Elsevier

Gene

Volume 408, Issues 1–2, 31 January 2008, Pages 27-36
Gene

Increasing genomic information in bivalves through new EST collections in four species: Development of new genetic markers for environmental studies and genome evolution

https://doi.org/10.1016/j.gene.2007.10.021Get rights and content

Abstract

The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300–3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.

Introduction

Bivalves are prominent members of marine communities, and have attracted the attention of both scientists and laymen for centuries, due to the conspicuous and often astonishingly beautiful structure in which they live: the shells. But bivalve species have also important roles in ecosystems. As filter feeders, they form a significant link with primary producers (mainly phytoplankton and bacteria), and also act as calcium and carbon accumulators, which they use in their shell construction. Some species thrive around deep-sea hydrothermal vents, where they have evolved intriguing life-styles based on symbioses with micro-organisms. Moreover, many bivalve species have an added commercial value as food (oysters, clams, scallops and mussels) or other uses (e.g.: pearl oysters). In spite of all this, bivalves have lagged behind other taxa with respect to studies of their genome biology (reviewed in Hedgecock et al., 2005, Saavedra and Bachère, 2006).

As of November 2006, the NCBI Entrez taxonomy browser retrieved 13,722 entries for “Ostreidae”, 673 for “Veneridae” and 7281 for “Mytilidae”. Among them, 4583 entries came from the Pacific oyster Crassostrea gigas, 29 for the venerid Ruditapes decussatus, 1066 for the blue mussel Mytilus edulis and 117 for the deep-sea mussel Bathymodiolus azoricus. In the particular case of hydrothermal vent species, available sequences from the Genbank database mostly consisted in mussel endosymbiont 16S sequences, mitochondrial cytochrome oxidase I, 18S/28S partial genes, and less than 10 functional genes. These data contrasted with other marine invertebrates when compared to the 21,400 entries obtained for Penaeus sp., the 27,000 entries for Carcinus sp. and the 15,000 entries for Daphnia sp., which are the main studied crustaceans and the availability of whole genomes such as the sea urchin Strongylocentrotus purpuratus, or the sea hare Aplysia california and the nematod Caenorhabditis elegans. Amongst bivalves, most of the sequencing effort was only restricted to the oysters C. gigas and C. virginica leading to a very partial and biased view of the codon usage and the mutation distribution in bivalves. When considering C. gigas, the complete mitochondrial genome is available together with 370 microsatellite-containing sequences. EST collection program for the Pacific oyster C. gigas mainly used hemocyte cDNA libraries (Gueguen et al., 2003; http://www.ifremer.fr/GigasBase), and substractive cDNA libraries dealing with the identification of genes related to stress response to pollutants and/or parasites (Boutet et al., 2004, Tanguy et al., 2004, Tanguy et al., 2005) or abiotic factors (David et al., 2005) but also in response to summer mortality effects (Huvet et al., 2004). Although these studies increased our knowledge by several hundreds of targeted genes, they greatly contrasted with a remarkable library of 4.6 million ESTs in C. gigas, available from a genome-wide scan of gene expression in inbred and hybrid individuals carried out by Lynx Therapeutics (http://www.lynxgen.com/), using Megaclone™ and massively parallel signature sequencing or MPSS™ technologies (Brenner et al., 2000a, Brenner et al., 2000b). cDNA libraries and their corresponding EST collections have been made available over the last few years in other bivalve species, such as the American oyster (C. virginica), the Mediterranean blue mussel (M. galloprovincialis), and the scallops Argopecten irradians and Pecten maximus (reviewed in Saavedra and Bachère, 2006). Since their discovery in 1977 (Corliss et al., 1979), invertebrate species from deep-sea hydrothermal vents, and in particular the Mytilidae, have attracted great attention due to their ability to live in extreme environments that are mainly characterised by high temperatures and depths, low pH, high toxic sulphide species levels (Blum and Fridovich, 1984), radionucleides, and naturally high concentrations of bioavailable metals (Desbruyères et al., 2001). Several studies have already dealt with the effect of these environmental parameters on B. azoricus physiology, such as heavy metal detoxification processes that occurred during the course of eukaryotic evolution (Rousse et al., 1998, Bebianno et al., 2005, Kádár et al., 2005) and oxidative stress response (Blum and Fridovich, 1984, Fridovich, 1998). Most of these studies are based on chemical or enzymatic activities approaches and very few molecular studies have been carried out. The blue mussel Mytilus spp. combines a significant economic importance and an equally relevant role as sentinel species for pollution in coastal waters in many areas of the world. They also proved to be good models to understanding speciation in the sea owing to their numerous hybrid zones observed worldwide (Rawson, 2005). On the other hand carpet shell clam aquaculture based on R. decussatus is one of the oldest aquaculture practices in Europe and used to be one of its most important marine resources until the 60's, when a series of diseases started to eradicate natural clam beds. In the beginning of the 70's some European countries started to import the Japanese clam R. philippinarum because of its natural resistance to some of the diseases that almost annihilated the native carpet shell clam. Nowadays carpet shell clam is still heavily harvested in many countries, and particularly in Portugal, due to its economic importance. According to the Food and Agriculture Organization, production of carpet shell clams represents between fifty and seventy percent of the 10,000 t of bivalves produced in Portugal since 2000. Despite the great importance of this living resource, very few studies were conducted on the molecular biology of this bivalve.

