猕猴桃性别相关基因的发掘及通用性别鉴定标记的开发

Kiwifruit (Actinidia chinenisis)is functional dioecious, and the male individuals which can not be identified during juvenile period waste cost in breeding. It is an urgent need to explore the genetic mechanism of sex determination for improving efficiency in breeding. In this project, based on kiwifruit high-density genetic maps, sex determination region will be narrowed down by linkage analysis in the regions that are suppressed for recombination. The comparative transcriptome method will be employed to detect differential gene expression in female and male flowers of monoecious and dioecious kiwifruit, respectively. Candidate sex-related genes will be identified in sex region, and gene function will be verified by gene editing. According to analysis of genetic variation in 4 kiwifruit species, allelic variation associated with sex will be detected to develop sex markers for early sex identification in kiwifruit. This project will facilitate to understand molecular mechanism of sex determination and breed hermaphrodite kiwifruit, and provide molecular tools for sex identification in kiwifruit breeding.

猕猴桃属于雌雄异株果树，雄株在苗期难以鉴定剔除，造成育种成本的浪费。解析猕猴桃的性别遗传机理是提升猕猴桃育种效率的迫切需求。本项目拟以雌雄同株特异猕猴桃和中华、软枣、毛花和山梨猕猴桃等物种为材料，在前期利用高密度遗传图谱定位性别区间的基础上，分析染色体重组抑制区的性别连锁，进一步缩小性别决定区间；采用比较转录组学方法，鉴定雌雄差异表达基因，结合区间定位结果发掘性别相关的候选基因，探究雌雄同株的突变机制，并通过基因编辑技术验证基因功能；研究性别决定区间的遗传变异，分析不同物种中性别连锁的核心等位变异，开发通用的猕猴桃性别鉴定标记。项目的开展有助于明确猕猴桃性别形成的分子机理，为雌雄同株猕猴桃育种提供理论依据，为猕猴桃杂交育种性别鉴定提供分子工具。

猕猴桃是功能性雌雄异株果树，研究猕猴桃性别遗传的分子机理，开辟遗传改良新途径，不仅对了解果树性别决定机制具有重要的理论意义，而且对提高猕猴桃育种效率，以提升猕猴桃产业经济效益具有现实的应用价值。本项目利用种间和种内杂交群体材料，计算图谱中SNP分子标记物理距离和重组率关系,发现性别区间在25号染色体亚端粒靠近核仁组织区，抑制重组区大约6Mb区间，进一步确定了性别决定区间。利用多组学相结合的方法，比较不同材料上雌、雄花的及不同时期雌、雄花材料的组学信息，筛选性别相关的差异表达基因；检测到8个雄性特异位点，通过群体进行验证筛选，在25号染色体上发掘到2个特异的性别变异位点在样本中能区分雌雄基因型。利用CRISPR/Cas9技术，构建双元表达载体，对性别基因SyGl和FrBy开展基因编辑，已成功转入毛花猕猴桃中获得到转基因植株；通过酵母文库筛选到与FrBy基因互作的因子为28个，与SyGl基因互作的因子为33个。经过点对点验证及LUC荧光素酶互补实验，检测到性别调控候选因子10个。猕猴桃遗传变异丰富，早期用于山梨和中华猕猴桃杂交群体的性别标记在其他种内迁移性较差，本项目开发通用性猕猴桃性别鉴定分子标记1个，可在中华猕猴桃、美味猕猴桃、大籽猕猴桃、软枣猕猴桃、山梨猕猴桃、毛花猕猴桃和黑蕊猕猴桃中早期鉴定性别，有利于高效辅助育猕猴桃育种。本项目的开展，有助于进一步解析猕猴桃性别调控机制，提高猕猴桃种间杂交育种效率，加速猕猴桃优质新品种的创制。