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.
Citation: Zhong X, Li Q, Yu H, Kong L (2014) SNP Mining in Crassostrea gigas EST Data: Transferability to Four Other Crassostrea Species, Phylogenetic Inferences and Outlier SNPs under Selection. PLoS ONE 9(9): e108256. https://doi.org/10.1371/journal.pone.0108256
Editor: Donald James Colgan, Australian Museum, Australia
Received: June 7, 2014; Accepted: August 28, 2014; Published: September 19, 2014
Copyright: © 2014 Zhong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: This study was supported by the grants from 863 Program (2012AA10A405-6), 973 Program (2010CB126406), and National Natural Science Foundation of China (31372524). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Oysters are widely distributed throughout tropical and subtropical regions, inhabiting near-shore areas, shallow waters, bays, and estuaries . Crassostrea oysters are important commercial species and account for most of the world's oyster production. Approximately 20 species make up the genus Crassostrea, of which C. gigas has become the leading species in world shellfish culture because of its rapid growth and capacity to adapt to various environmental conditions. Besides C. gigas, C. hongkongensis, C. ariakensis, C. sikamea and C. angulata are locally important species in China, Japan, Korea, the United States and some European countries. The rapid growth of the oyster aquaculture industry as well as intentional introduction or transplantation of oysters pressingly requires an appropriate understanding of the genetic variation within and among various oyster species. However, conventional taxonomic and phylogenetic studies based on morphology and geographic range information have proved problematic because of highly plastic shell patterns and overlapping geographic distributions –. There are ongoing debates as to the species designations in the genus Crassostrea, such as the specific status of C. gigas and C. angulata, and the nomenclature of C. hongkongensis and C. ariakensis. The ongoing confusion about oyster taxonomy and identification has become an impediment to further investigation of the genetics and conservation of oysters.
In recent years, relationships and identification of oyster species have been investigated by using allozymes, randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and DNA sequences such as mitochondrial and nuclear genes –. Particularly, the ability to sequence and compare whole mitochondrial genomes provides a new insight into phylogenetic relationships of oysters –. However, mtDNA loci are uniparentally inherited and cannot alone represent all historical and contemporary processes acting upon a population . Moreover, because mtDNA is fast evolving and nucleotide mutations may return to an earlier state, its sequences may not allow deep phylogenetic reconstruction . Hence, incorporating nuclear markers appears necessary to increase confidence in determining the relationships of Crassostrea species.
Single-nucleotide polymorphisms (SNPs) have become cornerstone markers for a wide variety of genetic applications because they are the most abundant class of polymorphisms in genomes, and can be genotyped cost-effectively , . Besides, SNP can be found within the genomic sequences of gene candidates for artificial or natural selection and therefore they might be more informative for evolutionary biology than markers such as microsatellites and AFLPs. They offer a wide range of applications such as association studies, high-density linkage maps, traceability of genealogies and phylogenetic inference , .
The rapid increase in the availability of EST sequences of Crassostrea gigas provides abundant resources for obtaining SNP markers –. To date, 320 SNPs have been developed for C. gigas by mining expressed sequence tags data, using the HRM method –. Nevertheless, SNP markers for C. hongkongensis, C. ariakensis, C. sikamea and C. angulata have not been documented. Transferred SNPs from C.gigas provide a valuable source of SNP markers for the four species. Such cross-species EST–SNPs will be useful for comparative mapping and phylogenetic studies among species in Crassostrea.
Here, 57 novel SNPs were developed from the NCBI EST database (http://www.ncbi.nlm.nih.gov/) of C. gigas and the cross-species transferability of 377 SNPs of C. gigas was tested among C. hongkongensis, C. ariakensis, C. sikamea and C. angulata. Meanwhile, through the use of the cross-species SNPs, we reconstructed the phylogenetic relationships among the five Crassostrea species. Moreover, through the use of Fst outlier analysis, we identified candidate SNPs that may have been targets of selection.
