Jihong Zhang, Na Liu, Shiwei Wang, Xiang Guo, Xinyu Sun, Haiyang Duan, Lianglei Zhang, Liang Yuan, Huiling Xie, Huili Yang, Xiaoyang Chen, Dong Ding, Jihua Tang, Xuehai Zhang
Accepted: 2025-06-10
Protein content (PC) in maize kernels is a key determinant of their nutritional quality, however, its genetic basis remains largely unexplored. In this study, we conducted a genome-wide association study (GWAS) using 264 maize inbred lines and 1.25 million single nucleotide polymorphisms (SNPs), applying six GWAS models: BLINK, FarmCPU, MLM, MLMM, SUPER, and 3VmrMLM. Kernel PC exhibited substantial variation, ranging from 9.26 to 20.94%, with a broad-sense heritability of 0.56. A total of 473 significant quantitative trait nucleotides (QTNs) were detected, each explaining 0.08 to 7.10% of the phenotypic variance. Among them, 115 QTNs were consistently detected across different models, environments and analytical methods. Notably, 3VmrMLM model identified 59 most significant QTNs, with 38 were QEIs, and the MLM model identified the fewest significant QTNs (8). We further identified 35 candidate genes located within or adjacent to the significant QTNs. Among these, four genes - Zm00001d033805, Zm00001d037565, Zm00001d052164 and Zm00001d031535 - were strongly associated with PC. These genes are implicated in critical biological pathways, including nitrogen metabolism, photosynthesis, and the tricarboxylic acid (TCA) cycle. Notably, Zm00001d037565, encoding a gibberellin 2-oxidase, plays a role in seed development and is likely involved in regulating protein accumulation in kernels. Haplotype analysis revealed that the HapA of Zm00001d037565 is significantly associated with higher PC. Selective sweep analysis indicated that this gene underwent selection during maize domestication from teosinte (Zea mays ssp. mexicana and Zea mays ssp. parviglumis), its adaptation from tropical/subtropical to temperate regions, and throughout modern breeding programs. Overall, this study advances our understanding of the genetic architecture of maize kernel PC and provides valuable candidate genes and haplotypes for marker-assisted selection, offering new targets for developing high-protein maize varieties.