Development of SNP-genotyping arrays in two shellfish species.

Use of SNPs has been favoured due to their abundance in plant and animal genomes, accompanied by the falling cost and rising throughput capacity for detection and genotyping. Here, we present in vitro (obtained from targeted sequencing) and in silico discovery of SNPs, and the design of medium-throughput genotyping arrays for two oyster species, the Pacific oyster, Crassostrea gigas, and European flat oyster, Ostrea edulis. Two sets of 384 SNP markers were designed for two Illumina GoldenGate arrays and genotyped on more than 1000 samples for each species. In each case, oyster samples were obtained from wild and selected populations and from three-generation families segregating for traits of interest in aquaculture. The rate of successfully genotyped polymorphic SNPs was about 60% for each species. Effects of SNP origin and quality on genotyping success (Illumina functionality Score) were analysed and compared with other model and nonmodel species. Furthermore, a simulation was made based on a subset of the C. gigas SNP array with a minor allele frequency of 0.3 and typical crosses used in shellfish hatcheries. This simulation indicated that at least 150 markers were needed to perform an accurate parental assignment. Such panels might provide valuable tools to improve our understanding of the connectivity between wild (and selected) populations and could contribute to future selective breeding programmes.

PCR survey of 50 introns in animals: cross-amplification of homologous EPIC loci in eight non-bilaterian, protostome and deuterostome phyla.

Exon Primed Intron Crossing (EPIC) markers provide molecular tools that are susceptible to be variable within species while remaining amplifiable by PCR using potentially universal primers. In this study we tested the possibility of obtaining PCR products from 50 EPIC markers on 23 species belonging to seven different phyla (Porifera, Cnidaria, Arthropoda, Nematoda, Mollusca, Annelida, Echinodermata) using 70 new primer pairs. A previous study had identified and tested those loci in a dozen species, including another phylum, Urochordata (Chenuil et al., 2010). Results were contrasted among species. The best results were achieved with the oyster (Mollusca) where 28 loci provided amplicons susceptible to contain an intron according to their size. This was however not the case with the other mollusk Crepidula fornicata, which seems to have undergone a reduction in intron number or intron size. In the Porifera, 13 loci appeared susceptible to contain an intron, a surprisingly high number for this phylum considering its phylogenetic distance with genomic data used to design the primers. For two cnidarian species, numerous loci (24) were obtained. Ecdysozoan phyla (arthropods and nematodes) proved less successful than others as expected considering reports of their rapid rate of genome evolution and the worst results were obtained for several arthropods. Some general patterns among phyla arose, and we discuss how the results of this EPIC survey may give new insights into genome evolution of the study species. This work confirms that this set of EPIC loci provides an easy-to-use toolbox to identify genetic markers potentially useful for population genetics, phylogeography or phylogenetic studies for a large panel of metazoan species. We then argue that obtaining diploid sequence genotypes for these loci became simple and affordable owing to Next-Generation Sequencing development. Species surveyed in this study belong to several genera (Acanthaster, Alvinocaris, Aplysina, Aurelia, Crepidula, Eunicella, Hediste, Hemimysis, Litoditis, Lophelia, Mesopodopsis, Mya, Ophiocten, Ophioderma, Ostrea, Pelagia, Platynereis, Rhizostoma, Rimicaris), two of them, belonging to the family Vesicomydae and Eunicidae, could not be determined at the genus level.

Variance in the reproductive success of flat oyster Ostrea edulis L. assessed by parentage analyses in natural and experimental conditions.

In order to document further the phenomena of variance in reproductive success in natural populations of the European flat oyster Ostrea edulis, two complementary studies based on natural and experimental populations were conducted. The first part of this work was focused on paternity analyses using a set of four microsatellite markers for larvae collected from 13 brooding females sampled in Quiberon Bay (Brittany, France). The number of individuals contributing as the male parent to each progeny assay was highly variable, ranging from 2 to more than 40. Moreover, paternal contributions showed a much skewed distribution, with some males contributing to 50-100% of the progeny assay. The second part of this work consisted of the analysis of six successive cohorts experimentally produced from an acclimated broodstock (62 wild oysters sampled in the Quiberon Bay). Allelic richness was significantly higher in the adult population than in the temporal cohorts collected. Genetic differentiation (F(st) estimates) was computed for each pair of samples and all significant values ranged from 0.7 to 11.9%. A limited effective number of breeders (generally below 25) was estimated in the six temporal cohorts. The study gives first indications of the high variance in reproductive success as well as a reduced effective size, not only under experimental conditions but also in the wild. Surprisingly, the pool of the successive cohorts, based on the low number of loci used, appeared to depict a random and representative set of alleles of the progenitor population, indicating that the detection of patterns of temporal genetic differentiation at a local scale most likely depends on the sampling window.

