Romeuf et al. BMC Genomics 2010, 11:185 http://www.biomedcentral.com/1471-2164/11/185 Blast searches against the probe data sets. However, sev- eral sequences correspond to the same Affymetrix ID so only 2,036 are unique. This information was added to wDBTF as it could facilitate the study of families or organ-specific TF expression as the expression informa- tion relating to each probe can be analyzed across all publicly available Affymetrix results. Plant material for C2C2-Dof expression analysis The wheat Dof TF sequences identified in silico were validated biologically by testing for their gene expression in seeds of T. aestivum line RE99006. Seeds were sown in 294-cm3 pots filled with a peat moss mix and kept in a greenhouse for 2 weeks. After 8 weeks of vernaliza- tion, the plants were transplanted to soil beds in a greenhouse with daily maximum/minimum air tempera- tures averaging 21°C/15°C and day/night relative humid- ity averaging 50%/60%. At anthesis, spikes were tagged when the anthers of the central florets appeared and the temperature was monitored every 30 min by four HOBO® H8 Indoor 8K Data Loggers (Onset Computer Corp., Bourne, MA). Two ears per plant were harvested every 50°C days from ovary stage until 600°C days after anthesis. All samples were taken at 11.00am to avoid possible diurnal effects on gene expression. Three inde- pendent biological replicates were used. The grains of central florets were collected, the embryo and the exter- nal pericarp were rapidly removed, and the endosperm was frozen in liquid nitrogen and stored at -80°C prior to RNA extraction. C2C2-Dof expression during seed development Total RNA was extracted from ovaries and developing grains (without the embryo and external pericarp) as described in Khaled et al. [29]. Dof transcripts were amplified by PCR with an ABI Prism 7900HT sequence detection system (Applied Biosystems) using Power SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Gene-spe- cific primer pairs used for Dof amplification were designed with Oligo6® and are listed in Additional file 2. Amplification of transcripts coding for actin, glyceralde- hyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (eF1a), b-tubulin, and 18S was used for internal controls. The geometric mean was calculated and the normalized quantity of each gene transcript and standard deviation of the three replicates was then cal- culated [30]. All primer pairs gave an amplification effi- ciency of 100% ± 10% and were comparable. The specificity of amplification products was assessed by analyzing their melting curves and by gel electrophoresis (giving a single band). Amplification plots and predicted threshold cycle values were obtained from three Page 7 of 15 independent biological replicates with the SDS software version 2.1 (Applied Biosystems). Utility wDBTF search wDBTF is an integrated database which compiles all wheat sequences identified as TFs from publicly avail- able expression data. Brought together in this form the data constitute a major resource for the study of gene expression in wheat and of plant TF families in general. The wDBTF home page is the main web interface for all information contained in the database (Figure 3). The TF family names link to individual TF family or subfamily pages. Each TF family has its own dedicated page that contains 2 tables, either contigs or singletons, with all TF sequence accessions from the group. The results page returns essential and comprehensive information, such as accession ID, best rice protein Blast hit, positions of Pfam domains and corresponding Affymetrix ID. In silico expression data is available for each accession referring to EST library sources. TF pro- tein and DNA sequences and expression data can be downloaded from the wDBTF web site. Therefore, each family’s results page integrates a large amount of data from multiple sources and is a good tool for research on the whole set of 7,112 TFs. Furthermore, classification associated with information on gene expression can be used to link candidate TFs to a given biological process. Analysis of C2C2-Dof family To illustrate our approach to in silico data analysis, we focused on the plant-specific Dof family. We identified 59 unique Dof sequences which are made of 186 expressed sequences in the above T. aestivum sequence output. In the wheat data set, 28 sequences cover Dof domains but only 22 cover it entirely. Dof domains of previously identified Dof genes from O. sativa japonica and Hordeum vulgare, described by Moreno-Risueno et al. [18], were downloaded from Genbank and were com- pared to the deduced amino acid sequences of the 22 wheat genes to establish relationships between the genes from different species. The conserved 50-amino acid Dof domains of the DOF proteins were aligned using CLUSTALW [31] and the resultant multiple alignment (Additional file 3) was represented as an unrooted neighbor-joining tree edited with the Treeview© (v1.6.6) program [32] (Figure 4). Wheat Dof were present in the seven groups observed for the Dof family thus Dof members from the three cereals appeared in all the sub- families independently of the species. Members were found for all major orthologous clusters (MCOG-A, -B, -C and -D) defined by Lijavetzky et al. [15]. Group I was shown to correspond to MCOG-B, group II to