120 individual, four-week old, greenhouse-grown plants of the winter oat varieties Gerald, 83-48 CH and the spring oat varieties Matilda and Birgitta were incubated in the dark at +4°C (± 0.5°C) for 12 and 24 h and thereafter moved to -15°C for 1, 2, 4, 8 or 16 h. Another 120 individual plants were transferred directly at -15°C for 1, 2 4, 8 or 16 h. After the incubation period the plants were transferred back to the greenhouse and allowed to recover for one week. Visible freezing damage was then scored on a scale from 1 to 5, where 1 means no visible damage and 5 means a dead plant (Figure 1A–D). The damage was less severe on the plants that were incubated at +4°C prior to freezing than on plants directly transferred to -15°C (Table 1). Thus, oat plants, as expected, are able to cold-acclimatise to some extent. A clear difference could be seen between summer and winter varieties, however, the latter being more cold adaptable and more winter hardy both before and after acclimation (Table 1).

To confirm cold acclimation on the molecular level a known marker for cold acclimation was investigated. Total RNA was isolated from 3 week-old plants of the winter variety Gerald after incubation at +4°C for 4, 16, 32 and 64 hours. An oat sequence, corresponding to the wheat COR410 gene, known to be cold inducible in wheat 17, was amplified by PCR from genomic oat DNA. A northern analysis using RNA isolated from cold induced plants and oat COR410 as a probe, showed that the COR410 gene expression could be detected after 4 h and then remained strongly expressed even after 64 h of cold incubation (Figure 1E).

Since we aimed for both early and late cold inducible genes in our EST analysis, a cDNA library was constructed from pooled RNA samples isolated from oat plants at different stages of cold acclimation. After plating the library, bacterial colonies were randomly picked and 9792 single-pass sequence reactions performed on cDNAs present in plasmids from these clones. This resulted in 8508 high-quality ESTs of 100 bp or longer, with an average sequence length of 710 bp. The 8508 ESTs were assembled into 1100 contigs and 2616 singletons, giving 3716 candidate genes. All ESTs that contained rRNA, mitochondrial or chloroplast sequences were excluded from further analysis. Finally, the number of redundant sequences in the candidate gene set was eliminated. This was done by a comparative BlastX analysis of the candidate gene set to the NCBI non-redundant (nr) protein database (see "Materials and Methods"). This reduced the candidate gene set to 2800 transcripts with an average sequence length of 800 bp (Figure 2). These final genes were denoted as the AsCIUniGene (Avena sativa cold induced UniGene) set.

The annotation of the AsCIUniGene set is based on homology. Each gene in the AsCIUniGene set inherited the annotation from the best match found after a BlastX search against nr protein database at NCBI. An expectation value (E value) threshold of 10^-10 was used. All sequences, in total 427, that had E values above this threshold were annotated as unknown.

The AsCIUniGene set was functionally classified by identifying every individual sequence in the set by the protein in the Munich Information Centre for Protein Sequences (MIPS) Arabidopsis database (MATDB) that gave the highest BlastX score. Since every gene in MATDB is assigned to at least one of the 28 different classes defined by the MIPS functional classification system, this proved to be a fruitful way to assign putative functions for sequences in the AsCIUniGene set. One drawback with the MIPS classification system is that it is somewhat crude since many of the functional classes were assigned automatically. Therefore, in addition to the automated process, we manually inspected the functional classification assignments for all the proteins and re-assigned them if necessary. In this way all of the 2373 oat sequences, out of which approximately 400 were manually assigned, were finally classified into the previously mentioned MIPS classes. The remaining 427 sequences (with E values > 10^-10 in the BLASTX search versus nr) could not be assigned either automatically or manually and where therefore classified as unknown proteins (Figure 3).