QTL for resistance to summer mortality and OsHV-1 load in the Pacific oyster (Crassostrea gigas).

Summer mortality is a phenomenon severely affecting the aquaculture production of the Pacific oyster (Crassostrea gigas). Although its causal factors are complex, resistance to mortality has been described as a highly heritable trait, and several pathogens including the virus Ostreid Herpes virus type 1 (OsHV-1) have been associated with this phenomenon. A QTL analysis for survival of summer mortality and OsHV-1 load, estimated using real-time PCR, was performed using five F(2) full-sib families resulting from a divergent selection experiment for resistance to summer mortality. A consensus linkage map was built using 29 SNPs and 51 microsatellite markers. Five significant QTL were identified and assigned to linkage groups V, VI, VII and IX. Analysis of single full-sib families revealed differential QTL segregation between families. QTL for the two-recorded traits presented very similar locations, highlighting the interest of further study of their respective genetic controls. These QTL show substantial genetic variation in resistance to summer mortality, and present new opportunities for selection for resistance to OsHV-1.

Combining two-stage testing and interval mapping strategies to detect QTL for resistance to bonamiosis in the european flat oyster Ostrea edulis.

We have identified quantitative trait loci (QTL) in the flat oyster (Ostrea edulis) for resistance to Bonamia ostreae, a parasite responsible for the dramatic reduction in the aquaculture of this species. An F(2) family from a cross between a wild oyster and an individual from a family selected for resistance to bonamiosis was cultured with wild oysters injected with the parasite, leading to 20% cumulative mortality. Selective genotyping of 92 out of a total of 550 F(2) progeny (i.e., 46 heavily infected oysters that died and 46 parasite-free oysters that survived) was performed using 20 microsatellites and 34 amplification fragment length polymorphism primer pairs. Both a two-stage testing strategy and QTL interval mapping methods were used. The two-stage detection strategy had a high power with a low rate of false positives and identified nine and six probable markers linked to genes of resistance and susceptibility, respectively. Parent-specific genetic linkage maps were built for the family, spanning ten linkage groups (n = 10) with an observed genome coverage of 69-84%. Three QTL were identified by interval mapping in the first parental map and two in the second. Good concordance was observed between the results obtained after the two-stage testing strategy and QTL mapping.

Identification and characterization of 18 novel polymorphic microsatellite makers derived from expressed sequence tags in the Pacific oyster Crassostrea gigas.

We report the development of 18 new polymorphic microsatellite DNA markers derived from Crassostrea gigas expressed sequences tags. Genotyping of 48 wild adult oysters sampled from Marennes-Oléron bay (France) revealed 12 to 48 alleles per locus. Observed and expected heterozygosity levels ranged from 0.64 to 1 and from 0.77 to 0.97, respectively. The development of these new markers creates a useful complementary tool for population genetics studies, parentage analysis and mapping in Pacific oyster, a species of major aquacultural and ecological importance.

A first-generation genetic linkage map of the European flat oyster Ostrea edulis (L.) based on AFLP and microsatellite markers.

