Molecular Marker Validation for Grain Iron, Zinc, and Protein Content in Rice Using Single Marker Analysis Approach

Abstract

Micronutrient malnutrition, particularly iron (Fe) and zinc (Zn) deficiencies, remains a major global health concern, highlighting the need for rice biofortification. Validating molecular markers linked to grain nutritional traits is essential for their effective use in marker-assisted breeding. In this study, a BC₁F₇ backcross inbred line (BIL) population comprising 158 lines derived from RNR15048 × Karuppunel was evaluated to validate simple sequence repeat (SSR) markers associated with grain Fe, Zn, and protein content (GPC) through single-marker analysis (SMA). A total of 196 SSR markers, including 118 previously reported QTL-linked markers, were screened for parental polymorphism; 89 polymorphic markers were subsequently used to genotype the BIL population. SMA identified 23 marker–trait associations across chromosomes 1, 2, 4, 6, 7, and 9, of which 18 exceeded the specified LOD threshold. Zinc content in polished rice had the highest number of significant associations (eight), followed by zinc content in brown rice (seven). Two markers, RM341 and RM11, were significantly associated with iron content in brown rice, while RM3524 was associated with GPC. The significant markers explained 3.83–10.68% of the phenotypic variation, indicating that these nutritional traits are influenced by multiple minor-effect loci. These validated SSR markers represent potentially useful molecular tools for marker-assisted selection aimed at developing rice cultivars with enhanced grain iron, zinc, and protein content for sustainable biofortification programmes.