Genomic Diversity of the Ostreid Herpesvirus Type 1 Across Time and Location and Among Host Species

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. This is particularly true for pathogens with low per-site mutation rates, such as DNA viruses, that do not exhibit a large amount of evolutionary change among genetic sequences sampled at different time points. However, whole-genome sequencing can reveal the accumulation of novel genetic variation between samples, promising to render most, if not all, microbial pathogens measurably evolving and suitable for analytical techniques derived from population genetic theory. Here, we aim to assess the measurability of evolution on epidemiological time scales of the Ostreid herpesvirus 1 (OsHV-1), a double stranded DNA virus of which a new variant, OsHV-1 μVar, emerged in France in 2008, spreading across Europe and causing dramatic economic and ecological damage. We performed phylogenetic analyses of heterochronous (n = 21) OsHV-1 genomes sampled worldwide. Results show sufficient temporal signal in the viral sequences to proceed with phylogenetic molecular clock analyses and they indicate that the genetic diversity seen in these OsHV-1 isolates has arisen within the past three decades. OsHV-1 samples from France and New Zealand did not cluster together suggesting a spatial structuration of the viral populations. The genome-wide study of simple and complex polymorphisms shows that specific genomic regions are deleted in several isolates or accumulate a high number of substitutions. These contrasting and non-random patterns of polymorphism suggest that some genomic regions are affected by strong selective pressures. Interestingly, we also found variant genotypes within all infected individuals. Altogether, these results provide baseline evidence that whole genome sequencing could be used to study population dynamic processes of OsHV-1, and more broadly herpesviruses

Alignments of C. gigas in vitro sequences

For the Pacific oyster, in vitro sequencing investigated 103 loci from ESTs retrieved from the Genbank database (http://www.ncbi.nlm.nih.gov/) or from specific libraries that had been obtained to detect genes differentially regulated during summer mortality events (Fleury et al. 2009). Primers were designed using the Primer3 software package (Rozen and Skaletsky 2000). For a first set of ESTs (n = 61), 24 oysters belonging to a third generation of selection for summer mortality resistance were used in the SNP discovery phase (Sauvage 2008; Sauvage et al. 2007). A second set of ESTs (n = 42) was then added and 10 of the 24 oysters were used for sequencing, as described in Sauvage et al. (2007), together with a third set of five SNPs from the 20 developed by Bai et al. (2009). Sequence alignment was performed with ClustalW via the BioEdit interface (Hall 1999) and DNAMAN version 4.1 (www.lynnon.com). The validity of each SNP was checked manually on the chromatograms and sequence alignments. A total of 321 in vitro SNPs were detected in the first dataset of 61 sequenced fragments, and 380 in the second dataset of 42 sequenced fragments. Among those 701 SNPs, 72 were selected (39 and 33 from the two datasets, respectively) because they had high functionality scores and no neighboring polymorphisms. However, as we wanted to be sure that some genes of interest were represented in the SNP dataset, for several ESTs we kept two SNPs. Therefore, our 72 selected in vitro SNPs were obtained from 65 different ESTs

Alignments of O. edulis in vitro sequences

For the European flat oyster, in vitro sequencing investigated 40 loci from two EST libraries (Morga et al. 2011, 2012). Primers were designed using Primer3 software package (Rozen and Skaletsky 2000). A total of 22 oysters, 16 from four different natural populations collected on the Atlantic and Mediterranean coasts and six belonging to the first generations of three selected families for resistance to bonamiosis were used to investigate polymorphisms. The PCR and sequencing protocols used were the same as those given in Harrang et al. (2013). Sequence alignment was performed with ClustalW via the BioEdit interface (Hall 1999). The validity of each SNP was checked individually on nucleotide sequences and sequence alignments. A total of 420 in vitro SNPs were detected in the dataset of 40 sequenced fragments. Among them, the indels (n = 34) were discarded. Moreover, 347 SNPs were also discarded because of neighboring polymorphisms or low functionality scores. However, as we wanted some genes of interest to be represented in the SNP dataset, we kept some (n = 13) that had neighboring polymorphisms. To favor genotyping, those polymorphic nucleotides were treated as degenerated nucleotides. In total, 52 in vitro SNPs were included in the array, representing 35 different gene fragments

Complete mitochondrial DNA sequence of the European flat oyster <it>Ostrea edulis </it>confirms Ostreidae classification

Abstract Background Because of its typical architecture, inheritance and small size, mitochondrial (mt) DNA is widely used for phylogenetic studies. Gene order is generally conserved in most taxa although some groups show considerable variation. This is particularly true in the phylum Mollusca, especially in the Bivalvia. During the last few years, there have been significant increases in the number of complete mitochondrial sequences available. For bivalves, 35 complete mitochondrial genomes are now available in GenBank, a number that has more than doubled in the last three years, representing 6 families and 23 genera. In the current study, we determined the complete mtDNA sequence of O. edulis, the European flat oyster. We present an analysis of features of its gene content and genome organization in comparison with other Ostrea, Saccostrea and Crassostrea species. Results The Ostrea edulis mt genome is 16 320 bp in length and codes for 37 genes (12 protein-coding genes, 2 rRNAs and 23 tRNAs) on the same strand. As in other Ostreidae, O. edulis mt genome contains a split of the rrnL gene and a duplication of trnM. The tRNA gene set of O. edulis, Ostrea denselamellosa and Crassostrea virginica are identical in having 23 tRNA genes, in contrast to Asian oysters, which have 25 tRNA genes (except for C. ariakensis with 24). O. edulis and O. denselamellosa share the same gene order, but differ from other Ostreidae and are closer to Crassostrea than to Saccostrea. Phylogenetic analyses reinforce the taxonomic classification of the 3 families Ostreidae, Mytilidae and Pectinidae. Within the Ostreidae family the results also reveal a closer relationship between Ostrea and Saccostrea than between Ostrea and Crassostrea. Conclusions Ostrea edulis mitogenomic analyses show a high level of conservation within the genus Ostrea, whereas they show a high level of variation within the Ostreidae family. These features provide useful information for further evolutionary analysis of oyster mitogenomes.</p