To analyze the most abundant ESTs from the cold induced cDNA library we grouped the sequences by means of the KOG (Clusters of Eukaryotic Orthologous Groups of Proteins) database. This database currently includes 7 eukaryotic genomes, including Arabidopsis. This gave a functional annotation of sequences based on orthologous proteins 18. In addition we used complementary databases to annotate our sequences like FOGs (Fuzzy Orthologous Group), which contains proteins with promiscuous domains that has not been assigned a KOG identity due to unclear orthologous relationships, TWOGs, which contains provisional clusters of proteins that are represented in two genomes and LSEs, which contains proteins that are lineage-specific expansions of paralogs present in the KOG database. The classification was based on best homology match of BlastX searches against Arabidopsis protein sequences where an expectation value (E value) threshold of 10^-10 was used. Proteins annotated in this way were termed "KOG-TWOG-LSE". Since not all Arabidopsis proteins are represented in the KOG database, not all ESTs could be annotated with a KOG-TWOG-LSE. Oat ESTs that had a homology match in Arabidopsis but not a KOG-TWOG-LSE annotation, inherited the annotation from MIPS annotation in MATDB. In addition, several of the sequences did not have an Arabidopsis homolog match with an E value above the threshold. These sequences were annotated with the best homolog match from a BlastX search against the nr protein database at NCBI. Again, an E value threshold of 10^-10 was used.

As a comparison, 2189 EST sequences from a non-induced oat leaf library 19 were analysed in the same way. As can be deduced from Table 2, the non-induced and the induced leaf libraries are quite different. In both libraries, chlorophyll a/b binding, ribulose 1, 5-bisphosphate carboxylase/oxygenase small chain and ribulose bisphosphate carboxylase/oxygenase activase (InterPro accession numbers IPR001344 and IPR000894) were the most expressed gene families, although in a different order (Table 2). The ribulose 1, 5-bisphosphate carboxylase/oxygenase sequence was the most common in the non-induced library, perhaps reflecting the higher photosynthetic activity in these plants. Interestingly, several cold related proteins were among the most abundant ESTs in the cold-acclimated library but were not represented among the most abundant ESTs from oat grown under optimal conditions. The cold-induced COR410 (Wcor410) is a dehydrin 17. Dehydrins are expressed during water-deficit and cold stress. The cold-responsive LEA/RAB-related COR protein (Wrab17) belongs to group-3 of LEA-proteins and has previously been established as induced by cold 20. Other interesting proteins in this context are the auqaporin PIP and hydroxyproline-rich glycoproteins. The aquaporins are membrane proteins that facilitate water transport across biological membranes. In Arabidopsis there are 13 members of aquaporins that belong to the plasma membrane intrinsic protein (PIP) subgroup. Recently, a study has shown that the PIP proteins are either up- or down-regulated in response to various abiotic stresses 21. Hydroxyproline-rich glycoproteins have a strong homology to OSR40 proteins in Oryza sativa, which have previously been shown to be up-regulated by salt stress 22. None of these genes were present in the non-induced library, indicating the great enrichment for cold stress related genes in the induced library. Since only genes that were annotated as cold induced, cold acclimated or as cold regulated were included, other stress related genes that indirectly also are involved in cold stress responses are most likely also enriched in this collection.

In the functional classification sequences belonging to four categories; "Cell Rescue, Defence and Virulence", "Cellular Communication/Signal Transduction Mechanism", "Metabolism" and "Transcription" were considered to be of great potential interest for cold acclimation. Together these categories were represented by 931 entries in the AsCIUniGene set, corresponding to more than 30% of all genes (Table 3). To increase the resolution of this analysis and to improve the identification of putative cold-regulated genes we built a separate database with proteins previously reported in the literature to be directly involved in cold stress-related processes 23242526. This "cold stress database" (CSDB) presently comprises 545 entries. In a BlastX search with the AsCIUniGene set against CSDB we identified 398 sequences in the AsCIUniGene set that showed significant homology (E ≤ 10^-10) to genes in the CSDB (Table 3). Thus, 14.2% of all the genes in the AsCIUniGene set seem to be cold stress related. Looking just at the two classes that we consider as most important for the cold acclimation process (Cell rescue, defence and virulence; Cellular communication and signal transduction) almost 40% of all sequences were homologous to potential cold stress related proteins. In the "Metabolism" and "Cellular Transport and Transport Mechanisms" classes, cold stress related genes were also overrepresented (approx. 25%).

