The bHLH transcription factor VfTT8 underlies zt2, the locus determining zero tannin content in faba bean (Vicia faba L.)

Faba bean (Vicia faba L.) is an important protein-rich fodder crop, which is widely cultivated in temperate areas. However, antinutritional compounds such as condensed tannins, limit the use of this protein source in monogastric feed formulations. Previous studies demonstrated that two recessive and complementary genes, zt1 and zt2, control absence of tannin and white flower colour in faba bean. An ortholog of the Medicago WD40 transcription factor TTG1 was reported to encode the zt1 phenotype, but the responsible gene for zt2 is still unknown. Here we used a candidate gene approach combined with linkage mapping, comparative genomics and gene expression to fine map the zt2 genomic region and to identify the regulatory gene controlling both traits. Seventy-two genes, including 23 MYB and bHLH regulatory genes predicted to be associated with anthocyanin expression together with WRKY proteins, were screened and genotyped in three mapping populations. The linkage groups constructed identified the regulatory gene, TRANSPARENT TESTA8 (TT8), encoding a basic helix-loop-helix (bHLH) transcription factor, as the candidate for zt2. This finding was supported by qPCR analysis and further validated in different genetic backgrounds. Accordingly, VfTT8 was downregulated in white flowered types while showing high levels of expression in wild genotypes. Our results provide new insights on the regulatory mechanisms of tannin biosynthesis in faba bean and will facilitate the development of an ultimate zt2 diagnostic marker for the fast generation of new value-added cultivars free of tannins and with improved nutritional value.