SNP Mining in Crassostrea gigas EST Data: Transferability to Four Other Crassostrea Species,Phylogenetic Inferences and Outlier SNPs under Selection

Oysters, with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. Our study is intended to generate new EST-SNP markers and to evaluate their potential for cross-species utilization in phylogenetic study of the genus Crassostrea. In the study, 57 novel SNPs were developed from an EST database of C. gigas by the HRM (high-resolution melting) method. Transferability of 377 SNPs developed for C. gigas was examined on four other Crassostrea species: C. sikamea, C. angulata, C. hongkongensis and C. ariakensis. Among the 377 primer pairs tested, 311 (82.5%) primers showed amplification in C. sikamea, 353 (93.6%) in C. angulata, 254 (67.4%) in C. hongkongensis and 253 (67.1%) in C. ariakensis. A total of 214 SNPs were found to be transferable to all four species. Phylogenetic analyses showed that C. hongkongensis was a sister species of C. ariakensis and that this clade was sister to the clade containing C. sikamea, C. angulata and C. gigas. Within this clade, C. gigas and C. angulata had the closest relationship, with C. sikamea being the sister group. In addition, we detected eight SNPs as potentially being under selection by two outlier tests (fdist and hierarchical methods). The SNPs studied here should be useful for genetic diversity, comparative mapping and phylogenetic studies across species in Crassostrea and the candidate outlier SNPs are worth exploring in more detail regarding association genetics and functional studies.     

A preliminary integrated genetic map distinguishes every chromosome pair and locates essential genes related to abiotic adaptation of <Emphasis Type="Italic">Crassostrea angulata</Emphasis>/<Emphasis Type="Italic">gigas</Emphasis>

Background

The re-sequencing of C. angulata has revealed many polymorphisms in candidate genes related to adaptation to abiotic stress that are not present in C. gigas; these genes, therefore, are probably related to the ability of this oyster to retain high concentrations of toxic heavy metals. There is, in addition, an unresolved controversy as to whether or not C. angulata and C. gigas are the same species or subspecies. Both oysters have 20 metacentric chromosomes of similar size that are morphologically indistinguishable. From a genomic perspective, as a result of the great variation and selection for heterozygotes in C. gigas, the assembly of its draft genome was difficult: it is fragmented in more than seven thousand scaffolds.

Results

In this work sixty BAC sequences of C. gigas downloaded from NCBI were assembled in BAC-contigs and assigned to BACs that were used as probes for mFISH in C. angulata and C. gigas. In addition, probes of H3, H4 histone, 18S and 5S rDNA genes were also used. Hence we obtained markers identifying 8 out the 10 chromosomes constituting the karyotype. Chromosomes 1 and 9 can be distinguished morphologically. The bioinformatic analysis carried out with the BAC-contigs annotated 88 genes. As a result, genes associated with abiotic adaptation, such as metallothioneins, have been positioned in the genome. The gene ontology analysis has also shown many molecular functions related to metal ion binding, a phenomenon associated with detoxification processes that are characteristic in oysters. Hence the provisional integrated map obtained in this study is a useful complementary tool for the study of oyster genomes.

Conclusions

In this study 8 out of 10 chromosome pairs of Crassostrea angulata/gigas were identified using BAC clones as probes. As a result all chromosomes can now be distinguished. Moreover, FISH showed that H3 and H4 co-localized in two pairs of chromosomes different that those previously escribed. 88 genes were annotated in the BAC-contigs most of them related with Molecular Functions of protein binding, related to the resistance of the species to abiotic stress. An integrated genetic map anchored to the genome has been obtained in which the BAC-contigs structure were not concordant with the gene structure of the C. gigas scaffolds displayed in the Genomicus database.

