Development and Application of KASP Markers for Candidate Glucosinolate Biosynthesis Genes in Broccoli

Broccoli is rich in glucosinolates (GSLs), secondary metabolites that contribute to both plant defense and human health. Optimizing the composition of major aliphatic GSLs is an important breeding objective, yet robust molecular markers for marker-assisted selection (MAS) remain limited. In this study, candidate gene-based kompetitive allele-specific PCR (KASP) markers were developed from conserved GSL biosynthesis genes, focusing on <i>AOP2</i> and <i>GSL-OH</i> selected from 19 GSL-related genes. Marker-trait associations were evaluated in a natural broccoli population and further validated in an independent F₂ population. Among the tested markers, S101, located in <i>AOP2</i>, exhibited consistent genotype-dependent effects on GNA and PRO across both populations, supporting its stable predictive value. Receiver operating characteristic (ROC) analysis further confirmed strong classification performance of S101 for distinguishing high- and low-content genotypes of these traits in the F₂ population. In contrast, S074 and <i>S035</i> showed population-dependent effects, with significant associations detected only in the natural population. Although association signals were reduced under mixed linear model (MLM) analysis with false discovery rate (FDR) correction, major loci identified under the general linear model (GLM) framework remained detectable. Overall, these results demonstrate the potential of candidate gene-based KASP markers for improving aliphatic GSL composition in broccoli through marker-assisted selection.