Genetic parallelism between European flat oyster populations at the edge of their natural range

Although all marine ecosystems have experienced global-scale losses, oyster reefs have shown the greatest. Therefore, substantial efforts have been dedicated to restoration of such ecosystems during the last two decades. In Europe, several pilot projects for the restoration of the native European flat oyster, Ostrea edulis, recently begun and recommendations to preserve genetic diversity and to conduct monitoring protocols have been made. In particular, an initial step is to test for genetic differentiation against homogeneity among the oyster populations potentially involved in such programs. Therefore, we conducted a new sampling of wild populations at the European scale and a new genetic analysis with 203 markers to (1) confirm and study in more detail the pattern of genetic differentiation between Atlantic and Mediterranean populations, (2) identify potential translocations that could be due to aquaculture practices and (3) investigate the populations at the fringe of the geographical range, since they seemed related despite their geographic distance. Such information should be useful to enlighten the choice of the animals to be translocated or reproduced in hatcheries for further restocking. After the confirmation of the general geographical pattern of genetic structure and the identification of one potential case of aquaculture transfer at a large scale, we were able to detect genomic islands of differentiation mainly in the form of two groups of linked markers, which could indicate the presence of polymorphic chromosomal rearrangements. Furthermore, we observed a tendency for these two islands and the most differentiated loci to show a parallel pattern of differentiation, grouping the North Sea populations with the Eastern Mediterranean and Black Sea populations, against geography. We discussed the hypothesis that this genetic parallelism could be the sign of a shared evolutionary history of the two groups of populations despite them being at the border of the distribution nowadays

Genetic characterization of cupped oyster resources in Europe using informative single nucleotide polymorphism (SNP) panels

International audienceThe 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

Is fertility of hybrids enough to conclude that the two oysters

The distinction of the two cupped oysters Crassostrea gigas (Thunberg, 1793) and Crassostrea angulata (Lamark, 1819) into two species was chiefly due to their differing geographical distributions, C. gigas being present in Asia and C. angulata in Europe. Today it is commonly accepted that C. angulata and C. gigas are a single species according to morphological, genetic and F1 hybridization data. However, the demonstration of the fertility of their hybrids and the absence of any reproductive isolation remained to be investigated. Consequently, we studied the fertility of hybrids and sperm competition by performing three different experiments and producing G1 and G2 hybrid progenies between wild populations of C. angulata and C. gigas. Progenies showed very close developmental yields, at 24 hours after fertilization, according to dam taxa suggesting a bold maternal transmission of oocyte quality, but no reproductive isolation was observed between the two taxa. Significant decreases of developmental yields were noticed in C. angulata females with sperm competition, most probably due to early larval mortality. The fertility of hybrids C. angulata × C. gigas was demonstrated, which is further evidence that they are the same species. To definitively state the precise taxonomic classification of C. angulata and C. gigas, further studies are needed to (i) identify geographical zones where these taxa are in contact and (ii) assess their level of hybridization in these zones

Can survival of European flat oysters following experimental infection with Bonamia ostreae be predicted using QTLs?

The present study identifies quantitative trait loci (QTLs) in response to an experimental infection with the parasite responsible for bonamiosis, Bonamia ostreae, in two segregating families of the European flat oyster, Ostrea edulis. We first constructed a genetic-linkage map for each studied family and improved the existing genetic-linkage map for the European flat oyster with a set of SNP markers. This latter map now combines the best accuracy and the best estimate of the genome coverage available for an oyster species. Secondly, by comparing the QTLs detected in this study with those previously published for O. edulis in similar experimental conditions, we identified several potential QTLs that were identical between the different families, and also new specific QTLs. We also detected, within the confidence interval of several QTL regions, some previously predicted candidate genes differentially expressed during an infection with B. ostreae, providing new candidate genome regions which should now be studied more specifically

Application of high-throughput sequencing to population differentiation in the cupped oysters Crassostrea angulata/C. gigas

The cupped oysters Crassostrea angulata and Crassostrea gigas have been sequentially introduced in Europe from their native Pacific area for aquaculture purposes. Ongoing viral epidemic disease is responsible for large losses in oyster aquaculture production throughout Europe. In parallel with current efforts in developing selection programs for disease resistance, it becomes critically important to characterize genetic diversity patterns in both native and introduced areas. With the advent of next generation sequencing techniques, the power to resolve the fine-scale genetic structure at a genome-wide scale opens to new perspectives towards genetic resource management. Here, we present a genome-wide screen of genetic variation that combines SNP genotyping and RADsequencing approaches. Our results, based on more than 18,000 SNP markers, improve our comprehension of the population structure and provide a powerful subset of highly informative markers for stock assignment. As these markers were also used in QTL mapping approaches, these new genomic resources will orientate the choice of breeders in selection programs in order to maximize the natural genetic variance involved in pathogen resistance