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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.

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 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 & Local Joint Engineering Laboratory of Ecological Mariculture | And 12 more authors.
Electronic Journal of Biotechnology | Year: 2016

Background: Reduced-representation sequencing technology is widely used in genotyping for its economical and efficient features. A popular way to construct the reduced-representation sequencing libraries is to digest the genomic DNA with restriction enzymes. A key factor of this method is to determine the restriction enzyme(s). But there are few computer programs which can evaluate the usability of restriction enzymes in reduced-representation sequencing. SimRAD is an R package which can simulate the digestion of DNA sequence by restriction enzymes and return enzyme loci number as well as fragment number. But for linkage mapping analysis, enzyme loci distribution is also an important factor to evaluate the enzyme. For phylogenetic studies, comparison of the enzyme performance across multiple genomes is important. It is strongly needed to develop a simulation tool to implement these functions. Results: Here, we introduce a Perl module named RestrictionDigest with more functions and improved performance. It can analyze multiple genomes at one run and generate concise comparison of enzyme performance across the genomes. It can simulate single-enzyme digestion, double-enzyme digestion and size selection process and generate comprehensive information of the simulation including enzyme loci number, fragment number, sequences of the fragments, positions of restriction sites on the genome, the coverage of digested fragments on different genome regions and detailed fragment length distribution. Conclusions: RestrictionDigest is an easy-to-use Perl module with flexible parameter settings. With the help of the information produced by the module, researchers can easily determine the most appropriate enzymes to construct the reduced-representation libraries to meet their experimental requirements. © 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.

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.

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.

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