Genetic Diversity of Marine Populations

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Population and Evolutionary Genetics and Genomics".

Deadline for manuscript submissions: closed (15 March 2020) | Viewed by 44087

Special Issue Editors


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Guest Editor
Ifremer, France
Interests: population genetics; marine species; aquaculture practices

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Co-Guest Editor
Ifremer, France
Interests: genetics; genomics; bivalves

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Co-Guest Editor
Université de Bretagne Occidentale, Brest, France
Interests: connectivity; adaptation; phylogeography; genetics; genomics; fishes; invertebrates

Special Issue Information

Dear Colleagues,

Marine biodiversity is declining at an unprecedented rate directly due to human activities such as fishing, coastal development, pollution, invasive species and climate change. Notwithstanding, the drivers of this decline are poorly understood and our ability to predict the future evolution of marine species is often limited. Notably, the processes shaping population genetic diversity and its possible erosion remain poorly understood in many marine species. However, such knowledge is critical to better preserve marine populations and related ecosystem services. This Special Issue will gather articles on the intraspecific component of this diversity under the title "Genetic Diversity of Marine Populations". Articles are expected to cover a broad range of native or naturalized marine species, cultivated or not, living in all types of marine ecosystems. This Special Issue of Genes aims at addressing a range of relevant topics in marine population genetics such as genetic diversity and resilience of marine populations, relationships between population genetic diversity and ecosystem services, the effects of global climate change and local environmental pressures, effects of fishing pressure and aquaculture practices.

Dr. Sylvie Lapègue
Dr. Pierre Boudry
Dr. Grégory Charrier
Guest Editors

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Keywords

  • marine diversity
  • population genetics
  • climate change
  • aquaculture practices

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Published Papers (11 papers)

