| 其他摘要 | 多倍化是指染色体组加倍的现象，即整个基因组的重复。多倍化在被子植物中普遍存在，而在脊椎动物中主要存在于硬骨鱼类和两爬类中的少量物种。在植物多倍体中的研究提示，基因组冗余和杂交效应引发的“基因组休克”很可能导致错误的同源染色体配对、异常四价体的产生、剂量不平衡效应以及高频率的基因突变和重组、丢失和重排等；此外，异源多倍体体系在杂交和基因组加倍发生过程中，还会经历由遗传不稳定到稳定的过程，而初期阶段基因组休克引发的综合效应，可能是多倍体是否能转向稳定进而存活的关键。相比之下，由于缺乏合适的研究对象，脊椎动物多倍化发生后物种经历怎样的遗传和表观遗传水平变化，仍未见系统的报道。为何多倍化现象在脊椎动物中相对罕见？前人的假设认为，由于动物具备更为复杂的生理和发育特征、性别决定机制等，多倍化可能造成胚胎死亡。对比植物，我们更为关注的问题是，脊椎动物基因组对多倍化造成的基因组和表观水平休克效应的容忍度是否有限，进而使个体难以存活？由此选择合适的脊椎动物多倍体模型，探究其多倍化发生初期的关键变化，可为解释多倍化在动物中较为罕见的原因提供清晰的线索。本论文选取人工繁育的鲫鲤异源多倍化杂交体系作为研究脊椎动物多倍化的模型。该体系利用红鲫 (n = 100) 作为原始母本，普通鲤 (n = 100) 作为原始父本，获得杂交鲫鲤F1，F2代 (n = 100)。由F2代不发生减数分裂的精卵细胞融合，繁育出四倍体F3代 (n = 200），经过连续繁殖，形成了一个遗传性状稳定、可育的四倍体鱼AT (n = 200) 群体 (F3–F17)，目前已到F24代。该体系由于遗传性状稳定，背景清晰，为脊椎动物多倍化发生初期遗传水平和表观遗传水平等方面的研究提供了绝佳模型。本项目旨在基于前期研究工作基础上，利用已获得的杂交鲫鲤体系原始亲本、二倍体及四倍体世代肝脏转录组数据和重测序数据，分别在转录水平和遗传水平探究该体系多倍化发生初期的关键改变，寻找发生关键变化的基因通路网络，为探讨多倍化在脊椎动物中相对罕见的原因提供线索。本研究第一部分，首先利用鲫鲤杂交体系的原始亲本及杂交4个世代 (F1，F2，F18和F22) 的肝脏转录组数据，分别采用从头组装和基于亲本参考基因组两种方法进行分析，通过比较亲本与子代的表达模式以及转录本变异规律，获得的结果提示：(1) 二倍体与父本鲤鱼的表达谱相似，而四倍体与母本红鲫的表达谱相似；(2) 二倍体世代发生差异表达的基因可能与突变的发生有关，并特异富集于细胞凋亡调控相关功能；(3) 四个世代中有约10%的基因为镶嵌基因 (即子代序列中出现亲本特异性变异位点交替出现)，且大部分为非对称式镶嵌 (即基因的两个拷贝中仅有一个发生镶嵌)，这些基因的功能显著富集于突变发生位点和疾病相关突变等功能，其中一些可能与细胞周期调控、通过重组进行DNA损伤修复等功能相关；(4) 约1%的基因存在杂交子代特异性变异。第二部分，本研究利用鲫鲤杂交体系的原始亲本及杂交6个世代 (F1，F2，F16，F18，F21和F22) 共20个个体的重测序数据，对照母本参考基因组进行分析，获得的结果提示：(1) 整体上，变异位点的分布更倾向于在外显子区累积；UTR区比CDS区积累了显著更多的变异；2nF1代变异位点数目存在个体差异；(2) 插入/缺失事件 (InDel) 在UTR区的分布显著高于内含子区和编码区；(3) 13个子代个体中发生镶嵌现象的基因比例约为9.75%–11.21%；2nF1代六个个体间仅有共享2,559个镶嵌基因，约为各个体镶嵌基因数目的50%，提示了该世代存在较大个体差异；不同世代间也仅有半数镶嵌基因相互覆盖；发生镶嵌和突变的热点区域主要集中在与生理机能调节以及免疫调节等相关功能，以及与细胞凋亡相关的调控过程。以上结果提示在多倍体化的初始阶段，基于转录组和重测序数据的分析提示多倍体鱼的基因结构、表达发生变化；F1中存在较大的个体差异，且不同世代之间也存在差异。这些变化可能是基因组休克效应造成的综合结果，可能会导致二倍体F2的存活率非常低。从鲫鲤品系基因组变异和表达量的变化看，杂交和多倍化初期该体系经历了剧烈的变化，且在基因组加倍后多个世代中仍有基因组水平结构变化的发生，这些变化在天然多倍体中很有可能是致死的。上述因素可能是导致脊椎动物中多倍体较少的原因之一。综上，本研究现阶段的工作仅对杂交鲫鲤体系多倍化发生初期的基因组变异和表达量改变规律进行初步的整体趋势的探究，但对遗传结构变化发生的机制等尚不能进行完善的阐述。进一步的工作将对异源多倍化后包括遗传水平和表观遗传水平改变对多倍体脊椎动物生存和基因组稳定性的影响，在基因组水平的基因区域和调控区域进行深入的探讨，以期为寻找脊椎动物多倍化发生困难的原因，并阐明脊椎动物多倍体基因组如何从最初不稳定到稳定的过程作出有意义的提示。; Polyploidization via whole-genome duplication (WGD) involves the integration of more than two complete sets of chromosomes in a cell. Polyploidization occurs most commonly in angiosperms. In plants, with different model systems, genome shock involves whole-genome wide genetic variants including chromosome rearrangement, DNA recombination, base and insert/deletion mutations, and also epigenetic changes. In contrast, polyploidization is relatively rare in animals and it mainly occurs in a handful of speices of insects and vertebrate taxa. Due to lack of suitable system, that why the occurring frequency in animal is much rarer than that in plants raised an open question for decades. Traditional explanations include barriers to sex-determination, physiological and developmental constraints, especially nuclear-cytoplasmic interactions and related factors, and genome shock or dramatic genomic restructuring. By ex situ, the bisexual, goldfish (Carassius auratus, ♀, n = 100) ×common carp (Cyprinus carpio, ♂, n = 100) hybrids allow for investigations into genomic consequences of allotetraploidization in vertetrates. This allopolyploidy system offers obvious advantages, e.g., their known parentage separates them from natural polyploids and it is easy to trace the fate of progenitor genes. In order to