Identification of key pathways and associated transcription factor-miRNA-gene regulatory networks driving heterosis in cotton (Gossypium spp.)

Heterosis, or hybrid vigor, represents a pivotal phenomenon in cotton (Gossypium spp.) breeding, enabling substantial advancements in yield, stress tolerance, and fiber quality. However, the underlying molecular mechanisms of this phenomenon are still largely unexplored. To address this issue, we performed RNA-seq meta-analysis using a P-value combination approach to identify key molecular signaling pathways associated with heterosis in root and bud tissues of hybrid and parental lines. In addition, the regulatory miRNA-transcription factor (TF) gene interactions associated with heterosis were further constructed and dissected. This comprehensive analysis identified 591 differentially expressed genes (DEGs) that were consistently observed in all datasets. In particular, 435 root-specific, 130 bud-specific, and 159 shared meta-DEGs were identified, revealing the intricate interplay between tissue-specific and shared molecular pathways. Functional enrichment analysis of identified meta-DEGs highlighted critical roles of specific biological processes, including circadian rhythm regulation and water transport, alongside essential metabolic pathways such as glutathione metabolism, and starch and sucrose metabolism in the heterosis phenomenon. Genes pivotal to growth and development, such as GhFT (flowering regulation), GhXTH9 (cell wall modification), and GhSUS4 (energy storage), were identified as key players in the heterosis phenomenon in cotton. The associations between several miRNA-TF-gene interaction networks such as Ghi-miR164-NAC and Ghi-miR166-HD-ZIP as heterosis driving regulatory interactions were highlighted by systems level analysis. This study provides a comprehensive framework for dissection of transcriptional regulatory mechanisms underlying heterosis in cotton and offers new insights for targeted breeding strategies to improve the performance of hybrids in modern cotton breeding programs.