This study presents the first genetic linkage map for the European flat oyster Ostrea edulis. Two hundred and forty-six AFLP and 20 microsatellite markers were genotyped in a three-generation pedigree comprising two grandparents, two parents and 92 progeny. Chi-square goodness-of-fit tests revealed high segregation distortion, which was significant for 32.8% of markers. Sixteen microsatellites and 235 AFLPs (170 type 1:1 AFLPs and 65 type 3:1 AFLPs) were used to build sex-specific linkage maps using crimap software. The first parental map (P(1)) consisted of 104 markers grouped in nine linkage groups, and spanned 471.2 cM with an average spacing of 4.86 cM. The second parental map (P(2)) consisted of 117 markers grouped in 10 linkage groups (which equals the haploid chromosome number), and covered 450.0 cM with an average spacing of 4.21 cM. The estimated coverage of the genome was 82.4% for the P(1) map and 84.2% for the P(2) map. Eight linkage groups that were probably homologous between the two parents contained the same microsatellites and 3:1 AFLPs (segregating through both parents). Distorted markers were not randomly distributed across the genome and tended to cluster in a few linkage groups. Sex-specific differences in recombination rates were evident. This first-generation genetic linkage map for O. edulis represents a major step towards the mapping of QTL such as resistance to bonamiasis, a parasitosis that has drastically decreased populations of flat oysters since the 1960s.

Single Nucleotide polymorphisms and their relationship to codon usage bias in the Pacific oyster Crassostrea gigas.

DNA sequence polymorphism and codon usage bias were investigated in a set of 41 nuclear loci in the Pacific oyster Crassostrea gigas. Our results revealed a very high level of DNA polymorphism in oysters, in the order of magnitude of the highest levels reported in animals to date. A total of 290 single nucleotide polymorphisms (SNPs) were detected, 76 of which being localised in exons and 214 in non-coding regions. Average density of SNPs was estimated to be one SNP every 60 bp in coding regions and one every 40 bp in non-coding regions. Non-synonymous substitutions contributed substantially to the polymorphism observed in coding regions. The non-synonymous to silent diversity ratio was 0.16 on average, which is fairly higher to the ratio reported in other invertebrate species recognised to display large population sizes. Therefore, purifying selection does not appear to be as strong as it could have been expected for a species with a large effective population size. The level of non-synonymous diversity varied greatly from one gene to another, in accordance with varying selective constraints. We examined codon usage bias and its relationship with DNA polymorphism. The table of optimal codons was deduced from the analysis of an EST dataset, using EST counts as a rough assessment of gene expression. As recently observed in some other taxa, we found a strong and significant negative relationship between codon bias and non-synonymous diversity suggesting correlated selective constraints on synonymous and non-synonymous substitutions. Codon bias as measured by the frequency of optimal codons for expression might therefore provide a useful indicator of the level of constraint upon proteins in the oyster genome.

AFLP-based genetic linkage maps of the blue mussel (Mytilus edulis).

We report the construction of the first genetic linkage map in the blue mussel, Mytilus edulis. AFLP markers were used in 86 full-sib progeny from a controlled pair mating, applying a double pseudo-test cross strategy. Thirty-six primer pairs generated 2354 peaks, of which 791 (33.6%) were polymorphic in the mapping family. Among those, 341 segregated through the female parent, 296 through the male parent (type 1:1) and 154 through both parents (type 3:1). Chi-square goodness-of-fit tests revealed that 71% and 73% of type 1:1 and 3:1 markers respectively segregated according to Mendelian inheritance. Sex-specific linkage maps were built with mapmaker 3.0 software. The female framework map consisted of 121 markers ordered into 14 linkage groups, spanning 862.8 cM, with an average marker spacing of 8.0 cM. The male framework map consisted of 116 markers ordered into 14 linkage groups, spanning 825.2 cM, with an average marker spacing of 8.09 cM. Genome coverage was estimated to be 76.7% and 75.9% for the female and male framework maps respectively, rising to 85.8% (female) and 86.2% (male) when associated markers were included. Twelve probable homologous linkage group pairs were identified and a consensus map was built for nine of these homologous pairs based on multiple and parallel linkages of 3:1 markers, spanning 816 cM, with joinmap 4.0 software.

Phylogeography of the green turtle, Chelonia mydas, in the Southwest Indian Ocean.

