MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences
- Paola Venier†1,
- Cristiano De Pittà†1, 2,
- Filippo Bernante1, 2,
- Laura Varotto1,
- Barbara De Nardi3,
- Giuseppe Bovo4,
- Philippe Roch5,
- Beatriz Novoa6,
- Antonio Figueras6,
- Alberto Pallavicini7Email author and
- Gerolamo Lanfranchi1, 2Email author
© Venier et al; licensee BioMed Central Ltd. 2009
Received: 10 November 2008
Accepted: 09 February 2009
Published: 09 February 2009
Although Bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of Mytilus galloprovincialis (the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria.
We have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes. A total of 24,939 clones were sequenced from these libraries generating 18,788 high-quality ESTs which were assembled into 2,446 overlapping clusters and 4,666 singletons resulting in a total of 7,112 non-redundant sequences. In particular, a high-quality normalized cDNA library (Nor01) was constructed as determined by the high rate of gene discovery (65.6%). Bioinformatic screening of the non-redundant M. galloprovincialis sequences identified 159 microsatellite-containing ESTs. Clusters, consensuses, related similarities and gene ontology searches have been organized in a dedicated, searchable database http://mussel.cribi.unipd.it.
We defined the first species-specific catalogue of M. galloprovincialis ESTs including 7,112 unique transcribed sequences. Putative microsatellite markers were identified. This annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis for investigations in the biology of Mediterranean mussels.
The marine mussel (Mytilus galloprovincialis, Lamark 1819) is commonly found in the Mediterranean Sea, Black Sea, and also intermixed with M. edulis along the Atlantic coasts of France, Britain and Ireland . Mussels are suspension feeders commonly living in dense masses at the intertidal and subtidal level, attached among themselves and to hard substrata by the fibrous threads of the byssus. As filter feeders, they are functionally linked with primary producers (mainly phytoplankton and bacteria), and also act as calcium and carbon accumulators, which they use for shell construction. Mytilus spp. combine a significant economic importance , and an equally relevant role as sentinel species for pollution in coastal waters in many areas of the world . Sessile mussels accumulate various water contaminants in their tissues hence react to environmental changes caused by natural and anthropogenic factors  with an assortment of physiological and genetic mechanisms, partly traceable with appropriated tests [5, 6].
Nucleotide and protein sequences belonging to all orders of the Bivalvia class.
Among mussel species, a high number of sequences are accessible for M. californianus (43,188) whereas the blue mussel M. edulis and the Mediterranean mussel M. galloprovincialis have 5,938 and 6,190 nucleotide sequences, respectively. Furthermore, only 409 amino acid sequences with a high rate of redundancy are present for M. galloprovincialis. They identify key proteins and enzymes of oxidative phosphorylation (NADH dehydrogenase subunit 1, 2, 3, 4, 4L, 5, 6; ATP synthase F0 subunit 6; cytochrome oxidase subunit I, II, III, cytochrome b), defence mechanisms (defensin MGD-1 precursor; myticin-A, B, C precursor; cathepsin L; lysozyme; MGD2 antimicrobial peptide precursor), adhesion and motility processes (twitchin; adhesive plaque matrix proteins; thread matrix proteins; precollagen-D; actin; paramyosin; tropomyosin; catchin) and stress response (methallothionein 20, 10B, 10 IIIB; heat shock protein 70, 27). Of note, the vitelline coat lysin M7, a protein found in sperm acrosomes of mussels that dissolves the egg vitelline coat permitting fertilization , is represented in public databases with a very high rate of redundancy (63 entries).
List of complete or nearly complete mitochondrial DNA sequences of the class Bivalvia.
Venerupis (Ruditapes) philippinarum
Current genomics technologies, like SAGE , differential display  and systematic sequencing of expressed sequence tags , are very useful approaches to rapidly identify protein coding genes on a large scale in model  and non-model organisms [32, 33]. Moreover, the frequency of a given sequence in the SAGE or cDNA libraries can be related to the relative abundance of the corresponding mRNA, giving an indication of the level of gene expression [34, 35]. EST analysis is also an effective approach for the identification of polymorphic cDNA markers such as microsatellites and single nucleotide polymorphisms [36–38].
