Showing papers by "Chinese Academy of Fishery Sciences published in 2013"

SLAF-seq: An Efficient Method of Large-Scale De Novo SNP Discovery and Genotyping Using High-Throughput Sequencing

Abstract: Large-scale genotyping plays an important role in genetic association studies. It has provided new opportunities for gene discovery, especially when combined with high-throughput sequencing technologies. Here, we report an efficient solution for large-scale genotyping. We call it specific-locus amplified fragment sequencing (SLAF-seq). SLAF-seq technology has several distinguishing characteristics: i) deep sequencing to ensure genotyping accuracy; ii) reduced representation strategy to reduce sequencing costs; iii) pre-designed reduced representation scheme to optimize marker efficiency; and iv) double barcode system for large populations. In this study, we tested the efficiency of SLAF-seq on rice and soybean data. Both sets of results showed strong consistency between predicted and practical SLAFs and considerable genotyping accuracy. We also report the highest density genetic map yet created for any organism without a reference genome sequence, common carp in this case, using SLAF-seq data. We detected 50,530 high-quality SLAFs with 13,291 SNPs genotyped in 211 individual carp. The genetic map contained 5,885 markers with 0.68 cM intervals on average. A comparative genomics study between common carp genetic map and zebrafish genome sequence map showed high-quality SLAF-seq genotyping results. SLAF-seq provides a high-resolution strategy for large-scale genotyping and can be generally applicable to various species and populations.

L_RNA_scaffolder: scaffolding genomes with transcripts

Abstract: Background Generation of large mate-pair libraries is necessary for de novo genome assembly but the procedure is complex and time-consuming. Furthermore, in some complex genomes, it is hard to increase the N50 length even with large mate-pair libraries, which leads to low transcript coverage. Thus, it is necessary to develop other simple scaffolding approaches, to at least solve the elongation of transcribed fragments.

Effects of bioflocs on water quality, and survival, growth and digestive enzyme activities of Litopenaeus vannamei (Boone) in zero‐water exchange culture tanks

Abstract: A 30-day experiment was performed to investigate the effects of bioflocs on water quality, and survival, growth and digestive enzyme activities of the white shrimp Litopenaeus vannamei. Altogether 28 shrimp (7.4 ± 0.1 g) were stocked in each 150 L tank. Two bioflocs treatments and one control were managed: ‘bioflocs 1’ and ‘bioflocs 2’ based on two different densities of the bioflocs, and clean water control without the bioflocs. Brown sugar was added to the bioflocs 1 and bioflocs 2 treatment tanks accounting for 28% and 80% of the shrimp feed respectively (corresponding to proximate C/N ratios of 10 and 14 in daily additions of organic matter respectively), so as to promote bioflocs production and approximately 14 mL L−1 in treatment bioflocs 1 and 20 mL L−1 in treatment bioflocs 2 were maintained from day 15. Monitoring of selected water quality parameters throughout the whole experiment period showed that all parameters remained within recommended levels for shrimp culture in the bioflocs treatments at zero-water exchange, especially low total ammonia nitrogen and nitrite nitrogen levels. By the end of the experiment, shrimp survival rates were above 86%, with no significant differences (P > 0.05) among the three groups. Both weight gain rate and special growth rate tended to increase in the bioflocs treatments compared to those in the control. Meanwhile, the overall specific activities of protease, amylase, cellulase and lipase of the shrimp in the bioflocs treatments were all higher than those in the control; and for the specific activity of the same digestive enzyme, the differences between the bioflocs treatments and the control performed inconsistently among different organs: hepatopancreas, stomach and intestine. Present results suggest that the bioflocs can not only maintain favourable water quality conditions for shrimp culture and help shrimp grow well in zero-water exchange culture systems, but may also have a positive effect on digestive enzyme activities of the shrimp.

Transcriptome analysis of Portunus trituberculatus in response to salinity stress provides insights into the molecular basis of osmoregulation.

