Dataset Information

Development of molecular resources for an intertidal clam, Sinonovacula constricta, using 454 transcriptome sequencing.

ABSTRACT:

Background

The razor clam Sinonovacula constricta is a benthic intertidal bivalve species with important commercial value. Despite its economic importance, knowledge of its transcriptome is scarce. Next generation sequencing technologies offer rapid and efficient tools for generating large numbers of sequences, which can be used to characterize the transcriptome, to develop effective molecular markers and to identify genes associated with growth, a key breeding trait.

Results

Total RNA was isolated from the mantle, gill, liver, siphon, gonad and muscular foot tissues. High-throughput deep sequencing of S. constricta using 454 pyrosequencing technology yielded 859,313 high-quality reads with an average read length of 489 bp. Clustering and assembly of these reads produced 16,323 contigs and 131,346 singletons with average lengths of 1,376 bp and 458 bp, respectively. Based on transcriptome sequencing, 14,615 sequences had significant matches with known genes encoding 147,669 predicted proteins. Subsequently, previously unknown growth-related genes were identified. A total of 13,563 microsatellites (SSRs) and 13,634 high-confidence single nucleotide polymorphism loci (SNPs) were discovered, of which almost half were validated.

Conclusion

De novo sequencing of the razor clam S. constricta transcriptome on the 454 GS FLX platform generated a large number of ESTs. Candidate growth factors and a large number of SSRs and SNPs were identified. These results will impact genetic studies of S. constricta.

SUBMITTER: Niu D

PROVIDER: S-EPMC3723811 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Development of molecular resources for an intertidal clam, Sinonovacula constricta, using 454 transcriptome sequencing.

Niu Donghong D Wang Lie L Sun Fanyue F Liu Zhanjiang Z Li Jiale J

PloS one 20130725 7

<h4>Background</h4>The razor clam Sinonovacula constricta is a benthic intertidal bivalve species with important commercial value. Despite its economic importance, knowledge of its transcriptome is scarce. Next generation sequencing technologies offer rapid and efficient tools for generating large numbers of sequences, which can be used to characterize the transcriptome, to develop effective molecular markers and to identify genes associated with growth, a key breeding trait.<h4>Results</h4>Tota ...[more]

PMID: 23935831

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Project description:The Chinese razor clam (Sinonovacula constricta) is an important for Chinese aquaculture marine bivalve that naturally occurs across intertidal and estuarine areas subjected to significant changes in salinity level. However, the information on the molecular mechanisms related to high salinity stress in the species remain limited. In this study, nine gill samples of S. constricta treated with 20, 30, and 40 ppt salinity for 24 h were used for whole-transcriptome RNA sequencing, and a regulatory network of competing endogenous RNAs (ceRNAs) was constructed to better understand the mechanisms responsible for adaptation of the species to high salinity. A total of 83,262 lncRNAs, 52,422 mRNAs, 2890 circRNAs, and 498 miRNAs were identified, and 4175 of them displayed differential expression pattern among the three groups examined. The KEGG analyses of differentially expressed RNAs evidenced that amino acid synthesis and membrane transport were the dominant factors involved in the adaptation of the Chinese razor clam to acute salinity increase, while lipid metabolism and signaling played only a supporting role. In addition, lncRNA/circRNA-miRNA-mRNA regulatory networks (ceRNA network) showed clearly regulatory relationships among different RNAs. Moreover, the expression of four candidate genes, including tyrosine aminotransferase (TAT), hyaluronidase 4 (HYAL4), cysteine sulfinic acid decarboxylase (CSAD), and ∆1-pyrroline-5-carboxylate synthase (P5CS) at different challenge time were detected by qRT-PCR. The expression trend of TAT and HYAL4 was consistent with that of the ceRNA network, supporting the reliability of established network. The expression of TAT, CSAD, and P5CS were upregulated in response to increased salinity. This might be associated with increased amino acid synthesis rate, which seems to play an essential role in adaptation of the species to high salinity stress. In contrast, the expression level of HYAL4 gene decreased in response to elevated salinity level, which is associated with reduction Hyaluronan hydrolysis to help maintain water in the cell. Our findings provide a very rich reference for understanding the important role of ncRNAs in the salinity adaptation of shellfish. Moreover, the acquired information may be useful for optimization of the artificial breeding of the Chinese razor clam under aquaculture conditions.

