基于基因组的矮牵牛重瓣花性状基因发掘及功能解析

Double flower is one of the most important traits of ornamental plants, the molecular mechanism underlying the trait has been the hot issue for research on flower development. However, there has been no report on the issue by forward genetics. As an important species for bed flower and model plant for flower development research, petunia is ideal for study of molecular mechanism of double flower formation. In the previous study, we mapped the trait with molecular marker by using near isogenic lines constructed by crossing of single flower and double flower petunia, and the gene controlling double flower trait was located in the range of 0.3 cM. In this project, the population for mapping will be increased and new molecular marker will be developed to fine localize the gene, meanwhile, the data of transcriptomes and genome re-sequences will be used to screen the genes that have differences between single and double flower plants, the candidate genes will be further determined by real-time quantitative PCR, the selected genes will be transferred to single flower petunia for complementation test. With the observation of phenotypes and molecular analysis of the transgenic plants, the genes that control double flower formation will be determined and the molecular mechanism of the process will be explained. With the implementation of the project, the pathway for artificial creation of double plants for ornamental plants could be possibly achieved.

重瓣花是观赏植物最重要的观赏性状之一，其形成机制一直是植物花发育研究的热点领域，至今尚未从正向遗传学的角度对其进行阐释。矮牵牛作为重要的花坛花卉和花发育研究的模式植物，对其单重瓣性状的机理研究，具有重要的科研与应用价值。项目组前期以单瓣和重瓣矮牵牛多代杂交构建的单重瓣近等基因系分离群体为试材，用分子标记将重瓣控制基因定位在0.3cM范围内。本项目在此基础上，进一步扩大作图群体、开发分子标记对其进行精细定位，同时结合转录组测序及重瓣基因组重测序数据，筛选在单重瓣中存在差异的基因，在实时定量PCR再次筛选后圈定候选基因，构建植物转化载体，通过对转基因植株的表型观察和分子分析，验证基因功能并解析其分子机理。本研究的实施，有望从遗传学和分子机理等方面明确重瓣基因及其功能，解析矮牵牛重瓣花形成机理，对人工创建重瓣花具有潜在的应用价值。

为进一步精细定位矮牵牛重瓣基因，本项目组从正向遗传学出发，构建了群体数量1956株的近等基因系群体，开发了19个多态性标记，共筛选到2株交换单株，将重瓣基因定位在1.82Mb区间内。后期通过GWAS和BSA联合分析，获得5个与重瓣性状相关的候选基因：AQN00014-RA（PhMFS）、AQN00023-RA（PhKP1）、AQN00099-RA（PhBTD）、AQN00135-RA（PhSSP）、AQN00206-RA（PhGS1），并构建候选基因的超量表达载体、干涉载体、病毒载体进行功能验证，建立了重瓣矮牵牛‘二重唱’高效再生体系，用5个候选基因的病毒载体注射重瓣矮牵牛‘二重唱’，表达量和花瓣数均出现不同程度的减少，但沉默PhGS1，花瓣数减少最多，并有形成单瓣的趋向。因此，超量表达PhGS1，获得两株阳性苗，但奇怪的是，与对照相比，转基因植株并无花瓣数的增加。结果表明，矮牵牛重瓣性状的形成可能涉及多个基因的相互调控，其研究成果有望丰富了花发育理论，为培育重瓣矮牵牛提供了理论基础。