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- Bräutigam, Marcus, 1968
(författare)
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Cold Acclimation in oats and other plants: Dissecting low temperature responses using a comparative genomic approach.
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cold acclimation protects plants from temperate regions of the world from the deleterious effects of low and freezing temperatures. This is through a series of transcriptional, regulatory and metabolic changes that enable continued growth and survival. The focus in this thesis is to increase our understanding of the cold acclimation process and there by open the door to development of cold hardy oat (Avena sativa) varieties for the Nordic climate conditions. We started by sequencing 9,792 oat ESTs from a cDNA library prepared from pooled total RNA extracted from cold induced oat plants. These sequences were assembled into a UniGene ser of 2,800 sequences, 398 displayed homology to genes previously reported to be involved in cold acclimation. The CBF factor family have a key regulatory role during cold acclimation and in our UniGene set we found four oat CBF sequences. To infer regulatory networks we developed a rule-based method, which combined data from microarrays with promoter sequences and known cis¬-elements. The method was tested on the cold acclimation process in Arabidopsis and could indentify both known and novel network connections. We also performed a comparative transcriptome study between rice and Arabidopsis during low temperature stress to explore the molecular differences between chilling sensitive and freezing tolerant plants. Interesting observations were that the dynamics of the response of key genes appears to be higher in Arabidopsis than in rice. Several important downstream genes encoding proteins with freezing protective activities in Arabidopsis are not present in rice or important cis-elements. Also stress mediated hormone signalling seem to be absent in rice. Together these observations partly explain why rice is unable to cold acclimate to the same extent as Arabidopsis. Finally we developed a TILLING (Targeting Induced Local Lesions IN Genomes) population in the oat consisting of 2,600 independent events. By random sequencing of two genes involved in the lignin (AsPAL1) and ß-glucan (AsClsF6) synthesis we estimated the mutation frequency in the population to be approximately 1 per 26,000 bp. This means that each gene is mutated ca 250 times looking at the entire population and assuming an average gene size of 2 kb. This TILLING population will now be an important tool for both breeding and genetic studies in oats.
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| 2. |
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| 3. |
- 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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| 4. |
- Bräutigam, Marcus, 1968, et al.
(författare)
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Generation and analysis of 9792 EST sequences from cold acclimated oat, Avena sativa
- 2005
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Ingår i: BMC Plant Biol. - : Springer Science and Business Media LLC. - 1471-2229. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Oat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required. RESULTS: From an oat cDNA library 9792 single-pass EST sequences were obtained. The library was prepared from pooled RNA samples isolated from leaves of four-week old Avena sativa (oat) plants incubated at +4 degrees C for 4, 8, 16 and 32 hours. Exclusion of sequences shorter than 100 bp resulted in 8508 high-quality ESTs with a mean length of 710.7 bp. Clustering and assembly identified a set of 2800 different transcripts denoted the Avena sativa cold induced UniGene set (AsCIUniGene set). Taking advantage of various tools and databases, putative functions were assigned to 1620 (58%) of these genes. Of the remaining 1180 unclassified sequences, 427 appeared to be oat-specific since they lacked any significant sequence similarity (Blast E values > 10(-10)) to any sequence available in the public databases. Of the 2800 UniGene sequences, 398 displayed significant homology (BlastX E values < or = 10(-10)) to genes previously reported to be involved in cold stress related processes. 107 novel oat transcription factors were also identified, out of which 51 were similar to genes previously shown to be cold induced. The CBF transcription factors have a major role in regulating cold acclimation. Four oat CBF sequences were found, belonging to the monocot cluster of DREB family ERF/AP2 domain proteins. Finally in the total EST sequence data (5.3 Mbp) approximately 400 potential SSRs were found, a frequency similar to what has previously been identified in Arabidopsis ESTs. CONCLUSION: The AsCIUniGene set will now be used to fabricate an oat biochip, to perform various expression studies with different oat cultivars incubated at varying temperatures, to generate molecular markers and provide tools for various genetic transformation experiments in oat. This will lead to a better understanding of the cellular biology of this important crop and will open up new ways to improve its agronomical properties.
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| 5. |
- 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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| 6. |
- 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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| 7. |
- 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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| 9. |
- Lindlöf, Angelica, et al.
(författare)
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Evaluation of combining several statistical methods with a flexible cutoff for identifying differentially expressed genes in pairwise comparison of EST sets
- 2008
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Ingår i: Bioinformatics and Biology Insights. - : Libertas Academica. - 1177-9322. ; 2, s. 215-237
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Tidskriftsartikel (refereegranskat)abstract
- The detection of differentially expressed genes from EST data is of importance for the discovery of potential biological or pharmaceutical targets, especially when studying biological processes in less characterized organisms and where large-scale microarrays are not an option. We present a comparison of five different statistical methods for identifying up-regulated genes through pairwise comparison of EST sets, where one of the sets is generated from a treatment and the other one serves as a control. In addition, we specifically address situations where the sets are relatively small (~2,000– 10,000 ESTs) and may differ in size. The methods were tested on both simulated and experimentally derived data, and compared to a collection of cold stress induced genes identified by microarrays. We found that combining the method pro- posed by Audic and Claverie with Fisher’s exact test and a method based on calculating the difference in relative frequency was the best combination for maximizing the detection of up-regulated genes. We also introduced the use of a flexible cutoff, which takes the size of the EST sets into consideration. This could be considered as an alternative to a static cutoff. Finally, the detected genes showed a low overlap with those identified by microarrays, which indicates, as in previous studies, low overall concordance between the two platforms.
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| 10. |
- 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
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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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