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Qi H.,CAS Qingdao Institute of Oceanology | Qi H.,Laboratory for Marine Biology and Biotechnology | Song K.,CAS Qingdao Institute of Oceanology | Song K.,Laboratory for Marine Fisheries and Aquaculture | And 10 more authors.
PLoS ONE | Year: 2017

Single nucleotide polymorphisms (SNPs) are widely used in genetics and genomics research. The Pacific oyster (Crassostrea gigas) is an economically and ecologically important marine bivalve, and it possesses one of the highest levels of genomic DNA variation among animal species. Pacific oyster SNPs have been extensively investigated; however, the mechanisms by which these SNPs may be used in a high-throughput, transferable, and economical manner remain to be elucidated. Here, we constructed an oyster 190K SNP array using Affymetrix Axiom genotyping technology. We designed 190,420 SNPs on the chip; these SNPs were selected from 54 million SNPs identified through re-sequencing of 472 Pacific oysters collected in China, Japan, Korea, and Canada. Our genotyping results indicated that 133,984 (70.4%) SNPs were polymorphic and successfully converted on the chip. The SNPs were distributed evenly throughout the oyster genome, located in 3,595 scaffolds with a length of [[amp]]sim;509.4 million; the average interval spacing was 4,210 bp. In addition, 111,158 SNPs were distributed in 21,050 coding genes, with an average of 5.3 SNPs per gene. In comparison with genotypes obtained through re-sequencing, [[amp]]sim;69% of the converted SNPs had a concordance rate of >0.971; the mean concordance rate was 0.966. Evaluation based on genotypes of full-sib family individuals revealed that the average genotyping accuracy rate was 0.975. Carrying 133 K polymorphic SNPs, our oyster 190K SNP array is the first commercially available high-density SNP chip for mollusks, with the highest throughput. It represents a valuable tool for oyster genome-wide association studies, fine linkage mapping, and population genetics. © 2017 Qi 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.


Wang J.,CAS Qingdao Institute of Oceanology | Wang J.,University of Chinese Academy of Sciences | Wang J.,Laboratory for Marine Biology and Biotechnology | Wang J.,National and Local Joint Engineering Laboratory of Ecological Mariculture | And 6 more authors.
G3: Genes, Genomes, Genetics | Year: 2016

Oysters are among the most important species in global aquaculture. Crassostrea gigas, and its subspecies C. angulata, are the major cultured species. To determine the genetic basis of growth-related traits in oysters, we constructed a second-generation linkage map from 3367 single-nucleotide polymorphisms (SNPs) based on genotyping-by-sequencing, genotyped from a C. gigas × C. angulata hybrid family. These 3367 SNPs were distributed on 1695 markers, which were assigned to 10 linkage groups. The genetic linkage map had a total length of 1084.3 cM, with an average of 0.8 cM between markers; it thus represents the densest genetic map constructed for oysters to date. Twenty-seven quantitative trait loci (QTL) for five growth-related traits were detected. These QTL could explain 4.2-7.7% (mean = 5.4%) of the phenotypic variation. In total, 50.8% of phenotypic variance for shell width, 7.7% for mass weight, and 34.1% for soft tissue weight were explained. The detected QTL were distributed among eight linkage groups, and more than half (16) were concentrated within narrow regions in their respective linkage groups. Thirty-eight annotated genes were identified within the QTL regions, two of which are key genes for carbohydrate metabolism. Other genes were found to participate in assembly and regulation of the actin cytoskeleton, signal transduction, and regulation of cell differentiation and development. The newly developed high-density genetic map, and the QTL and candidate genes identified provide a valuable genetic resource and a basis for marker-assisted selection for C. gigas and C. angulata. © 2016 Wang et al.


Huang B.,CAS Qingdao Institute of Oceanology | Huang B.,Laboratory for Marine Fisheries and Aquaculture | Zhang L.,CAS Qingdao Institute of Oceanology | Tang X.,CAS Qingdao Institute of Oceanology | And 5 more authors.
Marine Biotechnology | Year: 2016

