Integrated GWAS and BSA-seq Dissect the Genetic Architecture of Maize Leaf Morphology

Leaf morphology is a critical component of maize plant architecture, directly influencing photosynthetic efficiency and yield. To dissect the genetic basis of leaf length (LL), leaf width (LW), and leaf length-to-width ratio (RLW), we integrated genome-wide association studies (GWAS), bulk segregant analysis sequencing (BSA-seq), and the 3VmrMLM multi-locus model in a diverse maize panel. We observed high broad-sense heritability for LL (0.85), LW (0.84) and RLW (0.87), indicating these traits are largely genetically controlled. GWAS identified 49, 46, 40 significant SNPs for LL, LW, and RLW, across multiple environments, corresponding to 36, 36, and 25 quantitative trait loci (QTL), respectively. 3VmrMLM model further detected 448 quantitative trait nucleotides (QTNs), effectively capturing additive, epistatic, and QTN-by-environment interaction effects. We identified and functionally characterized two genes: Zm00001d026262 , which regulate LL through auxin-mediated cell elongation, and Zm00001d003176 , which influences LW via nitrogen assimilation, thereby influencing cell division and expansion. The convergence of evidence was strengthened by BSA-seq, which revealed 12 significant intervals co-localizing with GWAS signals. Haplotype analysis showed Hap1 of Zm00001d026262 (71.13 cm) was significantly associated with LL; compared with Hap1 (7.80 cm) of Zm00001d003176 , Hap2 (9.08 cm) exhibited a significant difference in LW. Furthermore, historical selection analysis indicated that both genes were strongly selected during early breeding, while their selection signatures have weakened in modern lines, reflecting a shift toward optimizing plant architecture for high-density planting. Collectively, our study provides novel insights into the genetic architecture of leaf morphology and delivers actionable targets for optimizing plant architecture for high-density maize breeding.