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A Microsatellite Genetic Map of the Turbot (Scophthalmus maximus)

A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 ± 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, ∼1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81–100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species.

Polymorphic microsatellite markers for blue mussels (Mytilus spp.)

Blue mussels of the genus Mytilus (M. edulis Linnaeus 1758; M. galloprovincialis Lamarck 1819; M. trossulus Gould 1850) are widely distributed in Southern and Northern hemispheres. This ecological plasticity together with the existence of interspecific hybridization in overlapping regions (Skibinski et al. 1978) makes them an interesting model for studies of population dynamics in marine habitats. Genetic marker surveys on Mytilus spp. have shown the genetic uniqueness of each species (McDonald and Koehn 1988). Several molecular markers have been developed over the last three decades to describe genetic polymorphisms in mussels. However most of them have a number of disadvantages for use as population markers, relating to data interpretation, technical difficulties, genotype discrimination, and restricted amount of polymorphism (Ohresser et al. 1997). In order to analyze intraspecific phenomena related to both natural genetic substructuring and anthropogenic changes, it is useful to develop highly informative markers, such as microsatellites.

Validation of a tRNA-Glu-cytochrome b Key for the Molecular Identification of 12 Hake Species (Merluccius spp.) and Atlantic Cod (Gadus morhua) Using PCR-RFLPs, FINS, and BLAST

The goal of this study was to develop a diagnostic key for hake meat to solve the limitations of previous identification methodologies, mainly related to the high degradation of the DNA recovered from processed foods. We describe the development of two molecular tools based on polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphisms of the cytochrome b gene, respectively, to identify DNA from 12 hake species in commercial products. The first assay is an exclusion test consisting of the PCR amplification of a 122 bp fragment using nested primers interspecifically conserved in Merluccius spp. and in Gadus morhua. This 122 bp amplicon, being the shortest one so far designed for hake DNA, is a useful traceability tool for highly degraded samples because its sequence contains enough interspecific diagnostic variation to identify 10 hake species and cod and has been successfully amplified from most commercial products so far tested. The second identification key follows a positive outcome of the exclusion test and consists of the PCR amplification of a 464-465 bp fragment and its digestion with three restriction enzymes whose targets map at interspecifically nonconserved sites of the cytochrome b. The key presented here has passed through a rigorous methodological calibration including its testing for genus specificity, its validation on a large number of authenticated sample types from each species range, and its implementation with a maximum likelihood method for the assignment of unknown samples. Together, these two procedures constitute the most complete molecular key so far developed for Merluccius spp., which is optimal for routine identification of hakes in large commercial samples at a reasonable cost-time ratio.

Development and characterization of 248 novel microsatellite markers in turbot (Scophthalmus maximus)

The turbot is a flatfish species of great relevance to marine aquaculture in Europe. Only a limited number of microsatellites have been isolated to date in this species. To increase the number of potentially useful mapping markers, we screened simple sequence repeat (SSR)--enriched genomic libraries obtained from several di-, tri-, and tetranucleotide tandem repeat motifs. A total of 248 new polymorphic microsatellites were successfully optimized. The efficiency of the protocol applied (6.4%) was higher than that in other studies of fish that used the same method. Dinucleotide and perfect microsatellites were predominant in this species; the (AC)n motif was the most frequent class of repeat. Polymorphism and structural properties at these loci, together with 30 variable loci previously reported in turbot, were evaluated in 6 wild individuals. The number of alleles per locus ranged from 2 to 10, with an average of 4.046. The microsatellite markers characterized in this study will contribute to the development of the turbot genetic map, which can be used for quantitative trait locus (QTL) identification, marker-assisted selection programs, and other applications to improve its culture.

Microsatellites of Mytilus chilensis: A Genomic Print of Its Taxonomic Status within Mytilus sp.

ABSTRACT The taxonomic status of the Chilean blue mussel Mytilus chilensis has been controversial for decades because of its phenotypic and genetic proximity to other species of the genus Mytilus from both hemispheres. This study reports the development of nine polymorphic microsatellite markers from the M. chilensis genome. The number of alleles per locus ranged between four and nine, and the observed heterozygosity ranged from 0.182–0.750 in a wild sample of 24 individuals from Caicaén (Chiloé Region, Chile). Lack of efficient amplification of many of these microsatellite loci in other Mytilus species suggests that M. chilensis is a valid, distinct species within the genus. These new markers would be useful in fine-scale population analyses of M. chilensis as well in the aquaculture management of this marine resource.