Patterns of mitochondrial DNA (mtDNA) variation were used to analyse the population genetic structure of southwestern Indian Ocean green turtle (Chelonia mydas) populations. Analysis of sequence variation over 396 bp of the mtDNA control region revealed seven haplotypes among 288 individuals from 10 nesting sites in the Southwest Indian Ocean. This is the first time that Atlantic Ocean haplotypes have been recorded among any Indo-Pacific nesting populations. Previous studies indicated that the Cape of Good Hope was a major biogeographical barrier between the Atlantic and Indian Oceans because evidence for gene flow in the last 1.5 million years has yet to emerge. This study, by sampling localities adjacent to this barrier, demonstrates that recent gene flow has occurred from the Atlantic Ocean into the Indian Ocean via the Cape of Good Hope. We also found compelling genetic evidence that green turtles nesting at the rookeries of the South Mozambique Channel (SMC) and those nesting in the North Mozambique Channel (NMC) belong to separate genetic stocks. Furthermore, the SMC could be subdivided in two different genetic stocks, one in Europa and the other one in Juan de Nova. We suggest that this particular genetic pattern along the Mozambique Channel is attributable to a recent colonization from the Atlantic Ocean and is maintained by oceanic conditions in the northern and southern Mozambique Channel that influence early stages in the green turtle life cycle.

Reduced female gene flow in the European flat oyster Ostrea edulis.

The geographical structure of 15 natural populations of the flat oyster (Ostrea edulis L.) was assessed by single-strand conformation polymorphism (SSCP) of a 313-base-pair (bp) fragment of the mitochondrial 12S-rRNA gene. Fourteen haplotypes were observed, with one being dominant in the Mediterranean samples and another one in the Atlantic populations. The geographically extreme populations sampled in Norway and the Black Sea appeared differentiated by exhibiting the dominance of a third group of haplotypes. The results were compared to available microsatellite data at five loci. The Atlantic/Mediterranean differentiation pattern was qualitatively the same with both types of markers, confirming an isolation-by-distance pattern. The average mitochondrial haplotypic diversity displayed a high among populations variance, reflecting small effective population size in some locations. Additionally, a 10-fold quantitative difference was observed in Fst between the mitochondrial and the nuclear genomes, which could be due to an unbalanced sex ratio or sex-biased differential reproductive success between males and females (or both).

Study of atrazine effects on Pacific oyster, Crassostrea gigas, haemocytes.

Shellfish farming is an important economic activity around the world. This activity often takes place in areas subjected to various recurring pollutions. The recrudescent use of herbicides in agriculture including atrazine implies pollutant transfer towards aquatic environment in estuarine areas. Harmful effects of such substances on animals in marine environment, particularly on cultured bivalves, are poorly documented. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate and concentrate contaminants within their tissues. Moreover, development of techniques allowing effect analysis of such compounds on bivalve biology may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, influence of atrazine on defence mechanisms was analyzed in Pacific oysters, Crassostrea gigas. Atrazine was tested in vitro and in vivo on oyster haemocytes, and its effects were analyzed by flow cytometry. Haemocyte viability, cell cycle and cellular activities were monitored. Atrazine induced no significant effect in oyster under tested conditions except for peroxidase activity.

Trans-atlantic distribution of a mangrove oyster species revealed by 16S mtDNA and karyological analyses.

Three species of mangrove oysters, Crassostrea rhizophorae, C. brasiliana, and C. gasar, have been described along the Atlantic shores of South America and Africa. Because the distribution of these molluscs is of great biological and commercial interest, their taxonomy and distribution deserve further clarification. Therefore, 15 populations were sampled from both continents. Their 16S mitochondrial polymorphism was studied by sequencing and PCR-RFLP analysis. Two haplotypes were identified. Haplotype a was the only one observed in Africa, but it was also observed in South America together with haplotype b. Because C. gasar is the only mangrove oyster identified on the west coast of Africa, haplotype a was attributed to this species, which has thus been shown to occur in South America. Haplotype b is attributed to C. rhizophorae. The karyotypes of specimens of C. gasar, from Africa and from South America, were very similar, and both species were observed at the same location in Brazil. The occurrence of C. gasar in South America adds a third species-in addition to C. rhizophorae and C. brasiliana-to the list of species present along these coasts. The predominant surface circulation patterns in this part of the Atlantic Ocean favor the hypothesis that C. gasar was transported from Africa to America. Finally, a phylogenetic tree built with seven 16S sequences from Crassostrea and Saccostrea species showed that C. gasar is intermediate between the American Crassostrea species (C. virginica and C. rhizophorae) and the Asian species (C. gigas and C. ariakensis).

