Identification of SNP markers linked to calcium and phosphorus accumulation in walnut (Juglans regia L.) fruit by GWAS

Walnut (Juglans regia L.) is a nut species with various applications worldwide and plays a significant role in human nutrition. The walnut kernel contains various essential nutrients crucial for regulating plant physiological functions and maintaining human health. The aim of the current study was to identify SNP markers associated with the accumulations of important macronutrients such as calcium (Ca) and phosphorus (P) in 154 walnut genotypes over two consecutive years (2021-2022) using the GWAS method. Concentrations of Ca and P ranged from 70.83 to 386.67 mg/100g and from 106.17 to 583.08 mg/100g, respectively, over the two-year period. Significant differences between genotypes (p <= 0.01) were observed for both macronutrients. High heritability values were obtained for Ca (0.994) and P (0.993) accumulations. The STRUCTURE analysis conducted using a total of 16,473 clean SNP data and 154 walnut genotypes resulted in the population being grouped into three clusters (K=3). The mean fixation index (Fst) and expected heterozygosity values were determined as 0.22 and 0.31, respectively. Using the MLM (Q+K) approach, a total of 59 significant associations (35 for Ca and 24 for P) exceeding the false discovery rate (FDR) threshold were identified (p < 0.01). Among these, 16 SNPs for Ca and 11 SNPs for P were found to be common across both years. One SNP (M14826) was common for Ca-2021, Ca2022, and P-2022, while two SNPs (M15542 and M576) were common for both macronutrients and both years. Analysis of the 59 SNP sequences using the Genome Data Viewer (GDV) in the NCBI database revealed that 16 SNPs were located in the J. regia genome. For nine SNPs associated with Ca-2021 and Ca-2022 accumulations, a total of 89 candidate genes were identified, while for five SNPs associated with P-2021 and P-2022 accumulations, 64 candidate genes were identified. Additionally, seven candidate genes were identified for one SNP associated with Ca-2021, Ca-2022, and P-2022, and five candidate genes were detected for one SNP associated with accumulations of Ca-2021, Ca-2022, P-2021, and P-2022. The results suggest that SNP markers associated with Ca and P accumulations in walnut kernels can be valuable for facilitating marker-assisted selection (MAS) strategies in future walnut breeding studies and for developing biofortified walnut genotypes.