Materials and Methods
The field studies did not involve endangered or protected species. No specific permissions were required for the locations. The locations are not privately-owned or protected in any way.
Oyster Materials and DNA Extraction
Thirty-two C. gigas individuals from 2 populations (Pop1: 16 individuals from Weihai, Shandong province, China; Pop2: 16 individuals from Rizhao, Shandong province, China) were used for validation of SNP polymorphisms. Five Crassostrea species collected from China were used for the examination of the transferability of SNPs, namely C. sikamea (from Nantong, Jiangsu Province), C. angulata (from Yueqing, Zhejiang Province), C. hongkongensis (from Xiamen, Fujian Province), C. ariakensis (from Shantou, Guangdong Province) and C. gigas (from Rushan, Shandong Province) (Table 1). A set of species-specific COI primers was used for species identification according to the study of Wang & Guo .
DNA was extracted from frozen adductor muscle tissue by a modification of the standard phenol–chloroform procedure previously described by Li et al.  and stored at −30°C prior to genetic analysis.
Data Mining for SNP Markers
Sequences containing SNPs were annotated using BLASTx software , and sequence homology was accepted based on a cut-off E value of 1.0×10−6. The informative strand and reading frame were identified by using the sequence with highest homology. The NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to determine whether SNPs were synonymous, non-synonymous or from untranslated regions (UTRs).
Primer Design and PCR Conditions
Primers were designed using the Primer Premier 5.0 program (PREMIER Biosoft International, Palo Alto, CA, USA). SNP markers were developed according to the procedure described by Zhong et al.  and genotyped using the high resolution melting (HRM) method on the LightCycler 480 real-time PCR instrument (Roche Diagnostics, Burgess Hill, UK). A total of 46,171 Pacific oyster EST sequences were downloaded from GenBank EST database (http://www.ncbi.nlm.nih.gov/). The sequences were assembled and clustered into contigs with SeqMan Pro software (DNASTAR Inc., Madison, WI, USA). A single-base mutation that occurred in four or more ESTs and that was surrounded by good flanking sequences was identified as a potential SNP for further analysis.
The 10-µl reaction mixture contained 0.25 U Taq DNA polymerase (Takara, Dalian, China), 10× PCR buffer, 0.2 mM dNTP mix, 0.2 µM of each primer set, 1.5 mM MgCl2, 5 µM SYTO9 (Invitrogen Foster City, CA, USA) and 10 ng template DNA. The concentration of DNA was measured by a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA). The PCR cycling conditions included an activation step at 95°C for 5 minutes followed by 45–50 cycles of 95°C for 20 seconds, a touch down of 68°C to 58°C for 20 seconds (0.5°C/cycle) and 72°C for 20 seconds. Following amplification, the products were denatured at 95°C for 1 min, and then annealed at 40°C for 1 min to randomly form DNA duplexes. Melting curves were generated by heating samples from 60°C to 90°C with 25 data acquisitions per degree. Data were analyzed using the LightCycler 480 Gene Scanning Software 1.5 (Roche Diagnostics).
Shannon's Information index, expected heterozygosity (He), observed heterozygosity (Ho) and Nei's genetic distance  were calculated using POPGENE 1.32 software . Phylogenetic trees were constructed using the neighbor joining (NJ) method implemented in MEGA 5.05 and POPTREE2 , . Bootstrap analyses with 1000 replicates were performed to test the support for the branches of a phylogenetic tree.
Arlequin version 184.108.40.206 software was used to calculate pairwise Fst between all pairs of species using 10000 permutations to test for significance (0.01). Outlier SNPs were tested using two island models, as implemented in Arlequin. We conducted 50000 coalescent simulations with 5 demes under a finite island-model. The analysis was also performed utilizing a hierarchical island model based on 3 groups of 3 demes with 50000 simulations to generate the joint distribution of Fst versus heterozygosity. Pre-defined population groupings were set as three groups (group 1: C. sikamea, C. angulata and C. gigas; group 2: C. hongkongensis; group 3: C. ariakensis) based on the pairwise Fst values. Loci that fall out of the 99% confidence intervals of the distribution were identified as outliers being putatively under selection. The putative function of genes with outlier SNPs was identified using the Gene Ontology (GO) annotation by mining the Swiss-Prot database.
