烟草叶片数杂种优势表现及其相关基因差异表达分析

【目的】探究烟草叶片数杂交优势表现，并分析烟草叶片数相关基因的差异表达情况及杂种优势形成的原因，为深入研究烟草叶片数的分子遗传基础和选育叶片数较多的杂交种提供理论依据。【方法】以叶片数差异较大的9个烟草品种（系）为亲本，按照NCⅡ遗传交配设计组配20个杂交组合，并测定亲本和杂交组合的叶片数，计算其杂种优势，从中筛选出强、弱优势组合，利用实时荧光定量PCR检测其叶片相关基因BRI1、BSK3、FLC、FPF1和PHYC的相对表达量。最后，对叶片数相关基因中亲表达优势间及其与叶片数中亲优势进行相关分析。【结果】9个亲本材料的叶片数为20.33～33.22片，以GDH94的叶片数最多，其次是南江三号和毕纳1号，三者间无显著差异（P> 0.05，下同），但GDH94显著高于其余6个亲本（P< 0.05，下同），表明供试亲本间的叶片数存在真实的遗传差异。20个杂交组合的叶片数存在明显差异，为20.89～31.33片，以GDH94×南江三号的叶片数最多，以NC82×青梗的叶片数最少，说明采用杂种优势育种方法可选育出烟草叶片数较多的杂交种。20个杂交组合叶片数的杂种优势差异较大，其中中亲优势为-14.71%～11.77%，表现为正向中亲优势和负向中亲优势的组合分别占25%和75%，其中，以K326×GDH88叶片数的正向中亲优势最强，为11.77%，以GDH94×湄潭大蛮烟叶片数的负向中亲优势最强，为-14.71%；NC82×南江三号叶片数的中亲优势最弱，为-0.22%，故选择K326×GDH88和GDH94×湄潭大蛮烟为强优势组合、NC82×南江三号为弱优势组合。不同叶片数相关基因的中亲表达优势之间存在一定的相关性，其中BRI1和BSK3基因的中亲表达优势之间存在显著正相关；FPF1和PHYC基因的中亲表达优势与叶片数杂种优势存在显著负相关。FPF1基因在正向强优势杂交组合K326×GDH88和弱优势组合NC82×南江三号中较其相应亲本下调表达，但负向强优势组合GDH94×湄潭大蛮烟较其亲本上调表达。PHYC基因在正向强优势组合K326×GDH88和弱优势组合NC82×南江三号中较其相应亲本下调表达，但在负向强优势组合GDH94×湄潭大蛮烟较其亲本上调表达。【结论】K326×GDH88组合的叶片数杂种优势最大，具有较大的高产潜力。FPF1和PHYC基因参与调控烟草叶片数杂种优势的形成，其下调表达是烟草叶片数性状杂种优势形成的分子基础，可指导亲本选配，提高烟草杂交选育效率。

Expression of leaf number heterosis and differential expression of related genes in tobacco

【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.