Alternative splicing (AS) is thought to enhance transcriptome diversity dramatically and play an important role in stress adaptation. While well studied in vertebrates, AS remains poorly understood in invertebrates. Here, we used high-throughput RNA-sequencing data to perform a genome-wide survey of AS in the Pacific oyster (Crassostrea gigas), an economically important mollusk that is cultivated worldwide. This analysis identified 8223 AS events corresponding to 4480 genes in the Pacific oyster, suggesting that about 16 % of oyster multiexonic genes undergo AS. We observed that a majority of the identified AS events were related to skipped exons (37.8 %). Then Gene Ontology analysis was conducted to analyze the function of the genes that undergo AS and the genes that produce more than five AS isoforms. After that, the expression of AS isoforms facing temperature, salinity, and air exposure challenge were examined. To validate our bioinformatic-predicted results and examine whether AS affects stress adaptation, we selected heat-shock protein 60 (HSP60) and HSP90 genes, both of which experience AS, for reverse transcription PCR (RT-PCR). We also performed quantitative real-time PCR (qRT-PCR) to determine the relative expression of each AS isoform among different stress adapted populations. Our study indicates that AS events are likely complex in the Pacific oyster and may be related to stress adaptation. These results will complement the predicted gene database of C. gigas and provide an invaluable resource for future functional genomic studies on molluscs. © 2016 Springer Science+Business Media New York


Sun X.,Chinese Academy of Fishery Sciences | Liu Q.-H.,Chinese Academy of Fishery Sciences | Liu Q.-H.,Laboratory for Marine Fisheries and Aquaculture | Yang B.,Chinese Academy of Fishery Sciences | And 3 more authors.
Fish and Shellfish Immunology | Year: 2016

WSSV is one of the most harmful pathogeny in the pacific white shrimp, and genetic variations caused the strains of different virulence. MicroRNAs (miRNAs) involved in the regulation of virus defense. To understand the different virulence of WSSV on miRNA expression in Litopeneaus vannamei, the deep sequencing was performed to compare two small RNA libraries prepared from hepatopancreas of Litopeneaus vannamei infected with normal-virulence or low-virulence WSSV. Approximately 29,398,623 raw reads from normal-virulence library and 35,291,803 raw reads from low-virulence library were obtained. There were about 37 miRNAs homologs identified. Sixteen miRNAs were significantly up-regulated and twenty-one miRNAs were significantly down-regulated in normal-virulence infection library compared with low-virulence infection library. Of these, Igi-miR-1175-3p was the most significant different miRNA, followed by bmo-miR-1175-3p and ipu-miR-26b, respectively. The putative target genes for differentially expressed miRNAs were concerned with biological processes, signal meditated, cell differentiation and apoptosis, immune recognition and other more functions. The results will help to understand the miRNAs response to different virulence WSSV infection. © 2016 Elsevier Ltd


Guan G.-K.,Chinese Academy of Fishery Sciences | Guan G.-K.,Shanghai Ocean University | Liu Q.-H.,Chinese Academy of Fishery Sciences | Liu Q.-H.,Laboratory for Marine Fisheries and Aquaculture | And 3 more authors.
Developmental and Comparative Immunology | Year: 2016

White Spot Syndrome Virus (WSSV) is currently the most serious shrimp pathogen, which has brought huge losses to shrimp industry worldwide. CD63 of shrimp belongs to the tetraspanin superfamily, which plays an important role in signal transduction and immune process. In this paper, CD63 cDNA sequence of Litopenaeus vannamei was cloned using RACE method. The amplified sequence is 1472 bp, with its ORF 744 bp, encoding 247 amino acids. Bioinformatics analysis showed that the sequence of LvCD63 has 93% similarity with Penaeus monodon and 92% similarity with Fenneropenaeus chinensis. Real-time PCR analysis showed that the mRNA levels of LvCD63 expressed in the tissues of hemocytes, gill, epithelial tissue, heart, lymphoid, hepatopancreas, stomach, intestines, muscle and nerve. Among these tissues the highest expression level was showed in the tissue of haemolymph, followed by epithelial tissue, hepatopancreas, and nerve. The lowest expression level of LvCD63 was appeared in the muscle tissue. After WSSV challenge, the expression levels of LvCD63 were both up-regulated in the tissues of gill and epithelial. However the expression level of LvCD63 in hepatopancreas was down-regulated. Far-western blot analysis showed that LvCD63 interacts with VP28, and both VP28N and VP28C fragments interact with LvCD63. Flow cytometry analysis showed that LvCD63 was present on the surface of hemocytes and it is required for binding of WSSV virions. Neutral experiments in vivo showed that LvCD63LEL delayed WSSV infection in shrimp. © 2016 Elsevier Ltd.