Development of microsatellite loci for the black-footed limpet, Patella depressa , and cross-amplification in two other Patella species

Limpets (Patella spp.) are suitable organisms to investigate the effects of climate change in marine systems. They are widespread over NE Atlantic intertidal rocky shores and have been extensively studied in terms of population dynamics and ecology. Within genus Patella, microsatellites have only been developed for Patella caerulea and cross-species tests are unknown. In this work, we describe 11 primer pairs for Patella depressa and the results of cross-species testing on Patella candei and Patella rustica.

Identification of South Atlantic Hakes (Merluccius australis and Merluccius hubbsi)in Processed Foods by PCR-RFLPs of Cytochrome bGene

Abstract The application of molecular genetics to the identification of fishes is an appropriate technology for quality control and commercial tracking. We have applied a DNA-based methodology to identify the exact species of Argentinean hake included in commercial products. The first test consisted in the PCR amplification of a 122 bp fragment from the cytochrome bgene to assess the presence of DNA from hakes. This test being positive, it was followed by a second test consisting of the amplification and digestion of a PCR fragment of 464 bp from the cytochrome bgene, to determine the identity of the commercialized species. Since a single restriction enzyme provided full diagnosis of the southern Atlantic hake species present in the samples, this methodology would be of great help in traceability across the food-chain that comprises (1) the authentication of Argentinean hake products, (2) the control of fraud to consumers, (3) the industrial advice regarding the labeling of commercial products, and (4) the fishery forensics of Argentinean hakes.

Experimental Assessment of a New rDNA-Based Method for the Identification of Merluccius capensisand Merluccius paradoxusin Commercial Products

Abstract There has been great interest in the identification of Cape hakes, Merluccius capensisand Merluccius paradoxus, by non-morphological methods. The information provided by a ribosomal DNA-based method will allow investigators to precisely identify the species of Cape hake in commercial samples. The first test consisted in the PCR amplification of a 193 bp fragment from the ITSl-rDNA to assess the presence of DNA from hakes. Positive tests were followed by a second test consisting of the amplification and digestion of the PCR fragment from the ITS1 sequence, to determine the identity of the commercial species. A single restriction enzyme provided full diagnosis of the species present in the samples. Since this new method is simple, reliable, and reproducible and can be performed in a working day, it allows the genetic traceability of Cape hakes across the food chain. This could also help management of the fisheries.

PERMANENT GENETIC RESOURCES: Development of microsatellite markers for the ecosystem bioengineer mussel Perumytilus purpuratus and cross‐priming testing in six Mytilinae genera

Eight microsatellite markers have been characterized from the Perumytilus purpuratus genome. Their gene diversity ranged from 0.057 to 0.873 and significant interpopulation genic heterogeneity was observed between two populations of southeastern Pacific (Chile) and southwestern Atlantic (Argentine). Distinct cross‐priming amplification rates were recovered on nine additional species belonging to six Mytilinae genera. The microsatellites developed herein would likely be a powerful intraspecific genetic tool to undertake fine population studies in the intertidal ecosystem bioengineer P. purpuratus along the South American shoreline.

A new multiplexed microsatellite tool for metapopulation studies in the overexploited endemic limpet Patella aspera (Röding, 1798)

Patellid limpets are ecologically important keystone grazers having a long history of overexploitation in the Macaronesian Archipelagos (NE Atlantic islands), where some species, such as Patella aspera, are under serious risk.[1, 2] Patella aspera is a protandric sequential hermaphrodite species with external fertilization, in which individuals start off as males but may undergo a sex reversal with age.[3] Hence, exploitation tends to focus on the larger females in the population as larger limpets (predominantly females) are selectively removed. Despite conservation legislation in Canaries, Madeira and Azores, limpets are under severe pressure and few individuals survive long enough to become females, a phenomenon that severely restricts the effective population size.[4] New conservation actions for the protection and sustainable use of limpets in Macaronesian Archipelagos are urgently needed and should be based on a multidisciplinary framework based on knowledge of the population dynamics and connectivity of this species.