Several EST collections have already been reported for commercial bivalves  but most of the sequencing effort was restricted to the oyster C. gigas [40, 41] and C. virginica [42, 43]. The Oyster Genome Consortium has integrated these EST resources for the construction of a publicly available cDNA microarray. This platform was used to evaluate the degree of cross-species hybridization between C. gigas and C. virginica . Different genomic approaches have been applied also to M. galloprovincialis. A number of cDNA libraries obtained from multiple mussel tissues  were sequenced. The resulting collection of independent 3'-end ESTs was assembled in MytArray 1.0 in order to analyze the tissue transcriptional signatures of Mediterranean mussel exposed to chemical mixtures in laboratory and in the Venice lagoon . Recently other cDNA libraries were constructed from haemolymph of immuno-stimulated mussels to better understand their immune response mechanisms . However, the EST resource of M. galloprovincialis remains too small compared to other bivalves such as oyster and M. californianus. To date, only 6,190 ESTs have been deposited in GenBank for the Mediterranean mussel. The aim of our study was to increase significantly the number of mussel genes in the public database. For this purpose, we have produced and massively sequenced a high-quality normalized cDNA library in order to generate new thousands of non-redundant ESTs and to analyze the ESTs for microsatellites. We have fully functionally annotated these sequences and we present the first knowledgebase of a mussel transcriptome.
Construction and content
Tissues samples and RNA purification
Mediterranean mussels (M. galloprovincialis) with a maximum shell length of 6–7 cm and mixed sex were obtained from commercial shellfish stocks from Chioggia, Venice, Trieste (North Adriatic Sea, Italy) and Ria de Vigo (Atlantic ocean, Spain). Bivalves were acclimatized in artificial sea water (Italy) and in tanks having an open-circuit of filtered seawater at 15°C with aeration in Vigo (Spain), and then subjected to different challenges. Selected tissues (gills, digestive gland, foot, anterior and posterior adductor muscles and mantle), essential for vital functions and potentially involved in stress responses, were dissected on ice, rapidly rinsed in sterile saline solution, frozen and stored in a large excess of Trizol reagent (Invitrogen, 15 ml for 0.5–1.5 g. of sample) at -80°C.
Haemolymph (1–2 ml) was withdrawn with a disposable syringe from the posterior adductor muscle of each animal treated with a mixture of heat-inactivated bacteria or a solution of poly I:C (Sigma) mimicking viral infection . Haemocytes were collected by centrifugation, lysed in a few ml of Trizol reagent and stored at -80°C.
Frozen tissues were minced and homogenized for 3–5 min using a Diax 900 (Heidolf, Germany) blender. Total RNA was isolated using the Trizol reagent following the manufacturer's instruction and further purified with LiCl 8 M in order to remove glucidic contaminants. All RNA samples were checked for quality by microcapillary electrophoresis (RNA 6000 Nano LabChip, Agilent Bioanalyzer 2100, Agilent Technologies).
Construction of cDNA libraries
Description of Mediterranean mussel cDNA libraries.
3 days of treatment with okadaic acid (January 2006 – Trieste, Italy)
Treatment with heat-inactivated bacteria (June 2005 – Padova, Italy)
Selected tissues: digestive gland, gills, foot, gonads, haemolymph and mantle (October 2000 – Padova, Italy)
Digestive gland and gills
Treatment with two mixtures of organic compounds and heavy metals (December 2002 – Padova, Italy)
Treatment with heat-inactivated bacteria (June 2005 – Padova, Italy)
Off-shore control mussels (June 2005 – Padova, Italy)
Treatment with heat-inactivated bacteria (June 2005 – Padova, Italy)
Treatment with heat-inactivated bacteria (June 2005 – Vigo, Spain)
Treatment with a solution of poly I:C mimicking viral infection (June 2005 – Vigo, Spain)
Control mussels (June 2005 – Vigo, Spain)
Gills, digestive gland, foot, anterior and posterior adductor muscles and mantle (June 2002 – Padova, Italy)
Gills, digestive gland, foot, anterior and posterior adductor muscles, mantle and haemolymph (October 2002 – Padova, Italy)
Selected tissues: gills, digestive gland, foot, anterior and posterior adductor muscles, mantle and haemolymph (October 2002 – Padova, Italy)
Treatment with heat-inactivated bacteria (June 2005 – Padova, Italy and Vigo, Spain)
Equal amount (333 ng) of cDNA from DiG01, DiG02, GDG01, Hae01, Hae02, Hae03, Hae04, Gll01, MxT04 have been pooled (April 2006 – Padova, Italy)
