The fresh and unique flavor of cucumber fruits, mainly composed of aldehydes and alcohols, is one of its most important fruit qualities. However, little is known about the genetic basis of aroma compounds in cucumber fruit and the related quantitative trait loci (QTLs). In this study, genomic screening of QTLs underlying aroma compounds was performed based on the genetic linkage map constructed using 1301 single-nucleotide polymorphism (SNP) markers from genotyping-by-sequencing of a recombinant inbred line (RIL) population developed from Q16 × Q24. Significant genetic variations of aroma compounds in the RIL population were observed, and a total of 28 QTLs were screened. A major QTL (qol8-2.1) related to (E,Z)-2,6-nonadien-1-ol was detected with a markedly high LOD score (10.97 in 2020 and 3.56 in 2019) between mk190 and mk204 on chromosome 2. Genome scans identified a cluster of nine lipoxygenase genes in this region. A significant positive correlation was detected between CsaV3_2G005360 (CsLOX08) and (E,Z)-2,6-nonadien-1-ol, and five amino acid variations were detected between the CsLOX08 protein sequences of the two parental lines. Based on the genome variation of CsLOX08, we developed an InDel marker. Genotyping of InDel markers was consistent with the content of (E,Z)-2,6-nonadien-1-ol in RILs, which were also verified in nine cucumber inbred lines. The results will give breeders guidance for obtaining better flavor in cucumber.

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