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28 pages, 1999 KiB  
Article
Seascape Genomics of the Sugar Kelp Saccharina latissima along the North Eastern Atlantic Latitudinal Gradient
by Jaromir Guzinski, Paolo Ruggeri, Marion Ballenghien, Stephane Mauger, Bertrand Jacquemin, Chloe Jollivet, Jerome Coudret, Lucie Jaugeon, Christophe Destombe and Myriam Valero
Genes 2020, 11(12), 1503; https://doi.org/10.3390/genes11121503 - 13 Dec 2020
Cited by 20 | Viewed by 4991
Abstract
Temperature is one of the most important range-limiting factors for many seaweeds. Driven by the recent climatic changes, rapid northward shifts of species’ distribution ranges can potentially modify the phylogeographic signature of Last Glacial Maximum. We explored this question in detail in the [...] Read more.
Temperature is one of the most important range-limiting factors for many seaweeds. Driven by the recent climatic changes, rapid northward shifts of species’ distribution ranges can potentially modify the phylogeographic signature of Last Glacial Maximum. We explored this question in detail in the cold-tolerant kelp species Saccharina latissima, using microsatellites and double digest restriction site-associated DNA sequencing ( ddRAD-seq) derived single nucleotide polymorphisms (SNPs) to analyze the genetic diversity and structure in 11 sites spanning the entire European Atlantic latitudinal range of this species. In addition, we checked for statistical correlation between genetic marker allele frequencies and three environmental proxies (sea surface temperature, salinity, and water turbidity). Our findings revealed that genetic diversity was significantly higher for the northernmost locality (Spitsbergen) compared to the southern ones (Northern Iberia), which we discuss in light of the current state of knowledge on phylogeography of S. latissima and the potential influence of the recent climatic changes on the population structure of this species. Seven SNPs and 12 microsatellite alleles were found to be significantly associated with at least one of the three environmental variables. We speculate on the putative adaptive functions of the genes associated with the outlier markers and the importance of these markers for successful conservation and aquaculture strategies for S. latissima in this age of rapid global change. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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29 pages, 3235 KiB  
Article
Trans-Atlantic Distribution and Introgression as Inferred from Single Nucleotide Polymorphism: Mussels Mytilus and Environmental Factors
by Roman Wenne, Małgorzata Zbawicka, Lis Bach, Petr Strelkov, Mikhail Gantsevich, Piotr Kukliński, Tomasz Kijewski, John H. McDonald, Kristil Kindem Sundsaasen, Mariann Árnyasi, Sigbjørn Lien, Ants Kaasik, Kristjan Herkül and Jonne Kotta
Genes 2020, 11(5), 530; https://doi.org/10.3390/genes11050530 - 10 May 2020
Cited by 40 | Viewed by 4384
Abstract
Large-scale climate changes influence the geographic distribution of biodiversity. Many taxa have been reported to extend or reduce their geographic range, move poleward or displace other species. However, for closely related species that can hybridize in the natural environment, displacement is not the [...] Read more.
Large-scale climate changes influence the geographic distribution of biodiversity. Many taxa have been reported to extend or reduce their geographic range, move poleward or displace other species. However, for closely related species that can hybridize in the natural environment, displacement is not the only effect of changes of environmental variables. Another option is subtler, hidden expansion, which can be found using genetic methods only. The marine blue mussels Mytilus are known to change their geographic distribution despite being sessile animals. In addition to natural dissemination at larval phase—enhanced by intentional or accidental introductions and rafting—they can spread through hybridization and introgression with local congeners, which can create mixed populations sustaining in environmental conditions that are marginal for pure taxa. The Mytilus species have a wide distribution in coastal regions of the Northern and Southern Hemisphere. In this study, we investigated the inter-regional genetic differentiation of the Mytilus species complex at 53 locations in the North Atlantic and adjacent Arctic waters and linked this genetic variability to key local environmental drivers. Of seventy-nine candidate single nucleotide polymorphisms (SNPs), all samples were successfully genotyped with a subset of 54 SNPs. There was a clear interregional separation of Mytilus species. However, all three Mytilus species hybridized in the contact area and created hybrid zones with mixed populations. Boosted regression trees (BRT) models showed that inter-regional variability was important in many allele models but did not prevail over variability in local environmental factors. Local environmental variables described over 40% of variability in about 30% of the allele frequencies of Mytilus spp. For the 30% of alleles, variability in their frequencies was only weakly coupled with local environmental conditions. For most studied alleles the linkages between environmental drivers and the genetic variability of Mytilus spp. were random in respect to “coding” and “non-coding” regions. An analysis of the subset of data involving functional genes only showed that