Gills, digestive gland, foot, anterior and posterior adductor muscles and mantle (October 2000 – Padova, Italy)
Gills, digestive gland, foot, anterior and posterior adductor muscles and mantle (November 2000 – Padova, Italy)
A normalized M. galloprovincialis library (Nor01) was produced to optimize the discovery rate of the random sequencing process by equilibrating the final representation of abundant and rare transcripts. This library was constructed by pooling equal amounts (333 ng) of cDNA from DiG01, DiG02, GDG01, Hae01, Hae02, Hae03, Hae04, Gll01, MxT04 libraries. This cDNA pool was concentrated by Microcon YM 30 (Millipore) and adjusted to a final concentration of 70 ng/μl. For cDNA normalization, 3 μl (about 200 ng) of purified double-strand cDNA plus 1 μl 4× Hybridization Buffer (200 mM HEPES-HCl, pH 8.0; 2 M NaCl) was overlaid with mineral oil, denatured at 98°C for 2 min and then allowed to anneal at 68°C for 5 h. The following pre-heated reagents were then added to the hybridization reaction kept at 68°C: 3.5 μl milliQ water; 1 μl of 5× DNAse buffer (500 mM Tris-HCl, pH 8.0; 50 mM MgCl2, 10 mM DTT); 0.5 μl double-strand nuclease (DSN) enzyme. After further incubation at 68°C for 30 min., the DSN enzyme was inactivated by adding 10 μl of 5 mM EDTA at 68°C for 10 min. The normalized cDNAs samples were diluted with 20 μl milliQ water and used for PCR amplification. The PCR reaction (50 μl) contained 1 μl diluted cDNA, 1 × Advantage 2 reaction buffer (BD Biosciences Clontech), 200 μM dNTPs, 0.15 μM attB1 and attB2 primers, 1 × Advantage 2 Polymerize mix (BD Biosciences Clontech). The amplification protocol consists of 21 cycles of the following consecutive steps: 7 s at 95°C, 20 s at 65°C and 3 min at 72°C. The amplified normalized cDNA was size-selected on SizeSep 400 Spun Columns (GE Healthcare) and directionally cloned into pDONR221 vector (Invitrogen) through BP recombinase.
The systematic sequencing of most recently produced cDNA libraries (DiG01, DiG02, GDG01, Gll01, Hae01, Hae02, Hae03, Hae04, Hae05, MxT04 and Nor01) was performed at the Sequencing Service of Max-Plank Institute for Molecular Genetics (Berlin, Germany). Libraries were arrayed on 384-well plates and single pass DNA sequencing from plasmids was performed by using the vector specific primer attB1_seq (5'-CTTTGTACAAAAAAGCAGGCT-3') and a modified Sanger dideoxy terminator cycle sequencing chemistry, the ABI BigDye kit version 3.1, on Capillary Sequencer systems (Applera ABI 3730 XL and GE Healthcare MegaBase 4500).
Sequence processing and analysis
Trace2dbest and Partigene  were used to process chromatograms, align and clusterize sequences and build an annotation database. Trace2dbest extracts sequences and quality information from traces (Phred algorithm), removes vector contamination and poly(A) and performs the trimming of low quality sequences. Sequences shorter than 150 bp were discarded. Partigene reads all sequence files and performs an assembling process in two step: 1) CLOBB software  clusterizes sequences on the basis of BLAST similarity; 2) Phrap  makes a consensus from each cluster.
Each consensus, converted into FASTA format, was searched locally in nucleotides database, downloaded from NCBI  and UniProtKB sources , using Blast-N and Blast-X, respectively. The first 5 High Scoring Pairs from each Blast result were collected and stored in a local PostgreSQL table as a collection of automatic annotations.
Each single annotation in our database was further manually examined to assign the best describing text to the correspondent cluster. Similarities with expectations values greater than e-6 for protein (Blast-X) and e-40 for nucleotide (Blast-N) were considered as poorly informative. Moreover, putative peptides identified by Prot4EST  where searched for protein domain in all available protein signature databases by means of InterproScan . Clusters, consensuses and related similarities were electronically organized and stored in a dedicated PostgreSQL database .
Gene Ontology annotation
To each UniProt ID taken from Blast-X description field, we have associated specific Gene Ontology annotations (GO) that integrate information about process, function, and component. The distribution of sequences in each of the main ontology categories was examined and percentages of unique sequences in each of the assigned GO terms was computed. In each of the three main categories of GO, namely Biological process, Molecular function, and Cellular component , 100% was considered as the total number of unique sequences having an assigned GO term. Thus, in each main category the percentages do not reach 100% because some deduced proteins result with more than one GO category assigned to them .
