番茄抗裂果主效基因CR3b的图位克隆及其功能分析

Fruit craking has caused serious economic losses in tomato in the whole world. The development of variety with high resistance to fruit cracking is hence one of the important goals of tomato breeding. Therefore, the cloning and functional analysis of the key genes that control fruit cracking resistance will not only help to clarify the molecular mechanism of tomato fruit development, but also mean important practical significance for tomato breeding. The project applicant has been studying the genetic analysis and genetic improvement of tomato fruit cracking resistance for many years. Recently, the applicant identified the fruit cracking resistance 3b (QTL cr3b), a major quantitative trait locus (QTL) controlling the resistance to fruit cracking in tomato, by using a Solanum pennellii LA716 introgression lines (ILs) that was developed by our group. QTL cr3b was located in a 380 kb region on chromosome 3. Subsequently the sub-ILs population of QTL cr3b have been developped by using the IL3-17 as male parent and elite breeding line 1502 as female parent. Four introgression lines of QTL cr3b and the elite breeding line 1502 have been sequenced. Based on the previous works, our present research is intending to clone the QTL cr3b and study on its cracking resistant mechanism. The main objectives of this research are: ⑴ Cloning of CR3b gene; ⑵ Analyze the cracking resistant mechanism of CR3b gene through comparing the transcriptome of the fruits of wild type and cr3b; ⑶ develop elite inbreed lines with high resistance to fruit cracking. The results of this project will play key roles in understand the molecular mechanism of fruit cracking and will also accelerate the tomato breeding process of fruit cracking resistance.

裂果严重地影响了番茄的经济产量，选育抗裂果品种是番茄育种的重要目标之一。因此，对控制果实抗裂性的关键基因进行克隆及功能分析，不但有助于阐明果实发育的分子机制，而且对于指导番茄育种也具有重要意义。申请人长期从事番茄果实抗裂性的遗传分析与分子改良研究。本课题组利用新构建的潘那利番茄渐参系群体定位了一个抗果实环裂的主效QTL cr3b，它位于第3染色体380 kb的区域内，随后开展了QTL cr3b亚渐参系群体构建和QTL cr3b渐参系及相关材料的测序工作。本项目在此基础上，拟以亚渐参系群体为主要研究材料对CR3b基因进行克隆，并研究其抗裂性机制，主要目的是：1）克隆CR3b基因，并鉴定其功能；2）初步探索CR3b基因的抗裂性机制；3）创制优良的抗裂果番茄育种材料，并开发其共显性分子标记。本项目的研究结果对于深入研究番茄抗裂果的分子机制和加速番茄抗裂果的育种进程均有极其重要的意义。

裂果严重地影响了番茄的经济产量，选育抗裂果品种是番茄育种的重要目标之一。因此，对控制果实抗裂性的关键基因进行克隆及功能分析，不但有助于阐明果实发育的分子机制，而且对于指导番茄育种也具有重要意义。本研究利用新构建的潘那利番茄渐渗系群体为主要材料，结合常规育种、分子标记、图位克隆、生物信息学等该法方法进行了近等基因系群体构建、番茄抗果实环裂基因CR3b的精细定位及克隆以及该基因的功能研究三部分工作，并取得以下结果：1）定位了一个抗果实环裂的主效QTL CR3b，它位于第3染色体380 kb的区域内；2）建立含抗番茄裂果基因CR3b的渐渗系IL3-17与其背景材料即骨干亲本l052杂交后的F2代群体，同时开发CR3b基因所在的380kb染色体片段两端的分子标记，利用两端的分子标记对F2代群体进行遗传分析，筛选出相应的重组单株；3）开发目的基因所在染色体片段上饱和的新标记，利用新标记对CR3b基因的亚渐渗系群体进行连锁遗传分析，综合分析遗传鉴定结果和多个世代的抗裂性鉴定结果从而对CR3b基因进行精细定位，并将物理距离缩短至26.97kb，并得到候选基因三个；4）利用CRISPR/Cas9对三个候选基因分别进行了功能验证工作，目前目的基因的cDNA序列、基因注册、相应的基因克隆的文章发表和开发与目的基因紧密连锁的分子标记的工作将在接下来的几个月内完成。项目执行期间发表学术论文4篇，会议论文2篇，专利1项，培养博士生2名，硕士7名，其中4名硕士生已毕业。