Amplification of oak DNA from ancient and modern wood.

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600-years-old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite-type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.

ARE CHLOROPLAST AND MITOCHONDRIAL DNA VARIATION SPECIES INDEPENDENT IN OAKS?

Extensive introgression of cytoplasmic genomes across oak species is now a well-established fact. To distinguish between ancient hybridization events and ongoing introgression, a direct test for the existence of local exchanges is proposed. Such local exchanges must be comparatively recent, that is, contemporaneous with or later than the last postglacial recolonization. The test is applied to an extensive set of data comprising 377 pure or mixed populations (1744 individuals) of four white oak species in southern France. After demonstrating that local exchanges have occurred frequently between all species pairs, another test is performed to check if species status does nevertheless play some role in restricting cytoplasmic gene flow. The results vary according to the species pairs considered, and the observed pattern may be related to the ecology and/or compatibility of interspecific crosses. It is also shown that, for some of these oak species, the presence of related species in a population significantly influences the intraspecific diversity. Altogether, the results demonstrate that (1) intraspecific cytoplasmic gene flow varies according to the species, (2) interspecific cytoplasmic gene flow varies according to the species pair, and (3) both components of gene flow are at least partly related.

Association between chloroplast and mitochondrial lineages in oaks.

Patterns of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) variation were studied in 378 populations of oak trees sampled throughout the southern half of France. Six cpDNA haplotypes detected in a previous European survey and three new cpDNA haplotypes were found in this region. Two mitochondrial polymorphisms detected earlier by restriction analysis of PCR-amplified fragments alone, or in combination with single-strand conformation polymorphism (SSCP), were compared with the cpDNA data. Sequencing revealed the nature of the two mitochondrial mutations: a single-base substitution and a 4-bp inversion associated with a 22-bp hairpin secondary structure. The single-base substitution was then analyzed by allele-specific amplification. Results for the two cytoplasmic genomes were combined, which allowed the identification of 12 cpDNA-mtDNA haplotypes. The 4-bp mtDNA inversion has appeared independently in different cpDNA lineages. Given the peculiar nature of this mtDNA mutation, we suggest that intramolecular recombination leading to repeated inversions of the 4-bp sequence (rather than paternal leakage of one of the two genomes) is responsible for this pattern. Furthermore, the geographic locations of the unusual cpDNA-mtDNA associations (due to the inversion) usually do not match the zones of contact between divergent haplotypes. In addition, in southern France, the groupings of populations based on the mtDNA substitution were strictly congruent with those based on cpDNA. Because many populations that are polymorphic for both cpDNA and mtDNA have remained in contact since postglacial recolonization in this area without producing any new combination of cytoplasms involving the mitochondrial substitution, we conclude that paternal leakage is not a significant factor at this timescale. Such results confirm and expand our earlier conclusions based on controlled crosses.

Phylogeographic structure of white oaks throughout the European continent.

Patterns of chloroplast DNA (cpDNA) variation were studied in eight white oak species by sampling 345 populations throughout Europe. The detection of polymorphisms by restriction analysis of PCR-amplified cpDNA fragments allowed the identification of 23 haplotypes that were phylogenetically ordered. A systematic hybridization and introgression between the eight species studied is evident. The levels of subdivision for unordered (GST) and ordered (NST) alleles are very high and close (0.83 and 0.85). A new statistical approach to the quantitative study of phylogeography is presented, which relies on the coefficients of differentiation GST and NST and the Mantel's test. Based on pairwise comparisons between populations, the significance of the difference between both coefficients is evaluated at a global and a local scale. The mapped distribution of the haplotypes indicates the probable routes of postglacial recolonization followed by oak populations that had persisted in southern refugia, especially in the Iberian peninsula, Italy and the Balkans. Most cpDNA polymorphisms appear to be anterior to the beginning of the last recolonization. A subset of the preexisting haplotypes have merely expanded north, while others were left behind in the south.