LsBADH1 is responsible for sweet fragrance in lettuce ( Lactuca sativa L.) through 2-acetyl-1-pyrroline biosynthesis

Fragrance is among the most valuable traits of high-quality crops and influences consumer preferences. Although 2-acetyl-1-pyrroline (2AP) is a key component of fragrant cultivars in several crops, its genetic mechanism in lettuce ( Lactuca sativa L.) remains poorly understood. The betaine aldehyde dehydrogenase ( BADH ) gene has been identified as causative for 2AP-derived fragrance in rice and soybean cultivars. Hence, we conducted a linkage analysis using an F ₂ population derived from a cross between 'Kukichisya' (fragrant) and 'Rennet' (non-fragrant) for three candidate genes of BADH orthologs in the lettuce genome. Analysis linked LOC111877932 　located in LG4 to the fragrance trait, and it was designated LsBADH1 . Comparison among 'Kukichisya', 'Salinas', and candidate BADH of sunflower ( Helianthus annuus L.) revealed three non-synonymous single-nucleotide polymorphisms (nsSNPs) in exons 1, 2, and 9, and suggested that nsSNP in exon 9 was strongly correlated with fragrance in 'Kukichisya.' A premature stop codon introduced in exon 5 of LsBADH1 using Target-AID base-editing technology resulted in truncated BADH1 and higher 2AP levels. Our results indicated that LsBADH1 is responsible for the 2AP-derived fragrance. Our findings can be applied to select cultivars based on a novel concept for the cooking process, providing a transformative platform to breed fragrant lettuce as a high-value-added product.