Romeuf et al. BMC Genomics 2010, 11:185 http://www.biomedcentral.com/1471-2164/11/185 Page 12 of 15 Figure 7 Relative expression of twelve Dof genes in developing seeds of T. aestivum cv. RE99006. Expression was monitored by qRT-PCR. Physiological stages of wheat seed formation: I, fertilization; II, cell expansion; III, cellularization and early grain filling; IV, completion of grain filling; V, desiccation. a) Expression pattern 1, b) expression pattern 2, c) expression pattern 3. TC315813 is considered separately because of its high expression level. Error bars represent the standard deviation of three replicates. similarity with known rice TF proteins and could be potential TFs. Although further experimental evidence is needed to validate whether they really belong to TF families, we decided to keep all sequences in wDBTF so users can themselves judge the sequences with no DNA-binding domain. The 3,820 wheat sequences identified as TFs represent 1.8% of all available unique wheat sequences. TF expres- sion is mostly specific to a particular tissue or develop- mental stage and most TFs are also known to be expressed at a low level. We have shown that almost 50% of sequences identified as wheat TFs are singletons and 70% of contigs comprised just a few ESTs. This confirms that wheat TFs are expressed at a low level and suggests that perhaps not all TF sequences are represented in the EST libraries available. Therefore in our analysis of wheat expressed sequences, the number of TF genes could be underestimated. Until the whole genome sequence is available, it is unlikely that all the TFs can be identified based only on transcript sequences. TFs have evolved by accumulating mutations, so the complexity in gene regulation patterns has increased. Much of the innovation in biological function has been attributed to events of gene duplication. Wheat appears to have more AUX/IAA, CCATT-Dr1, HMG, MBF, S1Fa-like, Whirly and VOZ compared with the pre- dicted number, i.e. three times more than in rice. How- ever, the largest families (EREBP, NAC, bHLH, R2R3- MYB, bZIP, WRKY and C3H zinc finger) do not exceed the proportional number predicted from rice. These pre- liminary results tend to confirm the coding theory that predicts that there are limits to the TF repertoire of cells. From each superfamily, the maximal number of TFs seems to correlate with the number of DNA bases effectively recognized by the binding mechanism of that superfamily. This limit of expansion is thought to mini- mize cross-binding errors between TFs [39].