异源杂交多倍化鲫鲤初期不同世代的遗传变化规律研究

Allopolyploidizaiton ocurrs recurrently in plants and lower vertebrates, and it can result in instantaneous speciation. Because new polyploids are immediately reproductively isolated from their non-polyploid congeners, polyploidization has played an important role in the diversification of the flowering plants and some vertebrates. Newly formed polyploids must respond to this instantaneous genomic change, which resembles “genome shock” syndrome, to survive and to reproduce successfully. Polyploidy cyprinids are useful models in vertebrate polyploidization investigations and in fisheries. In our recent work, we haved used the first sucessful allopolyploidy system in vertebrate, which was generated by goldfish (maternal) x common carp (parternal), as a model, to inverstigate early stage modifications in their genomes and gene pathways of the polyploidy offsprings with transcriptome data. We have found intriguingly high percentage of recombination rate and even mutation rate in the offspring. In order to fully use the obtained data and to conduct further work in this allopolyploidy system, we start sequencing its materal genome,the goldfish in our pilot work. First we have obtained gynogenetic tetraploid goldfish,by using normal goldfish eggs and stimulate them by heterologous sperms and cold shock. The gynogenetic goldfish has decreased heterozygosity comparing to that of the normal goldfish. After we obtained gynogenetic tetraploid goldfish, its genome were sequenced using the next generation sequencing techniques. We have obtained sequencing results from four short fragment pair-end libraries, and we conducted primitive assembly, In this project, firstly, we will add more longer fragment sequencing results from mate-pair libraries in order to obtain genome draft. Besides, to increase assembly rate and reliability for the tetraploid genome, new algorithms and programs will be developed. Secondly, based on the available goldfish genome, we plan to analyze both the obtained transcriptome data in our pilate work and additional transcriptome data from different tissues of the allopolyploidy offsprings comparing to their progenitors, and to identify the hot-change regions and pathways in the polyploidy offspring. We expect to find out possible key regions in the genomes and key changes in the gene pathways which are important to the formation of allopolyploidization. Lastly, we will also estimate recombination rate, genomic structural variation rate in the offsprings according to the genomes of two progenitors’. Thus, to find clues how of the vertebrate divercification is benefited from allopolyploidization, and in turn, how this process can be applied to fisheries more efficiently.

异源多倍化发生初期是形成多倍体的关键时期，此时期发生的基因组变化最终决定异源多倍体子代能否成功繁衍成为新物种。用鲫鱼和普通鲤鱼杂交获得的鲫鲤杂交体系，是目前世界上仅存的亲本背景清晰且杂交后代能稳定遗传的多倍化体系(F1-F21)。本项目拟综合应用新一代测序技术和生物信息学等手段，补充构建并测定mate-pair文库，继续完成获得雌核发育红鲫全基因组草图，以此为参考基因组，对鲫鲤杂交体系中亲本与多倍化初期不同世代(F1-F3; F18; F21)的重要组织进行转录本分析，全面而系统地分析研究异源多倍化发生后子代中的遗传水平改变规律，如重组、子代特异性改变等变化在不同世代中的规律，同时这些改变对基因网络改变的作用和对相关性状改变的贡献，以期了解脊椎动物异源多倍化初期发生的改变规律如何贡献于脊椎动物演化这一重要理论问题提供有意义的线索，也为渔业中更好地利用此现象奠定理论基础。

本项目主要以世界上首例人工杂交异源脊椎多倍化体系鲫鲤杂交体系为研究对象，探讨其如何克服异源基因组合并和冗余导致的剧变而幸存、并可能形成新的类群。在探究脊椎动物多倍化何以稀少、初期基因组事件及其研究方法上取得显著进展。主要开展和完成的研究如下：第一，利用mt全基因组和核基因发现10-12Ma 鲫和鲤共享的多倍化细节，这为更清晰地分析红鲫基因组提供了基础。第二，对遗传背景清晰的鲫鲤杂交体系母本类群的雌核发育红鲫的基因组进行了二代测序，获得233.4Gb的数据；同时对2个个体不同组织进行了重测序和转录组测序。拼装注释和比较基因组学分析初步发现其不同于植物和爪蟾的基因组演化模式。并进一步进行了三代测序，相关分析进展中。此外，第三，对鲫鲤杂交体系的不同世代和不同个体进行了转录组水平的研究，采用de novo及参考基因组策略对转录本进行了变异模式分析，结果表明杂交后代约10%基因表现为父母本嵌合的基因，并约有1%的子代基因存在特异性变异；此结果在子代基因组的重测序及其BAC文库测序中均得到证实。为何在如此短的时间内存在如此高的嵌合和子代特异性变异的相关机制正在深入研究中。第四，表达量的分析表明：单个组织的转录本子代表现为二倍体世代接近父本，而四倍体子代偏母本，并得到qRT-PCR实验证实。然而综合不同组织暂未发现规律。第五，本研究通过仔细分析重测序和不同组织的转录本，提示子代存在显著的转录编辑等现象，更明确的机制正在进一步研究中。第五，针对需要多个数据集聚类和图形化、定位和归类，研发了相应的软件并发表。本项目的实施，发现世界上目前世代最多的鲫鲤杂交体系不仅存在着结构水平的基因组迅速改变，同时转录组也处于休克状态的快速变化，显示了比植物多倍体更剧烈的变化，提示脊椎动物异源多倍化难以发生可能是由于初期其变化过于剧烈而难以幸存，并且此体系如何能幸存的基因组机制也基于此计划集成项目的支持而在深入探讨中。

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