辣椒始花节位调控基因CaFFN的精细定位、克隆及功能分析

First flower node is an important agronomic trait for pepper and closely related to precocity and early yield. To develop precocious cultivars requires a comprehensive understanding of the related genetic mechanisms. B9431 is a genetically stable precocious inbred line which can blossom and bear fruit at the first node of the main stem. Previous research has shown that the first-flower-node trait in B9431 is controlled by a recessive gene. In this study, we propose to fine map the CaFFN (Capsicum annuum First Flower Node) gene which is responsible for the first-flower-node trait based on BSR-Seq (Bulked Segregant RNA-Seq) method. RNA samples from the mutant and non-mutant individuals of the F2 population derived from a cross between B9431 and A145 will be combined into two separate pools and subjected to RNA-Seq. According to the allelic frequency between the two RNA-Seq pools, the genomic locus related with this trait will be identified. Genetic markers developed from the high-density SNPs (Single Nucleotide Polymorphisms) will be used to fine map CaFFN gene quickly and efficiently. Genetic and molecular approaches will be applied to clone CaFFN gene and to verify its function. This research will provide insights into the mechanism employed by CaFFN gene in the biological control of the first-flower-node trait in pepper.

始花节位是辣椒重要熟性性状，与辣椒采收期和早期产量显著相关。B9431为本团队创制的辣椒特早熟材料，具有在第1节位开花坐果的特性，该性状由1对隐性基因控制。本研究以目标性状差异显著的遗传材料B9431和A145为亲本，构建F2始花节位分离群体，应用BSR-Seq（Bulked Segregant RNA-Seq）技术，通过比较高、低始花节位池中RNA-Seq数据的等位基因频率，鉴定控制始花节位的染色体位点；基于高密度SNPs数据开发分子标记，快速高效精细定位始花节位调控基因CaFFN，并进一步运用分子生物学方法对该基因进行克隆和功能验证。研究结果将解析CaFFN基因时空表达特点，探明辣椒始花节位调控的分子机理，为在分子水平上实现辣椒始花节位遗传操纵及辣椒熟性分子标记辅助选择育种奠定坚实基础。

开花时间是植物的一个重要农艺性状，与植物的适应性密切相关。始花节位常被用来衡量辣椒的开花时间，但始花节位的遗传基础还不清楚。为了研究始花节位背后的遗传机制，我们用一个早花自然突变体和一个晚花自交系材料杂交构建F2群体。利用BSR-seq方法将始花节位的主效位点定位于2号染色体末端。该区间APETALA2（AP2）同源基因CaFFN与F2群体297株植株的始花节位共分离。对亲本间CaFFN基因序列进行比对，发现一个自然稀有突变（SNP2T > C）使早花突变材料中CaFFN基因的起始密码子丢失。进一步对该基因进行功能验证，CaFFN基因高表达的转基因本氏烟草开花延迟且花器官形态产生缺陷；沉默CaFFN基因的辣椒提前开花，始花节位降低。功能验证结果说明CaFFN是一个辣椒开花抑制因子且影响花器官发育。通过qRT-PCR实验揭示CaFFN可能作为转录激活因子以激活CaAGL15和miR156e的表达，作为转录抑制因子以抑制CaAG、CaAP1、CaSEP3、CaSOC1、和miR172b的表达。通过酵母单杂交试验证实CaFFN与CaAGL15、CaAG、CaAP1、CaSEP3和CaSOC1的顺式作用元件有直接互作，这与CaFFN调控开花时间和花器官发育的假说相一致。同时，CaFFN基因关联分析结果显示该基因与辣椒自然群体的始花节位显著相关，说明CaFFN基因对辣椒的开花时间有广泛的影响。本项目研究结果加深了我们对CaFFN参与调控辣椒开花时间的认识，揭示CaFFN是辣椒早熟育种的重要靶基因。