柑橘胞质杂种雄性不育性状形成的分子机制及相关基因发掘

Seedlessness is the most attractive agronomic trait for citrus fruit quality while male sterility is the main reason for fruit seedlessness. In our previous work, a male sterile somatic cybrid pummelo (Huayou No. 2) was produced by protoplast fusion, which was verified as a variant of male fertile pummelo with foreign mitochondrial genome from Satsuma mandarin, and is an ideal germplasm to reveal the mechanism of cytoplasmic male sterility in citrus. Previous transcriptomic and proteomic comparison between floral buds of this cybrid pummel and its mesophyll fusion parent (HB pummelo) profiled a large number of differentially expressed genes and proteins, from which genes potentially responsible for male sterility were identified. In this proposal, we plan to: (i) use laser microdissection and high throughput sequencing to identify the spectrum of genes and small RNAs present in the sterile stamen primodia; (ii) use high throughput sequencing to construct a fine map of citrus mitochondrial genome, and compare mitochondrial genome between male sterile cybrid and the corresponding fusion parents, to identify candidate male sterile genes and develop markers linked to male sterility; (iii) use the markers developed in item (ii) to predict in advance the male sterility segregation status of a previously constructed F1 hybrid population (over 900 individuals) between the male sterile cybrid and the fertile Shatian pummelo, and apply bulked DNA-Seq and RNA-Seq segregant analysis on the population upon flowering, to identify male-sterile genes; (iiii) carry out functional dissection of the candidate male sterile genes (like CitAP3 required for stamen development and CYP716 involved in pollen wall formation) and reveal the mechanisms of nucleo-cytoplasmic interactive male sterility in citrus. Based on a solid foundation established previously, and using this unique male sterile somatic cybrid pummelo, we expect the implementation of this proposal will provide knowledge of theoretical and practical importance, and will exploit gene resources for citrus seedless molecular breeding.

果实无核是柑橘的重要农艺性状和果实品质优良的重要标志，雄性不育是其果实无核的重要原因。申请人创制的雄性不育胞质杂种—华柚2号，遗传组成上已证实为其叶肉融合亲本—HB柚的雄性不育突变体，是研究柑橘雄性不育性状形成机制的理想试材。基于前期的比较转录组与蛋白质组研究，本申请项目拟开展：1）华柚2号及其双亲雄蕊原基的显微捕获和转录组、小RNA测序，筛选其雄蕊特异差异表达基因（包括育性的温度响应调控因子）；2）线粒体DNA提纯及基因组序列比较，发掘细胞质雄性不育基因；3）基于有性群体育性分离的BSA策略，结合DNA重测序和RNA-seq，开发雄性不育SNP标记和定位育性恢复基因，并筛选差异表达基因；4）雄性不育相关候选基因的功能解析，揭示柑橘雄性不育性状形成的分子机制，同时筛选用于柑橘无核分子改良的基因资源。本申请项目前期基础扎实，试材独特；其实施不仅具有理论意义，而且具有实际应用前景。

HB柚胞质杂种（即华柚2号）是研究柑橘雄性不育性状形成机理的理想试材。本项目创制的HB柚胞质杂种的有性杂交后代群体已部分开花结果，均表现雄性不育和果实无核，已评价筛选出无核优系5个；通过HB柚胞质杂种及其融合双亲的线粒体基因组序列比较，开发了柑橘种属特异的线粒体DNA标记5个，发掘出细胞质雄性不育候选基因orf88；采用激光显微捕获和组学分析，筛选了HB柚胞质杂种雄蕊和小孢子发育异常的候选基因（如BOP2）、小RNA（如miR399）；发现HB柚胞质杂种花药初生代谢异常可能与雄性不育有关；验证了20余个雄性不育候选基因的功能；解析了miR399通过抑制靶基因CsUBC24及其互作蛋白CsSEPs和CsICE1，从而调控柑橘花器官发育和雄性不育性的新功能。本项目发掘了柑橘雄性不育的关键胞质基因和核基因，揭示了柑橘胞质杂种雄性不育性状形成的分子机理，同时为柑橘无核分子改良提供了辅助分子标记、基因资源和种质材料。本项目发表论文13篇，其中Plant Physiology等SCI收录论文9篇；获湖北省技术发明一等奖1项，授权国家发明专利1项；受理国家发明专利1项，受理植物新品种权2项。