Zhu Q.,CAS Qingdao Institute of Oceanology | Zhu Q.,University of Chinese Academy of Sciences | Zhang L.,CAS Qingdao Institute of Oceanology | Zhang L.,Laboratory for Marine Biology and Biotechnology | And 6 more authors.
Marine Biotechnology | Year: 2016

As a characteristic sessile inhabitant of the intertidal zone, the Pacific oyster Crassostrea gigas occupies one of the most physically stressful environments on earth. With high exposure to terrestrial conditions, oysters must tolerate broad fluctuations in temperature range. However, oysters’ cellular and molecular responses to temperature stresses have not been fully characterized. Here, we analyzed oyster transcriptome data under high and low temperatures. We also identified over 30 key temperature stress-responsive candidate genes, which encoded stress proteins such as heat shock proteins and apoptosis-associated proteins. The expression characterization of these genes under short-term cold and hot environments (5 and 35 °C) and long-term cold environments (5 °C) was detected by quantitative real-time PCR. Most of these genes reached expression peaks during the recovery stage after 24 h of heat stress, and these genes were greatly induced around day 3 in long-term cold stress while responded little to short-term cold stress. In addition, in the second heat stress after 2 days of recovery, oysters showed milder expression in these genes and a lower mortality rate, which indicated the existence of plasticity in the oyster’s response to heat stress. We confirmed that homeostatic flexibility and anti-apoptosis might be crucial centers of temperature stress responses in oysters. Furthermore, we analyzed stress gene families in 11 different species and found that the linage-specific expansion of stress genes might be implicated in adaptive evolution. These results indicated that both plasticity and evolution played an important role in the stress response adaptation of oysters. © 2016, Springer Science+Business Media New York.


Wang X.-F.,Chinese Academy of Fishery Sciences | Wang X.-F.,Dalian Ocean University | Liu Q.-H.,Chinese Academy of Fishery Sciences | Liu Q.-H.,Laboratory for Marine Fisheries and Aquaculture | And 3 more authors.
Fish and Shellfish Immunology | Year: 2016

White spot syndrome virus (WSSV) is the main pathogen of shrimp culture, and has brought great losses of the shrimp aquaculture industry every year since it has been found. However, the specific mechanism of the virus into the cell is not very clear. Recent research suggests that clathrin-mediated endocytosis is involved in WSSV infection. By sequence analysis, clathrin coat AP17 is an σ subunit of AP-2 complex which is involved in clathrin-mediated endocytosis. To obtain the full-length sequence of Clathrin coat AP17 of Litopenaeus vannamei (LvCCAP17), the rapid amplification of cDNA ends (RACE) was performed to get the sequence of 3'and 5' end and splicing by DNAMAN. The full-length sequence of LvCCAP17 is 842 bp and expected to encoding 142 amino acids, and the amino acid sequence was analyzed by online software. The mRNA expression of LvCCAP17 in different tissues was carried out with quantitative real-time PCR and the LvCCAP17 was detected in all tested tissues of Litopenaeus vannamei. The transcriptional expression level of LvCCAP17 in epithelium and hepatopancreas was significantly up-regulated after WSSV infection. Far-Western blotting and ELISA assay showed that LvCCAP17 interacted with rVP26 and rVP37. Silencing of LvCCAP17 gene by double-strand RNA (dsRNA) interference significantly delay of cumulative mortality rate in WSSV infected shrimp and reduced the expression level of immediate early gene 1(ie1) and vp28. These results indicated that clathrin-meated endocytosis is responsible for WSSV infection. © 2016 Elsevier Ltd.


Wu Z.,Ocean University of China | Wu Z.,Murdoch University | Zhang X.,Ocean University of China | Zhang X.,Laboratory for Marine Fisheries and Aquaculture | And 2 more authors.
Estuarine, Coastal and Shelf Science | Year: 2016

This study evaluates the ecosystem structure and function of the nearshore reefs in the Lidao coastal ecosystem of northern China, a region of intensive kelp aquaculture, and fisheries enhancements, including the deployment of artificial reefs and release of cultured marine species. An Ecopath model, with 20 functional groups representing 81 species, was developed for a representative area in the region and Ecosim was used to explore two scenarios for alternative fishing practices and surrounding aquaculture activities. The mean trophic levels (TLs) of the functional groups ranged from 1.0 for the primary producers (phytoplankton, benthic algae and seagrass) and detritus to 4.14 for Type III fishes (fishes found in the water column above the artificial reefs, e.g., Scomberomorus niphonius). The mean transfer efficiency through the whole system was 11.7%, and the ecosystem had a relative low maturity, stability and disturbance resistance, indicating that it was at a developing stage. Nearly half of the total system biomass (48.9% of 620.20 t km−2 year−1), excluding detritus, was comprised of benthic finfish and invertebrates. The total yield from all fisheries (86.82 t/km2/year) was dominated by low trophic level herbivorous and detritivorous species, such as the sea cucumber Apostichopus japonicus (TL = 2.1, 46.07%), other echinoderms (sea urchins Asterias amurensis and Strongylocentrotus nudus, TL = 2.1, 34.6%) and abalone Haliotis discus hannai (TL = 2.0, 18.4%), and as a consequence, the mean TL of the catch was low (2.1). The results from the Ecosim simulation of closing all fisheries for 20 years predicted an increase of about 100% in the relative biomass of the main exploited species, A. japonicus and H. discus hannai. The simulated removal of all kelp farms over 10 years resulted in a two fold increase in the relative biomass of Type III fishes and a 120% increase in their main prey (i.e. Small pelagic fish), while the relative biomass of A. japonicus and Heterotrophic bacteria decreased by 31.4% and 12.7%, respectively. These predictions indicate that nearshore kelp cultivation favours benthic, rather than water column production, and is likely to be providing energy subsidies for the stock enhancement of benthic species in this region. © 2016


