柑橘离体培养30年过程中的基因组和表观基因组变异规律

Citrus is one of the most important economic fruit crops, and 70 percent of citrus varieties are produced through bud mutation (somatic variation of branch) breeding. Somaclonal variations arising from in vitro culture provide chances for selection of new genotype for asexually propagated crops, but have negative effects on the true-to-typeness of in vitro propagation, germplasm conservation, and genome editing process. Therefore, it is of great value to study the somaclonal variations of citrus. However, as the lack of high-throughput and high-resolution detection of somaclonal variations, little is known about the genetic landscapes of somaclonal variations in long time in vitro cultured plants as yet. In this proposal, the 30-year-old calli after in vitro preservation including diploid, tetraploid, and hexaploid callus lines, newly induced calli, tetraploid plant and in vivo explant of sweet orange were used as experimental materials, and the multi-moics analysis (Genome – Epigenome - Regulome) strategy was taken. We had identified the variation hotspots and rates, the whole genome duplications, the transposable element activations, and the dynamic changes of DNA methylation during the long time in vitro culture. Then, we further plan to explore the interaction between genomic and epigenomic variations during the long time in vitro culture, as well as the similarities and differences between in vitro and in vivo variations to elucidate the patterns of genomic and epigenomic in vitro variations. The implementation of this proposal will provide fundamental basis for citrus breeding programmes, and citrus functional genome researches.

柑橘是最重要的经济果树作物之一，约70%的柑橘品种通过芽变（枝条的体细胞变异）育种产生。无性系变异，即离体的体细胞变异，既对离体保存和基因编辑造成遗传变异等不利影响，但也可为创制新材料提供有利遗传基础。因此，柑橘无性系变异研究具有重要价值。然而，现有研究对无性系变异缺乏高通量高分辨率的检测，对综合的无性系变异规律仍不清楚。本项目针对离体保存30年的甜橙愈伤系（二倍体、四倍体和六倍体愈伤系）和新诱导的甜橙愈伤，以及二倍体和四倍体甜橙植株，采用“基因组-表观组-转录调控组”多组学联合分析的策略，已发现柑橘体细胞变异热点、全基因组复制、转座子激活和DNA甲基化修饰在离体保存过程中的动态变化，后期将进一步挖掘长期离体保存过程中基因组变异和表观修饰间相互关系，离体和活体体细胞变异的异同，阐明离体培养中基因组和表观基因组的变异规律。本项目前期基础扎实，其实施为柑橘育种和功能基因组研究提供理论基础。

基于组织培养过程的基因编辑和细胞工程技术被广泛应用，组织培养过程中发生的无性系变异引起人们的广泛关注。此前，鲜有研究系统揭示植物无性系变异的基因组规律。本项目以离体组织培养30年的甜橙愈伤系、新诱导愈伤和外植体为材料，进行基因组、DNA甲基化组和转录组多组学联合分析，定量鉴定和评估了SNP、结构变异和转座子活动，发现脱分化后出现变异爆发，在新诱导愈伤中检测到高频率的单核苷酸变异和转座子插入事件，但大多数变异在长期体外培养过程后被清除。CHH型甲基化水平首先在新诱导愈伤组织中显著降低，而在长期离体培养愈伤中CHH型甲基化水平增加，恢复至比外植体稍高的水平，主要是通过在基因上游的调控区域CHH岛甲基化修饰的变化，以此维持长期离体条件下基因组的稳定性。针对长期离体培养过程中发现的四倍体愈伤，本研究鉴定了甜橙两个亲本亚基因组（橘和柚）的特异性表达和拷贝数变异，发现相对于二倍体愈伤，四倍体愈伤表现出了亚基因组显性的现象，但是相对于四倍体甜橙植株，四倍体愈伤中亚基因组显性的程度更弱一些。结合多个组织和时期的转录组，发现离体培养过程中发现的转座活性变化和动态DNA甲基化修饰，均可能对离体培养后转录水平的重编码造成影响，并最终影响愈伤体细胞胚胎发生的能力。本研究可为适当的离体培养时间的选择提供指导，以避免或获得额外的无性系变异。通过对柑橘多组学数据进行系统搜集和重分析后，本研究构建了柑橘泛基因组数据库——CPBD，用于柑橘基因组和基因功能研究。已发表SCI论文2篇，授权专利1项。培养了2名博士/硕士研究生。项目主持人2021年获湖北省自然科学奖一等奖（序2），2022年获湖北省青年拔尖人才项目资助。