Identification of microsatellite containing ESTs
The unique consensus sequences were screened for microsatellites by using the MISA software . Only di-, tri-, tetra-, penta- and esanucleotide repeats were targeted, since mononucleotide repeats are not useful for mapping or population genetics due to difficulties in their genotyping. Strings of oligonucleotide sequences were used to search for microsatellites: 6 repeats for dinucleotide; 5 repeats for trinucleotide; 5 repeats for tetranucleotide, pentanucleotide and esanucleotide.
Utility and discussion
General characteristics of the cDNA libraries and EST assembly
Results of EST assembly for each Mediterranean mussel cDNA library.
# EST in cluster
% poly(A) detection
% gene discovery
We have decided to sequence intensely only the two cDNA libraries (MxT03, Nor01) that could contain a more general information of mussel transcriptome, since they were constructed by pooling equal amounts of RNA from different tissues (gills, digestive gland, foot, anterior and posterior adductor muscles and mantle). Instead, others cDNA libraries constructed from single tissues (DiG01, DiG02, Hae00, Hae05) or from mussels treated with organic compounds and heavy metals (GDG01) or heat-inactivated bacteria (Gll01, Hae01, Hae02, Hae03, Hae04) have been sequenced to a less extent because of the more committed nature of their transcriptome.
In the next future we are planning to apply a round of large-scale sequencing using second-generation of high throughput DNA sequencers (e.g. Roche 454) to the normalized cDNA library to fully exploit the information contained in it.
Functional annotation of ESTs and construction of the M. galloprovincialis transcript catalogue
Overall, 54% of non-redundant sequences (3,837 out of 7,112) identified by about 40% of total ESTs (7,694 out of 18,788) showed no or poor similarity with publicly available sequences. These unknown mussel transcripts support the discovery of new genes, and possibly new gene networks and metabolic pathways in mussel. Interestingly, three unknown transcripts are among the 20 most expressed genes: MGC00007 (188 ESTs), MGC00293 (61 ESTs) and MGC00279 (54 ESTs). However, we realize that some ESTs are relatively short and falling within the 3'-untranslated regions, thus their identities could probably not be easily revealed by sequence similarity comparison . A large number of ESTs with no similarity hit is a common feature of studies on mollusk species [39, 59, 60], probably because of the great level of amino acid divergence found between invertebrates and the reference taxa currently used in genomics.
A significant number of stress-, immune-, and defense-related transcripts were putatively identified from the systematic sequencing of five haemocyte cDNA libraries from healthy (Hae01, Hae05) bacterial-treated (Hae02, Hae03) or poly I:C treated mussels (Hae04). Small cationic antimicrobial peptides (AMPs) such as Myticin A, Myticin C, Mytilin B and Mytilin C were highly represented in our EST catalogue (0.4%, 0.8%, 1.0%, and 0.7% of total ESTs, respectively). We have also identified different types of lectins (C-type lectin, sialic acid binding lectin, fucolectin, galectin) that are molecules mediating agglutination processes acting as opsonins , thus playing a decisive role in the humoral defense against pathogenic organisms. Studies on the expression of these genes could improve the general understanding of the innate immune response and defense mechanisms in mussels.
Mussel of the Mytilus genus are sessile shells and, interestingly, all collagen precursors of the adhesive apparatus (byssus) such as the proximal collagen (PreCol-P), distal collagen (PreCol-D) and pepsin-resistant nongradient collagen (PreCol-NG) have been identified in our libraries. The elastic domains of PreCol-P, the silk fibroin-like domains of PreCol-D, and the plant cell wall-like domains of PreCol-NG characterize the unique, collagenous block copolymer found in the byssus threads of mussel. Moreover, we identified three members of the protein family secreted by the mussel foot (Mgfp3, Mgfp4, Mgfp6), that are located in the adhesive plaque, providing adhesiveness and strength to the fibrous collagen core of byssus threads .