Zhang P.,Ocean University of China | Zhang P.,Key Laboratory of Sustainable Development of Marine Fisheries | Li C.,Ocean University of China | Li W.,Ocean University of China | And 2 more authors.
Chinese Journal of Oceanology and Limnology | Year: 2016

Accordion-shaped traps are widely used in China to catch the Asian paddle crab Charybdis japonica but traps of conventional design often catch juvenile crabs. A new type of accordion-shaped trap with an escape vent (L×W=4.3 cm×3.0 cm) was designed and a comparative study between the newly designed and conventional traps was performed in the artificial reef area of Zhuwang, Laizhou Bay, China from June to August 2012. The mean catch per unit effort (CPUE) of undersized crabs was significantly lower in the vented traps than in the conventional traps (paired t -test, n=30, P<0.001), while the CPUE of marketable crabs was significantly higher in the vented traps (paired t-test, n=30, P<0.001). The mean size of crabs (carapace length) caught in the vented traps was significantly larger than in conventional traps (paired t-test, n=29, P<0.001). The ratio of undersized crabs was 35.05±2.57% in conventional traps and 12.53±0.69% in vented traps (significantly lower, paired t-test, n=29, P<0.001). Therefore, a 4.3 cm×3.0 cm escape vent was considered appropriate for C. japonica fishing in the artificial reef area. This finding will assist the development of more sustainable and efficient crab fishing methods using accordion-shaped traps. © 2016, Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg.


Chang Y.,Ocean University of China | Xing J.,Ocean University of China | Tang X.,Ocean University of China | Sheng X.,Ocean University of China | And 2 more authors.
Fish and Shellfish Immunology | Year: 2016

Haemocyanin (Hc) is frequently reported to vary significantly by physiological status and environmental stress in Crustaceans. In this paper, the shrimp Fenneropenaeus chinensis was infected with different concentrations of white spot syndrome virus (WSSV) and Vibrio harveyi. Then, the variation of Hc and total protein content of the haemolymph (TPCH) were investigated using the established double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and Coomassie brilliant blue method, respectively. The results showed that the Hc content peaked at 12 h post-infection (PI) in the 10-2, 10-4 and 10-6 viral supernatant (VS) groups, and the maximum was 93.03 ± 2.55 mg ml-1, 77.57 ± 6.02 mg ml-1 and 70.25 ± 3.96 mg ml-1, respectively. TPCH reached the maximum of 108.18 ± 1.36 mg ml-1 and 103.49 ± 1.33 mg ml-1 at 12 h PI in the 10-2 and 10-4 VS groups, respectively. The maximum was 96.94 ± 1.06 mg ml-1 at 24 h PI in the 10-6 VS group. In the V. harveyi infection groups, the Hc content reached a maximum of 87.97 ± 4.39 mg ml-1 at 36 h PI in the 106 CFU ml-1 group, 73.74 ± 4.38 mg ml-1 and 72.47 ± 2.09 mg ml-1 at 12 h PI in the 107 and 108 CFU ml-1 groups, respectively. TPCH reached a maximum of 111.16 ± 0.86 mg ml-1 at 36 h PI in the 106 CFU ml-1 group, 100.41 ± 0.51 mg ml-1 and 101.94 ± 0.47 mg ml-1 at 12 h PI in the 107 and 108 CFU ml-1 groups, respectively. These data showed that both Hc content and TPCH varied as the same extent after infection. The up-regulation of the Hc content at 6-36 h PI might be a reference threshold for shrimp infection. © 2015 Elsevier Ltd.

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