two SNPs at Hsp70 and ATPase genes correlated with environmental variables. Total predictive ability of the highest performing models (r2 between 0.550 and 0.801) were for alleles that discriminated most effectively M. trossulus from M. edulis and M. galloprovincialis, whereas the best performing allele model (BM101A) did the best at discriminating M. galloprovincialis from M. edulis and M. trossulus. Among the local environmental variables, salinity, water temperature, ice cover and chlorophyll a concentration were by far the greatest predictors, but their predictive performance varied among different allele models. In most cases changes in the allele frequencies along these environmental gradients were abrupt and occurred at a very narrow range of environmental variables. In general, regions of change in allele frequencies for M. trossulus occurred at 8–11 psu, 0–10 °C, 60%–70% of ice cover and 0–2 mg m−3 of chlorophyll a, M. edulis at 8–11 and 30–35 psu, 10–14 °C and 60%–70% of ice cover and for M. galloprovincialis at 30–35 psu, 14–20 °C. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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17 pages, 1263 KiB  
Article
Genetic Characterization of Cupped Oyster Resources in Europe Using Informative Single Nucleotide Polymorphism (SNP) Panels
by Sylvie Lapègue, Serge Heurtebise, Florence Cornette, Erwan Guichoux and Pierre-Alexandre Gagnaire
Genes 2020, 11(4), 451; https://doi.org/10.3390/genes11040451 - 21 Apr 2020
Cited by 3 | Viewed by 3902
Abstract
The Pacific oyster, Crassostrea gigas, was voluntarily introduced from Japan and British Columbia into Europe in the early 1970s, mainly to replace the Portuguese oyster, Crassostrea angulata, in the French shellfish industry, following a severe disease outbreak. Since then, the two [...] Read more.
The Pacific oyster, Crassostrea gigas, was voluntarily introduced from Japan and British Columbia into Europe in the early 1970s, mainly to replace the Portuguese oyster, Crassostrea angulata, in the French shellfish industry, following a severe disease outbreak. Since then, the two species have been in contact in southern Europe and, therefore, have the potential to exchange genes. Recent evolutionary genomic works have provided empirical evidence that C. gigas and C. angulata exhibit partial reproductive isolation. Although hybridization occurs in nature, the rate of interspecific gene flow varies across the genome, resulting in highly heterogeneous genome divergence. Taking this biological property into account is important to characterize genetic ancestry and population structure in oysters. Here, we identified a subset of ancestry-informative makers from the most differentiated regions of the genome using existing genomic resources. We developed two different panels in order to (i) easily differentiate C. gigas and C. angulata, and (ii) describe the genetic diversity and structure of the cupped oyster with a particular focus on French Atlantic populations. Our results confirm high genetic homogeneity among Pacific cupped oyster populations in France and reveal several cases of introgressions between Portuguese and Japanese oysters in France and Portugal. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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16 pages, 2746 KiB  
Article
Population Connectivity and Genetic Assessment of Exploited and Natural Populations of Pearl Oysters within a French Polynesian Atoll Lagoon
by Céline M. O. Reisser, Romain Le Gendre, Cassandre Chupeau, Alain Lo-Yat, Serge Planes and Serge Andréfouët
Genes 2020, 11(4), 426; https://doi.org/10.3390/genes11040426 - 15 Apr 2020
Cited by 12 | Viewed by 3032 | Correction
Abstract
In French Polynesia, the production and exportation of black pearls through the aquaculture of the black-lip pearl oyster Pinctada margaritifera provide the second largest economic income for the country after tourism. This industry entirely relies on the collection of natural spats from few [...] Read more.
In French Polynesia, the production and exportation of black pearls through the aquaculture of the black-lip pearl oyster Pinctada margaritifera provide the second largest economic income for the country after tourism. This industry entirely relies on the collection of natural spats from few highly recruiting lagoons. In recent years, pearl oyster producers have experienced variable success rates in spat collection, with significant spatial and temporal variability in spat supply, driving uncertainty in the future of pearl production. This study combines, for the first time in a farmed lagoon, genetic (SNPs), demographic (sex ratio, age), and biophysical data (larval dispersal modelling) to shed new light on population dynamics, connectivity, and spat recruitment in Ahe Atoll, a well-studied pearl farming site. Our results indicate that the geographical structuring of the natural populations and the contribution of both natural and exploited stocks to the production of spats result from the interaction of hydrodynamic features, life history traits and demographic parameters: the northeastern natural populations are older, not well connected to the southwestern natural populations and are not replenished by larvae produced by adjacent exploited populations. Moreover, we observe that the exploited populations did not contribute to larval production during our experiment, despite a sampling period set during the most productive season for spat collection. This is likely the result of a strong male bias in the exploited populations, coupled with a sweepstakes reproductive strategy of the species. Our results warrant further investigations over the future of the northeastern older natural populations and a reflection on the current perliculture techniques. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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17 pages, 2894 KiB  
