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.