The 3,837 consensus sequences with no significant similarities were locally searched for conserved protein domains using the InterPro tool  in order to find some clues for their possible biological role and generally to identify interesting candidates for functional studies in the next future. Interestingly, this approach has led us to the identification of 26 transcripts containing universal stress protein-like domains (Usp) and two transcripts (MGC03893 and MGC01634) that present a domain conserved in a number of proteins involved in heavy metal transport or detoxification . The study of these transcripts could be useful to increase the knowledge about the physiological and genetic mechanisms activated by mussels in response to toxic pollutants (heavy metals in particular).
Annotation of M. galloprovincialis ESTs
"Metabolic process" was the second most abundant GO subcategory in Biological Process, with 66% of annotated mussel transcripts assigned to it. A large majority of transcripts (48%) belonging to macromolecule metabolic process showed putative identity with ribosomal sequences and genes connected to the translation machinery. We found genes with regulative functions in the translational initiation, like translation factor SUI1, initiation factor 2A, 3D, 3E, 3M, 4E, 5A and elongation, like elongation factor 1α, 1γ, 2, Ts (mitochondrial). We were also able to identify some ubiquitin protein ligases (Bre1, MIB1, NRDP1, RING2, RNF19A, SIAH1, UBR2, UBR3, UBR5) and two members of the proteasome complex (subunit α and β) involved respectively in tagging and degradation of unneeded or damaged proteins . Other mussel transcripts appear to be involved in the mechanisms of DNA transcription (DNA polymerase II, mediator of RNA polymerase II, transcription elongation factor B, SPT5, SPT6, transcription initiation factor IIA, IID (subunit 1, 8, 9, 11) and transcription intermediary factor 1 β).
Eight percent of the annotated transcripts were assigned to the Biological Process subcategory named "response to stimulus". This class includes a set of genes recruited in stress responses and potentially useful in environmental studies such as heat shock proteins (HSP25, HSP60, HSP70, HSP71, HSP90), metallothioneins (MT10III, MT20II), ferritin, cytochrome P450 and glutathione S-transferase (GST). Ferritin in fact plays a key role in the metabolism of cellular iron including storage and detoxification [70, 71] and it is also involved in shell formation by iron storage . Metallothioneins are ubiquitous metal-binding proteins that function in the homeostasis of essential metals, such as zinc and copper, as well in detoxification mechanisms by sequestering toxic metals such as cadmium, lead, and mercury [73, 74]. Cytochrome P450 isoforms are involved in the metabolism of xenobiotics, such as polycyclic aromatic hydrocarbons [75, 76] while GST isoforms are important in the metabolism of organochlorinated compounds and other chemicals . Both enzymes have been used in mollusks as biomarkers for the assessment of coastal water contaminated by these pollutants . The systematic sequencing also identified some antioxidant enzymes such as thioredoxin, thioredoxin reductase, glutathione peroxidase and superoxide dismutase that are involved in the oxidative stress responses .
Some annotated transcripts such as matrix metalloproteinase 1, metalloproteinase inhibitor 2, and metalloproteinase inhibitor 3, were classified in the organism development GO subcategory. They are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes and their inhibitors .
"Protein binding" resulted as the most common GO term (24%) associated to the 2,266 consensus sequences which were assigned to the Molecular Function category in GO slims, followed by the "hydrolase" (17%) and "nucleotide binding" (15%) terms.
Identification of M. galloprovincialis microsatellites
Simple sequence tandem repeats (SSR), also known as microsatellites, are an excellent source of genetic markers to use in linkage mapping, parentage assignment and population genetics . Among the 7,112 non-redundant sequences examined in this study, we identified 154 (2.2%) consensuses containing SSR by using MISA software . Five of these present 2 or 3 distinct simple sequence repeats interrupted by more than 100 bp for a total of 159 identified SSR. The most frequent motifs are di- (27.7%) and tri- (61.0%) nucleotides with a prevalence of TA (15 out of 44), AT (15 out of 44), AGC (17 out of 97) and CAA (10 out of 97) respectively. The 5' and 3' SSR flanking regions have an average length of about 330 bp and only 33 repeats show flanking region shorter than 50 bp, making the design of primers difficult. A list of all microsatellite-containing ESTs is presented in the Additional file 2. Overall, 62,3% (99 out of 159) of non-redundant sequences containing microsatellites showed no or poor similarity in the nucleotide and protein databases. Sixteen sequences are similar to precollagen protein and share repetitive motifs generally found in this class of proteins . Comparing our microsatellite sequences with those described in a recent work on mussel EST-SSRs  we conclude that we have identified about 50 useful markers for genetic studies of mussel populations. Since our novel microsatellite markers were developed on the basis of expressed sequences and they are presumably conserved across other Mytilus species, they could also be useful for comparative mapping and for a molecular approach to mussel ecology.
