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

HighlightThe sweet fragrance of lettuce was attributed, for the first time, to the synthesis of 2-acetyl-1-pyrroline caused by a deficiency in the betaine aldehyde dehydrogenase gene. 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 F2 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.