Glucosinolates (GSLs) are sulfur-containing metabolites in Brassica species with dual roles in plant defense and human health. While glucoraphanin (GRA) offers anticancer benefits, progoitrin (PRO) poses risks due to goitrogenic effects. This study aimed to dissect the genetic basis of GRA, gluconapin (GNA), and PRO accumulation in florets of Brassica oleracea by integrating linkage mapping and quantitative trait locus (QTL) analysis using two F₂ populations (JB-F₂ and GJ-F₂) derived from crosses between broccoli, Chinese kale, and purple cauliflower. High-density linkage maps were constructed using a 10 K SNP array, and GSL profiles were quantified via high-performance liquid chromatography. QTL mapping identified 23 significant loci across both populations, with major-effect QTL clusters on chromosomes C3 and C9. Notably, epistatic analysis revealed strong interactions between major QTLs, particularly between loci on chromosomes C3 and C9, further emphasizing their central role in regulating GSL biosynthesis. Functional analysis prioritized BolC9t53177H (homologous to AOP2) and BolC3t13531H (homologous to GSL-OH) as key genes governing GRA-to-GNA and GNA-to-PRO conversions, respectively. Sequence variations in these genes explained parental GSL profiles: A 2-bp deletion causing a frameshift mutation in BolC9t53177H disrupted GRA metabolism in broccoli (B58-6), while defective BolC3t13531H in Chinese kale (J1402) abolished PRO synthesis. KASP markers developed for these loci enabled efficient genotyping of 104 B. oleracea accessions, revealing significant associations with GSL content. This study provides genetic insights and molecular tools to optimize GSL composition, facilitating the breeding of high-GRA/low-PRO Brassica varieties with enhanced nutritional value.

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