LsTT2encoding R2R3-MYB transcription factor is responsible for a shift from black to white in lettuce seed

Abstract

AbstractPrickly 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 F2population 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 theLG7_v8_49.251Mbp_HinfImarker, which employs a single nucleotide mutation in the stop codon ofLsat_1_v5_gn_7_35020.1was completely linked to the seed color phenotype. In addition, the sequences of the coding region for candidate genes except forLsat_1_v5_gn_7_35020.1were identical in the resequence analysis of ‘ShinanoPower’ (white seed) and ‘Escort’ (black seed). Therefore, we proposedLsat_1_v5_gn_7_35020.1, a gene located in the locus, as the candidate gene and designated it asLsTT2, an ortholog encoding the R2R3 MYB transcription factor inArabidopsis. When we validated the role ofLsTT2in seed color through genome editing,LsTT2knockout 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 inArabidopsis TRANSPARENT TESTA 2(TT2) mutants. Therefore,LsTT2was 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.