Whole genome sequencing-based multi-locus association mapping for kernel iron, zinc and protein content in groundnut

Malnutrition is a major global challenge, especially in the developing regions, where improving the nutritional content of staple crops is an important step towards alleviating hidden hunger. Groundnut, a nutrient rich legume, contains several essential nutrients, high protein, essential amino-acids and vitamins required for human health. In this study, multi-season phenotyping data for kernel iron (Fe), zinc (Zn) and protein content (PC), along with whole genome re-sequencing (WGRS) data from a mini-core collection, were used to perform genome-wide association study (GWAS) analysis. Phenotypic variability analysis revealed a large variation in Fe (7.6 - 42.8 ppm), Zn (10.9 - 62.4 ppm) and PC (12.7 - 33.6%). GWAS analysis identified a total of 15 marker-trait associations (MTAs) and 28 candidate genes for pooled season data, and 44 MTAs and 62 candidate genes for individual seasons. Key candidate genes like MYB transcription factor (Arahy.QI0PHV, Arahy.1I6ZSS), Zn finger MYM type protein, RING finger MYM type protein (Arahy.7P97F6, Arahy.9R964H, Arahy.I3B88T) and NAC domain protein (Arahy.LV3APC), were found to be associated with the Fe and Zn homeostasis pathway. In addition, genes related to protein homeostasis, such as Protein kinase family protein (Arahy.4D7KBI), and E3 ubiquitin-protein ligase (Arahy.PE3CF6), were identified within significant MTA regions. These findings provide basis for the detection and characterization of potential candidate genes associated with nutritional quality traits. Haplo-pheno analysis revealed that accessions with superior haplotypes for kernel Fe and Zn content were predominantly found in the Spanish Bunch/Valencia Bunch types, whereas the accessions with inferior haplotypes were more common in the Virginia Bunch/Virginia Runner types. Single nucleotide polymorphism (SNP)-based KASP (Kompetitive Allele Specific Polymerase Chain Reaction) markers for 9 MTAs were designed and validated for Fe and Zn. Among these, three markers (snpAH00636 and snpAH00641 for Fe, and snpAH00644 for Zn) showed polymorphism, and could be utilized in genomics-assisted breeding to develop nutrient-rich groundnut varieties.