Development and Transferability of SNPs
In the study, 262 putative SNPs were selected for validation. Among these, 57 SNPs (22%) were polymorphic and considered as validated. Information about the panel of loci is summarized in Table 2. The 57 substitutions included 41 transitions and 16 transversions. Of the polymorphic SNPs, 30 (52.6%) could not be annotated, 53 (93.0%) were located in the coding region, and 4 (7.0%) in the UTR. Eighteen of the 53 SNPs located within the coding region were nonsynonymous and 35 synonymous.
A total of 377 SNPs of C. gigas including 320 previously developed SNPs – and 57 new SNPs developed here were used to test the transferability in 4 other Crassostrea species: C. sikamea, C. angulata, C. hongkongensis and C. ariakensis. The basic information obtained with each SNP is shown in Table S1. Out of 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, 253 (67.1%) in C. ariakensis and 377 (100%) in C. gigas. Using the 377 primer pairs, 256 (67.9%) SNP loci were polymorphic in C. sikamea, 306 (81.2%) in C. angulata, 133 (35.3%) in C. hongkongensis, 119 (31.6%) in C. ariakensis and 335(88.9%) in C. gigas (Table 1). In total, 214 SNPs could give successful amplification in all the five Crassostrea species and 48 SNPs showed polymorphism in all the five species.
A total of 214 SNPs was used for the phylogenetic analysis. Information of the 214 SNPs evaluated from the 5 species is shown in Table 3. The values of observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.0792 (C. hongkongensis) to 0.2895 (C. gigas) and from 0.1026 (C. hongkongensis) to 0.3229 (C. gigas), respectively. Shannon's Information index and the number of polymorphic loci ranged from 0.1664 (C. ariakensis) to 0.4749 (C. gigas) and from 99 (C. ariakensis) to 201 (C. gigas). Nei's genetic distance values ranged from 0.0738 (C. angulata and C. gigas) to 0.2728 (C. hongkongensis and C. gigas) (Table 4). All Fst estimates were statistically significant (P<0.01). Pairwise Fst ranged from 0.1230 (C. angulata and C. gigas) to 0.5257 (C. hongkongensis and C. ariakensis). The phylogenetic tree separated the five species into two clusters (Figure 1). The first cluster included two species, C. hongkongensis and C. ariakensis. This clade was sister to the clade containing C. sikamea, C. angulata and C. gigas. In this clade, C. gigas and C. angulata had the closest relationship, with C. sikamea being the sister group. Phylogenetic analysis using the unweighted pair-group method with arithmetic mean (UPGMA) generated an identical topology with high support values (data not shown).
Numbers above branches indicate bootstrap values from NJ analysis using both MEGA 5.05 and POPTREE2 softwares.
Loci showing higher or lower differentiation with respect to the simulated confidence intervals are identified as candidates for positive or balancing selection . The Arlequin fdist method revealed 10 candidate SNPs (CgSNP28, CgSNP230, CgSNP273, CgSNP415, CgSNP420, CgSNP515, CgSNP524, CgSNP544, CgSNP669 and CgSNP805) for selection, including 7 for positive selection and 3 for balancing selection (Table 5 and Figure 2a). In addition, the hierarchical method detected 11 outlier loci (CgSNP14, CgSNP203, CgSNP803, CgSNP273, CgSNP415, CgSNP420, CgSNP515, CgSNP524, CgSNP544, CgSNP669 and CgSNP805) for selection, including 9 for positive selection and 2 for balancing selection (Table 5 and Figure 2b). Both approaches revealed 8 SNPs lying outside the 99% confidence region of the conditional joint distribution of Fst and heterozygosity, including 6 for positive selection and 2 for balancing selection. Among the 8 SNPs, 5 located within the coding region were synonymous and 3 nonsynonymous. The putative function of three genes (UPF0686 protein, ankyrin repeat domain-containing protein 60, and hypothetical protein CGI_10016494) could not be identified using GO searches. The other five proteins (endoglucanase, rho-related GTP-binding protein, flap endonuclease 1-A, hypothetical protein CGI_10023940 and Chlorophyllase-2) were respectively involved in carbohydrate metabolism, GTPase-mediated signal transduction, DNA repair, DNA binding and chlorophyll catabolic process.
