The incomplete dominance of gene expression controlled by trans-eQTL hotspots contributes to phenotypic heterosis in maize

Heterosis, or hybrid vigor, is a key phenomenon in genetics research and agricultural production, and has been primarily attributed to non-additive genetic effects --- a prevailing consensus shaped by decades of empirical research and theoretical debate. To address this long-standing question, particularly how non-additive genetic effects manifest at the transcriptomic level and contribute to phenotypic heterosis, we integrated transcriptomic and phenotypic data from a large population of maize hybrids and their inbred parents. We found that ~30% of the expressed seedling genes in a significant proportion of hybrids exhibited expression patterns deviating from the average of the two parents, indicative of non-additivity. Further analysis suggests that while hybrid gene expression per se is primarily regulated by cis-eQTLs, expression non-additivity (or incomplete dominance) is disproportionately controlled by trans-eQTLs. These trans-eQTLs cluster into hotspots that regulate the incomplete dominance of hundreds of target genes, mostly within co-expression networks, and are notably enriched for transcription factors (TFs). Focusing on one such hotspot, we functionally validated a classical maize gene ZmR1, a basic helix-loop-helix (bHLH) TF associated with multiple seedling trait heterosis, as a candidate regulator of incomplete dominance across hundreds of genes. Overexpression of ZmR1 increases phenotypic heterosis in both seedling and adult traits and confirms its direct regulatory role in modulating genes involved in anthocyanin biosynthesis and lignin metabolism, driving transcriptome-level non-additivity. These results provide empirical support for the modifier hypothesis under a dosage-sensitive trans-regulatory framework, suggesting that heterosis arises not from the modification of a single gene's inheritance but through the coordinated regulation of hundreds of phenotype-associated genes, thereby helping to reconcile the long-standing debate over the genetic basis of dominance in heterosis.