Monoterpenoids are vital compounds that impart a distinctive floral flavor. They exist in both glycosidic and free forms in grapes. The breeding of improved monoterpenoid varieties has consistently been a topic of interest, yet only a limited number of molecular markers have been documented. This study employed a genome-wide association study (GWAS) on an F₁ population crossed between a typical muscat variety (‘Muscat of Alexandria’) and a non-aromatic variety (‘Christmas Rose’), conducted over two consecutive years. A total of 4089 significant single nucleotide polymorphism sites (sigSNPs) and 892 candidate genes associated with monoterpenoids were identified. The sigSNPs corresponding to the glycosidic and total (glycosidic plus free) concentrations of various monoterpenoid compounds exhibited a high similarity. The majority of sigSNPs were located on chromosome 5, indicating the existence of a monoterpenoid-related marker cluster. Sixty-one lead SNPs located within the gene region and stably appearing in 2 years were selected and verified using a germplasm population. The alleles of the 25 lead SNPs were confirmed to be highly associated with monoterpenoid levels. The genes containing these lead SNPs were mainly glycoside hydrolase, ABC transporter, as well as the previously reported 1-deoxy-D-xylulose-5-phosphate synthase (VvDXS1) and geranylgeranyl pyrophosphate synthase large subunit (VvGGPPS-LSU). The function of VvGGPPS-LSU in regulating monoterpenoid levels was elucidated through in vivo overexpression, demonstrating the reliability of the marker cluster. The present study proposes a molecular marker set for the breeding with the objective of improving aroma, and a candidate gene network for the regulation of monoterpenoid synthesis in grapevine.

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