Abstract: Background The swimming crab, Portunus trituberculatus, which is naturally distributed in the coastal waters of Asia-Pacific countries, is an important farmed species in China. Salinity is one of the most important abiotic factors that influence not only the distribution and abundance of crustaceans, it is also an important factor for artificial propagation of the crab. To better understand the interaction between salinity stress and osmoregulation, we performed a transcriptome analysis in the gills of Portunus trituberculatus challenged with salinity stress, using the Illumina Deep Sequencing technology. Results We obtained 27,696,835, 28,268,353 and 33,901,271 qualified Illumina read pairs from low salinity challenged (LC), non-challenged (NC), and high salinity challenged (HC) Portunus trituberculatus cDNA libraries, respectively. The overall de novo assembly of cDNA sequence data generated 94,511 unigenes, with an average length of 644 bp. Comparative genomic analysis revealed that 1,705 genes differentially expressed in salinity stress compared to the controls, including 615 and 1,516 unigenes in NC vs LC and NC vs HC respectively. GO functional enrichment analysis results showed some differentially expressed genes were involved in crucial processes related to osmoregulation, such as ion transport processes, amino acid metabolism and synthesis processes, proteolysis process and chitin metabolic process. Conclusion This work represents the first report of the utilization of the next generation sequencing techniques for transcriptome analysis in Portunus trituberculatus and provides valuable information on salinity adaptation mechanism. Results reveal a substantial number of genes modified by salinity stress and a few important salinity acclimation pathways, which will serve as an invaluable resource for revealing the molecular basis of osmoregulation in Portunus trituberculatus. In addition, the most comprehensive sequences of transcripts reported in this study provide a rich source for identification of novel genes in the crab.

Comparative analysis of the transcriptome in tissues secreting purple and white nacre in the pearl mussel Hyriopsis cumingii.

Abstract: The triangle sail mussel Hyriopsis cumingii (Lea) is the most important mussel species used for commercial freshwater pearl production in China. Mussel color is an important indicator of pearl quality. To identify genes involved in the nacre coloring, we conducted RNA-seq and obtained 541,268 sequences (298 bp average size) and 440,034 sequences (293 bp average size) in secreting purple and white nacre libraries (P- and W-libraries), respectively. The 981,302 Expressed Sequence Tags (ESTs) were assembled into 47,812 contigs and 289,386 singletons. In BLASTP searches of the deduced protein, 22,495 were proteins with functional annotations. Thirty-three genes involved in pearl or shell formation were identified. Digital expression analysis identified a total of 358 differentially expressed genes, and 137 genes in the P-library and 221 genes in the W-library showed significantly higher expression. Furthermore, a set of SSR motifs and SNPs between the two samples was identified from the ESTs, which provided the markers for genetic linkage, QTL analysis and future breeding. These EST sequences provided valuable information to further understand the molecular mechanisms involved in the formation, color determination and evolution of the pearl or shell.

Effect of dietary carbohydrate on the growth performance, immune response, hepatic antioxidant abilities and heat shock protein 70 expression of Wuchang bream, Megalobrama amblycephala

Abstract: Summary To investigate the effects of different carbohydrate (CHO) levels in the diet of Wuchang bream Megalobrama amblycephala, the fish were randomly divided into six treatment groups. Five groups were fed 19, 25, 31, 38 and 47% CHO, respectively, for 8 weeks, and a control group was fed a diet with no CHO. Growth performance and feed utilization were significantly (P < 0.05) affected by the dietary carbohydrate level. Maximum weight gain and specific growth rate occurred at the 31% dietary carbohydrate level. Compared to the control, the 31% CHO group had a significantly increased serum total protein content, respiratory burst activity of leucocytes, serum complement 3 (C3) levels, serum lysozyme activity, serum alkaline phosphatase (AKP) activity and hepatic total antioxidative capacity (T-AOC), as well as a decrease in serum glutamic-oxaloacetic transaminase (GOT) activity. Compared to the control, the 47% CHO group had significantly increased serum GOT activity, and a tendency toward an increase in serum cortisol content and a decrease in serum lysozyme activity, hepatic superoxide dismutase (SOD) activity and T-AOC. The relative level of hepatic heat shock protein 70 (HSP70) mRNA in fish fed the 38% CHO diet was significantly higher than those of fish fed the 19, 25 and 31% CHO diets, respectively (P < 0.05). After challenge with Aeromonas hydrophila, fish fed the 47% CHO had the significantly lowest post-challenge survival, and fish fed the 31% CHO had the significantly highest post-challenge survival (P < 0.05). The results of this study suggest that ingestion of excessive dietary CHO can impact the non-specific immune responses, decrease the hepatic antioxidant abilities, and thus affect the health status of M. amblycephala.

