牡蛎全基因组测序与功能解析

许飞 孔令锋 张扬 黄辉洋 王威

摘 要：围绕牡蛎全基因组测序、基因组结构分析、基因注释、重要功能基因发掘和SNP标记开发几个目标展开了研究。采用fosmid克隆混池和等级组装策略成功解决了牡蛎高杂合度带来的基因组拼接困难，得到的基因组Contig和Scaffold N50分别达到19.4 Kb和401 Kb，整体测序深度大于800倍覆盖度。为后续的研究奠定了基础。对牡蛎基因组结构特征进行了分析。其基因组杂合度达到2.3%，重复序列丰富。在全基因组水平筛查到SNP位点312万个，其中基因区的SNP分布少于非基因区。共注释得到基因28 000多个。通过38个发育时期的表达谱测序，筛选到具有不同生物学含义的功能基因集，发掘了大量与生长发育等相关的基因，并对一些基因进行了详细研究。对牡蛎母本效应基因进行了发掘。鉴定出1 307个在雌性性腺中特异高度表达的基因，526个雄性性腺中特异高度表达的基因，包括一些重要的父本效应基因，如K81等。对性别决定基因进行了分析，发现Sox9、SRY等基因在牡蛎雄性性腺中特异表达，可能是决定牡蛎雄性转变的重要调控基因。对重要的发育基因家族进行了详细梳理和进化分析，包括130多种Homeobox基因、20多种Fox基因、10多种Wnt基因等。此外，对一些重要的功能基因如表皮生长因子受体（EGFR）和固醇调节原件结合蛋白（SREBP）等还进行了原味杂交、蛋白重组表达等功能研究。在全基因组范围以及转录区筛选到大量的SNP标记，在一些群体中验证了超过1 500个具有多态性的SNP。并把HSP、类胰岛素多肽等基因的SNP与表型性状进行了关联分析研究，发现一些与生长性状具有显著相关性的SNP标记。由于冠轮动物超门基因组数据缺少，牡蛎基因组的测序为生物多样性和进化生物学研究提供了重要数据。为牡蛎高度适应潮间带环境的分子机制研究提供了重要基础。项目的完成对贝类发育、贝壳形成、海洋无脊椎动物浮游幼虫的进化等机制有了更深入的理解。研究成果丰富了海洋基因资源，开发了大量分子标记，为分子育种的开展提供了条件。

关键词：牡蛎 基因组 功能解析 生长发育

Abstract：The following studies were conducted in this project：the oyster complete genome sequencing， structure analysis，gene annotation，functional genes identification，and single nucleotide polymorphism （SNP） discovery.We successfully assembled the oyster genome through a combined strategies of fosmid clones pooling and hierarchical assembly.The contig N50 and scaffold N50 of the genome were 19.4 Kb and 401 Kb respectively.The sequencing depth was as high as 800 fold of the genome size. The assembly of the genome provided basis for further biological studies. We then analyzed the oyster genome structure. The polymorphism across the whole genome was as high as 2.3%， while the repeat sequences was as rich as 36%. More than three million SNPs were identified. The SNP proportion from protein coding regions was far lower than that from noncoding regions.More than 28 000 genes were annotated. The gene function was primarily studied through the RNA-seq of samples from 38 development stages.Many important developmental gene families were identified， while some of them were further studied.A total of 1 307 and 526 genes were identified to be female and male specially expressed respectively， including some important maternal and paternal effect genes， such as the K81 gene. We also found that the SoxH gene specially expressed in male gonad，showing that this gene should be one of the key regulatory factor in male determination. Phylogeny analysis was conducted on some important developmental families， including ～130 Homeobox genes， ～20 Fox genes， ～10 Wnt genes. For some key genes such as epidermal growth factor receptor （EGFR） and Sterol Regulatory Element-Binding Proteins （SREBPs） were further studied using hybridization in situ and recombinant protein expression techniques. We identified large number of SNP markers across the whole genome and transcription regions. As many as 1 500 SNP markers were validated in some oyster populations. At the same time， the association analysis on the SNPs from heat shock proteins （HSP） and insulin-like peptide with phenotypes suggested some SNPs significantly correlated with the growth characters of oyster. As few genome data was available in Lophotrochozoans， the oyster genome sequence provided important data for the studies on biological diversity and evolutionary biology， as well as the study on the molecular mechanism of oyster adapting the intertidal environment. The study gave deep insight into the molecular mechanisms of molluscan development， shell formation， and larval evolution of marine invertebrates. It also enriched the marine gene resources， provided large number of SNP markers， and provided foundation for the oyster molecular breeding.

Key Words：Oyster；Genome；Functional analysis；Growth and development

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