A novel genetic framework reveals transcriptional "butterfly effect" underlying heterosis in maize.

Introduction: Maize is one of the first crops to benefit from heterosis, significantly enhancing commercial breeding. Despite extensive research, the molecular mechanisms of heterosis remain elusive.

Objectives: This study integrates a novel genetic framework with transcriptomic and phenotypic analyses to identify heterosis-related genes and uncover their regulatory mechanisms.

Methods: Single-segment substitution lines (SSSLs) were combined with a novel double single-factor differential subtraction (DSDS) strategy to identify heterosis-related differentially expressed genes (DEGs). Unlike traditional parent-hybrid comparisons, DSDS subtracts background expression noise and identifies heterosis-related genes within a controlled genomic context. Five hybrid groups, including mini-F and sub-F hybrids, were generated and analyzed. RNA sequencing was conducted at 15 days after pollination (DAP) to profile gene expression. Motif enrichment and dual-luciferase assays were used for functional validation.

Results: Heterosis exhibited a nonlinear trajectory with parental genetic distance, displaying a rapid increase at low genomic heterogeneity levels before reaching a saturation point at moderate divergence. DSDS identified 359 and 80 heterosis-related DEGs in two mini-F hybrids, with most DEGs located outside the substitution regions, supporting a trans-regulatory mechanism. GO (gene ontology) analysis revealed enrichment in transport and cell expansion pathways, aligning with key processes in early kernel development. Notably, ZmWRKY67 was the only DEG in the substitution segment of mini-F CL137 × Ye478. Initial functional assays confirmed its regulation of Incw1, a key sucrose metabolism gene. The findings suggest a transcriptional "butterfly effect", where minor genomic variations in heterozygous regions trigger genome-wide expression changes to drive heterosis.

Conclusion: This study demonstrates the effectiveness of SSSL-based DSDS in identifying heterosis-related genes and provides a framework for dissecting transcriptional regulation in heterosis. The observed transcriptional "butterfly effect" offers a novel perspective on how small genomic variations contribute to heterosis. Future research should explore additional substitution-region DEGs to further unravel the molecular basis of heterosis in maize.