Romeuf et al. BMC Genomics 2010, 11:185 http://www.biomedcentral.com/1471-2164/11/185 Comprehensive Affymetrix GeneChip platforms have now been developed for wheat based on extensive EST collections http://www.plexdb.org/index.php. The Affy- metrix wheat genome array can provide useful data on organ and developmental stage expression. We found 4,305 out of the 7,112 wDBTF wheat sequences repre- sented on the Affymetrix wheat genome array. However, several sequences correspond to the same Affymetrix ID and only 2,036 are unique. Thus, this result is in agree- ment with those of Wan et al. [40] who found about 2,000 probe sets for potential TFs. Analysis of expres- sion using this microarray platform can be difficult because of the often confounding presence of multiple splice forms, paralogs and orthologs, and, in the case of polyploids, homoeologs with near-identical sequences [41]. In general the wheat GeneChip is not able to dis- tinguish contributions from individual homoeologs. As the probe sequences were specifically taken from con- served regions of consensus sequences, the expression profiles measured represent a sum of the individual pro- files of the relevant homoeologs for ~90% of genes [42]. Furthermore, the expression measured could also be the sum of the expression of several splicing variants. TFs derived from the same precursor mRNA by alternative splicing may have distinct regulatory functions. In wheat, this kind of mRNA processing has been shown for some TF genes like the A and D copies of Vivipar- ous 1 (TaVp1) [43] and for wDreb2 [44]. Parameters used in TIGR assembly often place homoeologous genes and alternative splicing variants in separate contigs and thus the difference observed between the number of sequences matching Affymetrix ID probes and the num- ber of probes might be expected. On the other hand, singleton ESTs, relatively abundant in our study, tend to be shorter increasing the chances of confusion when try- ing to match them to a particular sequence present on GeneChip. We used the Dof TF family to validate our strategy for identifying wheat TFs. Based on sequence identity with rice gene models, 59 potential Dof genes were identified. Multiple alignment of the entire Dof domain showed that wheat sequences were distributed in seven groups among all four major orthologous clusters suggesting this TF family is organized similarly in the species studied. We used the database to select Dof involved in wheat grain development based on in silico expression criteria. First we verified these genes were indeed expressed in seed and then established the pattern of expression of 12 transcripts during seed development. For seven Dof (TC289916, TC277573, TC339845, CA484955, TC316155 and TC315813), expression reached a maxi- mum in the middle of the grain-filling phase when Page 13 of 15 starch and storage proteins accumulate. Accession TC315813 is wheat prolamin-box binding factor (WPBF), which was shown to be an activator of storage protein genes [24]. Its expression parallels the increase in abundance of transcripts encoding low molecular weight glutenin subunits and alpha gliadin genes consis- tent with a role in prolamin gene expression [40]. Thus, Dof TF expression profiles like that of Wpbf could be potential regulators of grain filling. In expressed sequences, there are few nucleotide variations between homoeologs. Thus the probability of designing primers specific to one or two copies is low. All the primers used amplified products from T. urartu, Aegilops spel- toides and A. taushii which confirms that potentially all copies which are expressed in hexaploid wheat are amplified. Thus, the expression profiles measured for any particular gene may represent the expression of all its homoeologs. Plant responses to allopolyploidy include unequal expression of duplicated genes and gene silencing. Studies in wheat [45,46] have shown that novel patterns of gene expression occur in polyploids that are not observed in diploid progenitor species. Furthermore, in silico analysis of gene expression has shown that there may be preferential expression of homoeologous genes between genomes and a uniform level of expression of all three homoeologs is only observed for about 20% of the genes [45]. A recent study on the wheat TF Spa has demonstrated that the three copies were expressed during all stages of grain development in similar patterns. However, there was more Spa transcript from B genome than A and D tran- scripts [47]. To study patterns of gene expression in bread wheat, it will be essential to develop an assay that allows genome/homoeolog-specific gene expression to be quantified. Our preliminary results provide an over- view of Dof expression and further research will confirm which homoeologous genes are implicated in the grain- filling mechanism. Conclusions The genome sequence of wheat is not yet available so analyzing the systematically produced sequences of cDNA libraries is a powerful way to identify many tran- scripts that can then be studied in terms of their gene expression and functional genomics. In our study, wheat TF sequences were identified based on their similarity to those in the model plant rice. Organized in a dedi- cated database available on our web site, and classified in families are 7,112 potential unique wheat TFs, 3,820 of which have a DNA-binding domain. For each sequence, in silico expression data is given. The database will therefore be a major resource for the plant research community for future studies of wheat TFs.