show Abstracthide AbstractHeterosis, a phenomenon where hybrids exhibit superior performance in various traits such as yield, quality, and resistance compared to their parents, has long been a focus of agricultural research. Although tobacco biomass significantly affects tobacco yield and economic profitability, the underlying reasons for the dominance of tobacco biomass hybrids and their formation remain unclear. To address this, we selected 5 tobacco varieties (lines) with large differences in tobacco biomass as the parents and formulated F1 hybrids to analyze the expression of tobacco biomass heterosis. Our findings demonstrate that tobacco leaf biomass exhibits a clear heterosis advantage. In particular, the hybrid Va116×GDH94 showed strong heterosis, with 76.69% of differentially expressed genes (DEGs) displaying Overdominant expression pattern. Notably, these Overdominant DEGs were significantly enriched in biological processes such as photosynthesis and respiration. During photosynthesis, transgressive up-regulation of Lhc improved light-harvesting efficiency, while the up-regulation of Psa and Psb in the photosystem enhanced the defense of chloroplasts against strong light. Additionally, the up-regulation of rbcl promoted the increase of photosynthetic products in the hybrids. During respiration, the down-regulation of MDH, ACO, and OGDH suppressed the tricarboxylic acid cycle (TCA cycle) and weakened respiratory consumption in the hybrids. Furthermore, we observed that the net photosynthetic rate and intercellular CO2 concentration of the hybrids were significantly higher than those of the parents. Taken together, our results suggest that the overdominant expression effect of DEGs plays a crucial role in the formation of tobacco biomass hybrid advantage. The overdominant expression effect of photosynthesis and respiration-related genes enhanced the photosynthetic capacity of the hybrids, reduced respiration consumption, and promoted the increase of tobacco biomass, thus demonstrating the obvious hybrid advantage. Overall design: We subsequently selected the hybrid combination Va116 × GDH94, with the highest heterosis value 45 days after transplantation, for transcriptome sequencing analysis.