Article
Within-Generation Polygenic Selection Shapes Fitness-Related Traits across Environments in Juvenile Sea Bream
by Carine Rey, Audrey Darnaude, Franck Ferraton, Bruno Guinand, François Bonhomme, Nicolas Bierne and Pierre-Alexandre Gagnaire
Genes 2020, 11(4), 398; https://doi.org/10.3390/genes11040398 - 7 Apr 2020
Cited by 7 | Viewed by 3284
Abstract
Understanding the genetic underpinnings of fitness trade-offs across spatially variable environments remains a major challenge in evolutionary biology. In Mediterranean gilthead sea bream, first-year juveniles use various marine and brackish lagoon nursery habitats characterized by a trade-off between food availability and environmental disturbance. [...] Read more.
Understanding the genetic underpinnings of fitness trade-offs across spatially variable environments remains a major challenge in evolutionary biology. In Mediterranean gilthead sea bream, first-year juveniles use various marine and brackish lagoon nursery habitats characterized by a trade-off between food availability and environmental disturbance. Phenotypic differences among juveniles foraging in different habitats rapidly appear after larval settlement, but the relative role of local selection and plasticity in phenotypic variation remains unclear. Here, we combine phenotypic and genetic data to address this question. We first report correlations of opposite signs between growth and condition depending on juvenile habitat type. Then, we use single nucleotide polymorphism (SNP) data obtained by Restriction Associated DNA (RAD) sequencing to search for allele frequency changes caused by a single generation of spatially varying selection between habitats. We found evidence for moderate selection operating at multiple loci showing subtle allele frequency shifts between groups of marine and brackish juveniles. We identified subsets of candidate outlier SNPs that, in interaction with habitat type, additively explain up to 3.8% of the variance in juvenile growth and 8.7% in juvenile condition; these SNPs also explained significant fraction of growth rate in an independent larval sample. Our results indicate that selective mortality across environments during early-life stages involves complex trade-offs between alternative growth strategies. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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20 pages, 3319 KiB  
Article
Population Genetic Structure Is Unrelated to Shell Shape, Thickness and Organic Content in European Populations of the Soft-Shell Clam Mya Arenaria
by Michele De Noia, Luca Telesca, David L. J. Vendrami, Hatice K. Gokalp, Grégory Charrier, Elizabeth M. Harper and Joseph I. Hoffman
Genes 2020, 11(3), 298; https://doi.org/10.3390/genes11030298 - 11 Mar 2020
Cited by 8 | Viewed by 4443
Abstract
The soft-shell clam Mya arenaria is one of the most ancient invaders of European coasts and is present in many coastal ecosystems, yet little is known about its genetic structure in Europe. We collected 266 samples spanning a latitudinal cline from the Mediterranean [...] Read more.
The soft-shell clam Mya arenaria is one of the most ancient invaders of European coasts and is present in many coastal ecosystems, yet little is known about its genetic structure in Europe. We collected 266 samples spanning a latitudinal cline from the Mediterranean to the North Sea and genotyped them at 12 microsatellite loci. In parallel, geometric morphometric analysis of shell outlines was used to test for associations between shell shape, latitude and genotype, and for a selection of shells we measured the thickness and organic content of the granular prismatic (PR), the crossed-lamellar (CL) and the complex crossed-lamellar (CCL) layers. Strong population structure was detected, with Bayesian cluster analysis identifying four groups located in the Mediterranean, Celtic Sea, along the continental coast of the North Sea and in Scotland. Multivariate analysis of shell shape uncovered a significant effect of collection site but no associations with any other variables. Shell thickness did not vary significantly with either latitude or genotype, although PR thickness and calcification were positively associated with latitude, while CCL thickness showed a negative association. Our study provides new insights into the population structure of this species and sheds light on factors influencing shell shape, thickness and microstructure. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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24 pages, 5559 KiB  
Article
Seascape Genetics and the Spatial Ecology of Juvenile Green Turtles
by Michael P. Jensen, Mayeul Dalleau, Philippe Gaspar, Maxime Lalire, Claire Jean, Stéphane Ciccione, Jeanne A. Mortimer, Mireille Quillard, Coralie Taquet, Andrew Wamukota, Géraud Leroux and Jérôme Bourjea
Genes 2020, 11(3), 278; https://doi.org/10.3390/genes11030278 - 5 Mar 2020
Cited by 19 | Viewed by 5100
Abstract
