四季草莓夏季开花主效基因FaPFRU鉴定和分子遗传调控机理

Perpetual-flowering (PF) strawberry is a new member of cultivated strawberries (Fragaria x ananassa Duch.), which can initiate floral bud and keep fruiting under long-day condition in summer. Benefited from PF strawberry breeding and suitable climate, Yunnan has become a main cultivation area of PF strawberry, providing high-quality fresh fruits in summer and autumn for the whole China market. Molecular regulation mechanism of PF trait in PF strawberry is the basis of molecular breeding in the near future. A series of recent researches reveal that a gene, FaPFRU, regulates PF trait in PF strawberry, located in a segment of chromosome Ⅳ with a genetic distance of 5.6 cM. However, it is unclear that FaPFRU, regulating not only PF trait but also runnering trait, is a single gene or a tightly linked gene cluster. The molecular network and molecular regulation mechanism of FaPFRU is obscure, which hampers the molecular breeding for PF strawberry. This proposed research, based on the analysis of inheritance model, transcriptome sequencing (accomplished) and the genome sequencing of PF strawberry (to be accomplished soon), the FaPFRU will be located on the genome of PF strawberry. Then, according to the comprehensive analysis of the genes annotated and the gene expression differences in the transcriptome of shoot and leaf tissues, the candidate FaPFRU will be acquired and the sequence of cDNA will be cloned, too. After that, over expression vector and RNAi vector of FaPFRU will be constructed and transferred to strawberry plants to verify the gene function of FaPFRU. Finally, the molecular network of FaPFRU , related to the regulation of flowering and runnering, will be predicted and the expression of the associated genes will be quantified to elucidate the molecular regulation mechanism of FaPFRU.

四季草莓是凤梨草莓中的新成员，能在夏季长日照条件下完成花芽分化并开花结果。云南是我国四季草莓主要种植区和夏秋季鲜果优质产区。四季草莓夏季开花分子调控机理是分子育种的基础。系列研究表明FaPFRU基因调控夏季开花性状，其被定位在4号染色体上5.6cM片段内。但是，具有控制开花和匍匐茎发生双重功能的FaPFRU是单一基因还是紧密连锁的基因族并不清楚。FaPFRU基因分子调控网络和机理也仍不明确，妨碍了分子育种实践。本项目在已完成遗传模型分析、转录组测序分析和即将完成四季草莓基因组精细图构建等基础上，将FaPFRU定位到四季草莓基因组精细图谱上，结合基因功能注释和转录组差异表达基因分析获取FaPFRU候选基因，克隆cDNA序列；构建FaPFRU过量表达载体和RNAi载体，验证基因功能；预测FaPFRU分子调控网络，分析FaPFRU与相关调控基因表达量关系，阐述FaPFRU基因分子调控机理。

在四季性草莓4号染色体上存在一主效位点（FaPFRU）调控夏季开花性状；但该主效位点分子调控机理目前尚不清楚。本课题结合了遗传图谱分析、基因组学分析、转录组学分以及qPCR验证等方法探究四季性草莓连续开花的遗传调控基础。得到的主要结果如下：.通过PacBio环形一致性测序技术测序，利用软件HiFiasm组装，再利用ALLHiC软件进行染色体的挂载，构建2个四季性品种，2个季节性品种，1个智利草莓 (F. chiloensis (L.) Duch.) 株系和1个弗州草莓(F. virginiana Duch.) 株系染色体水平基因组，为基因定位和鉴定等研究提供基因组学支撑。比较基因组学分析结果显示，凤梨草莓中FaFT在不同开花习性的品种间存在拷贝数差异，四季性（PF）草莓中FT基因的拷贝数为3个，比季节性（SF）草莓‘Camarosa’中（2个）多一个，并且季节性草莓‘Camarosa’中一个FT拷贝由于序列缺失导致翻译提前终止。由此推测FaFT基因可能参与了四季性草莓连续开花性状的调控。对2个季节性草莓和2个四季性草莓夏冬两季的茎尖和叶片分别做了转录组测序与分析表明：四季性草莓和季节性草莓的遗传和表达差异较明显，茎尖和叶片中均有1万多个差异表达基因，并且茎尖和叶片中分别有200多个表达量存在差异的基因是与开花相关的；在200个差异基因中，FT基因在冬夏两季存都在表达差异，在不同开花习性的草莓品种中表达量也存在差异，具体表现为夏季时在四季性草莓中表达量略高于季节性草莓。转录组分析结果进一步说明FT基因可能是调控四季性草莓连续开花的重要基因。通过qRT-PCR对FT基因中一个拷贝（FaPFRU片段内）进行表达量的测定，结果显示夏季时在四季性草莓中FT基因的表达量略高于季节性草莓。该结果与转录组数据所得结果一致，说明FT基因或FT基因家族可能是调控四季性草莓连续开花性状的关键基因。此外，项目组还开展了栽培草莓遗传转化和基因编辑的探索性研究，便于后续基因功能验证工作的开展。本研究发现虽然在八倍体栽培草莓连续开花的调控中可能包含很多基因，FT或者FT基因家族可能是最重要的调控基因。本研究通过初步鉴定连续开花的候选基因，将为后期进行开花调控机制的深入研究奠定基础。