苹果自交亲和性特异资源的评价与基因挖掘

Apple generally exhibits self-incompatibility even though it is hermaphroditic, and self-compatible resources are very rare. Based on this situation, pollination tree is needed to plant for biotic-pollination or handpollination in order to guarantee the yield. However, this increases producing costs and difficulties for management. Therefore, self-compatible resources are needed to solve this problem. Our group has been studying on special self-compatible resources for many years. To date, we have evaluated apple resources and obtained 4 apple self-compatible cultivars. For S factor variation, we are going to mine self-compatible genes based on these 4 apple self-compatible cultivars and determine the molecular mechanism of self-compatibility. First, we will clone S-RNase or SLF/SFB from a self-compatible apple which is on the S-locus, and then compare their gene sequences, expression profiles and promoter sequences with those from a self-incompatible cultivar which has the same S-genotype. For non-S factor variation, we will use Bulk Segregant DNA-seq and Bulked Segregant RNA-Seq to excavate candidate genes controlling self-compatibility. Finally, we will design different strategies to verify the gene functions for different types of candidate genes. This study will on one handdetermine the mechanism of self-compatibility,on the other hand provide gene resources and theoretical guidance for creating self-compatible apple cultivars.

苹果虽雌雄同花却自花授粉不能结实，自交亲和性的资源非常稀少。对此，生产上多采用配置授粉树通过虫媒传粉或人工授粉以获得果实。这种做法会增加生产成本和管理难度。因此，要彻底解决这一问题，需要挖掘和创制自交亲和性品种资源。本课题组多年从事苹果自交亲和性特异资源的评价与研究，目前已筛选出4个自交亲和性特异资源。本研究拟在此基础上，深入挖掘控制自交亲和性的基因，即以筛选的自交亲和性资源为试材，首先克隆苹果S基因座上的花柱S-RNase和花粉SFB/SLF基因，与相同S等位基因的自交不亲和性材料比较编码区、启动子区和翻译后修饰等，获得S因子变异位点；其次利用后代自交亲和与不亲和极端性状混池基因组重测序结合转录组高通量测序，寻找S基因座以外的非S因子变异位点；最后根据不同变异情况设计不同策略研究候选基因或元件的性质和功能。项目将率先阐明苹果自交亲和性分子机制，为创制自交亲和性品种提供基因储备和理论指导。

苹果自花授粉不能结实，生产上须配置授粉品种、饲养蜜（壁）蜂或购买花粉人工授粉，造成生产成本提升，效益下降。因此，利用自交亲和特异资源挖掘自交亲和基因，阐明自交亲和形成机制，对创制和选育自交亲和性品种具有重大意义。本项目通过田间自花授粉实验、授粉后花柱花粉管荧光观察等评价出‘寒富’（S1S9）、‘惠’(S2S9)两个苹果自交亲和性特异资源，克隆‘寒富’、‘惠’中花柱自交不亲和决定基因S1-、S2-、S9-RNase全长及启动子序列，与自交不亲和品种‘富士’（S1S9）和‘金星’（S2S9）比对发现序列不存在差异，初步判定‘寒富’、‘惠’不属于S因子变异。正反交授粉、杂（自）交群体S基因型鉴定进一步确认‘寒富’自交亲和性属于自我识别型变异，‘惠’属于花柱侧变异，二者均由非S因子变异导致。利用自交亲和与不亲和极端性状后代个体重测序获得的高质量SNPs和Indels构建高密度遗传图谱，经全基因组关联分析和QTL定位发现‘寒富’和‘惠’自交亲和性位点分别位于苹果第4号染色体930 kb和第12号染色体2.74 Mb范围内，并开发出Indel和SSR两个高效分子标记，验证准确率大于80%。目标区域内SNPs及Indels的变异分析筛选得到8个‘寒富’自交亲和候选基因和12个‘惠’候选基因，进一步通过“SNP变异效应分析-LD block连锁不平衡分析-亲本和后代群体授粉后花柱表达量-变异启动子元件活性分析”确定‘寒富’HF15792启动子-1056bp的GT/T变异和‘惠’MD1200032516启动子-1598bp的CTT插入变异导致启动子活性下降，基因表达降低。HF15792定位于内质网上，HF15792+S2-RNase蛋白酶K降解实验、变性S-RNase+HF15792复性pull-down实验证明该蛋白具有辅助S-RNase正确折叠的功能，变性S1-/S9-RNase+HF15792共孵育分别培养‘寒富’、‘富士’花粉管，发现HF15792具有恢复S-RNase抑制花粉管能力的功能，且90min时‘富士’花粉管相对生长量为‘寒富’的80.2%，证明‘寒富’自交亲和性由HF15792启动子元件变异导致表达失常无法辅助S-RNase正确折叠所致。项目成果发表研究论文13篇，获批国家发明专利7项，植物新品种权1项，获神农中华农业科技奖一等奖1项。