Assessing genetic diversity and gene pool structure is crucial for biofortification, which can address micronutrient deficiencies in underdeveloped countries. This study evaluated grain iron (Fe) and zinc (Zn) content in 67 wheat genotypes from diverse origins over three years (2017-2019). To quantify genetic diversity, three marker techniques were employed: 15 simple sequence repeats (SSR), 20 start codon targeted (SCoT), and 20 intron-targeted amplified polymorphisms (ITAP). The study revealed significant variation and high heritability in grain Fe and Zn content each year. Some wheat genotypes consistently exhibited high Fe and Zn content despite year-genotype interactions. In 2018 and 2019, Pearson correlation tests indicated significant positive associations between Fe and Zn content. Cluster analysis identified distinct genotype groupings based on grain Fe-Zn content, with certain high-content genotypes forming separate clusters. The study highlighted substantial genetic diversity among wheat genotypes, with SSR markers exhibiting the highest polymorphism (100%), while ITAP markers were less informative (89.57%). AMOVA analysis demonstrated significant variability within and between subpopulations for all marker types. SSRs and SCoTs exhibited weak, non-significant Mantel correlations with Fe-Zn content, whereas ITAPs showed a significant negative correlation, suggesting inconsistency with Fe-Zn content. Nonetheless, strong correlations were identified among SSRs, SCoTs, and ITAPs. Spearman correlation analysis revealed several SSR, SCoT, and ITAP amplicons correlated with high grain Fe and Zn content, some displaying consistent stability over multiple years. These findings provide vital insights for wheat breeding and biofortification, enhancing efforts to improve nutritional content through understanding population structure and genetic diversity.
