The study assessed genetic diversity and trait relationships in 64 safflower genotypes obtained from the National Plant Gene-Bank of Iran. Employing an alpha lattice design with two replications, various agro-morphologic characteristics were measured. Analysis of pairwise association’s unveiled positively significant interrelationships of yield performance with the other measured characteristics, except for 1000-seed weight. This suggested that these traits generally moved in the same direction, positively influencing seed yield. To gain a more nuanced understanding of trait interdependencies, sequential path analysis was employed. This analysis identified the capitula of each plant and the seeds of each capitulum as crucial first-order characteristics significantly impacting seed yield. All direct effects identified through sequential path analysis were deemed significant, emphasizing the robustness of the findings. The analysis further categorized characteristics as second or third order variables according to impacts on seed yield. Leaf area, plant’s dry weight and 1000-seed weight were identified as second-order variables, indicating their indirect influence on seed yield. Additionally, height of plant, oil percentage, diameter of lateral capitulum and biological yield were categorized as third-order characteristics, further expanding the understanding of the complex trait relationships. The key takeaway from the study is the potential use of the capitula of each plant and the seeds of each capitulum as effective selection criteria for enhancing safflower yield performance. These findings provide valuable insights for safflower breeding programs, guiding the selection of genotypes with improved seed yield based on these identified influential traits.
