Sequencing and characterization of striped venus transcriptome expand resources for clam fishery genetics

Background: The 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 Findings: The 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/Significance: This 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

Variability assessment of 14 SSR loci of <i>Chamelea gallina</i>.

<p>Variability, expressed in terms of number of different alleles, was assessed on 12 individuals collected in Chioggia in 2010 (off Venice lagoon, Italy). The table reports the name of each locus, taken from the contig number, the repeat content, the forward (F) and reverse (R) primer sequences, the fluorescent label, the annealing temperature (Ta) of PCR amplification, the size range of amplified fragments in bp, the allelic range in repeats, the number of alleles (Na) detected and the Hardy-Weinberg probability (pHWE). Significant p-values in bold (α = 0.05). Mean values for allele number, observed and expected heterozygosity are reported in the last row. Standard Deviation is reported in brackets (± SD).</p>*<p>Loci putatively affected by null alleles following MICRO-CHECKER 2.2.3. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044185#pone.0044185-vanOosterhout1" target="_blank">[51]</a>.</p>a<p>p-values were calculated based on a limited number of individuals (n = 12).</p

<i>Chamelea gallina</i> SSR mining results.

<p>The table reports the main results provided by MISA for SSR detection.</p

Length and quality of <i>Chamelea gallina</i> transcriptome contigs.

<p>Panel histograms report the contig's length (a) and average quality (b) distribution, while panel c shows the relationship between length and quality.</p

Putative SNPs identified from <i>Chamelea gallina</i> transcriptome database.

<p>More than 20,000 SNPs were identified out of 36,422 contigs and meta-contigs.</p

Set of annotated contigs of <i>Chamelea gallina</i> trascriptome with nucleotide and protein BLAST hits.

<p>The Venn diagram reports the intersection between number of contigs with BLAST hits in nr protein and nt nucleotide databases.</p

Summary of SSR types and frequency in <i>Chamelea gallina</i> transcriptome.

<p>Summary of SSR types and frequency in <i>Chamelea gallina</i> transcriptome.</p

ChameleaBase.

<p>The screenshots report the <i>Chamelea gallina</i> database online version homepage (on the upper right side), the search facility (bottom right) and an example of the gene-like entry (on the left).</p