黄瓜近缘野生种酸黄瓜重要抗病基因的深度发掘

Cucumber is a crop with narrow genetic base and susceptible to many diseases and prone to insect damage for lack of important desease resistant genes. It is necessary to broaden the gene pool of cucumber by introgressing resistance genes from wild relative species to deal with the challenges facing in cucumber production. Besides, as the first sequenced horticultural crop, cucumber becomes an important model crop for gene exploration research..Sour cucumber (C. hystrix Chakr.), being native to China, is the only wild relative species found possessing cross compatibility with cucumber. With the inter-specific introgression lines developed, the the genetic mapping of downy mildew, gummy stem blight and root knot nematode resistant genes were finished. However, the genetic distance between resistant gene locus and linked markers is still far and the high quality genome physical maps of the introgression lines are difficult to construct by using traditional methods. These are big challenges to clone and identify the disease resistant genes. To solve the problems, we have constructed BAC library of sour cucumber and sequenced the wild cucumber genome de novo. We have also developed a chromosome painting method for single-copy gene pools in Cucumis sativus. With those progress newly made, the project proposed herein aim at screening molecular markers that tightly linked with the resistant genes base on the genome sequence data of sour cucumber; accurate assembly of introgression high-resolution physical map by re-sequencing and single-copy gene-based chromosome painting (ScgCP); cloning and functional analysis of resistance genes to downy mildew, gummy stem blight and root knot nematode with plant transformation and VIGS technique. .Expected results could be the breakthrough of cucumber disease-resistant breeding, and therefore maintain our advantage in cucumber inter-specific genetics and cultivar improvement.

黄瓜遗传基础狭窄，种内变异率低，缺乏重要抗性基因，阻碍了其遗传研究与品种改良。发掘利用野生种优异基因，能够突破黄瓜遗传改良瓶颈，同时作为首个完成基因组测序的园艺作物，对其它作物品种改良有重要指导意义。申请者在黄瓜与野生种酸黄瓜种间杂交基础上，创制种间渐渗系，实现了霜霉病等三种抗病基因定位。但定位区域较大、渐渗系基因组物理图谱构建难等问题阻碍了进一步发掘。近年来项目组构建了酸黄瓜BAC文库，与中国农科院蔬菜花卉所合作完成了酸黄瓜全基因组测序，同时在单拷贝基因染色体涂染技术等方面取得突破，为深入发掘抗病基因创造了必要条件。本项目提出基于基因组de novo测序获得紧密连锁的抗病基因分子标记；结合重测序和单拷贝基因染色体涂染作图技术绘制高精度的渐渗系物理图谱；利用遗传转化等技术进行鉴定和分子机理研究。本项目将有力推动栽培黄瓜品种改良进程，保持我国在基于种间杂交的黄瓜遗传与品种改良的特色和优势。

黄瓜是重要的蔬菜作物，但由于遗传基础狭窄，其种内变异率低，缺少重要抗性基因，因此黄瓜生产和育种改良受到了严重的阻碍，因此发掘利用野生种优异基因有重要意义。前期研究中，项目组通过种间杂交和染色体加倍，人工合成了黄瓜-酸黄瓜异源四倍体，首次实现了黄瓜与酸黄瓜的基因交流，这个“桥梁”作物为突破黄瓜遗传改良瓶颈奠定了重要的基础。然而由于缺乏酸黄瓜基因组信息，酸黄瓜优异基因的遗传定位和克隆研究进展较慢。本项目根据研究计划顺利完成了酸黄瓜特异性标记开发、渐渗系高精度物理图谱绘制、抗病基因图位克隆和抗病分子机制等4个方面的任务，无调整内容，总体上达成了研究目标。项目组利用二代、三代测序技术结合的策略，完成了酸黄瓜和种间异源四倍体基因组的de novo测序，开发了大量酸黄瓜特异性的细胞分子标记，全面解析了酸黄瓜丰富的抗性基因，揭示了黄瓜-酸黄瓜异源四倍体基因组变异导致的遗传基础扩增现象，同时绘制了高精度的渐渗系物理图谱；利用酸黄瓜特异性分子标记，结合群体基因组重测序、BSA-seq等技术完成了酸黄瓜抗霜霉病、蔓枯病、南方根结线虫等抗病基因的图位克隆；采用转录组、蛋白质组和代谢组等多组学联合分析的方式，从细胞分子多个层面揭示了酸黄瓜抗病基因的作用机制，为这些基因的育种应用提供了指导；开发了稳定可靠的育种分子标记，建立了黄瓜种间优异基因转育的分子标记辅助育种体系；采用远缘杂交、胚胎拯救和FISH等细胞学分析技术，持续系统地创制和鉴定黄瓜-酸黄瓜种间杂种、异源四倍体、异附加系和渐渗系材料，进一步显著扩大了栽培黄瓜的遗传基础。相关成果发表第一标注论文28篇，其中在Plant journal、Plant Physiology等国际著名期刊上发表SCI论文20篇；申报专利7项，授权1项；荣获农业部中华农业科技一等奖1项，江苏省科学技术二等奖1项。本项目的实施推动了栽培黄瓜品种改良进程，进一步保持我国在基于种间杂交的黄瓜遗传与品种改良的特色与优势。