To analyze whether this high proportion of cold regulated genes in relation to the total number of genes in the AsCIUniGene set was similar to other EST collections derived from cold acclimated plants, we downloaded and analysed EST datasets from cold acclimated wheat and barley. The datasets were clustered and assembled with the TGI clustering tool (see Material and Methods). This resulted in a TaCIUniGene set of 2894 genes and a HvCIUniGene set of 3932 genes. In addition, as a comparison the same oat ESTs collection derived from leaves of plants grown under optimal conditions (Table 2) was clustered and assembled into a AsNIUniGene set of 1445 sequences. These three UniGene sets were then searched against the CSDB. This showed that the proportions of cold stress related genes were 9.6% in the TaCI candidate gene set and 11.1% in the HvCI, but only 5.1% in the AsNI UniGene set (Table 4). Thus the AsCI UniGene set is quite different from previous oat EST collections and also the EST collection that contains the highest proportion of cold induced genes of all cereals.

Among the 190 sequences in the transcription class (Table 3), 107 were found to be homologous to transcription factors. Remarkably, 51 of these 107 sequences were homologous to genes in the CSDB, representing almost 48% of all transcription factors found in the AsCIUniGene set. To investigate this a bit more, the transcription factors were further classified into 14 different transcription factor families (Table 5). Dominating among these were genes encoding AP2 domain, homeodomain and zink finger proteins. Proteins belonging to the CBF/DREB1 transcription factor family have previously been shown to be the regulators of the majority of cold-response genes. The CBF/DREB1 family belongs to the AP2/ERF super family 27 and in Arabidopsis the AP2/ERF super family comprises 145 proteins. Based on similarities in their DNA-binding domains, these proteins have been subdivided into the AP2, RAV, DREB and ERF subfamilies and one family with the remaining proteins. In the AsCIUniGene set, we found 11 sequences belonging to the AP2/ERF superfamily (Table 5). The AP2/ERF domain of these 11 Avena proteins were more closely analysed and also compared to 45 previously described AP2/ERF containing proteins 28. The resulting phylogenetic tree revealed that 4 of the oat AP2/ERF proteins belonged to the DREB subfamily, 2 to the AP2/RAV subfamily and the remaining 5 sequences to the ERF subfamily (see Figure 4 and Table 6). From the analysis it can also be deduced that the oat CBF/DREB1 proteins are most closely related to the monocot CBF/DREB1 proteins (Figure 4).