Transcriptome Analysis of Crucian Carp (Carassius auratus), an Important Aquaculture and Hypoxia-Tolerant Species

Abstract: The crucian carp is an important aquaculture species and a potential model to study genome evolution and physiological adaptation. However, so far the genomics and transcriptomics data available for this species are still scarce. We performed de novo transcriptome sequencing of four cDNA libraries representing brain, muscle, liver and kidney tissues respectively, each with six specimens. The removal of low quality reads resulted in 2.62 million raw reads, which were assembled as 127,711 unigenes, including 84,867 isotigs and 42,844 singletons. A total of 22,273 unigenes were found with significant matches to 14,449 unique proteins. Around14,398 unigenes were assigned with at least one Gene Ontology (GO) category in 84,876 total assignments, and 6,382 unigenes were found in 237 predicted KEGG pathways. The gene expression analysis revealed more genes expressed in brain, more up-regulated genes in muscle and more down-regulated genes in liver as compared with gene expression profiles of other tissues. In addition, 23 enzymes in the glycolysis/gluconeogenesis pathway were recovered. Importantly, we identified 5,784 high-quality putative SNP and 11,295 microsatellite markers which include 5,364 microsatellites with flanking sequences ≥50 bp. This study produced the most comprehensive genomic resources that have been derived from crucian carp, including thousands of genetic markers, which will not only lay a foundation for further studies on polyploidy origin and anoxic survival but will also facilitate selective breeding of this important aquaculture species.

MiR-125b acts as an oncogene in glioblastoma cells and inhibits cell apoptosis through p53 and p38MAPK-independent pathways.

Abstract: Background: We have recently identified miR-125b upregulation in glioblastoma (GMB). The aim of this study is to determine the correlation between miR-125b expression and malignant grades of glioma and the genes targeted by miR-125b.

Transcriptome Analysis of Androgenic Gland for Discovery of Novel Genes from the Oriental River Prawn, Macrobrachium nipponense, Using Illumina Hiseq 2000

Abstract: Background The oriental river prawn, Macrobrachium nipponense, is an important aquaculture species in China, even in whole of Asia. The androgenic gland produces hormones that play crucial roles in sexual differentiation to maleness. This study is the first de novo M. nipponense transcriptome analysis using cDNA prepared from mRNA isolated from the androgenic gland. Illumina/Solexa was used for sequencing. Methodology and Principal Finding The total volume of RNA sample was more than 5 ug. We generated 70,853,361 high quality reads after eliminating adapter sequences and filtering out low-quality reads. A total of 78,408 isosequences were obtained by clustering and assembly of the clean reads, producing 57,619 non-redundant transcripts with an average length of 1244.19 bp. In total 70,702 isosequences were matched to the Nr database, additional analyses were performed by GO (33,203), KEGG (17,868), and COG analyses (13,817), identifying the potential genes and their functions. A total of 47 sex-determination related gene families were identified from the M. nipponense androgenic gland transcriptome based on the functional annotation of non-redundant transcripts and comparisons with the published literature. Furthermore, a total of 40 candidate novel genes were found, that may contribute to sex-determination based on their extremely high expression levels in the androgenic compared to other sex glands,. Further, 437 SSRs and 65,535 high-confidence SNPs were identified in this EST dataset from which 14 EST-SSR markers have been isolated. Conclusion Our study provides new sequence information for M. nipponense, which will be the basis for further genetic studies on decapods crustaceans. More importantly, this study dramatically improves understanding of sex-determination mechanisms, and advances sex-determination research in all crustacean species. The huge number of potential SSR and SNP markers isolated from the transcriptome may shed the lights on research in many fields, including the evolution and molecular ecology of Macrobrachium species.

