelling the complexity of cold acclimation in plants
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many plants respond to low non-freezing temperatures by increasing their freezing tolerance in a process known as cold acclimation. Microarray studies have shown that hundreds of genes are differentially expressed during the cold acclimation process in Arabidopsis. To predict the gene regulatory interactions amongst these differentially expressed genes a rule based bioinformatics model was developed. The inferred regulatory network correctly identified several previously characterized interactions and predicted several new interactions under combinatorial control of many TF families (Chapter 3.1, Paper I). As a continuation of this work, detailed combinatorial studies on promoters were done to understand weather the key regulon DREB1/CBFs in turn is regulated by several other TFs in Arabidopsis and rice (Oryza Sativa L.). The results showed that bioinformatics can correctly predict combinatorial regulation and can be used to identify previously known promoters motifs and predict new ones involved in co-regulated genes (Chapter 3.1, Paper II). In Sweden, cultivated oat (Avena sativa L.) is grown only as a spring crop as no suitable winter oat exists. To develop such a cultivar, a model system to detect differences between spring and winter oats on the molecular level is required. To this end 294 winter oat lines from throughout the world were collected, tested in the field in Sweden and rated based on their survival and vigor. The best performing lines were further characterized in the laboratory by physiological, biochemical and molecular analysis. The tests showed that while the German cultivar LPWH992209 performed best in the field, the American cultivar Win/Nor-1 outperformed the others in the controlled tests. Six cultivars including two spring, two intermediate and two winter cultivars were finally selected to make up the winter oat model system. Metabolic analysis revealed several metabolites such as sugars, amino acids as well as unknown metabolites that were differentially expressed in the winter oat model lines (Chapter 3.2, Paper III). Finally, an EMS mutagenized oat TILLING (Target Induced Local Lesions In Genomes) population consisting of 2,500 different mutated lines was generated. The genetic variation of the library was verified by various molecular analysis and proven by the identification of mutations in the AsPAL1 and AsCslF6 genes. Several mutants producing low levels of lignin in their husk were identified by biochemical analysis. This TILLING population will now be used to identify mutants with increased freezing tolerance (Chapter 3.3).
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| 6. |
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| 7. |
- Lindlöf, Angelica, et al.
(författare)
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Identification of Cold-Induced Genes in Cereal Crops and Arabidopsis Through Comparative Analysis of Multiple EST Sets
- 2007
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Ingår i: S. Hochreiter and R. Wagner (Eds.): BIRD, LNBI. - Berlin, Heidelberg : Springer. ; 4414, s. 48-65, s. 48-65
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Tidskriftsartikel (refereegranskat)abstract
- Freezing tolerance in plants is obtained during a period of low nonfreezing temperatures before the winter sets on, through a biological process known as cold acclimation. Cold is one of the major stress factors that limits the growth, productivity and distribution of plants, and understanding the mechanism of cold tolerance is therefore important for crop improvement. Expressed sequence tags (EST) analysis is a powerful, economical and timeefficient way of assembling information on the transcriptome. To date, several EST sets have been generated from cold-induced cDNA libraries from several different plant species. In this study we utilize the variation in the frequency of ESTs sampled from different cold-stressed plant libraries, in order to identify genes preferentially expressed in cold in comparison to a number of control sets. The species included in the comparative study are oat (Avena sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), rice (Oryza sativa) and Arabidopsis thaliana. However, in order to get comparable gene expression estimates across multiple species and data sets, we choose to compare the expression of tentative ortholog groups (TOGs) instead of single genes, as in the normal procedure. We consider TOGs as preferentially expressed if they are detected as differentially expressed by a test statistic and up-regulated in comparison to all control sets, and/or uniquely expressed during cold stress, i.e., not present in any of the control sets. The result of this analysis revealed a diverse representation of genes in the different species. In addition, the derived TOGs mainly represent genes that are long-term highly or moderately expressed in response to cold and/or other stresses.
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| 8. |
- Lindlöf, Angelica, et al.
(författare)
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In silico analysis of promoter regions from cold-induced genes in rice (Oryza sativa L.) and Arabidopsis thaliana reveals the importance of combinatorial control
- 2009
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811. ; 25:11, s. 1345-1348
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Tidskriftsartikel (refereegranskat)abstract
- Motivation: Cold acclimation involves a number of different cellular processes that together increase the freezing tolerance of an organism. The DREB1/CBFs are transcription factors (TFs) that are prominent in the regulation of cold responses in Arabidopsis thaliana, rice and many other crops. We investigated if the expression of DREB1/CBFs and co-expressed genes relies on combinatorial control by several TFs. Our results support this notion and indicate that methods for studying the regulation of complex cellular processes should include identification of combinations of motifs, in addition to searching for individual overrepresented binding sites.
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