Ruth Freire

The 5S rDNA of mussels Mytilus galloprovincialis and M. edulis: sequence variation and chromosomal location

The 5S ribosomal DNA of the mussels Mytilus galloprovincialis and M. edulis was amplified by PCR using contiguous primers. The most general 5S rDNA amplification pattern consisted of several products in both mussels. Two main PCR products of about 250 bp and 760 bp were cloned and sequenced, revealing two classes of 5S rDNA units. These were characterized as containing an identical coding region of 119 bp but with highly divergent spacers. Clones of each unit type exhibited minimal differences except those of the large unit of M. edulis. The sequences analysed of the two mussels possess the same coding region and only six fixed base changes on the spacers. FISH, carried out with specific probes, consistently showed hybridization signals on the largest metacentric pair (two differentiated sites) and with variable frequency on two other metacentric pairs (one site on each pair). Differences in the 5S rDNA distribution between both mussels were not found. In M. edulis, chromosomes carrying 18S-28S rDNA were also identified by FISH. These correspond to two submetacentric–subtelocentric pairs, as was previously reported in M. galloprovincialis, demonstrating that the two rDNA multigene families are located on different chromosome pairs in these mussels.

The 5S rDNA of the bivalve Cerastoderma edule: nucleotide sequence of the repeat unit and chromosomal location relative to 18S–28S rDNA

The whole 5S rDNA repeated unit of the bivalve Cerastoderma edule was amplified by PCR and several clones were sequenced. In addition, the PCR product from several individuals was digested with restriction enzymes. The results obtained indicate that 5S rDNA is organized in tandem repeats of 544-546 bp, 120 of which could represent the coding region and 424-426 the spacer region. Minimal intra- and inter-individual variation was detected, always within the spacer region. In comparison to the published 5S rRNA sequences of three other bivalves, C. edule displays a maximum of four different nucleotide positions. A specific probe of C. edule 5S rDNA was generated by PCR and used for FISH. Five chromosome pairs were identified that carried a cluster of 5S rDNA at the telomere of the long arm. After performing FISH with a heterologous 18S-28S rDNA probe and C-banding, absence of linkage between 5S and 18S-28S rDNA was demonstrated.

Evolutionary Dynamics of the 5S rDNA Gene Family in the Mussel Mytilus: Mixed Effects of Birth-and-Death and Concerted Evolution

In higher eukaryotes, the gene family encoding the 5S ribosomal RNA (5S rRNA) has been used (together with histones) to showcase the archetypal example of a gene family subject to concerted evolution. However, recent studies have revealed conspicuous features challenging the predictions of this model, including heterogeneity of repeat units, the presence of functional 5S gene variants as well as the existence of 5S rDNA divergent pseudogenes lacking traces of homogenization. In the present work, we have broadened the scope in the evolutionary study of ribosomal gene families by studying the 5S rRNA family in mussels, a model organism which stands out among other animals due to the heterogeneity it displays regarding sequence and organization. To this end, 48 previously unknown 5S rDNA units (coding and spacer regions) were sequenced in five mussel species, leading to the characterization of two new types of units (referred to here as small-β 5S rDNA and γ-5S rDNA) coexisting in the genome with α and β rDNA units. The intense genetic dynamics of this family is further supported by the first description of an association between γ-5S rDNA units and tRNA genes. Molecular evolutionary and phylogenetic analyses revealed an extensive lack of homology among spacer sequences belonging to different rDNA types, suggesting the presence of independent evolutionary pathways leading to their differentiation. Overall, our results suggest that the long-term evolution of the 5S rRNA gene family in mussels is most likely mediated by a mixed mechanism involving the generation of genetic diversity through birth-and-death, followed by a process of local homogenization resulting from concerted evolution in order to maintain the genetic identities of the different 5S units, probably after their transposition to independent chromosomal locations.

Identification of razor clams Ensis arcuatus and Ensis siliqua by PCR–RFLP analysis of ITS1 region

Ensis arcuatus and Ensis siliqua are the most economically important species of razor clams in the European Union. Due to similarities between their shell morphology, and the differing retail value, these species are often misidentified. Therefore, it is necessary to develop an appropriate protocol to allow accurate differentiation between these species of razor clam in order to protect consumer rights, avoid commercial fraud (whether intentional or unintentional), and to enforce labeling and safety regulations. With the aim of developing a rapid and reliable method of differentiation, individuals of E. arcuatus and of E. siliqua were examined by polymerase chain reaction restriction fragment polymorphism (PCR-RFLP) using the internal transcribed spacer region 1 (ITS1). A species-specific restriction endonuclease pattern was found with the enzyme Kspl for both species, allowing their exact identification. Thus, this work provides a simple, reliable and rapid protocol for accurate discrimination between E. arcuatus and E. siliqua, which proves useful for traceability and enabling the enforcement of labeling regulations.

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.

