中国南瓜光周期不敏感基因Ppd的精细定位与候选基因分析

Cucurbita moschata, a monoecious short-day plant, is sensitive to photoperiod which induced the serious restrict for its planting region and sowing time. In our former study, we generated a photoperiod insensitive self-crossed cultivar PPIS that the female flower differentiation was not affected by long daylight treatment. The inheritance of the photoperiod response trait was studied, which showed a pair of dominant nuclear gene, which is named Photoperiod-insensitivity gene (Ppd), determines the photoperiod-insensitive female flowering. F2 population derived from the cross breeding between PPIS and a photoperiod sensitive cultivar PPS was analyzed by Specific-Locus Amplified Fragment Sequencing (SLAF-seq) and the Bulked Segregant Analysis (BSA). A total amount of 186,284 SLAFs were generated. In this study, further bioinformatics analysis based on the SLAFs and C. moschata genome information obtained from cooperation unit will be done to preliminarily map the Ppd gene in the genome. Furthermore, using greater F2 population as materials, the flanking markers linked to Ppd will be used to screen recombinant plants and the markers of SSR, InDel and SNP generated from whole genome resequencing will be used to narrow the scope of candidate genes to eventually fine map Ppd gene. Gene annotation, structure variation and expressing profiles of candidate genes combining with previous transcriptomes analysis of the two parents will be used to identify the candidate genes of Ppd. Ultimately, fine mapping and detecting of candidate genes for Ppd is achieved. The results will provide research foundation for marker-assisted selection and gene cloning of Ppd, which has important theoretical and practical significance.

中国南瓜属短日植物，对光周期敏感导致其种植地域和播种时间受到严格限制。本项目组经多年选育获得1份光周期不敏感中国南瓜纯系材料PPIS，其在长日照下仍能正常进行雌花分化，该光周期不敏感性状受一对显性核基因控制，命名为Ppd；为精细定位Ppd基因，构建了以PPIS和光周期敏感材料PPS为亲本的F2群体，通过简化基因组测序结合混池分组分析法获得186,284个序列标签及标签与该性状的连锁值。本项目将以前期工作为基础，利用南瓜全基因组信息，将标签定位到基因组上，根据连锁值得到Ppd基因的关联区域；进一步加大次级分离群体，利用关联区域两侧连锁分子标记进行重组株筛选及利用全基因组重测序进行标记加密实现Ppd基因的精细定位；最后，利用基因注释、基因结构、前期亲本表达谱分析和基因表达差异分析等进行候选基因预测。本研究结果不仅为Ppd基因的克隆和功能研究奠定基础，也为光周期不敏感南瓜的分子育种提供技术支持。

中国南瓜属于短日植物，其中，蜜本类型中国南瓜对光周期较敏感，其种植地域和播种时间受到严格限制。为了提高蜜本南瓜的广适性，本项目利用一份光周期不敏感高代自交材料PPIS采用SLAF-seq结合BSA方法和构建高密度遗传图谱的方法对光周期不敏感性状进行精细定位研究。通过定位研究在10号染色体上获得了一个94kb的主效位点区间，定位区间共包含15个基因，进一步利用全基因组重测序开发了紧密连锁分子标记。同时，通过比较两个材料在长日照和短日照下的转录组结合激素处理及含量分析发现两个材料的光周期敏感性与赤霉素和乙烯通路有关，这为后续进行后续基因的筛选提供了重要信息。结合上述结果，本项目筛选了一个候选基因AP2，为乙烯ERF转录因子，该基因主要在花器官中表达，且在不敏感材料中表达量高于敏感材料。通过本研究精细定位了中国南瓜的光周期不敏感性状，获得一个候选基因，且明确了光周期不敏感性与赤霉素和乙烯通路相关调控相关。共发表期刊论文4篇，其中SCI论文2篇，获得授权专利2项，指导3名研究生毕业。本研究工作将为后续改善蜜本类型南瓜的光周期敏感性打破种植地域限制奠定了良好的工作基础。