     

Differential invasion success in aquatic invasive species: the role of within- and among-population genetic diversity

Despite a well-developed theoretical basis for the role of genetic diversity in the colonization process, contemporary investigations of genetic diversity in biological invasions have downplayed its importance. Observed reductions in genetic diversity have been argued to have a limited effect on the success of establishment and impact based on empirical studies; however, those studies rarely include assessment of failed or comparatively less-successful biological invasions. We address this gap by comparing genetic diversity at microsatellite loci for taxonomically and geographically paired aquatic invasive species. Our four species pairs contain one highly successful and one less-successful invasive species (Gobies: Neogobius melanostomus, Proterorhinus semilunaris; waterfleas: Bythotrephes longimanus, Cercopagis pengoi; oysters: Crassostrea gigas, Crassostrea virginica; tunicates: Bortylloides violaceous, Botryllus schlosseri). We genotyped 2717 individuals across all species from multiple locations in multiple years and explicitly test whether genetic diversity is lower for less-successful biological invaders within each species pair. We demonstrate that, for gobies and tunicates, reduced allelic diversity is associated with lower success of invasion. We also found that less-successful invasive species tend to have greater divergence among populations. This suggests that intraspecific hybridization may be acting to convert among-population variation to within-population variation for highly successful invasive species and buffering any loss of diversity. While our findings highlight the species-specific nature of the effects of genetic diversity on invasion success, they do support the use of genetic diversity information in the management of current species invasions and in the risk assessment of potential future invaders.     

Relationship Among Intron Length,Gene Expression,and Nucleotide Diversity in the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

Crassostrea gigas is a model mollusk, but its genetic features have not been studied comprehensively. In this study, we used whole-genome resequencing data to identify and characterize nucleotide diversity and population recombination rate in a diverse collection of 21 C. gigas samples. Our analyses revealed that C. gigas harbors both extremely high genetic diversity and recombination rates across the whole genome as compared with those of the other taxa. The noncoding regions, introns, intergenic spacers, and untranslated regions (UTRs) showed a lower level diversity than the synonymous sites. The larger introns tended to have lower diversity. Moreover, we found a negative association of the non-synonymous diversity with gene expression, which suggested that purifying selection played an important role in shaping genetic diversity. The nucleotide diversity at the 100- and 50-kb levels was positively correlated with population recombination rates, which was expected if the diversity was shaped by purifying selection or hitchhiking of advantageous mutants. Our work gives a general picture of the oyster’s polymorphism pattern and its association with recombination rates.     

Phosphoglucose isomerase and esterase phenotypes in Crassostrea angulata and C. gigas

The phenotype distributions and the allele frequencies of the phosphoglucose isomerase and esterase loci examined in the samples of Crassostrea angulata (Essex, England) and C. gigas (Brittany) do not differ significantly and the two populations as such are indistinguishable. The validity of the species C. angulata is questioned and it is postulated that the two samples may be geographic isolates of the same species, i.e. C. gigas. The hatchery reared population of C. gigas from Conway is distinguishable from the other samples of Crassostrea examined. The lack of phenotype diversity is attributed to founder effects of the small parental stock imported in 1965. The distributions of all phenotypes are in agreement with Hardy-Weinberg expectations. Phosphoglucose isomerase (E.C. 5.3.1.9.) is a dimer governed by at least four alleles in C. angulata and five alleles in C. gigas. The slower (Es-S) zone of the esterase electrophoretogram would appear, in both species to be governed by four alleles at a single locus. There was no esterase banding which was specific to either species of Crassostrea.     

