LsTT2 encoding R2R3-MYB transcription factor is responsible for a shift from black to white in lettuce seed

Prickly lettuce ( Lactuca serriola ), which is considered the wild ancestor of lettuce, has black seeds, whereas the major seed color of domesticated lettuce is black or white. The successfully-selected white seed trait is a key domestication trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black to white seeds remains to be clarified. We aimed to identify the gene/s responsible for white seed trait in lettuce. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F2 population derived from a cross between ‘ShinanoPower’ (white seed) and ‘Escort’ (black seed). The white seed trait was controlled by a single recessive locus (48.055–50.197 Mbp) in linkage group 7. Narrowing down using five PCR-based markers and 84 cultivars, eight candidate genes were mapped in the locus. Only the LG7\_v8\_49 . 251Mbp\_HinfI marker, which employs a single nucleotide mutation in the stop codon of Lsat\_1\_v5\_gn\_7\_35020 . 1 was completely linked to the seed color phenotype. In addition, the sequences of the coding region for candidate genes except for Lsat\_1\_v5\_gn\_7\_35020 . 1 were identical in the resequence analysis of ‘ShinanoPower’ (white seed) and ‘Escort’ (black seed). Therefore, we proposed Lsat\_1\_v5\_gn\_7\_35020 . 1 , a gene located in the locus, as the candidate gene and designated it as LsTT2 , an ortholog encoding the R2R3 MYB transcription factor in Arabidopsis . When we validated the role of LsTT2 in seed color through genome editing, LsTT2 knockout mutants harboring an early termination codon showed a change in seed color from black to white. White seeds accumulated less proanthocyanidins than black seeds, which was similar to the phenotype observed in Arabidopsis TRANSPARENT TESTA 2 ( TT2 ) mutants. Therefore, LsTT2 was the allele responsible for the shift in seed color from black to white. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds has implications for future breeding technology and physiological analysis. ### Competing Interest Statement The authors have declared no competing interest.