异源四倍体青花菜/黑芥体细胞杂种及后代的基因组结构和DNA甲基化变异研究

Somatic hybridization technique is an effective way for the introduction of heterologous excellent genetic resources. Allopolyploids formation of somatic hybridization have an important value for crop genetic and breed. Moreover, they are unique materials for epigenetic research because of the large number of non-Mendelian inheritance of phenotypic variation. Broccoli is one of the world's important vegetable crops. However, its germplasm resource-poor and narrow genetic base can not meet the demand for production and breeding. In this study, based on the synthesis of broccoli/black mustard somatic hybrids and selfed generations materials, we compared the changes of chromosome structure and parental genome specific DNA sequence of different materials. Then we revealed the characteristics and mechanisms of genome structural changes in the process of genetic stability of broccoli/black mustard somatic hybrids. On the other hand, using the techniques of MASP, cDNA-AFLP and qRT-PCR, we compared the changes in methylation patterns and gene expression of different materials in order to prove the correlation of DNA methylation and hybrid phenotypic variation. In short, the implementation of the project will be able to promote broccoli breeding process and lay the theoretical foundation for the effective import of the heterologous excellent gene. Moreover, it is of great significance in riching the theory of polyploid evolution.

体细胞杂交技术是一种引入异源优异基因资源的有效方法。体细胞杂交形成的异源多倍体不仅对于作物的遗传和品种改良具有重要的价值，同时由于其存在大量非孟德尔式遗传的表型变异，是进行表观遗传学研究的独特材料。青花菜是全世界重要的蔬菜作物之一，但其种质资源贫乏，遗传基础狭窄，已不能满足生产及育种的需求。本研究基于实验室已合成的青花菜/黑芥体细胞杂种及不同自交世代材料，将染色体行为观察和基因组位点检测相结合，比较不同材料染色体结构变化和基因组特异DNA序列的变化，以揭示杂种在遗传稳定过程中的基因组结构变化特点及机制；同时，将采用MASP、cDNA-AFLP、qRT-PCR等技术，比较不同材料的甲基化模式变化特点及基因表达特征，探明DNA甲基化与杂种表型变异的相关性。总之，项目的实施将能够促进青花菜体细胞杂交育种进程，为外源优异基因的有效导入奠定理论基础；同时，对于丰富多倍体物种进化理论也具有重要意义。

体细胞杂交技术是一种引入异源优异基因资源的有效方法，但体细胞杂交形成的后代植株表型发生广泛变异的分子机理很少了解。本项目基于已经获得的青花菜-黑芥体细胞杂种（S0）及不同自交世代材料（S1-S4），利用细胞学、分子标记和RNA-测序等技术，研究结果一致表明体细胞杂交诱发的遗传和表观遗传变异在S0和S1代表现最为激烈，随着杂种不断自交，其染色体减数分裂异常行为、基因组位点变异频率及DNA甲基化频率逐步降低。对S4-S0代植株的差异表达基因进行GO功能分类注释和代谢通路富集，发现大部分差异表达基因与植物信号转导及响应逆境胁迫相关，表明这些基因在青花菜和黑芥体细胞杂种的基因组遗传稳定和环境适应性中具有重要作用。本研究结果不仅为青花菜体细胞杂交育种提供理论基础，同时也为其它作物利用体细胞杂交技术进行品种改良提供指导。