Enoyl‐ <scp>CoA</scp> hydratase genes underlie natural variation of lactones in cultivated and wild strawberries

Strawberry flavor is a key factor in consumer acceptance and is highly influenced by volatile aroma compounds. Multiple consumer and trained panel sensory studies indicate that γ-lactones are among the most important aroma compounds in strawberry, imparting sweet, fruity aromas. We quantified γ-lactones in Fragaria × ananassa and Fragaria vesca to explore the genetic basis for lactone production in the two species using genome wide association study (GWAS). We developed a haplotype-phased genome assembly for a high lactone cultivar to explore previously published expression quantitative trait locus and fruit ripening RNA sequencing data to identify candidate genes in Fragaria × ananassa. We leveraged knowledge from Fragaria × ananassa to identify candidate genes in F. vesca and validated one gene from each strawberry species using fruit transient gene expression assays. We found that a novel Enoyl CoA hydratase/isomerase gene in Fragaria × ananassa, FaECH, increases concentration of γ-lactones while a similar but nonhomologous gene, FvECH-like, increases γ-lactones in F. vesca. Additionally, the major gene shown to control γ-decalactone production in Fragaria × ananassa, FaFAD1, has a homolog in F. vesca, FvFAD1, that underlies a strong GWAS. These results validate key γ-lactone biosynthesis steps in both strawberry species and provide tools to breed more flavorful strawberries.