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

Establishment of highly efficient callus proliferation, plant regeneration and micro-tillering systems in commercial oat cultivars

Different explants (seeds, mature embryos, 3-day and 6-day old leaf/mesocotyl joints) of seven commercial summer oat varieties (Adamo, Belinda, Birgitta, Freja, Matilda, Petra and Stork) and one winter line, Pen #65, were cultivated on 2, 4-D containing callus induction medium. After about 4 weeks, explants were evaluated for callus growth. In all varieties maximum callus growth was obtained from mature embryos. Efficient shoot regeneration could be obtained from Belinda (mature embryos, 3-day and 6- day old leaf/mesocotyl joints), Birgitta (3-day and 6-day old leaf/mesocotyl joints), Freja (seeds), Matilda (3-day old leaf/mesocotyl joints) and Stork (mature embryos, 3-day and 6-day old leaf/mesocotyl joints) and Pen #65 (3- day old leaf/mesocotyl joints) in a medium with a specific auxin/cytokinin combination. Plant regeneration occurred via either organogenesis or somatic embryogenesis. Root initiation and further growth took place in a hormone free medium. Cultivating 9-day old leaf/mesocotyl joints of winter oat varieties 83-48-CH and Gerald on 2,4-D and BAP containing MS2m medium suppressed apical growth and induced lateral enlargement of the apical domes. This led to the formation of multiple meristems and resulted in vigorous microtiller forming cultures. These oat tissue culture protocols have now established a base towards development of genetically engineered winter oat for Swedish conditions.vokMyynti MTT tietopalvelu

Development of a Scandinavian winter oat by molecular breeding and tissue culture techniques

During the winter 2002/2003, 174 different oat cultivars were planted in the field of Landskrona, Sweden to identify cold hardy lines. The germplasm represented the best winter hardy oats available in the world, originating from seed collections in the US (USDA-NSGC), the Vavilov Institute in St Petersburg, Lochow-Petkus in Germany and IGER in Great Britain. Spring oat (Belinda) and winter barley (SW Hampus) were also included in the trial as references. Weather data was continuously monitored. In December, triplicates of all field-grown lines were established in the green house from field selections. In January, a second batch of plants of the most promising accessions was collected in the field. Parameters like vigour, time of flowering and height of plants at bolting were recorded. Some of the lines were also established in tissue culture. Due to the severe winter in south Sweden in 2003, all plants except the repeats from one single line died. This surviving line came from the Pennsylvania winter oat-breeding program and was denoted as Pen#65. Pen#65 plants were crossed with German winter oats and segregating F2 populations will be tested in field trials during the coming winters. During the winter 2003/2004 a second field trial on winter oat was done. In this trial, leaf samples from several individual plants were collected at different time and temperature intervals. Total RNA was extracted, and global expression studies were performed by micro-arrays using an oat biochip of 2866 different genes, developed by us. From these studies key genes up- and down regulated in the field during winter stress conditions are identified. This information will be used to develop new molecular markers for an efficient selection of cold tolerant oat varieties in segregating breeding populations. Work is also in progress to establish an Agrobacterium mediated genetic transformation protocol for the Pen#65 line. Key genes, will then be introduced to the Pen#65 line by genetic transformation.vokMyynti MTT tietopalvelu

Analysis of 9703 expressed sequence tags in cold acclimated oat

Winter oat, Avena sativa cv. Gerald, was cold acclimated at +4oC for 4, 6, 8 and 32 hours. Total RNA was prepared from these plants, pooled and a cDNA library was constructed. From this library 9792 expressed sequence tags (EST) were sequenced. The average sequence length after vector clipping was 626 bp and the longest sequences were over 900 bp. Clustering and assembly of the these 9792 EST sequences resulted in a set of 4543 sequences. Clustering, assembly and filtering of these sequences resulted in a set of 2866 unique transcripts, denoted the "UniGene" set. Homology searches on publicly available sequence data allowed the assignment of a tentative function to 1622 (57.57%) of these transcripts. Out of the remaining 1246 unclassified sequences, 494 appeared to be oat specific since they lacked any significant sequence similarity (E-values > 1e-10 after BlastX search) to any sequence presently available in the public databases. Genes active in photsynthesis were most commonly found, but genes involved in metabolism, signal transduction and abiotic stress were also well represented. Interestingly, 398 sequences displayed strong homologies (E-values ? 1e-10 after BlastX search) to genes previously reported to be involved in cold stress related processes. Of particular importance for the regulation of coldacclimation are cold induced transcription factors. We found 47 such genes, including 4 CBF transcription factor sequences, a known cold regulatory gene class (see other abstract by Bräutigam et al). The set of 4543 clustered and assembled sequences (in total 5.3 Mb) was searched for microsatellites (SSRs) and 596 di- to pentanucleotide SSRs were found. 83% of these SSRs occurred in non-coding sequences. Work is now in progress to identify the particular SSRs from this collection that give reliable PCR products and are polymorphic. The best SSR markers will then be mapped to the oat genome and linked to valuable genetic traits like lipid biosynthesis, antioxidant activity and cold hardiness.vokMyynti MTT tietopalvelu

Development of Swedish winter oat with gene technology and molecular breeding

In Sweden, oat (Avena sativa) is only grown as a spring crop. A Swedish winter oat, on theother hand, would give increased yields and would secure oat in Swedish agriculture. Duringthree consecutive winters we performed field trials with oat aiming at identifying potentialwinter material. More than 300 varieties, originating from breeding programs all over theworld, were tested. Plants were rated according to winter survival, vigour and generalperformance during the following growth season and more than 20 lines were identified thatwere cold hardier than present commercial oat varieties. In parallel experiments a cDNAlibrary was constructed from cold induced English winter oat (Gerald) and ca 10000 ESTsequences 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 advancedbioinformatics, gene interactions in the complex cold induced signal transduction pathwaywere deduced. By comparison to the oat UniGene set, several genes potentially involved inthe regulation of cold hardiness in oat were identified. An Agrobacterium mediatedtransformation protocol was developed for one oat genotype. Key regulatory genes in coldacclimation 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 tocommercial oat