In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs of commercial marine mollusk species and more particularly in the less studied but ecologically and commercially important groups, such as mussel and clam genera.

In this paper, we present the results of a collaborative research of 7 research groups aimed at increasing genomic information in bivalves and developing tools for general use, such are cDNA libraries and EST collections. Our research was performed in the framework of the Marine Genomics Europe (MGE) Network of Excellence (http://www.marine-genomics-europe.org). This network is devoted to the development, utilization and spread of high-throughput approaches for the investigation of the biology of marine organisms and the functioning of marine ecosystems. This involves the dedication and the development of common research infrastructures, both in genomics and in marine biology. This effort parallels others carried out elsewhere to push forward research on marine biology from a genomic perspective, such as the Marine Genomics organization led by Hollings Marine Laboratory, Charleston, South Carolina in the USA (McKillen et al., 2005). We report here the production of new cDNA libraries from bivalves, which have resulted in a total of 13,013 EST sequences from 4 species of special interest due to their biological role in the ecosystems they inhabit or to fisheries and aquaculture production: the Pacific oyster C. gigas, the carpet-shell clam R. decussatus, the blue mussel M. edulis and the deep-sea mussel B. azoricus. Multiple normalized libraries were constructed for this sequencing effort and are still available for further sequencing in order to increase the EST collections. This database will complement previous ESTs available in GenBank and was developed with the aim of sharing the largest information between the European laboratories of the “Fish & Shellfish” MGE node to perform joint studies in the fields of both environmental and functional genomics in marine bivalves. Data obtained will be thus used to develop tools for: 1) transcriptomics using micro-arrays for environmental studies and RNAi as promising functional tools 2) population genetics through the discovery of new polymorphism markers (SNP, microsatellites) and, 3) genome evolution based on the comparison of multiple sets of orthologous genes.

Section snippets

Oyster samples

A mixture of several individuals of C. gigas representing different physiological or stressful conditions has been used to prepare mixed RNA extracts from different tissues in order to diversify and maximize expression of various genes. RNAs used for the “stressed” library construction were extracted from the digestive gland of oysters submitted to hydrocarbons (Boutet et al., 2004), pesticides (Tanguy et al., 2005) and hypoxia (David et al., 2005) stress that were used in previous studies.

EST sequencing and general characteristics of the cDNA libraries

About 1200 to 3200 clones were randomly selected from each cDNA library and single-pass sequenced, resulting in the characterization of a total of 13,013 ESTs that coalesced into 124 contigs and 1814 singletons for the R. decussatus, 374 contigs and 5228 singletons for C. gigas, 362 contigs and 1918 singletons for B. azoricus and 60 contigs and 2865 singletons for M. edulis. The average size of sequenced inserts was comprised between 500 and 700 pb according to the species. A variable number of

Conclusion

The increasing number of mollusk EST databases available in GENBANK can now serve as a starting point to perform new promising gene comparisons with other related genomes, and especially Molluska genomes for a better understanding of their evolution in the marine fauna. Phylogenetic studies can also be envisaged in order to distinguish evolutionary history from adaptation and to help in the characterization of putative function of specific proteins in mollusk species by identifying conserved

Acknowledgements

The research presented in this paper was funded by the European Commission through the Marine Genomics Network of Excellence (GOCE-CT-2004-505403). C.S. wishes to acknowledge additional funding from the “Ramón y Cajal” Program and grants AGL2003–04143 and AGL2006–08944 from the Spanish government.

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