Estimates of Heritability for Growth and Shell Color Traits and Their Genetic Correlations in the Black Shell Strain of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The Pacific oyster Crassostrea gigas has been introduced widely and massively and became an economically important aquaculture species on a global scale. We estimated heritabilities of growth and shell color traits and their genetic correlations in black shell strain of C. gigas. Analyses were performed on 22 full-sib families in a nested mating design including 410 individuals at harvest (24 months of age). The parentage assignment was inferred based on four panels of multiplex PCR markers including 10 microsatellite loci and 94.9% of the offspring were unambiguously assigned to single parent pairs. The Spearman correlation test (r = ? 0.992, P < 0.001) demonstrated the high consistency of the shell pigmentation (SP) and L* and their same efficacy in shell color measurements. The narrow-sense heritability estimated under the animal model analysis was 0.18 ± 0.12 for shell height, 0.25 ± 0.16 for shell length, 0.10 ± 0.09 for shell width, 0.42 ± 0.20 for total weight, 0.32 ± 0.18 for shell weight, and 0.68 ± 0.16 for L*, 0.69 ± 0.16 for shell pigmentation, respectively. The considerable additive genetic variation in growth and shell color traits will make it feasible to produce genetic improvements for these traits in selective breeding program. High genetic and phenotypic correlations were found among growth traits and among shell color traits. To optimize a selection strategy for both fast growth and pure dark shell strain of C. gigas, it is proposed to take both total weight and black shell as joint objective traits in selective breeding program. Our study offers an important reference in the process of selective breeding in black shell color stain of C. gigas and will facilitate to develop favorable breeding strategies of genetic improvements for this economically important strain.     

Genetic characterization of <Emphasis Type="Italic">Wolbachia</Emphasis> from Great Salt Lake brine flies

Wolbachia are intracellular prokaryotic endosymbionts associated with a wide distribution of arthropod and nematode hosts. Their association ranges from parasitism to mutualism, and there is growing evidence that Wolbachia can have dramatic effects on host reproduction, physiology, and immunity. Although all Wolbachia are currently considered as single species, W. pipientis, phylogenetic studies reveal about a dozen monophyletic groups, each designated as a supergroup. This study uses 16S rRNA gene sequences to examine the genetic diversity of Wolbachia present in three species of Great Salt Lake brine flies, Cirrula hians, Ephydra gracilis, and Mosillus bidentatus. The brine fly Wolbachia sequences are highly similar, with an average nucleotide sequence divergence among the three species of 0.00174. The brine fly Wolbachia form a monophyletic group that is affiliated with a subset of supergroup B, indicating that this supergroup may be more diverse than previously thought. These findings expand the phylogenetic diversity of Wolbachia and extend their host range to taxa adapted to a hypersaline environment.     

Trojan Horse Strategy for Non-invasive Interference of <Emphasis Type="Italic">Clock</Emphasis> Gene in the Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.     

Mitochondrial DNA sequence analysis from multiple gene fragments reveals genetic heterogeneity of Crassostrea ariakensis in East Asia

The native Asian oyster, Crassostrea ariakensis is one of the most common and important Crassostrea species that occur naturally along the coast of East Asia. Molecular species diagnosis is a prerequisite for population genetic analysis of wild oyster populations because oyster species cannot be discriminated reliably using external morphological characters alone due to character ambiguity. To date there have been few phylogeographic studies of natural edible oyster populations in East Asia, in particular this is true of the common species in Korea C. ariakensis. We therefore assessed the levels and patterns of molecular genetic variation in East Asian wild populations of C. ariakensis from Korea, Japan, and China using DNA sequence analysis of five concatenated mtDNA regions namely; 16S rRNA, cytochrome oxidase I, cytochrome oxidase II, cytochrome oxidase III, and cytochrome b. Two divergent C. ariakensis clades were identified between southern China and remaining sites from the northern region. In addition, hierarchical AMOVA and pairwise Φ _ST analyses showed that genetic diversity was discontinuous among wild populations of C. ariakensis in East Asia. Biogeographical and historical sea level changes are discussed as potential factors that may have influenced the genetic heterogeneity of wild C. ariakensis stocks across this region.     

Molecular evolution of glutathione peroxidase (<Emphasis Type="Italic">Gpx</Emphasis>) family genes in desert poplar (<Emphasis Type="Italic">Populus euphratica</Emphasis> Oliv.)

