Sequencing and de novo Analysis of Crassostrea angulata (Fujian Oyster) from 8 Different Developing Phases Using 454 GSFlx

et al. (2012) Sequencing and de novo Analysis of Crassostrea angulata (Fujian Oyster) from 8 Different Developing Phases Using 454 GSFlx. PLoS ONE 7(8): e43653. doi:10.1371/journal.pone.0043653 Sequencing and de novo Analysis of Crassostrea angulata (Fujian Oyster) from 8 Different Developing Phases Using 454 GSFlx Ji Qin 0 Zixia Huang 0 Jun Chen 0 Quan Zou 0 Weiwei You 0 Caihuan Ke 0 Timothy Ravasi, King Abdullah University of Science and Technology, Saudi Arabia 0 1 State Key Laboratory of Marine Environmental Science, Xiamen University , Xiamen , China , 2 College of Ocean and Earth Sciences, Xiamen University , Xiamen , China , 3 College of Information Science and Technology, Xiamen University , Xiamen , China Research on the mechanism for early development of shellfish, such as body plan, shell formation, settlement and metamorphosis is currently an active research field. However, studies were still limited and not deep enough because of the lack of genomic resources such as genome or transcriptome sequences. In the present research, de novo transcriptome sequencing was performed for Crassostrea angulata, the most economically important cultured oyster species in China, at eight early developmental stages using the 454 sequencing technology. A total of 555,215 reads were produced with an average length of 309 nucleotides that were then assembled into 10,462 contigs. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes recovered diverse biological functions and processes. Six unique sequences related to settlement, metamorphosis and growth were subsequently analyzed by real-time PCR. Given the lack of whole genome information for oysters, transcriptome and de novo analysis of C. angulata from the eight different developing phases will provide important and useful information on early development mechanism and help genetic breeding of shellfish. - Funding: This study was funded by The National Basic Research Program of China (No.2010CB1264000), Hi-Tech Research and Development (863) Program of China (No. 2010AA10A110) and Shellfish Modern Agro-industry Technology Research System (No. nycytx-47). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Lophotrochozoans usually show similar early development processes, but body plan and behavior pattern for the adults are very different. Mollusks are the most diverse animal phylum and the representative lophotrochozoan phylum which exhibits two ancient developmental features: spiral cleavage and trochophore larva. Bivalves, which include clams, oysters, mussels, and scallops, are the second largest group of mollusks [1]. Therefore, research on early developmental mechanism on bivalves will provide useful information on evolution, phylogeny and diversification for lophotrochozoan. Apart from common developmental issues to the model animals, such as cleavage, gastrulation formation and organogenesis, there are other specific developmental issues for invertebrate such as shell formation, settlement and metamorphosis. For instance, metamorphosis, which involves transformations of many organs during the transition from a free-swimming larva to a benthic juvenile that usually occurs within a short period of time. Metamorphosis is also the critical phase in terms of mortality in the life cycle for bivalves [2]. In the past years, progresses have been made in the mechanism of metamorphosis. Related genes and proteins have been identified and their functions were analyzed on abalones, ascidians, corals and so on. The spatial expression patterns of five anterior Hox genes during larval development of the abalone Haliotis asinina were reported and Hox genes were suggested to play an important role in gastropod shell formation [3]. The expression of Hemps, an EGF-like signaling peptide required for the induction of ascidian Herdmania metamorphosis, increases in competent larvae, and gene expression patterns in pre-competent and competent stages are markedly different [4]. Gene expression microarray analyses were performed in the scleractinian coral to elucidate the molecular mechanisms underlying coral metamorphosis and early stages of calcification [5]. However, studies were still limited and not deep enough to form integrated theories about the developmental biology of invertebrate. Because this field has long suffered from the challenges of lack of genomic resources such as genome or transcriptome sequences. Oysters are widely distributed along the coast of China and are the most important commercial shellfish group cultured in China. The annual production of oysters was about 3.5 million tonnes in 2009 and accounted for more than 20% of mariculture total production in China [6]. Due to its economic value and ecological role, in recent years many studies have been carried out in its genetics [7,8], breeding [911], disease control [12,13] and so on. Fujian oyster, Crassostrea angulata, is the main oyster species in coastal river mouths and estuaries of southern China, ranging from Zhejiang Province to Hainan Province. The production for Fujian oyster usually accounts for about 50% of total oyster production in China. A recent study found that C. angulata should be considered as a subspecies of C. gigas by sequencing 16 S rRNA and COI genes [14]. The sequencing and analysis of expressed sequence tags (ESTs) has been a primary tool for the discovery of novel genes in terrestrial animals, especially in non-model species for which full genome sequences are not currently available. The recent highthroughput sequencing technologies, which can effectively speed up genomic studies on non-model animals, provide a great potential for bivalve research. The increased throughput of nextgeneration sequencing technologies, such as the massively parallel 454 pyrosequencing, enables the rapid generation of transcriptomes for non-model species and allows increased sequencing depth and coverage, while reducing the time, labor, and cost. According to this technology, transcriptome has been used in shellfish research in the past several years for solving some basis scientific problems, such as immune response, adaption under environmental pressure, and shell formation on C. gigas [15], Mytilus galloprovincialis [16] Sinonovacula constricta [17], Laternula elliptica [18], and H. midae [19]. By sequencing cDNA libraries for Aplysia, a well-established model organism for cellular and systems neural science, over 175,000 ESTs have been identified, and 19,814 are unique neuronal gene products which represent 50%70% of the total Aplysia neuronal transcriptome [20]. High throughput EST pyrosequencing was also undertaken on the calcifying mantle, combined with a proteomic analysis of the shell for Pinct (...truncated)