The upper and lower lines are the 99% confidence intervals.
A total of 48769 potential SNPs were detected by mining the C. gigas EST database . In our studies, the 1283 putative SNPs selected for validation allowed the development of 57 new SNPs bringing the total to 377 SNPs that have been validated in this species –,. Among the 377 SNPs, 66 SNPs are known to be distributed in 8 linkage groups of C. gigas . Compared to the use of several (often partial) genes, the adequate number of EST–SNPs, distributed in almost all linkage groups of C. gigas, may provide more genetic information which is valuable for phylogenetic analyses. The high cross-species transferability of the set of 377 EST-SNPs of C. gigas tested in four other Crassostrea species also suggests their potential utilization in evolutionary analysis across taxa of the genus Crassostrea. Moreover, mutations resulting in some SNPs can be responsible for an adaptive phenotype or the direct target of selection. Studies have shown that variation in allele frequencies at some outlier SNP loci can be correlated with environmental variables, such as salinity and temperature , . Consequently, the SNP markers offer a valuable opportunity to understand the genetic basis of phenotypic variation in relation to environmental variation.
In general, the more evolutionarily distant the taxa, the less successful is cross amplification , . In a previous study, 15 EST-SSRs developed for C. gigas amplified successfully in at least one species, with C. sikamea sharing 14 (93.3%) primer pairs, C. hongkongensis 12 (80.0%), and C. ariakensis 11(73.3%) . Hedgecock et al.  tested 86 genomic SSRs developed for C. gigas in cross-species amplification, 83 (96.5%) were likely useful for C. angulata, 71 (82.6%) for C. sikamea and 31 (36.0%) for C. ariakensis. Our data also showed C. angulata (93.6%) and C. sikamea (82.5%) had higher cross-amplification rates than both C. hongkongensis (67.4%) and C. ariakensis (67.1%). These results suggest that C. gigas has a closer relationship with C. angulata and C. sikamea than with C. hongkongensis and C. ariakensis.
The taxonomy of Crassostrea has been studied for many years, but confusions still exist. There is an open debate as to whether C. gigas and C. angulata are distinct species , , , . Some experts have argued that they are different species but genetically closely related , , , but other phylogenetic analyses suggest that the two should be considered one species , . In our study, C. gigas and C. angulata were recovered as separate clades, suggesting that C. gigas and C. angulata may be two distinct species. However, the low Nei's genetic distance value between C. angulata and C. gigas (0.0738) indicates a very close relationship between them. Furthermore, C. angulata and C. gigas can cross-fertilize without any difficulty in the laboratory and form viable, fertile offspring –. Therefore, we still can not conclude that C. gigas and C. angulata are two distinct species. A large amount of the two species sampled from a wide geographic range and the same locations are required to better resolve this problem. Another species, C. hongkongensis has been routinely misidentified as C. ariakensis for a long time. In our study, C. hongkongensis and C. ariakensis were recovered as separate clades. Moreover, the Nei's genetic distance between C. hongkongensis and C. ariakensis (0.1396) was a little higher than that observed between two closely related sister species (between C. angulata and C. sikamea, 0.1327). The above data suggest that C. hongkongensis and C. ariakensis are two distinct species. Yu & Li  analyzed the complete mitochondrial DNA sequence and determined that C. hongkongensis and C. ariakensis are two separate species. Reece et al.  also suggested that the C. ariakensis sequences formed a distinct clade from C. hongkongensis in the COI tree. Therefore, we can conclude that C. hongkongensis and C. ariakensis are two separate species.
