166 and MYB-like protein (Reddy et al. 1998). The WRKY factors encode the seed coat development gene of Arabi- dopsis (Johnson et al. 2002), and several MADS box transcription factors are expressed preferentially in flowers and cause early flowering when ectopically expressed in rice plants (Jeon et al. 2000). Papillomavirus binding factor (PBF) regulates expression of cereal storage protein, which interacts with Opaque-2 and Related to ABI3/VP1 1 (RAV) factors, to act as negative regulators of growth. APETALA2 (AP2) is related to seed development, ethylene-responsive expression, and floral morphogenesis (Jofuku et al. 1994; Shukla et al. 2006). Unknown genes involved in anthocyanin biosynthesis Among the 137 transcription factor genes identified above, the expression of 24 unknown and hypothetical genes was J Appl Genetics (2011) 52:161–169 found at each seed developmental stage. Among these, 14 showed a pattern of upregulation, and 10 genes were down- regulated. Therefore, this method is able to efficiently screen the unknown genes associated with specific biological process. Finally, 17 unknown and hypothetical genes differed between the Donjin cultivar and the two black cultivars at all seed developmental stages. Of the 17 candidate genes, 10 showed a pattern of upregulation and 7 appeared to be downregulated genes. These genes most likely play either a regulatory role in the anthocyanin production process or are related to anthocyanin metabolism during flavonoid biosyn- thesis. Additional function studies of these genes are necessary to further characterize possible regulatory factors of the anthocyanin pathway and pigmentation metabolism. Nevertheless, these 17 transcription factor genes play a potential role in anthocyanin production. This study provides valuable insight into anthocyanin pigmentation production Fig. 2 RT-PCR analysis result of 15 candidate genes, repre- senting nine up-regulated and six down-regulated genes. A total 27 RNA samples were isolated from three cultivars and three developmental stages, in triplicate. Actin was used as a control. DAF = Day after head- ing, WT = Dongjin (white color seed of no anthocyanin), B1 = Heugjinju (black color seed of lower anthocyanin), B2 = Heugseol (black color seed of higher anthocyanin)