Pathway analysis for alanine and beta-alanine metabolism.

<p>The map displays selected steps from KEGG pathways map00473, ‘D-Alanine metabolism’, and map00410, ‘beta-Alanine metabolism’. A red rectangle indicates increased expression of genes compared with salt 30, while blue indicates decreased expression of genes. The EC number with an underline was validated via Q-RT PCR. The transcriptome data and Q-RT PCR data were displayed in the form of columnar diagram. For Q-RT PCR data, vertical bars represent the mean ± S.D (N  =  3). Significant differences between salt stress treatment (salt 5, 10, 15, 20, 25 and 40) and control (salt 30) are analyzed by one-way ANOVA (**<i>P</i><0.01, *<i>P</i><0.05).</p

Marker genes search under different salinity treatments.

<p>(A) Venn diagram of gene sets identified under various salt stress conditions. The green region indicates ‘extreme low salt stress’ (salt 5); The yellow indicates ‘low salt stress’ (salt 10 and 15); The blue indicates ‘moderate low salt stress’ (salt 20 and 25); The red indicates ‘moderate high salt stress’ (salt 40). <b>(</b>B, C) The expressions of <i>HSP beta 1</i> (B) and <i>Glycine transporter</i> (C) in transcriptome data and Q-RT PCR. For Q-RT PCR data, vertical bars represent the mean ± S.D (N  =  3). Significant differences between salt stress treatment (salt 5, 10, 15, 20, 25 and 40) and control (salt 30) are analyzed by one-way ANOVA (**<i>P</i><0.01, *<i>P</i><0.05).</p

Global metabolic pathway (iPath) analysis for expanding KOs.

<p>Green lines represent expanded pathways in intertidal animals (<i>C. gigas, Capitella capitata, Lottia gigantean, Strongylocentrotus purpuratus and Helobdella robusta</i>) compared with terrestrial animals (<i>Drosophila melanogaster, Apis mellifera and Homo sapiens</i>), and the red line represents the oyster (<i>C. gigas</i>) expanded pathways compared with seven other species.</p

Molecular model of the oyster long-term salt stress response.

<p>The map describes salt stress-related signal transduction, effectors and physiological changes in the oyster. The italicized characters in a rectangle with a white border indicate enriched genes under the salt stress response. PLA2: Phospholipase A2, CT: Calcitonin receptor, CAM: Calmodulin-like protein, GPCR: G-protein coupled receptor, TAUT: Taurine transporter, APOL: Apolipoprotein-L, APLP: The amyloid precursor-like protein, CDO: Cysteine dioxygenase, CSAD: Cysteine sulfinic acid decarboxylase, GLDC: Glycine dehydrogenase, AMT: Aminomethytransferase, P5CS: Δ-1- pyrroline-5-carboxylate synthase, P5CR: Δ-1-pyrroline-5-carboxylate reductase, ALT: Alanine transaminase, AGT: Alanine-glyoxylate transaminase, GAD: Glutamate decarboxylase, SHMT: Serine hydroxymethyl transferase, GPX: Glutathione peroxidase, GST: Glutathione S-transferase, MT: Metallothionein, IAP: Inhibitor apoptosis protein, CYP450: Cytochrome P450, FMO: Flavin-containing monooxygenase, ALDH: Aldehyde dehydrogenase.</p

Pathway analysis of arginine and proline metabolism.

<p>The map displays selected steps from KEGG map00330, ‘Arginine and proline metabolism’. A red rectangle indicates increased expression genes compared with salt 30, while blue indicates decreased expression of genes, and the gray rectangle indicates no significant changes. The EC number with an underline was validated via Q-RT PCR. The transcriptome data and Q-RT PCR data were displayed in the form of columnar diagram. For Q-RT PCR data, vertical bars represent the mean ± S.D (N  =  3). Significant differences between salt stress treatment (salt 5, 10, 15, 20, 25 and 40) and control (salt 30) are analyzed by one-way ANOVA (**<i>P</i><0.01, *<i>P</i><0.05).</p

FAAs contents in gills of oysters after 7 days different salinities treatments.

<p>Values of FAAs contents are in gram/kgram dry weight. The content values represent the mean value in three different individuals. Salt 5, 10, 15, 20 and 25 were hypo-osmotic stress, salt 40 was hyper-osmotic stress, and salt 30 was the normal salinity.</p

Pathway analysis of glycine metabolism.

<p>The map displays selected steps from KEGG map00260, ‘Glycine, serine and threonine metabolism’. A red rectangle indicates increased expression compared with salt 30, and blue indicates decreased expression compared with salt 30. The EC number with an underline was validated via Q-RT PCR. The transcriptome data and Q-RT PCR data were displayed in the form of columnar diagram. For Q-RT PCR data, vertical bars represent the mean ± S.D (N  =  3). Significant differences between salt stress treatment (salt 5, 10, 15, 20, 25 and 40) and control (salt 30) are analyzed by one-way ANOVA (**<i>P</i><0.01, *<i>P</i><0.05).</p

Selection acting on the NE1 locus.