Identifying the regions of the genome that are shaped by adaptation to different environments can be relevant to answering several important questions in evolutionary biology. Among many selection detection strategies, Fst outlier approaches are becoming widely used in identifying genes without known phenotypes that are under selection , , . These methods can identify relatively highly differentiated markers (so-called outlier loci) in comparison to expected levels under neutrality inferred from coalescent simulations , . Strong outlier patterns have been classically interpreted as being caused by divergent selection affecting the loci themselves or genes strongly linked with them . Indeed, an alternative explanation for strong genetic divergence at some loci exists and is difficult to rule out when the tests are being made on comparisons of distinct species. Bierne et al.  advocate the role of pre- or postzygotic genetic barriers in genetic divergence. Such endogenous barriers could be the consequence of incompatibilities between combinations of alleles, established through selective mechanisms that are independent from adaptation to habitats . To increase confidence in the conclusions reached, two-island models and a high confidence level (99%) were used in the Fst outlier analysis.
Eight loci were identified as being possible targets of selection following two Fst outlier tests. Among the 8 SNPs, 5 located within the coding region were synonymous and 3 nonsynonymous. While nonsynonymous outlier SNPs are particularly interesting due to the potential effect of amino acid changes on protein structure and function, synonymous SNPs should not be simply dismissed as false-positives. This is because natural selection may affect synonymous codon usage in some genes, leading to codon usage bias , . Furthermore, there is increasing evidence that silent mutations may have functional effects either on translational efficiency and accuracy, or on mRNA stability and splicing. Another explanation is that they might carry the footprint of selection on a beneficial allele that is closely linked to the outlier SNP.
In marine environments, environmental factors such as temperature, salinity, pH and dissolved oxygen often interact in complex ways leading to a complicated ‘fitness landscape’. In our study, C. angulata and C. gigas were sampled from coastal zones, whereas C. sikamea, C. hongkongensis and C. ariakensis were sampled from estuarine zones. Moreover, the five species were collected from 5 sites across 13° of latitude along the coast of China. Therefore, water temperature and salinity may be environmental variations relevant to fitness. The importance of the cytoskeleton in the adaptation to water temperature and salinity is well known –. Major players during cytoskeletal remodeling are rho-GTPases, upstream molecular switches triggering signaling cascades that target cytoskeletal effector proteins to induce morphological change . Another key aspect of the cell stress response is modulation of pathways of energy metabolism . The data presented here reveal that two genes with outlier SNPs (endoglucanase and rho-related GTP-binding protein) are involved in carbohydrate metabolism and GTPase-mediated signal transduction. Furthermore, the ankyrin repeat domain-containing protein 60 may be involved in cytoskeletal motility regulation . Although the genomic scan provides an encouraging result, association genetics and functional studies are ultimately required to confirm that particular loci are involved in responding to environmental variations.
In summary, a total of 57 SNPs from EST sequences in C. gigas were developed using HRM method. The study confirmed a high cross-species transferability of the set of 377 EST-SNPs of C. gigas tested in four other Crassostrea species. Additionally, the current study represents an initial attempt at resolving phylogenetic relationships in Crassostrea species, using a large collection of cross-species SNP markers. The NJ analysis revealed two main groups of the five Crassostrea species. The first clade included C. hongkongensis and C. ariakensis. C. hongkongensis was a sister species of C. ariakensis. This clade was sister to the clade containing C. sikamea, C. angulata and C. gigas. C. gigas and C. angulata had the closest relationship, with C. sikamea being the sister group. Finally, the work, using Fst outlier approaches, presented evidence for adaptive genetic divergence in Crassostrea species. Further functional studies are needed to confirm the role of these outlier loci or genome segments in Crassostrea species.
Conceived and designed the experiments: XZ QL. Performed the experiments: XZ. Analyzed the data: XZ. Contributed reagents/materials/analysis tools: XZ QL HY LK. Contributed to the writing of the manuscript: XZ QL.
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