VC2regulates baseline vicine content in faba bean

Faba bean ( Vicia faba ) is a valuable legume crop desired globally for its high nutritional composition. However, the seed vicine and convicine (v-c) content reduces the nutritional quality of faba bean protein and can induce favism in individuals with glucose-6-phosphate dehydrogenase deficiency. Recently, VC1 gene, encoding a bi-functional riboflavin protein, was reported to be responsible for initiating the biosynthetic pathway in V. faba . In low v-c cultivars, a 2 bp insertion in this gene results in a loss of function, but the mutation only partially eliminates v-c biosynthesis, indicating the involvement of other genes. Here, we demonstrate that a novel V. faba riboflavin gene, VC2 , is responsible for the residual v-c contents in faba bean. VC2 shares nearly identical functional domains with VC1 and has GTP cyclohydrolase II activity, catalyzing the conversion of GTP into an intermediate molecule in the biosynthetic pathway. Gene expression analysis reveals that VC2 contributes a minor effect to the trait, accounting for approximately 5-10% of total riboflavin gene transcripts which significantly correlates with the baseline contents in low v-c cultivars. Our results illustrate that cultivars carrying the 2 bp inactivating insertion in VC1 still have residual v-c levels due to VC2 activity. Furthermore, we find that VC1 has multiple alleles and exhibits copy number variations, complicating molecular marker development. Conversely, single nucleotide polymorphisms within VC2 provide a reliable alternative for marker-assisted selection in faba bean breeding. In conclusion, our study elucidates the complex genetic regulation of v-c biosynthesis and provides valuable insights to facilitate its elimination in faba bean.