鲫鲤体系杂交初期转录调控变化研究

Polyploidization or genome duplication triggers vast genome instability including random gene loss, accelerated mutations, chromosomal rearrangements and failed paring of homologous chromosomes, and these synthetic effects are defined as genome shock. The outcome of combining two genomes in vertebrates remains unpredictable especially because polyploidization seldom shows positive effects, and more often results in lethal consequences because viable gametes fail to form during meiosis. Fortunately, goldfish (maternal♀) × the common carp (paternal♂) hybrids have reproduced successfully up to generation 26 and this permits an investigation into the genomics of hybridization and polyploidization. The first two generations of these hybrids are diploids and subsequent generations are tetraploids. The fertilities and survival rates of embryos in first three generations showing dramatic decrease and subsequent recovery indicated the nascent generations might be experiencing rapid, acute alterations, and what the detailed variations of expressional and regulatory networks behind are still unresolved. Based on the previous works of the allopolyploid system, the chimeric genes (≥10%) in both diploids and tetraploids are with higher frequencies comparing to the plant systems (≤7%). Interestingly, the patterns of chimeras in mRNA are quite different with the ones in DNA with the same individual, which suggested different mechanisms of chimera in DNA and mRNA, respectively. In addition, the chimeric patterns of different individuals within one generation are also surprisingly different, especially in 2nF1, which indicated multiple potentials of variations might be caused by the genomic shock syndrome in vertebrates..In order to explore the formation of chimeric genes in mRNA, the immediate alternations of expressional and regulatory networks after the hybridization and genome doubling, in this project, we are going to 1) detect the RNA editing patterns of both parents and 2nF1 in ovary/testis and embryos, respectively, and figure out how many differences of chimeric patterns in 2nF1 between DNA and mRNA are contributed by the RNA editing process; 2) identify the alterations of expressional and regulatory networks in 2nF1, including coding and non-coding RNAs (lncRNA and miRNA) at the meiosis and mitosis levels, respectively. Based on above works, we will find clues of what happened in the processes of transcription, expression and regulation after the hybridization, and lay the foundation for further exploring of both hybridization and polyploidization. In summary, these works provides better understanding of the genome shock and related syndrome in vertebrates, which might include the higher frequencies of RNA editing and more complicated alterations of expression and regulations in both meiosis and mitosis processes comparing to the diploid.

多倍体发生初期经历的即时急剧变化可能是其是否能成功存活的关键，而在脊椎动物中鲜有报道。本项目前期利用人工繁育的鲫鲤异源多倍化杂交体系作为研究模型，伴随着杂交和多倍化的发生，该体系初期世代先后经历育性和胚胎存活率的急剧下降而后又恢复。前期利用该体系亲本和不同倍性子代研究发现高频率的基因镶嵌和突变(PNAS,2016,共同一作)，进一步工作提示杂交初期幸存和未幸存子代群体在编码和非编码RNA可能发生更多变化。基于此，本项目拟利用前期已获得的杂交鲫鲤体系亲本、2nF1代性腺组织和胚胎群体转录组、重测序数据，加测非编码RNA数据(包括lncRNA和miRNA)，1) 在减数分裂和有丝分裂水平，探究杂交初期基因镶嵌现象的模式差异、RNA编辑对该现象发生的贡献，2) 寻找与育性和胚胎发育相关的编码和非编码RNA表达调控网络改变，为进一步寻找杂交初期育性和胚胎存活率显著改变的遗传机制提供有意义的线索。

多倍体发生初期经历的即时急剧变化可能是其是否能成功存活的关键，而在脊椎动物中鲜有报道。本项目利用人工繁育的鲫鲤异源多倍化杂交体系作为研究模型，在前期研究基础上，着眼于杂交初期基因镶嵌现象的模式差异、RNA编辑对该现象发生的贡献，并寻找与育性和胚胎发育相关的编码和非编码RNA表达调控网络改变，为进一步寻找杂交初期育性和胚胎存活率显著改变的遗传机制提供有意义的线索。在项目执行期间，研究工作主要进展和成果如下：① 完成了杂交体系的双亲自交系和杂交2nF1代的胚胎样品收集工作；顺利开展了杂交鲫鲤2nF2代的自交系构建；完成了杂交鲫鲤亲本及2nF1代卵子及不同胚胎发育时期的非编码RNA补充测序工作。② 解析了杂交鲫鲤亲本红鲫、鲤鱼的异源多倍化起源亚基因组的识别、演化模式分析，提示同源基因对的演化命运的多样性，在此基础上提示了胚胎发育进程中亲本和杂交初代的祖先基因拷贝的表达模式变化以及基因组休克效应引发改变的关键通路。③完成了转录水平的DNA-RNA碱基差异分析工作，解析了包括RNA编辑和非忠实转录等转录水平变异的类型和比例，发现杂交初代从胚胎发育早期就开始经历了更多的DNA-RNA碱基替换，提示可能在“基因组休克”效应的影响下，转录过程中的非忠实转录现象。④ 开展了杂交鲫鲤体系亲本及杂交初代不同发育时期胚胎的非编码RNA表达量比较和靶基因预测工作。以上工作围绕转录组水平的系列变化，包括异源多倍化起源的基因对的演化命运解析、时空表达模式改变、RNA的非忠实转录和RNA编辑、编码和非编码RNA的差异表达模式及调控预测，追踪了鲫鲤体系在杂交初代胚胎发育进程中的变异来源和调控规律，验证异源多倍化体系在杂交初代经历的“转录组休克”效应的具体过程、关键的通路以及可能的调控机制。本项目通过开展扎实的基础数据积累和分析工作，为揭示杂交初代的高频变异发生来源、调控网络的改变提供了研究基础，为理解杂交与多倍化的综合效应在转录组水平发生何种规律性改变提供了线索。