Populus euphratica Oliv. is a poplar species that is distributed mainly in deserts, making it an interesting model in which to investigate molecular mechanisms underlying different stress responses. Here, we used molecular population genetic methods to detect potential selection in candidate genes belonging to the P. euphratica glutathione (GSH) peroxidase (Gpx) family, which are associated with an enzymatic mechanism that combats oxidative damage caused by reactive oxygen species (ROS) in plant cells; earlier studies have shown that Gpx proteins play important roles in coping with increased ROS levels during biotic and abiotic stresses in plants. We analyzed the nucleotide diversity and divergence patterns of five loci encoding Gpx genes, and 16 reference loci used as controls, in order to detect departures from the neutral expectation. Gpx1 has an excess of mid-frequency alleles; high intraspecific nucleotide diversity, distributed in the upper tail of the simulated neutral model; and extensive LD, showing strong evidence of balancing selection/local adaptation. The Gpx3.2 gene exhibits very low nucleotide diversity and divergence, suggesting that it has evolved under strong purifying selection. We failed to detect any evidence for natural selection at the other loci (Gpx2, Gpx4, and Gpx5) compared with the reference loci. The results show that nucleotide diversity and/or divergence differ greatly between members of the Gpx gene family, resulting from differential selective pressure acting on genes, and that adaptive evolution influenced the distribution of P. euphratica in desert regions.     

Hybrid plants preserve unique genetic variation in the St Helena endemic trees <Emphasis Type="Italic">Commidendrum rotundifolium</Emphasis> DC Roxb. and <Emphasis Type="Italic">C. spurium</Emphasis> (G.Forst.) DC

The island of St Helena in the South Atlantic Ocean has a rich endemic flora, with 10 endemic genera and 45 recognised endemic species. However, populations of most endemic species have undergone dramatic reductions or extinction due to over-exploitation, habitat destruction and competition from invasive species. Consequently, endemic species are likely to have lost genetic variation, in some cases to extreme degrees. Here, the entire extant wild populations and all planted trees in seed orchards, of two critically endangered species in the endemic genus Commidendrum (Asteraceae), C. rotundifolium and C. spurium, were sampled to assess levels of genetic variation and inbreeding. Six new microsatellite loci were developed from next-generation sequence data, and a total of 190 samples were genotyped. Some seed orchard trees contained alleles from both wild C. rotundifolium and C. spurium indicating they could be hybrids and that some backcrossing may have occurred. Some of these trees were more similar to C. rotundifolium than C. spurium both genetically and morphologically. Importantly, allelic variation was detected in the putative hybrids that was not present in wild material. C. rotundifolium is represented by just two individuals one wild and one planted and C. spurium by seven, therefore the seed orchard trees comprise an important part of the total remaining genetic diversity in the genus Commidendrum.     

<Emphasis Type="Italic">Otidea</Emphasis> species from China,three new species with comments on some previously described species

The genus Otidea was recently monographed and studied phylogenetically, but knowledge of the diversity and distribution of Otidea species in China is fragmentary. In this study, collections from China were examined morphologically and included in phylogenetic analyses. Using LSU, TEF1-α, and RPB2 new species were placed within previously recognized clades in the genus. The results agree with both Genealogical Concordance Phylogenetic Species Recognition (GCPSR) and genetic divergence as previously reported. Three new species, Otidea hanseniae, Otidea korfii and Otidea purpureogrisea are recognized based on phylogenetic reconstruction using ITS, LSU, TEF1- α and RPB2. Comments on some incompletely known species are added. With the discovery of these three new species, the genus Otidea in China proves to be more diverse than previously recognized.     

Effects of nonnative invertebrates on two life stages of the native eastern oyster <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