<p>(A) Maximum likelihood tree based on select NE1 <i>(red)</i> and nonNE1 <i>(blue)</i> haplotypes, with the chimpanzee haplotype as an outgroup. The gray-box indicates the estimated interval for the Human-Neandertal divergence between 400,000–800,000 years ago <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003404#pgen.1003404-Eriksson1" target="_blank">[51]</a>. Note that the coalescence at this locus is extremely long and very unlikely to have evolved under neutral conditions as modeled here. (B) Comparison of <i>F_ST</i> and Tajima's <i>D</i> values of 10 kb intervals across the human genome. The red to dark blue gradient indicates decreased density of observed events at a given location in the graph. The NE1 locus, and other loci with similar profiles, are highlighted in white.</p

Ancient African origins of the NE1 haplogroup.

<p>(A) Models of scenarios that could lead to NE1 haplotypes observed in humans and Neandertals. The frequency of the NE1 haplogroup is depicted in red and the frequency of the nonNE1 haplogroup in yellow. The red corresponds to higher frequencies, whereas yellow corresponds to lower frequencies of the NE1 haplotypes in the population. The arrows represent the direction of possible admixture events. The left panel represents a model, under which the NE1 haplotypes admixed into Eurasian populations (Asn and Eur) after Human-Neandertal divergence. The second model, which is depicted in the central panel, is similar to the first model, except with the addition of more recent back migration of Eurasian NE1 haplotypes into Africa (Afr). The right panel shows the third model, under which the NE1 haplotypes among humans are explained by persistence of ancient African substructure. All these scenarios were based on the assumption that the NE1 haplotype occurs at high frequency or is fixed in the Neandertal population given that the available Neandertal sequences align well to the NE1 haplotype. (B) Geographical distribution of the NE1 haplogroup. We estimated the proportion of chromosomes that carry the CNVR8163.1 deletion from various sources described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003404#s4" target="_blank"><i>Materials and Methods</i></a>. The dark red portion of each circle represents the frequency of the homozygous nonNE1 genotypes, the white represents the homozygous NE1 genotypes and the light red represents the frequency of heterozygote genotypes. Note the existence of the NE1 haplotypes (i.e., as heterozygotes, <i>light</i> red) among sub-Saharan African populations (e.g., LWK and ASW) as well as the high frequency of heterozygotes (<i>light red</i>) in the European populations. (C) The pairwise distances between the African (Afr) NE1 haplotypes, the Asian (Asn) NE1 haplotypes, and the European (Eur) NE1 haplotypes, calculated using phase 1 data from the 1000 Genomes Project. p-values were calculated by the Mann-Whitney test.</p

The regulatory functions of NE1 locus.

<p>(A) The LTR region was determined using the Repeat Masker Track version 3.2.7 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003404#pgen.1003404-Jurka1" target="_blank">[63]</a>. Dark red boxes indicate the location of amplicons for ChIP qPCR. TFBS-1 and TFBS-2 refers to the two predicted transcription factor binding sites that were predicted by the latest ENCODE project data release, available in UCSC Genome Browser for the hg19 assembly. The CNVR8163.1 deletion polymorphism is flanked by black vertical lines. The blue vertical lines indicate the approximate locations of SNPs that differentiate between NE1 and nonNE1 haplogroups and overlap the TFBS-1 and TFBS-2 transcription factor binding sites. The 8 SNPs that overlap with TFBS-2 are from 5′ to 3′, rs132525, rs4306795, rs4434085, rs5750701, rs35853418, rs6001308, rs6001309, and rs9622868) (B) Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) results across the NE1 locus for representative samples NA12155 and NA10851 that belong to NE1 and nonNE1 haplogroups, respectively. The locations of the amplified segments (P1–P6) are shown in dark red rectangles in (A). The positive control primers amplify a segment within <i>BCL6</i> gene on chromosome 3 that is known to have high H3K4me2 occupancy. The blue stars indicate significant differences in qPCR amplification between NE1 and nonNE1 haplotypes (p<0.01). The brown and blue arrows indicate qPCR primers that are closest to the predicted transcription binding sites (P1, P2, P3 for TFBS-1 and P7 for TFBS-2). Overall, our results demonstrate that H3K4me2 is enriched in NA12155 cells, which harbor the NE1 deletion as compared to NA10851 cells which do not have the deletion (data plotted represents the average of four replicate experiments ± Std. Error).</p