Identification of four Donax species by PCR–RFLP analysis of cytochrome c oxidase subunit I (COI)

Four Donax species, D. semistriatus, D. trunculus, D. variegatus and D. vittatus, are found on European coasts. Nevertheless, despite their economic importance there is not a reliable method to differentiate these species independently of their size or condition. Such a method could help to protect consumer rights and avoid commercial fraud due to the replacement of valuable species by less valuable ones with similar morphological traits. In this work, the sequence of the mitochondrial cytochrome oxidase subunit I region was examined in individuals of these species to identify restriction site variation and develop polymerase chain reaction–restriction fragment length polymorphisms assays. Species-specific restriction endonuclease patterns were found with the enzymes AluI, HaeIII and MspI, allowing an exact identification of Donax species. This methodology provides simple, reliable and cost-effective identification of four Donax species and may be useful to prevent commercial fraud and to increase food traceability.

Sequence characterization and phylogenetic analysis of the 5S ribosomal DNA in some scallops (Bivalvia: Pectinidae)

This study was designed to characterize the 5S ribosomal DNA repeat unit and to evaluate its phylogenetic informativeness in Mimachlamys varia, Hinnites distortus, Aequipecten opercularis and Pecten maximus, scallops of the bivalve family Pectinidae. The repeat unit was PCR amplified and several products cloned and sequenced. The deduced coding region covered 120 bp, showed 52-53% GC content and an identifiable internal promoter. The spacer region was 313-345 bp in length with 30-40% GC and the ends contained elements showing similarity with upstream and downstream sequences involved in the transcription. The sequences of P. maximus were identical and those of M. varia and H. distortus differed only at 2-3 positions. However, the sequences of A. opercularis showed a percentage of differences of 0.69-9.15%, distinguishing two types of units differing in sequence and length of the spacer region. All scallops displayed an identical gene sequence, except M. varia that differed by one nucleotide substitution, but a highly variable spacer. In the phylogenetic trees the sequences grouped by species with two well supported clades, one containing the sequences of M. varia and H. distortus and the other those of P. maximus and A. opercularis, the latter grouped according to the unit type. The topology obtained was in accordance with scallop evolutionary relationships inferred from previous phylogenetic studies.

Intron characterization and their potential as molecular markers for population studies in the scallops Aequipecten opercularis and Mimachlamys varia.

Exon-primed intron-crossing PCR was used on the European commercial scallops Aequipecten opercularis and Mimachlamys varia to characterize introns of four nuclear genes and to identify DNA markers useful for population studies. The primers used yielded the expected product, except those for the lysozyme gene that failed to work in M. varia and amplified a fragment of a proteasome subunit gene (APSM) in A. opercularis. According to the sequences characterized, A. opercularis has at least four calmodulin genes, one of arginine kinase and two of beta-tubulin, and M. varia five, one and one, respectively. Length polymorphism or/and restriction fragment length polymorphism was detected at two loci of A. opercularis (arginine kinase and APSM) and four of M. varia (calmodulin and beta-tubulin), distinguishing in each case two or three alleles. The polymorphic loci were not closely linked. The population survey included four localities from Spain and one from Northern Ireland for A. opercularis and two Spanish localities for M. varia. Observed heterozygosity (H(o)) per locus was 0.276 and 0.296 in A. opercularis. The Northern Ireland sample had the lowest H(o) value (0.200) and the Mediterranean Spanish sample the highest (0.350). In M. varia, H(o) per locus ranged from 0.172 to 0.391 and the two localities showed similar H(o) values (0.255 and 0.293). All population-locus combinations were in agreement with Hardy-Weinberg equilibrium, except two loci of M. varia that showed a strong heterozygote deficit in the two localities examined. Evidence for genetic differentiation among samples was not found.

Identification, Inheritance, and Variation of Microsatellite Markers in the Black Scallop Mimachlamys varia

Five polymorphic microsatellite loci were identified in the black scallop Mimachlamys varia after construction of a genomic library enriched for (GT)n. To examine the transmission pattern of microsatellite alleles, several families were created and genotypes scored for three loci. The expected Mendelian ratios were found in 12 of 14 segregations examined. Unexpected segregations may be explained by a genotyping error (allelic dropout), given that when a specific allele was treated as dominant, the phenotypic ratios conformed to Mendelian expectations. The five loci were also examined in two samples from the Spanish coast. The two localities displayed similar mean values for the number of alleles per locus (7.2–8.4), allelic richness (7.2–7.9), and observed (0.389–0.484) and expected heterozygosity (0.545–0.618). Significant Hardy–Weinberg deviations were observed at three loci, with heterozygote deficiency occurring in all cases. Global multilocus θ value and allele frequencies at one locus revealed significant differentiation between the two localities.

Isolation of twelve microsatellite markers in the pullet carpet shell Venerupispullastra (Bivalvia: Veneridae)

In this paper we report 12 polymorphic microsatellite loci isolated for the pullet carpet shell Venerupis pullastra, a commercial important clam in Europe. All loci were tested on a minimum of 45 individuals from O Barqueiro (NW Spain). The number of alleles ranged from three to 28. Observed and expected heterozygosity ranged from 0.074 to 0.727 and from 0.073 to 0.958, respectively. No linkage disequilibrium between loci was detected and nine of them conformed to Hardy–Weinberg equilibrium. These 12 loci will be useful for future population genetic studies in V. pullastra.