Identification and genomic characterization of a novel fish reovirus, Hubei grass carp disease reovirus, isolated in 2009 in China

Abstract: A novel fish reovirus, Hubei grass carp disease reovirus (HGDRV; formerly grass carp reovirus strain 104, GCRV104), was isolated from diseased grass carp in China in 2009 and the full genome sequence was determined. This reovirus was propagated in a grass carp kidney cell line with a typical cytopathic effect. The total size of the genome was 23 706 bp with a 51 mol% G+C content, and the 11 dsRNA segments encoded 12 proteins (two proteins encoded by segment 11). A nucleotide sequence similarity search using blastn found no significant matches except for segment 2, which partially matched that of the RNA-dependent RNA polymerase (RdRp) from several viruses in the genera Aquareovirus and Orthoreovirus of the family Reoviridae. At the amino acid level, seven segments (Seg-1 to Seg-6, and Seg-8) matched with species in the genera Aquareovirus (15–46 % identities) and Orthoreovirus (12–44 % identities), while for four segments (Seg-7, Seg-9, Seg-10 and Seg-11) no similarities in these genera were found. Conserved terminal sequences, 5′-GAAUU----UCAUC-3′, were found in each HGDRV segment at the 5′ and 3′ ends, and the 5′-terminal nucleotides were different from any known species in the genus Aquareovirus. Phylogenetic analysis based on RdRp amino acid sequences from members of the family Reoviridae showed that HGDRV clustered with aquareoviruses prior to joining a branch common with orthoreoviruses. Based on these observations, we propose that HGDRV is a new species in the genus Aquareovirus that is distantly related to any known species within this genus.

Toxic effects of Triclosan on the detoxification system and breeding of Daphnia magna.

Abstract: The toxic effects of different concentrations of Triclosan (TCS) (1-128 μg/L) on Daphnia magna (D. magna) were investigated by acute (48 h) and chronic (21-day) toxicity tests. The response of antioxidase system and Phase I metabolism process of D. magna exposed to TCS were investigated by measuring a series of biomarkers including glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), 7-ethoxyresorufin O-deethylase (EROD), Erythromycin N-demethylase (ERND) and Aminopyrine N-demethylase (APND). The 48 h LC50 of TCS was 330 μg/L for D. magna. In the chronic test, total number of neonates per female, body length and the intrinsic rate of natural increase (r) of D. magna increased at the low exposure concentrations (1-16 μg/L) and decreased at the high concentrations (64-128 μg/L), while the total number of molting per adult decreased continually. The GST and CAT activities showed no significant increase in all treatments, and SOD activities were induced after 24-h exposure and inhibited after 48-h exposure at 4-128 μg/L of concentrations. The MDA content increased after 6-h exposure but decreased after 48-h exposure at 4-128 μg/L. EROD activities initially increased after 6-h exposure, but decreased after 24 and 48-h exposure, ERND and APND activities showed a similar temporal pattern among different treatments groups. SOD, MDA and APND were sensitive to TCS, thus they are suitable as potential biomarkers for the exposure to TCS.

Complete sequence and analysis of plastid genomes of two economically important red algae: Pyropia haitanensis and Pyropia yezoensis.

Abstract: Background Pyropia haitanensis and P. yezoensis are two economically important marine crops that are also considered to be research models to study the physiological ecology of intertidal seaweed communities, evolutionary biology of plastids, and the origins of sexual reproduction. This plastid genome information will facilitate study of breeding, population genetics and phylogenetics.

Transcriptome profiling and digital gene expression analysis of Nile tilapia ( Oreochromis niloticus ) infected by Streptococcus agalactiae

Abstract: Tilapia is an important freshwater aquaculture species worldwide. In recent years, streptococcal diseases have severely threatened development of tilapia aquaculture, while effective prevention and control methods have not yet been established. In order to understand the immunological response of tilapia to infection by Streptococcus agalactiae (S. agalactiae), this study employed Solexa/Illumina RNA-seq and digital gene expression (DGE) technology to investigate changes in the tilapia transcriptome before and after S. agalactiae infection. We obtained 82,799 unigenes (mean size: 618 bp) using de novo assembly. Unigenes were annotated by comparing against databases including Nr, Swissprot, cluster of orthologous groups of proteins, Kyoto encyclopedia of genes and genomes, and gene ontology. Combined with DGE technology, transcriptomic changes in tilapia before and after bacteria challenging were examined. A total of 774 significantly up-regulated and 625 significantly down-regulated unigenes were identified, among which 293 were mapped to 181 signaling pathways including 17 immune-related pathways involving 65 differentially expressed genes. We observed a change in the expression of six genes in the Toll-like receptor signaling pathway, and this was subsequently confirmed via quantitative real-time PCR. This comparative study of the tilapia transcriptome before and after S. agalactiae infection identified important differentially-expressed immune-related genes and signaling pathways that will provide useful insights for further analysis of the mechanisms of tilapia defense against S. agalactiae infection.

