牡蠣全基因組測序與功能解析

許飛 孔令鋒 張揚 黃輝洋 王威

摘 要：圍繞牡蠣全基因組測序、基因組結構分析、基因注釋、重要功能基因發掘和SNP標記開發幾個目標展開了研究。采用fosmid克隆混池和等級組裝策略成功解決了牡蠣高雜合度帶來的基因組拼接困難，得到的基因組Contig和Scaffold N50分別達到19.4 Kb和401 Kb，整體測序深度大于800倍覆蓋度。為后續的研究奠定了基礎。對牡蠣基因組結構特征進行了分析。其基因組雜合度達到2.3%，重復序列豐富。在全基因組水平篩查到SNP位點312萬個，其中基因區的SNP分布少于非基因區。共注釋得到基因28 000多個。通過38個發育時期的表達譜測序，篩選到具有不同生物學含義的功能基因集，發掘了大量與生長發育等相關的基因，并對一些基因進行了詳細研究。對牡蠣母本效應基因進行了發掘。鑒定出1 307個在雌性性腺中特異高度表達的基因，526個雄性性腺中特異高度表達的基因，包括一些重要的父本效應基因，如K81等。對性別決定基因進行了分析，發現Sox9、SRY等基因在牡蠣雄性性腺中特異表達，可能是決定牡蠣雄性轉變的重要調控基因。對重要的發育基因家族進行了詳細梳理和進化分析，包括130多種Homeobox基因、20多種Fox基因、10多種Wnt基因等。此外，對一些重要的功能基因如表皮生長因子受體（EGFR）和固醇調節原件結合蛋白（SREBP）等還進行了原味雜交、蛋白重組表達等功能研究。在全基因組范圍以及轉錄區篩選到大量的SNP標記，在一些群體中驗證了超過1 500個具有多態性的SNP。并把HSP、類胰島素多肽等基因的SNP與表型性狀進行了關聯分析研究，發現一些與生長性狀具有顯著相關性的SNP標記。由于冠輪動物超門基因組數據缺少，牡蠣基因組的測序為生物多樣性和進化生物學研究提供了重要數據。為牡蠣高度適應潮間帶環境的分子機制研究提供了重要基礎。項目的完成對貝類發育、貝殼形成、海洋無脊椎動物浮游幼蟲的進化等機制有了更深入的理解。研究成果豐富了海洋基因資源，開發了大量分子標記，為分子育種的開展提供了條件。

關鍵詞：牡蠣 基因組 功能解析 生長發育

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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