Project description:Bone morphogenetic proteins (BMPs) not only play essential roles in bone development but also are involved in embryonic growth, organogenesis cell proliferation and differentiation. However, the previous studies on the functions of shellfish BMPs genes are still very limited. To better understand its molecular structure and biological function, BMP7 of the razor clam Sinonovacula constricta (Sc-BMP7) was cloned and characterized in this study. The full length of Sc-BMP7 is 2252 bp, including an open reading frame (ORF) of 1257 bp encoding 418 amino acids. The protein sequence included a signal peptide (1-32 aa), a prodomain (38-270 aa) and a TGF-β domain (317-418 aa). The quantitative expression of eleven adult tissues showed that Sc-BMP7 was significantly higher expressed in the gill, foot, and mantle (P < 0.05), but lower in hemocytes and hepatopancreas. In the early development stages, low expression was detected in the stages of unfertilized mature eggs, fertilized eggs, 4-cell embryos, blastula, gastrulae, whereas it increased after the stage of trochophore and demonstrated the highest expression in umbo larvae (P < 0.01). In shell repair experiment, Sc-BMP7 showed increasing expression level after 12 h. The higher expression of Sc-BMP7 was detected while Ca2+ concentration was reduced in seawater. After inhibiting Sc-BMP7 expression using RNA interference (RNAi) technology, expression of Sc-BMP7 mRNA and protein were significantly down-regulated (P < 0.05) in the central zone of mantle (nacre formation related tissue) and the pallial zone of mantle (prismatic layer formation related tissue). Association analysis identified two shared SNPs in exon of Sc-BMP7 gene from 246 individuals of two groups. These results indicated that BMP7 might be involved in shell formation and growth. These results would contribute to clarify the role of Sc-BMP7 in the regulation of growth and shell formation, and provide growth-related markers for molecular marker assisted breeding of this species.

Project description:BackgroundThe striped venus Chamelea gallina clam fishery is among the oldest and the largest in the Mediterranean Sea, particularly in the inshore waters of northern Adriatic Sea. The high fishing pressure has lead to a strong stock abundance decline, enhanced by several irregular mortality events. The nearly complete lack of molecular characterization limits the available genetic resources for C. gallina. We achieved the first transcriptome of this species with the aim of identifying an informative set of expressed genes, potential markers to assess genetic structure of natural populations and molecular resources for pathogenic contamination detection.Methodology/principal findingsThe 454-pyrosequencing of a normalized cDNA library of a pool C. gallina adult individuals yielded 298,494 raw reads. Different steps of reads assembly and filtering produced 36,422 contigs of high quality, one half of which (18,196) were annotated by similarity. A total of 111 microsatellites and 20,377 putative SNPs were identified. A panel of 13 polymorphic transcript-linked microsatellites was developed and their variability assessed in 12 individuals. Remarkably, a scan to search for contamination sequences of infectious origin indicated the presence of several Vibrionales species reported to be among the most frequent clam pathogen's species. Results reported in this study were included in a dedicated database available at http://compgen.bio.unipd.it/chameleabase.Conclusions/significanceThis study represents the first attempt to sequence and de novo annotate the transcriptome of the clam C. gallina. The availability of this transcriptome opens new perspectives in the study of biochemical and physiological role of gene products and their responses to large and small-scale environmental stress in C. gallina, with high throughput experiments such as custom microarray or targeted re-sequencing. Molecular markers, such as the already optimized EST-linked microsatellites and the discovered SNPs will be useful to estimate effects of demographic processes and to detect minute levels of population structuring.

Dataset Information

Development of molecular resources for an intertidal clam, Sinonovacula constricta, using 454 transcriptome sequencing.

Background

Results

Conclusion

Publications

Development of molecular resources for an intertidal clam, Sinonovacula constricta, using 454 transcriptome sequencing.

Similar Datasets

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