Abstract: 【Objective】To explore the performance of heterosis of tobacco leaf number, analyze the differential expression of leaf number related genes and the causes of heterosis, so as to provide a theoretical basis for further research on the molecular genetic basis of tobacco leaf number and breeding of hybrid varieties with more leaves.【Method】Nine tobacco varieties(lines) with large difference in leaf number were as parents, 20 hybrid combinations were set according to the NCⅡgenetic mating, and leaf number of parents and hybrid combinations were measure.Calculated the heterosis, the strong and weak dominant combinations were selected.The relative expression of leaf genes BRI1, BSK3, FLC, FPF1 and PHYC were detected using real-time fluorescent quantitative PCR.Finally, the correlation between leaf number related gene expression dominance and leaf number over-parent heterosis was analyzed.【Result】The number of leaves of the nine parents was 20.33-33.22, and GDH94 had the most, followed by Nanjiang No.3 and Bina No.1.There was no significant difference among the three parents(P> 0.05, the same below), but GDH94 was significantly higher than that of the other six parents(P< 0.05, the same below), indicating that there was real genetic difference in the number of leaves among the tested parents.There were significant differences in the number of leaves among the 20 hybrid combinations, ranging from 20.89 to 31.33, with the highest number of leaves in GDH94×Nanjiang No.3 and the lowest number of leaves in NC82×Qinggeng, indicating that the hybridization method could be used to breed the hybrid with the appropriate number of leaves.Among the 20 hybrid combinations, the heterosis of the number of leaves was significantly different, of which the over-parent heterosis was-14.71%-11.77%, and the combinations showing positive over-parent heterosis and negative over-parent heterosis accounted for 25% and 75%, respectively.The positive over-parent heterosis of the number of leaves of K326×GDH88 was the strongest(11.77%), and the negative over-parent heterosis of the leaf number of GDH94×Meitandaman tobacco was the strongest(-14.71%).Over-parent heterosis of the leaf number of NC82×Nanjiang No.3 was the weakest(-0.22%).Therefore, K326×GDH88 and GDH94×Meitandaman tobacco were selected as the representative combinations of strong dominance, while NC82×Nanjiang No.3 was selected as the representative combination of weak dominance.There was a certain correlation in the over-parent heterosis among different leaf number related genes, and there was a significant positive correlation between the over-parent heterosis of BRI1 and BSK3 genes.There was a significant negative correlation between the over-parent heterosis of FPF1 and PHYC genes and leaf number heterosis.The expression of FPF1 gene was down-regulated in positive strong dominant hybrid K326×GDH88 and weak dominant hybrid NC82×Nanjiang No.3 compared with their parents, but up-regulated in negative strong dominant hybrid GDH94×Meitandaman tobacco compared with their parents.The PHYC gene expression was down-regulated in positive strong dominance combination K326×GDH88 and weak dominant combination NC82×Nanjiang No.3 compared with their parents, but up-regulated in negative strong dominant combination GDH94×Meitandaman tobacco compared with their parents.【Conclusion】The combination of K326×GDH88 has the highest leaf number heterosis and high yield potential.FPF1 and PHYC genes are involved in the regulation of the formation of tobacco leaf number heterosis, and their down-regulated expression is the molecular basis for the formation of tobacco leaf number heterosis, which can guide the selection of parents and improve the efficiency of tobacco hybrid breeding.