Since their recent introductions into Florida waters, three sessile invertebrates [Perna viridis (Asian green mussel), Mytella charruana (charru mussel) and Megabalanus coccopoma (pink titan acorn barnacle)] have expanded their range along the Atlantic coast in estuarine waters. Little research has been done to understand how these nonnative species interact with the ecologically and economically important eastern oyster Crassostrea virginica. To assess the potential effects of P. viridis, M. charruana and M. coccopoma on C. virginica, the following questions were addressed in manipulative experiments. (1) Does the presence of nonnative species decrease oyster larval settlement? (2) Do oyster larvae avoid settling on nonnative species? (3) Do nonnative species decrease survival of juvenile oysters (spat)? (4) Do nonnative species hinder spat growth? We included two controls: absence of nonnative species and presence of the native mussel Geukensia demissa. The nonnative species influenced settlement, growth and survival of C. virginica in different ways. M. coccopoma and P. viridis negatively influenced larval settlement, whereas M. charruana had no influence on the total number of settled larvae. Oyster larvae avoided settling on all three nonnative species and the native G. demissa. Both nonnative mussels negatively affected survival of juvenile oysters but only M. charruana also reduced spat growth. The native mussel, G. demissa, had no negative impacts on total settlement, survival and growth of C. virginica; in fact, it increased larval settlement in some trials. These three nonnative species should be classified as invasive because all had negative effects on native C. virginica.     

<Emphasis Type="Italic">Cantharellus</Emphasis> sect. <Emphasis Type="Italic">Amethystini</Emphasis> in Asia

In this contribution on the genus Cantharellus in Asia, C. subvaginatus is described from the Republic of Korea as a close relative to the Chinese C. vaginatus, which is here reported for the first time from India. Both species are here placed in Cantharellus subg. Cantharellus sect. Amethystini, together with the Indian C. pseudoformosus (syn.: C. umbonatus) and the Malayan C. subamethysteus. As such, Asia has suddenly become the continent with the highest diversity for Amethystini. Species delimitation in sect. Amethystini is molecularly supported by a combined phylogenetic analysis of rDNA sequences obtained for LSU and ITS and additionally suggests the existence of a still undescribed species in North America. Character variability is discussed for all known members of Amethystini, including atypical specimens of the North American C. lewisii that are morphologically more reminiscent of the South Korean C. subvaginatus.     

Diversity of fungi associated with roots of <Emphasis Type="Italic">Calanthe</Emphasis> orchid species in Korea

While symbiotic fungi play a key role in the growth of endangered Calanthe orchid species, the relationship between fungal diversity and Calanthe species remains unclear. Here, we surveyed root associated fungal diversity of six Calanthe orchid species by sequencing the internal transcribed spacer (ITS) region using 454 pyrosequencing. Our results revealed that Paraboeremia and Coprinopsis are dominant fungal genera among Calanthe species. In terms of overall relative abundance, Paraboeremia was the most common fungal genus associated with Calanthe roots, followed by Coprinopsis. Overall fungal diversity showed a significant degree of variation depending on both location and Calanthe species. In terms of number of different fungal genera detected within Calanthe species, C. discolor had the most diverse fungal community, with 10 fungal genera detected. This study will contribute toward a better understanding of those fungi that are required for successful cultivation and conservation of Korean Calanthe species.     

Distribution of <Emphasis Type="Italic">Divo</Emphasis> in <Emphasis Type="Italic">Coffea</Emphasis> genomes,a poorly described family of angiosperm LTR-Retrotransposons

Coffea arabica (the Arabica coffee) is an allotetraploid species originating from a recent hybridization between two diploid species: C. canephora and C. eugenioides. Transposable elements can drive structural and functional variation during the process of hybridization and allopolyploid formation in plants. To learn more about the evolution of the C. arabica genome, we characterized and studied a new Copia LTR-Retrotransposon (LTR-RT) family in diploid and allotetraploid Coffea genomes called Divo. It is a complete and relatively compact LTR-RT element (~5 kb), carrying typical Gag and Pol Copia type domains. Reverse Trancriptase (RT) domain-based phylogeny demonstrated that Divo is a new and well-supported family in the Bianca lineage, but strictly restricted to dicotyledonous species. In C. canephora, Divo is expressed and showed a genomic distribution along gene rich and gene poor regions. The copy number, the molecular estimation of insertion time and the analysis at orthologous locations of insertions in diploid and allotetraploid coffee genomes suggest that Divo underwent a different and recent transposition activity in C. arabica and C. canephora when compared to C. eugenioides. The analysis of this novel LTR-RT family represents an important step toward uncovering the genome structure and evolution of C. arabica allotetraploid genome.