Insertion of ten amino acids into 13S globulin zero-repeat subunit improves trypsin digestibility in common buckwheat (Fagopyrum esculentum Moench) seeds

The 13S globulin zero-repeat subunit is resistant to trypsin and may have higher allergenicity than the 1–6 tandem repeat subunits in common buckwheat (Fagopyrum esculentum Moench). To explore alleles useful for lowering allergenicity, amplicon deep sequencing targeting the zero-repeat subunit gene was conducted in bulked genomic DNA from eight cultivars and landraces. The analysis identified a unique allele encoding a zero-repeat subunit with 10 amino acid insertion (10aa) at a position equivalent to the tandem repeat insertion. Prediction of its 3-D structure suggested that 10aa changes the β-hairpin structure in the non-10aa (native) subunit to a random coil, which is also found in 1- and 3- repeat subunits. Homozygotes of the 10aa allele were developed and showed that the 10aa subunit was more digestible than the native subunit. However, the 10aa subunit was still less digestible than the 1–6 repeat subunits, suggesting needs to explore unfunctional alleles.