Genetic variability in Ruditapes decussatus clam combined with Perkinsus infection level to support founder population selection for a breeding program.

Clam farmers worldwide face several challenges, including irregular seed supply and high mortalities due to pathogenic organisms such as Perkinsus olseni. In Europe, there is a high unmet consumer demand for native clam species such as Ruditapes decussatus. The high market value of R. decussatus makes the culture of this species potentially more attractive than that culture of the alien species Ruditapes philippinarum. Thus, there is a market opportunity in breeding and producing R. decussatus at an industrial scale. A selective breeding program to improve R. decussatus performance will be carried out in Portugal; and the first critical step to develop such a breeding program is the establishment of a founder population. In this study, intra- and interpopulation genetic diversity was assessed using 13 microsatellite markers in eight natural beds located in Portugal, Spain and Italy. Also, allele and genotypic frequencies of each microsatellite locus were assessed discriminating between clams infected and non-infected by P. olseni. All locations showed similar values for several genetic diversity parameters. Analyses of population differentiation (F ST, Bayesian clustering and AMOVAs) revealed five genetically differentiated regions: Rías Altas and Rías Baixas (NW Spain), North/Central Coast of Portugal, Gulf of Cadiz and Adriatic Sea. Significant differences in the allelic and genotypic frequency distribution between infected clams and non-infected ones at four microsatellite loci are reported suggesting that resistance to the disease could have a genetic basis. Moreover, a positive or negative relationship between the frequency of certain alleles and the parasite infection was inferred. Further studies should confirm the potential use of those alleles as genetic markers for P. olseni infection. Integrating results of genetic diversity within and between populations and Perkinsus infection levels, a founder population for a R. decussatus breeding program is proposed, composed by individuals from Barallobre (Rías Altas), Pontevedra or Cangas (Rías Baixas), Óbidos (North/Central Coast of Portugal), Algarve (Gulf of Cadiz) and Venice (Adriatic Sea).

Sequence characterization of the 5S ribosomal DNA and the internal transcribed spacer (ITS) region in four European Donax species (Bivalvia: Donacidae).

BACKGROUND: The whole repeat unit of 5S rDNA and the internal transcribed spacer (ITS) of four European Donax species were analysed. After amplifying, cloning and sequencing several 5S and ITS units, their basic features and their variation were described. The phylogenetic usefulness of 5S and ITS sequences in the inference of evolutionary relationships among these wedge clams was also investigated. RESULTS: The length of the 5S repeat presented little variation among species, except D. trunculus that differed from the rest of the Donax species in 170-210 bp. The deduced coding region covered 120 bp, and showed recognizable internal control regions (ICRs) involved in the transcription. The length of non-transcribed spacer region (NTS) ranged from 157 bp to 165 bp in Donax trunculus and from 335 bp to 367 bp in the other three species. The conservation degree of transcriptional regulatory regions was analysed revealing a conserved TATA-like box in the upstream region. Regarding ITS sequences, the four Donax species showed slight size differences among clones due to the variation occurring in the ITS1 and ITS2, except Donax variegatus did not display size differences in the ITS2. The total length of the ITS sequence ranged between 814 and 1014 bp. Resulting phylogenetic trees display that the two ribosomal DNA regions provide well-resolved phylogenies where the four European Donax species form a single clade receiving high support in nodes. The topology obtained with 5S sequences was in agreement with Donax evolutionary relationships inferred from several sequences of different nature in previous studies. CONCLUSIONS: This is not only a basic research work, where new data and new knowledge is provided about Donax species, but also have allowed the authentication of these wedge clams and offers future applications to provide other genetic resources.

First complete female mitochondrial genome in four bivalve species genus Donax and their phylogenetic relationships within the Veneroida order.

BACKGROUND: Four species of the genus Donax (D. semistriatus, D. trunculus, D. variegatus and D. vittatus) are common on Iberian Peninsula coasts. Nevertheless, despite their economic importance and overexploitation, scarce genetic resources are available. In this work, we newly determined the complete mitochondrial genomes of these four representatives of the family Donacidae, with the aim of contributing to unveil phylogenetic relationships within the Veneroida order, and of developing genetic markers being useful in wedge clam identification and authentication, and aquaculture stock management. PRINCIPAL FINDINGS: The complete female mitochondrial genomes of the four species vary in size from 17,044 to 17,365 bp, and encode 13 protein-coding genes (including the atp8 gene), 2 rRNAs and 22 tRNAs, all located on the same strand. A long non-coding region was identified in each of the four Donax species between cob and cox2 genes, presumably corresponding to the Control Region. The Bayesian and Maximum Likelihood phylogenetic analysis of the Veneroida order indicate that all four species of Donax form a single clade as a sister group of other bivalves within the Tellinoidea superfamily. However, although Tellinoidea is actually monophyletic, none of its families are monophyletic. CONCLUSIONS: Sequencing of complete mitochondrial genomes provides highly valuable information to establish the phylogenetic relationships within the Veneroida order. Furthermore, we provide here significant genetic resources for further research and conservation of this commercially important fishing resource.

Characterization of nineteen microsatellite markers and development of multiplex PCRs for the wedge clam Donax trunculus (Mollusca: Bivalvia).

The wedge clam Donax trunculus is an Atlantic-Mediterranean warm-temperate species found from Senegal to the northern coast of France, including the Mediterranean and Black Sea. It is commercially exploited in several European countries and constitutes an important fishing resource due to its high economical value. To contribute to its conservation and management, nineteen microsatellite markers were isolated from two enriched genomic libraries. These loci were characterized in 30 clams from a single population from northwest Spain. The number of alleles per locus ranged from 2 to 17 and observed and expected heterozygosity varied from zero to 0.714 and from 0.078 to 0.950, respectively. Linkage disequilibrium was not detected and nine loci were in agreement with Hardy-Weinberg equilibrium. Fifteen polymorphic markers were arranged into three multiplex PCR sets to reduce both time and cost of microsatellite genotyping. This is the first time that polymorphic microsatellite markers have been reported for D. trunculus. These new markers provide a valuable resource for future population genetics studies and management and culture of this species.