To further analyse relations between oat and other monocot CBF/DREB1 proteins, a multiple alignment of AP2/ERF domains from AsCBF1, AsCBF2, AsCBF3, AsCBF4, OsDREB1A, OsDREB1B, HvCBF1, HvCBF2, HvCBF3, ScCBF and TaDREB was made. In addition, the Arabidopsis CBF/DREB sequences AtCBF1, AtCBF2 and AtCBF3 were included to further elucidate the relation between dicots and monocots in this respect (Figure 5). In previous studies it has been reported that in particular two amino acids, a valine at position 19 (V19) and a glutamic acid at position 24 (E24) (Figure 5) in the AP2/ERF domains of Arabidopsis have important roles in determining DNA-binding specificity 27. The AsCBF proteins have the conserved valine in the V19 position but not the glutamic acid in the E24 position (Figure 5). Instead, a valine is conserved in this position. This feature is in fact shared among all included monocot CBF/DREB proteins (Figure 5). The monocot CBF/DREB1 proteins could be further divided into three subgroups (Figure 4). The first subgroup (G1) contained the AsCBF3, HvCBF1, HvCBF2, ScCBF, and TaDREB proteins, the second subgroup (G2) contained the AsCBF1, AsCBF2 and HvCBF3 proteins and the third subgroup the remaining AsCBF4, OsDREB1 and OSDREB2 proteins (Figure 4). This grouping is based on differences in aa at positions 10, 18 and 39 between the different proteins (Figure 4, Figure 5). At position 10 the G1 proteins have a basic arginine (R) residue, the G2 proteins a hydrophilic asparagine (N) and the G3 proteins N, hydrophilic serine (S) or glycine (G) residues (Figure 5). In position 18 there is a basic residue, an arginine (R) in G2 and G3 proteins whereas G1 proteins have a hydrophilic residue, a glutamine (Q) (Figure 5). Finally G2 proteins have a hydrophilic tyrosine (Y) in position 39 while in G1 and G3 proteins this position is occupied by a hydrophobic phenylalanine (F) except for AsCBF4, which has a basic histidine (H) (Figure 5).

To explore whether the four identified oat AsCBF genes were cold induced with similar kinetics as other previously described CBF genes an expression analysis was performed. Multiplex RT-PCR was run on total RNA isolated from leaves of three weeks old plants cold induced (to +4°C) at time points between 15 min to 24 h using gene specific primers for AsCBF1, AsCBF2, AsCBF3 and AsCBF4 respectively. Total RNA isolated from untreated plants at the same time points were used as a comparison. An oat actin gene (AsActin) was also amplified from the same RNA samples as a loading and RNA quality control. To define conditions where the PCR amplification was in the exponential phase, several experiments with 30, 35 or 40 cycles were performed. This showed that the different AsCBF genes all were induced by the cold treatment but that they were differentially regulated (Figure 6). The AsCBF1 gene was not detectable at time 0, was induced after about 30 min, peaked at 4 hours and was completely shut off after 24 h. The AsCBF3 gene had quite a different expression pattern. It was weakly expressed also in non-induced plants, but was in addition rapidly induced already at the first time point after 15 min. The expression levels continued thereafter to increase, peaked after about 4 hours but still showed an elevated expression at 24 h. The AsCBF4 gene was slowly induced and not detected at all until after 4 hours. Unlike the others its expression peaked a bit later, after 8 hours and then had completely declined after 24 h. Despite several attempts, using different primer pairs we could not obtain a reproducible expression pattern of the AsCBF2 gene (data not shown). Thus, the different oat AsCBF genes seem to be active during different phases of the cold acclimation process and therefore perhaps induce different downstream gene programs. We are presently addressing this issue more specifically.

Using the Sputnik program and threshold values as specified in the Methods section, we searched for potential microsatellite (SSR) sequences in the 3716-candidate gene set. In total, 399 di- to pentanucleotide SSRs that fulfilled the criteria of the search were identified. This corresponds to approximately one microsatellite per 13 kb of sequence. Using the same methods and thresholds, Cardle et al. (2000) found on average one SSR per 14 kb of EST data in Arabidopsis. This indicates that the SSR frequency in oat is similar to that in Arabidopsis. In the oat sequence collection, tri-nucleotide repeats were the most commonly found followed by di-nucleotide repeats (Figure 7), which again matches the results from Arabidopsis 29. Except for two exceptional TA/AT SSRs of length 45 and 55 bp, SSR lengths ranged from 15 to 25 bp, with 16 and 17 bp being the most common.

Work is now in progress to determine which of these SSRs can be reproducibly amplified by PCR, are polymorphic, and can be linked to a phenotypic marker. The vast majority of the oat SSRs were found in non-coding DNA. Since they nevertheless represent actively transcribed genes, we expect that several of these will turn out to be useful markers for breeding.

