The oyster genome reveals stress adaptation and complexity of shell formation

ARTICLE doi:10.1038/nature11413 The oyster genome reveals stress adaptation and complexity of shell formation Guofan Zhang1*, Xiaodong Fang2*, Ximing Guo3*, Li Li1*, Ruibang Luo2,4*, Fei Xu1*, Pengcheng Yang2*, Linlin Zhang1*, Xiaotong Wang1*, Haigang Qi1, Zhiqiang Xiong2, Huayong Que1, Yinlong Xie2,4, Peter W. H. Holland5, Jordi Paps5, Yabing Zhu2, Fucun Wu1, Yuanxin Chen2, Jiafeng Wang1, Chunfang Peng2, Jie Meng1, Lan Yang2, Jun Liu1, Bo Wen2, Na Zhang1, Zhiyong Huang2, Qihui Zhu1, Yue Feng2, Andrew Mount6, Dennis Hedgecock7, Zhe Xu8, Yunjie Liu2, Tomislav Domazet-Lošo9, Yishuai Du1, Xiaoqing Sun2, Shoudu Zhang1, Binghang Liu2,4, Peizhou Cheng1, Xuanting Jiang2, Juan Li1, Dingding Fan2, Wei Wang1, Wenjing Fu2, Tong Wang1, Bo Wang2, Jibiao Zhang1, Zhiyu Peng2, Yingxiang Li1, Na Li2, Jinpeng Wang1, Maoshan Chen2, Yan He3, Fengji Tan2, Xiaorui Song1, Qiumei Zheng2, Ronglian Huang1, Hailong Yang2, Xuedi Du1, Li Chen2, Mei Yang1, Patrick M. Gaffney10, Shan Wang3, Longhai Luo2, Zhicai She1, Yao Ming2, Wen Huang1, Shu Zhang2, Baoyu Huang1, Yong Zhang2, Tao Qu1, Peixiang Ni2, Guoying Miao1, Junyi Wang2, Qiang Wang1, Christian E. W. Steinberg11, Haiyan Wang1, Ning Li2, Lumin Qian3, Guojie Zhang2, Yingrui Li2, Huanming Yang2, Xiao Liu1, Jian Wang2, Ye Yin2 & Jun Wang2,12,13 The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster’s adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa. Oceans cover approximately 71% of the Earth’s surface and harbour most of the phylum diversity of the animal kingdom. Understanding marine biodiversity and its evolution remains a major challenge. The Pacific oyster C. gigas (Thunberg, 1793) is a marine bivalve belonging to the phylum Mollusca, which contains the largest number of described marine animal species1. Molluscs have vital roles in the functioning of marine, freshwater and terrestrial ecosystems, and have had major effects on humans, primarily as food sources but also as sources of dyes, decorative pearls and shells, vectors of parasites, and biofouling or destructive agents. Many molluscs are important fishery and aquaculture species, as well as models for studying neurobiology, biomineralization, ocean acidification and adaptation to coastal environments under climate change2,3. As the most speciose member of the Lophotrochozoa, phylum Mollusca is central to our understanding of the biology and evolution of this superphylum of protostomes. As sessile marine animals living in estuarine and intertidal regions, oysters must cope with harsh and dynamically changing environments. Abiotic factors such as temperature and salinity fluctuate wildly, and toxic metals and desiccation also pose serious challenges. Filter-feeding oysters face tremendous exposure to microbial pathogens. Oysters do have a notable physical line of defence against predation and desic- cation in the formation of thick calcified shells, a key evolutionary innovation making molluscs a successful group. However, acidification of the world’s oceans by uptake of anthropogenic carbon dioxide poses a potentially serious threat to this ancient adaptation4. Understanding biomineralization and molluscan shell formation is, thus, a major area of interest5. Crassostrea gigas is also an interesting model for developmental biology owing to its mosaic development with typical molluscan stages, including trochophore and veliger larvae and metamorphosis. A complete genome sequence of C. gigas would enable a more thorough understanding of oyster biology and the evolution of Lophotrochozoa. One of the main challenges, however, is the high levels of polymorphism present in oysters and many marine invertebrates6–8. To overcome this, an oyster derived from four generations of full-sibling mating (coefficient of inbreeding, F 5 0.59) was used for genome sequencing and assembly (Supplementary Text B1) through fosmid pooling, next-generation sequencing (NGS) and hierarchical assembling. Combining these genomic data with transcriptomes from different organs, different developmental stages and adults challenged with stressors, in addition to mass spectrometric analysis of shell proteins, allowed us to explore characteristics of the oyster genome 1 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. 2BGI-Shenzhen, Shenzhen 518083, China. 3Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University, Port Norris, New Jersey 08349, USA. 4HKU-BGI Bioinformatics Algorithms and Core Technology Research Laboratory, Hong Kong. 5Department of Zoology, University of Oxford, Oxford OX1 3PS, UK. 6Department of Biological Sciences, Clemson University, South Carolina 29634, USA. 7Department of Biological Sciences, University of Southern California, Los Angeles, - Bošković Institute, Bijenička cesta 54, P.P. 180, HR-10002, California 90089, USA. 8Atlantic Cape Community College, Mays Landing, New Jersey 08330, USA. 9Laboratory of Evolutionary Genetics, Ruder Zagreb, Croatia. 10School of Marine Science and Policy, University of Delaware, Lewes, Delaware 19958, USA. 11Institute of Biology, Humboldt Universität zu Berlin Arboretum, Späthstraße 80/81, 12437 Berlin, Germany. 12Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark. 13The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, DK2200 Copenhagen, Denmark. *These authors contributed equally to this work. 4 O C T O B E R 2 0 1 2 | VO L 4 9 0 | N AT U R E | 4 9 ©2012 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE and key aspects of molluscan biology related to stress response and shell formation. Sequencing and hierarchical assembly NGS technology has been successfully applied for de novo genome sequencing and assembly using whole-genome shotgun strategies9–13. We initially generated 155-fold Illumina whole-genome shotgun reads (Supplementary Table 1), but could not adequately assemble them owing to high levels of polymorphism and abundant repetitive sequences (Supplementary Text B2 and Supplementary Fig. 1). As possible alternat (...truncated)