Understanding how ocean currents impact the distribution and connectivity of marine species, provides vital information for the effective conservation management of migratory marine animals. Here, we used a combination of molecular genetics and ocean drift simulations to investigate the spatial ecology of juvenile [...] Read more.
Understanding how ocean currents impact the distribution and connectivity of marine species, provides vital information for the effective conservation management of migratory marine animals. Here, we used a combination of molecular genetics and ocean drift simulations to investigate the spatial ecology of juvenile green turtle (Chelonia mydas) developmental habitats, and assess the role of ocean currents in driving the dispersal of green turtle hatchlings. We analyzed mitochondrial (mt)DNA sequenced from 358 juvenile green turtles, and from eight developmental areas located throughout the Southwest Indian Ocean (SWIO). A mixed stock analysis (MSA) was applied to estimate the level of connectivity between developmental sites and published genetic data from 38 known genetic stocks. The MSA showed that the juvenile turtles at all sites originated almost exclusively from the three known SWIO stocks, with a clear shift in stock contributions between sites in the South and Central Areas. The results from the genetic analysis could largely be explained by regional current patterns, as shown by the results of passive numerical drift simulations linking breeding sites to developmental areas utilized by juvenile green turtles. Integrating genetic and oceanographic data helps researchers to better understand how marine species interact with ocean currents at different stages of their lifecycle, and provides the scientific basis for effective conservation management. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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16 pages, 1907 KiB  
Article
Genetic Differentiation in Hatchery and Stocked Populations of Sea Trout in the Southern Baltic: Selection Evidence at SNP Loci
by Rafał Bernaś, Anita Poćwierz-Kotus, Mariann Árnyasi, Matthew Peter Kent, Sigbjørn Lien and Roman Wenne
Genes 2020, 11(2), 184; https://doi.org/10.3390/genes11020184 - 10 Feb 2020
Cited by 8 | Viewed by 3074
Abstract
The impacts and interactions between hatchery-bred fish and wild fish populations has been a topic of active investigation in recent decades. In some instances, the benefits of stocking can be overshadowed by negative effects such as genetic introgression with natural populations, loss of [...] Read more.
The impacts and interactions between hatchery-bred fish and wild fish populations has been a topic of active investigation in recent decades. In some instances, the benefits of stocking can be overshadowed by negative effects such as genetic introgression with natural populations, loss of genetic diversity, and dilution of local adaptations. Methods that facilitate the identification of stocked fish enable us to estimate not only the effectiveness of stocking but also the level of natural reproduction and the degree of hybridization. The longest Baltic river, the Vistula, also has the second highest discharge. Historically, it hosted numerous populations of the anadromous form of brown trout (sea trout); however, dam construction has since interfered with and reduced spawning migration to a rate that is much lower than before. Reduced spawning has resulted in a population collapse and a negative flow-on effect on commercial catches. In response, Poland (along with many other Baltic countries) initiated an intensive stocking program which continues today and which sees the average annual release of 700,000 smolts. As a consequence, today’s main-river and inshore catches come from stock-enhanced populations. High-throughput single-nucleotide polymorphism (SNP) genotyping was performed on samples of sea trout from southern Baltic populations; results suggest that a significant portion of the sea trout catches in the Vistula mouth region have direct hatchery origin and indicate the presence of Pomeranian specimens. SNP loci identified as outliers indicate a potential selection pressure that may be related with effects of hatchery breeding and mixing with natural populations. The brown trout SNP array applied in this study showed high effectiveness not only for population differentiation, but more importantly, it emerged as a sensitive tool to provide evidence of detection selection. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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23 pages, 3699 KiB  
Article
Population Genomics of an Anadromous Hilsa Shad Tenualosa ilisha Species across Its Diverse Migratory Habitats: Discrimination by Fine-Scale Local Adaptation
by Md Asaduzzaman, Yoji Igarashi, Md Abdul Wahab, Md Nahiduzzaman, Md Jalilur Rahman, Michael J. Phillips, Songqian Huang, Shuichi Asakawa, Md Moshiur Rahman and Li Lian Wong
Genes 2020, 11(1), 46; https://doi.org/10.3390/genes11010046 - 30 Dec 2019
Cited by 15 | Viewed by 6216
Abstract
The migration of anadromous fish in heterogenic environments unceasingly imposes a selective pressure that results in genetic variation for local adaptation. However, discrimination of anadromous fish populations by fine-scale local adaptation is challenging because of their high rate of gene flow, highly connected [...] Read more.
