异源杂交鲫鲤体系不同世代基因组初期变化及其遗传稳定性研究

Polyploidization via whole-genome duplication (WGD) involves the integration of more than two complete sets of chromosomes in a cell. It occurs in many eukaryotes either by autopolyploidization or allopolyploidization. Polyploidization occurs most commonly in angiosperms, and possibly 70 % species were thought to have experienced one or more WGD events, and also give rise to most crops and fiber species. WGD may cause immediate genome doubling and provide more genetic materials, yet it creates genomic redundancy and causes so called genome shock. In plants, genome shock involves whole-genome wide genetic variants including chromosome rearrangement, DNA recombination, base and insertion/deletion mutations, and also epigenetic changes including DNA methylation, miRNA and long non-coding RNA regulation. In contrast, polyploidization is relatively rare in animals and it mainly occurs in some species of insects and a few vertebrate groups. Due to lack of suitable system, what causes the occurring frequency in animal is much rarer leaves an open question for decades. . By ex situ, the bisexual, goldfish (Carassius auratus, ♀ chromosome = 100) × common carp (Cyprinus carpio, ♂, chromosome = 100) hybrids allow for investigations into genomic consequences of allo-octaploidization in vertetrate. This allopolyploidy system offers several advantages, e.g., their known parentage separates them from natural polyploids and it is easy to trace the fate of progenitor genes. The parental species appear to have originated from the same allopolyploidization event (Ma et al., 2014). Supported by the pilot project (91331105), we have found intriguingly high percentage of chimeric genes (>9%) and even high percentage offspring-specific mutation (>1%) in different generations; meanwhile, some of the chimeric and differentially expressed genes relate to mutagenesis, repair, and cancer-related pathways in 4nF1. Erroneous DNA excision between homeologous parental genes may drive the high percentage of chimeric genes. These discoveries reveal that fast changes are mainly deleterious at the level of transcriptomes, although some offspring still survive their genomic abnormalities, which provide allopolyploidization hinders genomic functions in vertebrates, and this may extend to all animals (PNAS, 2016). . In order to fully use the system to investigate how the allopolyploidy offspring survive from the more severe genome shock comparing the plants, when bigger is really better? We plan to conduct the following tasks, firstly, by employing the third generation sequencing platform with longer sequencing reads, we will obtain fine and well-assembled maternal gynogenetic tetraploid goldfish; to increase assembly rate and reliability for the tetraploid genome, specific programs will be developed to test its reliability by excluding artificial chimeric regions. Secondly, based on the available goldfish genome, we plan to analyze both the obtained transcriptomic data and to re-sequence individuals from different generations of offspring, to compare to their progenitors, and to identify the hot-change regions and pathways both for coding region and noncoding regions (including UTR, MiRNA, lncRNA) after genome shock. Then we will compare methylation pattern alternation in the embryogenesis of offspring to that of the progenitor. Lastly, we will combine both genomic structural change and epigenetic alternation to find clues how the vertebrate allopolyploids survive from severe genome shock, how they live along with allopolyploidization by deep learning technology, that is, when bigger is really better.

鲫鲤杂交体系是通过以母本红鲫鱼和父本普通鲤鱼杂交得到的异源多倍化体系，其中F1-2倍性与亲本相同，F3代起基因组比亲本加倍。此体系遗传背景清晰，能代代繁育，为研究脊椎动物异源多倍化后基因组初期变化的珍贵模型。脊椎动物多倍化问题之前因缺少适合的模型，对多倍化后产生的基因组变化究竟是何时稳定并能持续稳定遗传，一直是悬而未决的问题。基于此研究体系，我们前期的研究发现明显有害的基因组变化，包括子代高频率的基因重组、特异性突变、表达水平的整体改变，并提示比植物更长的基因组休克期 (PNAS, 2016)。在其后代又能生存繁育并为生产所使用的基础上，本研究拟继续使用该体系为模型，获得亲本的精细基因组为参考，在全基因组学水平，包括编码区、相关的非编码调控（包括UTR区、MiRNA、lncRNA等）和表观遗传改变等层次对鲫鲤杂交体系不同世代进行系统而深入的探讨，为阐释其基因组如何稳定提供较为清晰的线索。

异源多倍化研究中的关键科学问题在于：合并了不同基因组来源的子代如何克服基因组休克并能幸存适应环境。围绕此核心问题，本项目主要以新生异源多倍体鲫鲤杂交体系（其中F1-F2染色体数与亲本一致，F3起基因组加倍），辅以其母本——古异源四倍体红鲫为研究对象，探讨其如何克服基因组休克（不稳定性），采取怎样的策略，最终实现从不稳定到稳定以及适应环境的可能性。在遗传、表达调控和表观遗传学层面主要开展和完成的研究内容如下：其一，在DNA水平，新生异源多倍化体系——鲫鲤杂交体系不同世代的核基因组发生9.75–11.21%基因镶嵌、1.16–2.27%子代特异性突变，线粒体基因组发生重组、子代特异性突变，在RNA水平发生非忠实转录、表达模式差异、lncRNA调控差异，综合不同组学水平的结果提示，新生多倍体在G2/M存在多倍化checkpoint，基因组长期不稳定，至今观察到的最新世代均未达到稳定适应；其二，对古老的异源多倍化体系——红鲫及已发表的金鱼基因组、可能同时发生异源四倍化的近缘四倍体鲤鱼的两两亚基因组之间进行了比较基因组学分析，红鲫和鲤鱼50条染色体有非常好的共线性，而两个物种在基因组加倍后亚基因组间演化模式的差异可能是两个物种分歧的原因之一。红鲫基因组两个亚基因组的基因分布无显著bias，演化速率Ka/Ks无显著差异，呈现对称演化的模式；在转录水平，大部分组织及发育时期中，亚基因组之间整体表达水平无显著差异，只是在某些特定的发育时期呈现优势表达和表达优势切换，CNEs、lncRNA能在一定程度上和发育时期基因对的优势表达和表达优势切换相关，部分解释了古多倍体红鲫现在稳定遗传的原因，但表观遗传层次的三维基因组compartment A/B和甲基化水平的研究并不能解释亚基因组表达模式。本项目在遗传、表达调控、表观遗传层面深入了解新生/古老异源多倍体在生存及适应性基础上，为更好地利用异源多倍化现象进行遗传育种奠定组学的基础，对异源多倍体后代的选育和品质改善提供理论支撑。同时为应用云南及其周边有数十种天然多倍化鱼类相关的遗传资源奠定理论基础。