A bHLH transcription factor TsMYC2 is associated with the blue grain character in triticale (Triticum × Secale)

RNA-Seq was employed to compare the transcriptome differences between the triticale lines and to identify the key gene responsible for the blue aleurone trait. The accumulation of anthocyanins in the aleurone of triticale results in the formation of the blue-grained trait, but the identity of the genes associated with anthocyanin biosynthesis in the aleurone has not yet been reported. In this manuscript, RNA-Seq was employed to compare the transcriptome differences between the triticale lines HM13 (blue aleurone) and HM5 (white aleurone), and to identify the key genes responsible for the blue aleurone trait. There were 32,406 differentially expressed genes between HM13 and HM5. Seventy-three unigenes were homologous to the structural genes related to anthocyanin biosynthesis, and the average transcript level of the structural genes was higher in HM13 than in HM5, so that quantitative differences between the two lines in transcription rates could be the cause of the blue aleurone. The MYB and bHLH transcription factors had two homologous unigenes, but contained only one differentially expressed unigene each. The relative transcript level of bHLH Unigene5672_All (TsMYC2) in HM13 was 42.71 times that in HM5, while the relative transcript level of the MYB transcription factor Unigene12228_All in HM13 was 2.20 times that in HM5. qPCR experiments determined the relative transcript level of TsMYC2 in developing grain, with the expression of TsMYC2 in grain being the highest compared with that in root, stem or leaf tissue. TsMYC2 was homologous to the bHLH transcription factor regulating anthocyanin biosynthesis and contained three entire functional domains: bHLH-MYC_N, HLH and ACT-like, which were important for exercising regulation of anthocyanin biosynthesis as a bHLH transcription factor. Transient expression of ZmC1 and TsMYC2 could induce anthocyanin biosynthesis in white wheat coleoptile cells, demonstrating that TsMYC2 was a functional bHLH transcription factor. These results indicated that TsMYC2 was associated with the blue aleurone trait and could prove to be a valuable gene with which to breed new triticale cultivars with the blue aleurone trait.