Evaluation of Combining Several Statistical Methods with a Flexible Cutoff for Identifying Differentially Expressed Genes in Pairwise Comparison of EST Sets

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 proposed 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

Putative cold acclimation pathways in Arabidopsis thaliana identified by a combined analysis of mRNA co-expression patterns, promoter motifs and transcription factors

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Generation and analysis of 9792 EST sequences from cold acclimated oat, Avena sativa

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°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 ≤ 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

Development and characterization of an oat TILLING-population and identification of mutations in lignin and β-glucan biosynthesis genes

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

Development of a Model System to Identify Differences in Spring and Winter Oat

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

A two-component response regulator is involved in cold sensing in oat

Plants have developed a number of different physiological and developmental responses to abiotic stress. One important process is acclimation, where mild stress conditions greatly enhance tolerance to later and more severe conditions. During the acclimation changes in gene expression patterns occur, which leads to a plant response involving the necessary modifications of growth, development and cellular homeostasis. During the last years, six different cellular signal transduction pathways between the initial cold-stress perception and the gene expression response have been documented. Here we suggest an additional pathway. Two component systems, first described in prokaryotes, have also been identified in yeast and plant systems, but not in animals. They are characterised by a phosphotransfer reaction between two types of signal transducers and involves a sensory histidine kinase receptor and a response regulator. In plants, two-component systems play important roles in ethylene and cytokinin signalling and osmosensing but have not been directly coupled to cold signalling so far. From a subtractive oat cDNA library enriched in cold induced sequences we isolated a full-length clone, denoted AsDP5. Northern and RT-PCR analysis showed that the AsDP5 was induced at +4oC in less than 1 h. Analysis of the deduced 621 amino acids long protein revealed that it had a N-terminal two component response regulator domain, putative nuclear localisation signals and a zink finger DNA binding domain at the C-terminus. Thus, the AsDP5 protein strongly resembles the response regulator protein of two-component systems. This cold signalling pathway is now carefully analysed in oat and in the model plant Arabidopsis, using various knock-out mutants and transgenic systems.vokMyynti MTT tietopalvelu

Development of an oat database

We have recently built a new database that contains information about oat gene sequences, gene expression profiles, protein structure and activities and primers for cloning and mapping purposes. The database is the property of AvenaGene AB and is denoted AGOD for AvenaGene Oat Database. It is accessible at http://www.agod.org. The database was built using PostgreSQL, which is an object-relational database management system. It is combined with a web-based user-friendly interface developed with the PHP scripting language. There is also a webbased BLAST engine connected to the database, which gives the user the opportunity to search AGOD with own sequence data. Our long-term aim is to make AGOD the primary repository for information about oat-related issues and the major part of AGOD will therefore be made publicly available. At present, this database contains more than 10,000 EST sequence entries, full-length cDNA sequences and information about deduced protein activities. OatDB will be continuously updated with new sequence data, as well as with new results derived from various biological experiments, advanced bioinformatics analysis, various protocols, literature references and oat-related web links.vokMyynti MTT tietopalvelu

Molecular characterization of CBF transcription factor genes in oat

By EST sequencing we identified 2866 different genes from a cold induced cDNA library from the British winter oat variety Gerald (see other abstract by Bräutigam et al). Among these there are several genes similar to cold and/or drought-induced genes previously identified in rice, wheat, rye, barley and Arabidopsis. More than 100 genes encoded putative transcription factors. Of particular importance for the regulation of cold acclimation is the CBF transcription factor family genes, which have important regulatory roles in the cold signalling pathway. In all CBF proteins analysed so far, a characteristic AP2-binding domain has been found. This interacts with a DRE/CRT regulatory element and activates transcription of downstream cold and dehydration responsive genes. Four CBF factor sequences were found in the oat EST collection, belonging to at least 2 different gene families. A full-length cDNA clone from one such gene, denoted AsCBF1, was isolated and sequenced. This revealed a 909 bp long open reading frame (ORF) encoding a putative protein of 303 aa that contain an AP2-binding domain. By semiquantitative RT-PCR analysis we showed that expression of the AsCBF1 gene was induced already 30 minutes after cold induction at +4oC. By chromosomal walking an additional 457 bp upstream of the AsCBF1 translational start codon was isolated. A TATAA-box was then found as well as several other potential regulatory elements including a Myc-recognition site. These elements will now be further characterised both in vitro and in vivo. CBF genes will also be used as genetic engineering tools to develop more cold tolerant oat.vokMyynti MTT tietopalvelu