Oat plants, Avena sativa v. Gerald, 83-48 CH, SW Matilda and SW Birgitta were obtained from the SW-collection (Svalöf Weibull AB, Landskrona, Sweden). Gerald and 83-48 are English winter varieties while Matilda and Birgitta are Swedish spring varieties. Seeds were germinated in 2-liter pots filled with fertilized and pressed peat. Plants were cultured in a greenhouse under natural light supplemented with metal halogen lamps, giving a photon flux density of 240 μmol per m² per sec. The photoperiod was 18 h, the day/night temperature was 20/12°C and the relative humidity about 70%. The plants were watered as needed.

To investigate the cold acclimation capacity of the chosen oat varieties, 24 pots with 10 seeds each of Gerald, 83-48 CH, Matilda, and Birgitta were prepared. About three weeks after germination, when each plant had produced 3 – 4 leaves, pots were moved to a dark cold room at +4°C (± 0.5°C) and incubated for 12 and 24 hours. After this period the pots were moved to -15°C (± 1°C) for 3 h, 6 h and 12 h. In addition, plants were moved from the greenhouse directly to -15°C, and incubated for 3 h, 6 h and 12 h. After the cold incubation period, the plants were moved back to the greenhouse for recovery. One week later the cold damage was visually scored.

Winter oat (Gerald) was germinated and grown for three weeks in the greenhouse. They were then incubated in the dark at +4°C (± 0.5°C) for 4, 16, 32 or 48 hours. At every timepoint, leaves were randomly picked from several individual plants and pooled. RNA was extracted from pooled leaves essentially as described by Chang et al. (1993). Tissue isolates were ground in liquid nitrogen, transferred to 65°C CTAB extraction buffer (2% CTAB [hexadecyltrimethylammonium bromide] [Sigma], 2% PVP [polyvinyl pyrrolidone, intrinsic viscosity 29–32] [Sigma], 100 mM Tris-HCl [pH 8.0], 25 mM EDTA, 2.0 M NaCl, 0.5 g per L spermidine, 2% β-mercaptoethanol), and extracted twice in equal volumes of chisam (phenol:chloroform:iso-amyl-alcohol 1:1:24). RNA was precipitated overnight at 4°C by adding 0.25 v/v 10 M LiCl. The precipitate was dissolved in 1 × SSTE (1.0 M NaCl, 0.5% SDS, 10 mM Tris-HCl, 1 mM EDTA), pH 8.0, extracted with an equal volume of chisam, precipitated with two volumes 99.5% ethanol, and re-suspended in distilled water treated with DEPC. Total RNA of each sample was quantified spectrophotometrically at OD₂₆₀. An OD₂₆₀ of 1 corresponded to 40 μg/ml RNA. Subsequently, the RNA was precipitated and re-suspended in DEPC-treated distilled water to a final concentration of 1 mg/ml.

Ten μg of total RNA were denatured with glyoxal/DMSO 43 and separated on a 1% agarose gel. The RNA was blotted onto a nylon membrane (Boeringer-Mannheim) and hybridized in Church hybridization buffer 44. An oat sequence, similar to the wheat COR410 gene was used as a probe. This was isolated by PCR amplification from oat genomic DNA using the forward primer 5'-ATGGAGGATGAGAGGAGCAC-3' and the reverse primer 5'-TTTCTTCTCCTCCTCGGGC-3'. Primer design was based on the wheat sequence. Amplification resulted in an 530 bp sequence which was verified by DNA sequencing (data not shown). The fragment was labelled with ³²P-dCTP (Amersham), using a random hexanucleotide mix and labelling-grade Kleenow enzyme (Boeringer-Mannheim). Stringency washes were performed at 65°C for 2 × 5 min in 2 × SSC, 0.5% SDS and for 4 × 5 min in 0.2 × SSC, 0.1% SDS. Membranes were exposed to X-ray film (Du Pont Medical Scandinavia AB).

