利用野生山金柑资源发掘控制多胚性状的关键基因

Polyembryony has long been an obstacle to cross breeding of citrus, however, it guarantees the consistent production of rootstock. Polyembryony of citrus is a stable sporophytic apomixes of nucellar cells, typical among the over 400 apomictic plants in nature. China possesses abundant citrus germplasm, and is the origin of Fortunella hindsii Swingle. Our research group has investigated and collected 853 wild Fortunella hindsii materials from southern provinces of China including Fujian, Zhejiang, Guangdong and Jiangxi etc. Nearly all of the materials were polyembryonic, however, among them the only one monoembryonic Fortunella hindsii germplasm was identified. Like the annual plants, Fortunella hindsii flowers and sets fruits in the same year of sowing, which makes it an ideal material for study of citrus polyembryony. Based on our previous preparation and preliminary study of the hybrid population (1354 progeny individuals) between the poly- and mon-embryonic parents, citrus polyembryony was identified as a qualitative trait controlled by one major locus. In this project, we plan to further utilize the hybrid population to fine-map the polyembryony locus, and to conduct genomics-assisted approach to identify the key gene controlling polyembryony. In order to explore genes responsible for nucellar embryogenesis and embryogenesis of callus, i.e. the in vivo and in vitro somatic embryogenesis, transcriptome analysis will be conducted on the Laser Microdissection (LMD)-collected initial cells and developmental stages of nucellar embryo and in vitro somatic embryo. Gene function will be validated using genetic transformation system of citrus callus and Fortunella hindsii plants, to evaluate the contribution of the key genes to polyembryony and to elucidate the functional mechanism of the key genes. The present project aims to reveal the genetic and molecular mechanisms of the unique apomixes phenomenon of citrus, based on the autonomously explored wild germplasms specific to China. The project is not only of theoretical value to understanding the diversity of plant reproduction, but also applicable to rootstock improvement of fruit trees like citrus, and even is instructive to heterosis fixation of other crops.

柑橘多胚性属于珠心细胞发育的孢子体无融合生殖类型，长期以来影响杂交育种的开展，也为生产整齐一致的砧木提供保障。山金柑播种当年开花结果，是研究柑橘多胚性状的理想材料。研究小组前期调查收集到野生山金柑材料853份；其中发掘了世界迄今唯一的1份单胚山金柑资源。经过多年的单多胚杂交群体（1354株）配制和研究，初步表明柑橘多胚性状是质量性状，受1个主效位点控制。本项目拟进一步利用大规模有性分离群体精细定位多胚位点，结合基因组学手段发掘控制多胚性状的关键基因；利用激光显微切割术分离珠心起始细胞，结合RNA-Seq鉴定珠心胚发生相关基因；利用柑橘愈伤组织和山金柑活体验证候选基因的功能，并解析其作用机制。本项目以自主发掘的我国特有野生资源为材料，针对独特的生殖现象研究其遗传和分子机制，对认识植物生殖多样性具有理论价值，对柑橘等果树砧木改良以及其他作物固定杂种优势具有重要应用价值。

柑橘多胚性属于珠心细胞发育成不定胚的孢子体无融合生殖类型，长期以来影响柑橘杂交育种效率，也被用于繁育整齐一致的实生砧木。山金柑播种当年开花结果，是研究柑橘多胚性状的理想材料。本项目以单胚山金柑的自交纯系为材料，测序组装了高质量基因组序列，构建了山金柑生活史13个组织的基因转录谱，建立了基因编辑体系；构建单胚×多胚山金柑的杂交群体，并构建高密度遗传图谱，为柑橘功能基因研究提供了模式材料体系。利用分离群体的遗传定位和自然群体的关联分析，将柑橘属和金柑属控制多胚性状的关键位点定位到4号染色体，挖掘到了与多胚性状紧密连锁的基因CitRWP。根据CitRWP启动子区有/无微小型转座子（MITE）插入，开发了鉴别柑橘单/多胚性状的分子标记。细胞学观察确定珠心胚起始发育关键时期在开花前及花后2-3 d，于此时挑取胚珠和采用激光显微切割术精细取样珠心胚起始细胞，通过转录组和DNA甲基化组测序，发掘到了参与珠心胚发育的生长素和细胞分裂素信号途径以及关键基因CitRWP和CitC2H2转录因子，提出了珠心胚起始发生的工作模型。筛选到CitRWP基因上游的互作转录因子CitARID，并分别通过遗传转化获得了CitRWP与CitARID的基因编辑山金柑植株。利用离体愈伤组织的遗传转化体系，解析了miR156-CsSPL3/14调控柑橘体细胞胚发生的功能。本项目建立了以短童期山金柑为材料的柑橘模式研究体系，挖掘到了柑橘控制多胚性状的关键基因CitRWP，开发了鉴别单/多胚柑橘的分子标记，解析了关键基因调控珠心胚和离体胚发生的功能，为提高柑橘育种效率和砧木繁育提供了基因资源和辅助标记。

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