萝卜与芸薹属远缘杂种属间染色体高效重组新技术研究及新种质创制

Distant hybridization is an important technology for germplasm innovation. The Raphanus vegetable radish and three Brassica vegetables, Chinese cabbage, cabbage, and mustard, occupied one-third of China's total vegetable acreage. The disease resistance, metabolic components, and nutritional compositions diverged among these species. New germplasm can be generated by interspecific hybridization of crops in Brassica. However, intergeneric gene exchange was blocked due to the serious barrier to the homeologous chromosome recombination between the R genome of Raphanus and the A/B/C genome of Brassica. Therefore, develop a new technology to promote efficient intergeneric chromosomal recombination can greatly improve the germplasm innovation and crop breeding of cruciferous vegetables. The introduction of targeted DNA double-strand break is a potential method to enhance the intergeneric chromosomal recombination. The present project aimed to establish a high efficient intergeneric chromosomal recombination technique. We first improve the chromosome cutting tool by enhance the CRISPR/Cas9 with the replicon of CbLCV virus. Subsequently select the best karyogram that suitable for intergeneric homeologous recombination by testing in AR, CR, AARR, CCRR hybrids and AACCR+ radish disomics addition lines. Then optimize the operation process and build a pipeline for quick isolation of the recombination events and precise detection of the recombinant fragments. At the same time, we will transfer the anti cyst nematode gene from radish to Brassica vegetables by using this technique. This ample can prove the useful of this technology in introduction of intergeneric gene exchange; broaden the genetic background, and innovation of germplasm of cruciferous vegetables.

远缘杂交是种质创新的重要方法。萝卜属的萝卜和芸薹属的白菜、甘蓝和芥菜共同占据了我国蔬菜种植面积的三分之一。这些蔬菜具有不同的抗性、代谢组分和营养成分。芸薹属蔬菜种间杂交可以创造新种质。但是萝卜属的R基因组与芸薹属的A、B、C基因组之间存在严重的重组障碍，制约了优异基因属间交流。因而，开发促进属间染色体高效重组的新技术，对十字花科蔬菜种质创新和遗传育种有重要意义。染色体定点断裂可能是促进属间染色体重组的有效方式。本项目用CbLCV病毒复制子增强CRISPR以作为染色体定点剪切工具，以芸薹属与萝卜杂交合成的AR、CR、AARR、CCRR整倍体杂种及AACCR+萝卜附加系为实验材料，通过筛选最适受体、优化操作流程、建立重组子快速筛选和重组位点精准鉴定方法，创建一套属间染色体高效重组新技术。并以抗包囊线虫基因属间转移为例，证明该技术可用于加强属间基因交流，拓宽蔬菜遗传背景，创造十字花科蔬菜新种质。

远缘杂交是物种形成和种质创新的重要途径。萝卜属蔬菜和芸薹属蔬菜在我国蔬菜产业中占有重要地位，其各自进化出不同的抗性、代谢组分和营养成分。萝卜属和芸薹属蔬菜远缘杂交具有广阔的应用前景，但是，远缘杂种中属间染色体之间存在的严重重组障碍阻遏了优异基因的属间交流，制约了其在育种中的利用。因而，开发出促进属间染色体高效重组的新技术，对十字花科蔬菜种质创新和遗传育种具有重要意义。.本项目按计划足额完成了CRISPR/Cas9载体的改造和筛选、最适受体基因型的筛选、遗传转化技术的建立和优化、剪切位点的设计、转基因植株的创制、重组子筛选和重组位点鉴定等研究工作。在萝卜与甘蓝异源四倍体（RRCC基因组）中创建了高效的遗传转化和基因编辑技术。成功实现了萝卜与甘蓝属间染色体的定点重组。通过在萝卜×甘蓝（RRCC）远缘杂种中编辑同源重组途径上的关键负调控基因，创制了属间染色体重组效率大幅提高的新种质。通过本项目研究，发表了研究论文6篇，其中SCI期刊论文1篇，中文核心期刊论文3篇，会议论文2篇；申请国家发明专利2项；培养研究生4名，完成硕士学位论文2篇。.总之，本项目建立了萝卜与芸薹属蔬菜属间染色体高效重组新技术，并用该技术创制了一批优良新种质。超额完成了研究目标。本项目创建的萝卜与甘蓝异源四倍体属间染色体定点重组新技术及属间染色体高效重组新种质在研究物种进化、基因组演化等方面具有重要科学意义。在十字花科作物分子设计、种质创新、多倍体育种等方面具有广阔的应用前景。