Total RNAs isolated from plants incubated at +4°C for 6, 12 and 24 hours were pooled. The RNA pooled preparation was sent to MWG Biotech (Germany) where cDNA libraries were constructed, cDNA cloned into the pSPORT1 vector 45 and EST sequencing was performed.

All similarity searches were batch executed locally using the BlastN, BlastX or TBlastX tools 46, all included in the BLASTALL program package 47. Transeq, a program from the EMBOSS package 4849 was used to translate DNA sequences into protein sequences. Conserved domain search (CD-search) was performed against the Conserved domain database (CDD) at NCBI 47 using the Reversed position specific Blast (RPS-BLAST) algorithm with translated ESTs. InterProScan 5051 was used to scan translated ESTs for protein signatures in the InterPro member databases. For the multiple alignments we used ClustalW 52, included in the MacVector 7.2.2 package (Accelrys Inc). The phylogenetic tree was constructed by means of the MacVector 7.2.2 tool kit using the neighbour-joining (NJ) algorithm. Appropriate PERL scripts were written in order to pipeline the process of running tools in sequence, parsing result files and loading the results into the database. All data and results are stored in a PostgreSQL database.

In this paper started off with four different primary sequence data sets. The first set was the 9792 ESTs from cold acclimated oat, which was denoted the Avena sativa Cold Induced (AsCI) data set. The second data set comprises 2189 ESTs 53, which originated from untreated green leafs of 3 weeks old oat plants and was denoted the Avena sativa NonInduced (AsNI) data set. The third data set, which contains 4337 sequences originates from cold acclimated wheat 53 was denoted the Triticum aestivum Cold Induced (TaCI) data set and the final data set comprises 5418 sequences from cold stressed barley plants 53 and was denoted the Hordeum vulgare Cold Induced (HvCI) data set.

The AsCI data set was filtered, clustered and assembled with the Paracel Transcript Assembler™ (PTA) program (Paracel, Pasadena, CA), which integrates quality filtering, clustering, and assembly into a single pipeline. The filtering step includes masking of vector sequence, low-complexity, low-quality, repeats and poly(A) regions. In the next step clustering was performed. Here PTA utilizes the Haste algorithm in an all-versus-all sequence comparison. The criterion set for clustering sequences together was an alignment of a minimum of 100 bases and with at least 93% similarity between the aligned sequences. Sequences that did not fit into such clusters were defined as singlets. In the assembly step PTA uses CAP4, which is a refinement of the CAP3 algorithm 54. Sequences that did not fit into a contig were also defined as singlets. Finally, ESTs in singlets that had passed the filters but had an unmasked sequence < 100 bases were discarded. The resulting singlets and contigs represented the AsCI candidate gene set.

The other data sets where clustered and assembled into candidate gene sets using the TGI clustering tool 55. The clustering was performed by a slightly modified version of NCBI's MegaBlast program 56 and the resulting clusters were assembled using CAP3.

Individual ESTs were first annotated by the best BlastX homolog match against Arabidopsis thaliana, where an E value of < 10^-10 was used. The A. thaliana proteins were retrieved from the MIPS Arabidopsis database (MATDB). Thereafter the annotation given in the KOG database 18 was retrieved for each A. thaliana protein. For those A. thaliana homologs that did not have a KOG annotation, the EST sequence inherited the annotation from MATDB. Those sequences that did not have an A. thaliana homolog above the threshold were annotated with the best homolog match from a BlastX search versus the nr database at NCBI. Again an E value of < 10^-10 was used.

Non-redundant sets of genomic singlets and contigs (UniGene sets) were created in a two-step procedure. First sequence information derived from rRNA, chloroplastic DNA or mitochondrial DNA was identified by comparison to homologous Arabidopsis sequences (accession nr. X52322, AP000423, and Y08501/Y08502 respectively) using BlastN. In this way, sequences containing rRNA or mitochondrial DNA were separated from the genomic sequences.

