Gwas-based identification of heterosis-associated genes for yield traits in maize testcross populations

Heterosis is central to breeding high-yielding maize ( Zea mays L.). To dissect the genetic basis of heterosis for yield-related traits, we constructed four testcross populations using four diverse inbred lines as testers and an association mapping panel of 368 inbred lines. We systematically investigated ear diameter, ear length, kernel row number, kernels per row, and kernel weight per ear, as well as their mid-parent heterosis values, across two environments. Using whole-genome sequencing and genome-wide association analysis, we mapped genomic regions and identified candidate genes associated with these traits and their heterosis. We detected a total of 94 genomic regions containing 389 candidate genes, of which Zm00001d051889 ( ETHYLENE INSENSITIVE 4 [ EIN4 ]), involved in ethylene signaling, was a key candidate locus. We identified a 59-bp insertion–deletion (InDel) variant in its promoter region, and EIN4 showed non-additive expression in hybrids. Haplotype analysis revealed that the stacking of Hap 3 of EIN4 in hybrids is associated with greater ear diameter and kernel row number. EMS-induced loss-of-function mutation of EIN4 resulted in altered ear length in hybrids, indicating an impact on ear length heterosis. These findings demonstrate that promoter variation, haplotype changes, and EMS-induced premature termination of EIN4 all affect heterosis or yield-related traits, providing convergent genetic evidence for the contribution of EIN4 to yield-related heterosis in maize. The identification of EIN4 and its natural polymorphisms provides a useful molecular resource for maize hybrid breeding, facilitating marker-assisted selection and the development of high-yielding and stable maize varieties.