The migration of anadromous fish in heterogenic environments unceasingly imposes a selective pressure that results in genetic variation for local adaptation. However, discrimination of anadromous fish populations by fine-scale local adaptation is challenging because of their high rate of gene flow, highly connected divergent population, and large population size. Recent advances in next-generation sequencing (NGS) have expanded the prospects of defining the weakly structured population of anadromous fish. Therefore, we used NGS-based restriction site-associated DNA (NextRAD) techniques on 300 individuals of an anadromous Hilsa shad (Tenualosa ilisha) species, collected from nine strategic habitats, across their diverse migratory habitats, which include sea, estuary, and different freshwater rivers. The NextRAD technique successfully identified 15,453 single nucleotide polymorphism (SNP) loci. Outlier tests using the FST OutFLANK and pcadapt approaches identified 74 and 449 SNPs (49 SNPs being common), respectively, as putative adaptive loci under a divergent selection process. Our results, based on the different cluster analyses of these putatively adaptive loci, suggested that local adaptation has divided the Hilsa shad population into two genetically structured clusters, in which marine and estuarine collection sites were dominated by individuals of one genetic cluster and different riverine collection sites were dominated by individuals of another genetic cluster. The phylogenetic analysis revealed that all the riverine populations of Hilsa shad were further subdivided into the north-western riverine (turbid freshwater) and the north-eastern riverine (clear freshwater) ecotypes. Among all of the putatively adaptive loci, only 36 loci were observed to be in the coding region, and the encoded genes might be associated with important biological functions related to the local adaptation of Hilsa shad. In summary, our study provides both neutral and adaptive contexts for the observed genetic divergence of Hilsa shad and, consequently, resolves the previous inconclusive findings on their population genetic structure across their diverse migratory habitats. Moreover, the study has clearly demonstrated that NextRAD sequencing is an innovative approach to explore how dispersal and local adaptation can shape genetic divergence of non-model anadromous fish that intersect diverse migratory habitats during their life-history stages. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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10 pages, 840 KiB  
Communication
A Useful SNP Panel to Distinguish Two Cockle Species, Cerastoderma edule and C. glaucum, Co-Occurring in Some European Beds, and Their Putative Hybrids
by Francesco Maroso, Celia Pérez de Gracia, David Iglesias, Asunción Cao, Seila Díaz, Antonio Villalba, Manuel Vera and Paulino Martínez
Genes 2019, 10(10), 760; https://doi.org/10.3390/genes10100760 - 27 Sep 2019
Cited by 11 | Viewed by 3249
Abstract
Cockles are highly appreciated mollusks and provide important services in coastal areas. The two European species, edible (Cerastoderma edule) and lagoon (Cerastoderma glaucum) cockles, are not easily distinguishable, especially when young. Interestingly, the species show different resistance to Marteilia [...] Read more.
Cockles are highly appreciated mollusks and provide important services in coastal areas. The two European species, edible (Cerastoderma edule) and lagoon (Cerastoderma glaucum) cockles, are not easily distinguishable, especially when young. Interestingly, the species show different resistance to Marteilia cochillia, the parasite responsible for marteiliosis outbreaks, which is devastating cockle production in some areas. C. edule is severely affected by the parasite, while C. glaucum seems to be resistant, although underlying reasons are still unknown. Hybrids between both species might be interesting to introgress allelic variants responsible for tolerance, either naturally or through artificial selection, from lagoon into edible cockle. Here, we used 2b restriction site-associated DNA sequencing (2b–RAD) to identify single nucleotide polymorphisms (SNP) diagnostic for cockle discrimination (fixed for alternative allelic variants). Among the nine diagnostic SNPs selected, seven were validated using a SNaPshot assay in samples covering most of the distribution range of both species. The validated SNPs were used to check cockles that were suggested to be hybrids by a claimed diagnostic tool based on the internal transcribed spacers of the ribosomal RNA. Although these were shown to be false positives, we cannot rule out the fact that hybrids can occur and be viable. The SNP tool here developed will be valuable for their identification and management. Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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2 pages, 463 KiB  
Correction
Correction: Reisser, C.M.O. et al. Population Connectivity and Genetic Assessment of Exploited and Natural Populations of Pearl Oysters within a French Polynesian Atoll Lagoon. Genes 2020, 11, 426
by Céline M. O. Reisser, Romain Le Gendre, Cassandre Chupeau, Alain Lo-Yat, Serge Planes and Serge Andréfouët
Genes 2020, 11(11), 1358; https://doi.org/10.3390/genes11111358 - 16 Nov 2020
Cited by 1 | Viewed by 1375
Abstract
The authors wish to make the following corrections to this paper [...] Full article
(This article belongs to the Special Issue Genetic Diversity of Marine Populations)
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