24nt siRNA介导DNA甲基化对美洲黑杨子代生长优势形成的调控机理研究

DNA methylation is an important epigenetic modification which plays a key role in the formation of dominant trait. Previous studies found that different DNA methylation patterns appeared with different growth potential among hybrids and their parents. Small non-coding RNA(sncRNA) is involved in regulating plant growth and development mediated by DNA methylation. Among them, 24nt siRNA-directed DNA methylation was closely related to traits of hybrids. However, literature has not yet been reported in forest trees. Due to the great breeding value of Populus deltoides, different growth potential hybrids and their parents will be used to study in this project. Firstly, next-generation sequencing technology, and independent bioinformatics tools (Pln24NT and PoplarGene) are used to analyze the different expression of sncRNA and screen the 24nt siRNA associated with growth. Secondly, we will identify the different 24nt siRNA and their producing sites, and obtain methylation difference sites mediated by 24nt siRNA with correlation analysis. Thirdly, 24nt siRNA-mediated methylation of target genes and adjacent genes will be performed the functional annotation and enrichment analysis and then screen the genes related to growth and development. Finally, the expression relationship between 24nt siRNA and their target genes are used to verify with qRT-PCR analysis etc. We aim to reveal the regulation mechanism of DNA methylation directed by 24nt siRNA in the formation of growth vigor. The results will provide a new perspective for analyzing the mechanism of tree traits dominance formation, and lay a foundation for further development of high yield hybrid breeding and germplasm innovation.

DNA甲基化是表观遗传修饰重要途径，对性状优势形成具有重要作用。研究发现不同生长势杂交子代与其亲本具有不同DNA甲基化模式。sncRNA可通过介导DNA甲基化调节植物生长发育。其中24nt siRNA介导DNA甲基化与植物杂交子代性状表现关系密切，但在林木中未见报道。由于美洲黑杨育种价值巨大，以其不同生长势子代及亲本为材料，采用高通量小RNA测序技术和自主研发的Pln24NT等生物信息工具，分类注释sncRNA，筛选与生长优势有关24nt siRNA；鉴定差异24nt siRNA及其靶基因，与全基因组甲基化差异关联，获得其介导的甲基化靶基因及邻近基因，通过功能注释等分析，筛选与生长相关功能基因；结合24nt siRNA表达量与靶基因甲基化关系验证，揭示24nt siRNA介导DNA甲基化调控生长优势形成机理，为林木性状优势形成解析提供新的视角，为深入开展林木高产杂交育种和种质创新奠定基础。

24nt siRNA介导DNA甲基化与植物杂交子代性状表现关系密切，但林木中未见报道。由于美洲黑杨育种价值巨大，以其不同生长势子代及亲本为材料，采用小RNA高通量测序、多组学联合分析等技术，研究美洲黑杨不同生长势子代与其亲本的24nt siRNA差异，关联全基因组水平DNA甲基化差异，明确不同生长势子代与其亲本24nt siRNA介导DNA甲基化差异模式，揭示24nt siRNA介导DNA甲基化对美洲黑杨杂交子代生长优势形成的调控机理。1）通过不同生长势杂交子代与亲本间24nt siRNA差异表达模式分析，筛选鉴定出328个与杂交子代生长优势形成紧密关联的非加性表达24nt siRNA，其中108个24nt siRNA预测到818个靶基因，主要富集在植物-病原菌互作、淀粉和蔗糖代谢、植物MAPK信号途径等代谢通路，参与调控生长发育进而形成不同生长势。2）根据产生24nt siRNA相位基因的位置与亲本间共有甲基化区域位置信息比对选择有重叠区域相位基因，鉴定出206个24nt siRNA介导DNA甲基化，涉及62个甲基化区域及86个差异表达基因，其中有43个非加性24nt siRNA介导DNA甲基化调控37个基因表达，发现24nt siRNA可能主要通过介导亲本间相似甲基化区域的CHG和CHH型DNA甲基化来调控生长相关基因表达进而形成不同生长势。3）通过受24nt siRNA介导DNA甲基化基因与生理生长性状显著相关基因联合分析，筛选出11个受34个24nt siRNA介导的DNA甲基化水平调控的重要功能基因，其中高生长势中24nt siRNA的表达水平与基因甲基化水平趋势一致，并整体负向调控基因表达，且对CHG和CG型甲基化更为明显，低生长势中24 siRNA表达水平与基因甲基化水平相对较低，基因表达水平相对较高。4）通过DNA甲基化调控的54个关键基因和11个受24nt siRNA介导DNA甲基化调控基因共表达网络分析，筛选出10个调控杨树生长杂种优势形成关键基因，其所在DMR区甲基化水平及基因表达量分析显示，高生长势子代甲基化水平和基因表达量均偏向父本，表现出高甲基化低表达调控模式，而低生长势子代偏向母本，表现出低甲基化高表达水平。研究为林木性状优势形成解析提供新的视角，为深入开展林木高产杂交育种和种质创新奠定基础。