Characterization of High-Artemisinin Yielding Artemisia annua Bioecotypes Using Gene-Specific STS Markers and HPLC

Artemisia annua is the sole natural source of artemisinin, an essential compound for malaria treatment. However, variable yields among bioecotypes hinder consistent production. This study aimed to characterize 17 A. annua biotypes using gene-specific STS markers targeting key enzymes in the artemisinin biosynthesis pathway and to evaluate their association with artemisinin content. Eleven STS primer pairs were designed for genes including ADS , CYP71AV1 , DBR2 , ALDH1 , HMGR , TTG1 , and others. PCR amplification, gel electrophoresis, and HPLC quantification of artemisinin were performed. Molecular marker efficiency indices were calculated. Hierarchical clustering, PCA, correlation heatmaps, and radar chart visualizations were employed to assess genotype–metabolite relationships. A total of 10 markers were polymorphic across biotypes, with significant correlation to artemisinin content (R² > 0.85 for key markers). Cluster and PCA analyses grouped biotypes into high-, medium-, and low-producing clusters. Biotypes 260 and 316 consistently displayed superior marker profiles and artemisinin content (> 2.7 mg/g DW). Marker indices such as PIC, MI, and EMR highlighted TTG1 , DXR , and ADS as the most informative loci. Radar chart analysis confirmed these markers and genotypes as optimal targets for selection. Gene-targeted STS markers can effectively distinguish elite A. annua biotypes with high artemisinin content. This integrated molecular-metabolite framework supports marker-assisted breeding and transgenic improvement for enhanced artemisinin production.