Comparative analysis of M. galloprovincialis and M. californianus EST sequences
Recently, Gracey and colleagues have deposited 22,836 5'- and 3'-end ESTs of Mytilus californianus in the EBI-GenBank-DBJ database. Taking advantage of this data, we compared the transcribed genomes of M. californianus and M. galloprovincialis in order to verify the level of divergence between these two occasionally sympatric species (i.e. California Bay, North America). For this purpose, following the process described in the Methods, we have aligned 22,836 M. californianus ESTs sequences together with our 18,788 Mediterranean mussel ESTs. Since this process generated only 1,054 hybrid clusters, we can assume that, at nucleotide level, the two mussel datasets seem to be very different. However, the number of hybrid clusters may be influenced by some technical issues such as the different methodology for cDNA library construction and different parameters applied for base calling and trimming of M. galloprovincialis or M. californianus chromatogram traces. Recently, the full protein sequences for preCol D, NG and P from M. californianus, M. edulis and M. galloprovincialis have been compared. In agreement with our evidences, the preCols from M. californianus are more divergent from the other two closely related species . The genetic divergence between M. californianus and M. galloprovincialis is supported also by the clearly different external morphology and by the lack of cross-hybridization in spite of sympatric localizations, in contrast of M. trossulus and M. galloprovincialis species [85, 86]. Furthermore, analysis of mitochondrial DNA sequences has revealed that M. californianus is the most divergent of mussel species, whereas M. edulis and M. galloprovincialis are the most similar . Analysis of 18S rDNA sequences again showed that M. californianus is the most divergent species .
The genome sequence of Mediterranean mussels is not yet available and therefore the systematic sequencing of cDNA libraries of these invertebrates represents a powerful approach to identify large numbers of transcripts that could be used in gene expression and functional genomics studies  and also a first step toward the deciphering of the complete mussel genome. We have produced and sequenced 17 cDNA libraries from different M. galloprovincialis tissues, obtaining 18,788 high-quality ESTs that identify 7,112 unique transcribed sequences. In particular, a highly effective normalized cDNA library (Nor01) was constructed, as demonstrated by its high gene discovery rate (65.6%). Over 54% of the M. galloprovincialis transcribed sequences resulted in no BLAST matches with published sequences and they probably represent novel genes that could be targeted for functional studies. Of the 7,112 unique sequences, the majority (5,400) were novel ESTs for this species. Moreover, the alignment of sequences from M. californianus and M. galloprovincialis EST collections resulted in only 1,054 clusters composed by ESTs from both species. Despite possible difference in sample origin and sequence processing, this data has two implications. First, despite the evolutionary and geographical vicinity, these two species appear transcriptionally different. Second, global transcriptome analysis in mussels could make use of specie-specific microarray platforms. In oyster species the level of cross-hybridization between C. gigas and C. virginica was shown to be 30–40% using a microarray platform with sequences derived from cDNA libraries of both species . Therefore, our collection represents a significant addition to the existing genomic resources for the Mediterranean mussel and generally for Mytilus species. All sequencing data have been organized in a dedicated database available from our web site . This EST collection is also a potential source for the development of genetic markers including microsatellite and single nucleotide polymorphisms. Among the 7,112 unique sequences, 159 (2%) unique microsatellite containing ESTs were identified by using MISA software. On the basis of the cluster consensus sequences, we are now producing a M. galloprovincialis microarray platform with transcript-specific oligonucleotide probes. The information contained in our database will therefore provide a valuable resource for future studies of mussel transcriptional responses to various biological conditions such as environmental challenges , morphological development and bacterial or viral infections.
Availability and requirements
Project name: Generation and analysis of ESTs from the Mediterranean mussel (Mytilus galloprovincialis);
Project home page: http://mussel.cribi.unipd.it;
Operating system(s): Debian GNU/Linux;
Programming language: PHP;
Any restrictions to use by non-academics: none. Users can obtain a personal account and full access to MytiBase by free subscription.
This work was supported by the EC Integrated Project IMAQUANIM (FOOD-CT-2005-007103), Quicksilver Biotech Action II (CIPE 20/04; DGR 643/04.03.2005 Veneto Region) and Biotech Action III bis (CIPE 3/06 DGR 4073 19/12/2006 Veneto Region).
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