Effect of size-fractionated fish protein hydrolysate on growth and feed utilization of turbot (Scophthalmus maximus L.)

Abstract: Four experimental diets were fed to turbot to examine the effect of fish hydrolysate and ultra-filtered fish hydrolysate on growth performance, feed utilization and non-specific immune response. Fish hydrolysate was produced by enzymatic treatment and size fractionated using ultra-filtration (UF). The permeate (molecular weight <1000 Da) after UF and the non-ultra-filtered fish hydrolysate (NUF) were tested as feed ingredients. Diets UF1, UF2 contained 3.7%, 1.2% ultra-filtered fish hydrolysate to replace fish meal protein respectively. The diets UF1, NUF were identical in composition except that the molecular weight of fish hydrolysate in the diet. Fish meal was used in the control diet. All diets were made equal in protein, lipid and energy. Each experimental diet was fed to juvenile turbot (27.87 ± 0.04 g) in triplicate for 8 weeks. Results of this study indicate that the best overall growth and feed utilization of turbot juveniles were obtained with a diet containing higher dose of the small molecular weight compounds in fish hydrolysate. Acid phosphatase, alkaline phosphatase, lysozyme and superoxide dismutase activity in serum were not affected by diet. Total antioxidant capacity was improved with increasing level of low molecule weight fish hydrolysate (UF1).

Effect of dietary vitamin E and selenium supplementation on growth, body composition, and antioxidant defense mechanism in juvenile largemouth bass (Micropterus salmoide) fed oxidized fish oil

Abstract: Six oxidized fish oil contained diets were formulated to investigate the effect of graded levels of vitamin E (V(E)) (α-tocopherol acetate: 160, 280, and 400 mg kg(-1)) associated with either 1.2 or 1.8 mg kg(-1) selenium (Se) on growth, body composition, and antioxidant defense mechanism of juvenile largemouth bass. Another control diet containing fresh fish oil with 160 mg kg(-1) V(E) and 1.2 mg kg(-1) Se was also prepared. Over a 12-week feeding trial, about 5 % of Micropterus salmoide fed diet OxSe1.2/V(E)160 showed inflammation and hemorrhage symptoms at the base of dorsal, pectoral, and tail fin. Fish in all treatments survived well (above 90 %). Feed intakes (88.42-89.58 g fish(-1)) of all treatments were comparable. Growth performances (weight gain and specific growth rate) and feed utilization (feed and protein efficiency ratio) were significantly impaired by dietary oil oxidation, and they did not benefit from neither V(E) nor Se supplementation. Regardless of dietary V(E) and Se supplementation, oxidized oil ingestion resulted in markedly decreased hepatosomatic index and intraperitoneal fat ratio. Oxidized oil ingestion also induced markedly lower liver and muscle lipid contents, and these effects could be alleviated by dietary Se supplementation. Dietary oil oxidation stimulated hepatic catalase activities relative to the control, and supplementation of V(E) abrogated this effect. Hepatic reduced glutathione content in the control was markedly higher than that of treatment OxSe1.2/V(E)160, without any significant differences comparing with the other oxidized oil receiving groups. Hepatic glutathione peroxidase activity and liver Se concentration reflected dietary Se profile, whereas liver V(E) level reflected dietary V(E) profile. Compared with the control, fish fed diet OxSe1.2/V(E)160 obtained markedly higher serum, liver and muscle malondialdehyde contents, which droppe significantly with increasing either V(E) or Se supplementation. In conclusion, the overall results in this study suggested that both V(E) and Se inclusion could protect largemouth bass from the oxidative damage challenged by dietary oil oxidation.