甜瓜苗期低温耐受性相关位点的全基因组发掘及候选基因鉴定

Melon is a kind of plants with high sensitive to low temperature. The seedling period of melon frequently suffers low-temperature weather prolonging seedling stage and further delaying the fruit maturity and listing. Genetic research on low-temperature tolerance (LTT) of melon and then exploitation and utilization of the LTT related genes are extremely important to enhancement of melon production benefit. There are abundant melon resources in China with obvious LTT variation, however, the weak research on the genetic basis limits the application of molecular breeding in LTT improvement of melon. Previously, our research group constructed two melon core collections and optimize the method of LTT examination at seedling stage. In the present subject, the integrated melon core sample is used as natural population and genome-wide association study is planned to discover the LTT related loci in the melon genome. Then, the important candidate genes in the loci were identified and cloned in combination with several approaches, QTL linkage analysis, transcriptome analysis, and prediction of LTT genes of model plants in the melon genome. Finally, the functions of the genes are further verified by qRT-PCR, homologous gene comparison, genetic transformation, etc. The expected findings not only reveal the genetic basis of LTT of melon seedlings but also obtain the genes and markers of value, which will offer the gene resources for molecular breeding of cold resistance in melon.

甜瓜是低温敏感植物，苗期经常发生的低温天气延长了其育苗期，进而推迟了果实成熟和上市。开展甜瓜低温耐受性(low-temperature tolerance, LTT)遗传研究，挖掘和利用LTT相关基因，对提高其生产效益具有重要意义。我国甜瓜种质资源丰富，且LTT变异明显，但其遗传基础研究十分薄弱，制约了分子育种在LTT遗传改良中应用。本课题组前期构建了2个甜瓜核心种质，并优化了苗期LTT表型鉴定方法。本项目拟以整合后的核心种质为自然群体，采用全基因组关联分析发掘甜瓜苗期LTT相关位点，结合QTL连锁定位、转录组分析、模式植物LTT基因在甜瓜基因组中的预测等方法，鉴定和克隆重要的候选基因，最后通过qRT-PCR、同源基因比较、遗传转化等方法予以功能验证。预期结果不仅可以揭示甜瓜苗期LTT遗传基础，还可以挖掘到有价值的基因和标记，为甜瓜耐冷性分子育种提供基因资源。

本项目旨在对调控甜瓜苗期低温耐受性（low-temperature tolerance，LTT）位点进行全基因组鉴定，从中挖掘重要的候选基因，并对其进行功能验证，为甜瓜耐冷性分子改良提供基因资源。首先，对200份甜瓜核心种质进行深度测序（平均测序深度18.2×），采用全基因组关联分析（GWAS）检测到15个LTT相关位点，并结合转录组分析候选到44个差异表达基因，其中与模式植物冷响应有关的基因17个。其次，采用简化基因组测序（ddRAD-seq）技术构建了一张甜瓜高密度遗传图谱，利用多环境下LLT表型数据定位到6个调控甜瓜苗期LTT的QTL位点，并结合转录组数据从2个稳定的主效QTL中候选到21个差异表达基因，其中5个基因是调控LTT的重要候选基因。对GWAS和QTL-mapping筛选到的关键基因如DREB、RR等，进行了基因家族分析和表达分析，从中鉴定到6个调控LTT的基因，即3个CmCBFs、2个CmRRs和1个CmEAF7基因，并克隆了这些基因的cDNA序列。最后，对这6个基因进行了功能验证。对CmCBF1/2/3进行异源转化，发现这3个CmCBFs能够调控番茄和拟南芥的耐冷性，但调控模式并不相同；VIGS沉默试验发现CmRR6和CmPRR3能负调控甜瓜苗期LTT；瞬时过表达和VIGS沉默试验证明，CmEAF7不仅能增强甜瓜苗期LTT，还能提高成熟期果实含糖量，是一个具有较大育种价值的基因。本项目发表学术论文7篇，申请发明专利2项，授权实用新型专利2项，获得软件著作权2项，国内作学术报告3次，培养硕士研究生3人、博士研究生1人。