The second step was a BlastX search of the non-redundant (nr) protein database. The accession numbers and E values of the best matches were extracted from the result file. The criterion used for a sequence to be identified as non-redundant was based on a unique best match based on the accession numbers from the BlastX search. If two or more query sequences resulted in best matches with identical accession numbers they were sorted according to their E values. Only the sequence with the lowest E value was included in the UniGene set.

The UniGene sets were annotated based on the results of BlastX searches of the nr database. The definition line of the Blast match was used as a description of the putative function of the UniGene gene. An E value threshold of 10^-10 was used and UniGene genes that did not meet this requirement were annotated as unknown.

Our functional classification of individual genes followed the functional categories as defined in the Munich Information Centre for Protein Sequences (MIPS) Arabidopsis thaliana functional catalogue (MATDB; downloaded from http://mips.gsf.de). To create a semi-automated functional classification pipeline a two-step procedure was developed. First, a BlastX search was performed with the UniGene set versus the MATDB, requiring an E value of < 10^-10. Locus name and E value of the best match for each gene were extracted from the result file. Secondly, the functional classification was identified by a search with the locus name in the Arabidopsis functional catalogue. Genes that did not meet the criteria for being functionally classified based on the semi-automated procedure were classified manually based on the annotation and the result from a conserved domain search versus the conserved domain database (CDD) downloaded from the NCBI web site.

A set of 3716 sequences resulting from the clustering and assembly steps, comprising a total of 5.3 Mb of sequence, was searched for microsatellites (simple sequence repeats; SSRs) in the form of mononucleotide repeats of > 15 bp, dinucleotide repeats of > 14 bp, trinucleotide repeats of > 15 bp, tetranucleotide repeats of > 16 bp, and pentanucleotide repeats of > 20 bp, as previously described by 29. To better locate di- to pentanucleotide repeats, we also used the program Sputnik, developed at the Washington University 57. This program allows minor imperfections on the SSRs by implementing a scoring system for insertions, mismatches and deletions. To locate mononucleotide repeats we used a simple PERL script developed by ourselves.

Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) were performed on total RNA prepared from leaves of three weeks old oat plants (variety Gerald), incubated for 0 min, 15 min, 30 min, 45 min, 1 h, 2 h, 8 h and 24 h at +4°C, using the SuperScript™ III One-Step RT-PCR system (Invitrogene™). RNA samples were first DNase treated using the Dnase I Amplification Garde from Invitrogene™. To amplify the different AsCBF genes the following primers were used:

AsCBF1 forward primer 5'-CCACAGTCCACCGTATCAGCAAG-3'

AsCBF1 reverse primer 5'-CGTCTCCTTGAACTTGGTGCG-3'

AsCBF3 forward primer 5'-CGGGCAAAGTTGAGGCAGGC-3'

AsCBF3 reverse primer 5'-TAGGCTCTGGCTCGGCACCTTC-3'

AsCBF4 forward primer 5'-CCCAGCCTTCAGCAGCGTC-3'

AsCBF4 reverse primer 5'-TCTCCACAGTCTCCTCCGTGC-3'

For the AsCBF1 gene a product size of 174 bp was expected, for AsCBF3 104 bp and for AsCBF4 172 bp. The AsActin gene used as a control and was amplified using the forward primer 5'-GCGACAATGGAACTGGC-3 and the reverse primer 5'-GTGGTGAAGGAGTAACCTCTCTCG-3'. In this case the expected product size was 580 bp. The RT-PCR reactions were run according to the manufacturer's instructions and 100 ng total RNA were used in each reaction. A 30-min reverse transcription at 55°C followed by a PCR amplification step with 30, 35 or 40 cycles were used. To verify the outcome of the RT-PCR reactions, equal amounts (30%) of the corresponding RT-PCR reaction mixes were applied on 1% agarose gels containing ethidium bromide (0.5 ng per ml).