fully use the system to investigate how the allopolyploidy offspring survive from the more severe genome shock comparing the plants, by analyzing the RNA-seq and resequencing data, the expression alternations and genomic variations were investigated. Firstly, we employed two strategies for transcriptomic data processing, including de novo assembly and reference-based mapping strategies. The results showed that, (1) The expression of tetraploid offspring showed paternal-biased pattern, while the octaploids showed maternal-biased expression; (2) some differentially expressed genes were specifically enriched in mutagenesis site, regulation of cell death and apoptosis in tetraploids; (3) the chimeric genes (the genes in offspring with alternating parental-specific variants) comprised about 10%, most chimeras of maternal/paternal origin do not overlap, and this phenomenon indicates nonreciprocal structural change; the genes with offspring-specific mutations were 1.02%–1.16% in different generations; meanwhile, some of the chimeric and differentially expressed genes relate to mutagenesis, repair, and cancer-related pathways in 2nF1. Erroneous DNA excision between homologous parental genes may drive the high percentage of chimeric genes.Next, for the analyses of resequencing data from parents and six generations (20 samples in total), the genome of goldfish was used as reference genome. The results showed that, (1) variations distributed in untranslated regions (UTR) were significantly more abundant than the ones in coding regions (CDS) and intron; the six individuals of first generation 2nF1 were with the difference of possessing variations; (2) Insertions/Deletions (InDels) distributed in UTR regions were with significantly higher ratios than in intron and CDS; (3) within 13 offspring, chimeric genes comprised about 9.75%–11.21%; six individuals of 2nF1 shared only 2,559 chimeric genes, which indicated the individual difference of variation pattern within this generation; the chimeric genes with mutations were enriched in the regulation of physiological functions and immunoreactivity, and the regulation of programmed cell death and apoptosis.The discoveries yieled by both transcriptomes and resequencing data indicated fast changes including the genomic variations and expression changes, especially in 2nF1. These syndrome effects caused by genome shock might be the reasons of the greatly reduced viability in 2nF2 hybrid offspring, which might be lethiferous in natural polyploids. The results might help explain why polyploid are much rarer in vertebrates than in plants.Although the initial research documented that rapid and extensive genomic changes following tetraploidization, many questions about allopolyploidization remain unanswered. Ultimately, further work remains on exactly how polyploid plants and animals survive from genome shock, which happens more frequently in allopolyploid plants than in animals. Additional functional analyses at the genomic (genetic/epigenetic) level are also await. In the future work, we expect to find out possible key regions in the genomes and key changes in the gene pathways and regulatory elements which are most importantly contributed to the survival and genomic stability for the allopolyploidy. |
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