ABSTRACT
Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer.
Subject(s)
Chromosomes/genetics , DNA, Neoplasm/genetics , Glioblastoma/genetics , Oncogenes/genetics , Carcinogenesis/genetics , Chromatin/genetics , HumansABSTRACT
BACKGROUND: Sex determination mechanisms in teleost fish broadly differ from mammals and birds, with sex chromosomes that are far less differentiated and recombination often occurring along the length of the X and Y chromosomes, posing major challenges for the identification of specific sex determination genes. Here, we take an innovative approach of comparative genome analysis of the genomic sequences of the X chromosome and newly sequenced Y chromosome in the channel catfish. RESULTS: Using a YY channel catfish as the sequencing template, we generated, assembled, and annotated the Y genome sequence of channel catfish. The genome sequence assembly had a contig N50 size of 2.7 Mb and a scaffold N50 size of 26.7 Mb. Genetic linkage and GWAS analyses placed the sex determination locus within a genetic distance less than 0.5 cM and physical distance of 8.9 Mb. However, comparison of the channel catfish X and Y chromosome sequences showed no sex-specific genes. Instead, comparative RNA-Seq analysis between females and males revealed exclusive sex-specific expression of an isoform of the breast cancer anti-resistance 1 (BCAR1) gene in the male during early sex differentiation. Experimental knockout of BCAR1 gene converted genetic males (XY) to phenotypic females, suggesting BCAR1 as a putative sex determination gene. CONCLUSIONS: We present the first Y chromosome sequence among teleost fish, and one of the few whole Y chromosome sequences among vertebrate species. Comparative analyses suggest that sex-specific isoform expression through alternative splicing may underlie sex determination processes in the channel catfish, and we identify BCAR1 as a potential sex determination gene.
Subject(s)
Ictaluridae/genetics , Sex Determination Processes/genetics , Y Chromosome , Animals , Chromosome Mapping , Female , Genetic Linkage , Genome , Male , Sequence Analysis, DNAABSTRACT
Galectins are lectins possessing an evolutionarily conserved carbohydrate recognition domain (CRD) with affinity for ß-galactoside. The key role played by innate immunity in invertebrates has recently become apparent. Herein, a full-length galectin (ScGal) was identified in razor clam (Sinonovacula constricta). The 528 bp open reading frame encodes a polypeptide of 176 amino acids with a single CRD and no signal peptide. ScGal mRNA transcripts were mainly expressed in hemolymph and gill, and were significantly up-regulated following bacterial challenge. Recombinant rScGal protein binds to and aggregates various bacteria, and has affinity for peptidoglycan, lipoteichoic acid and d-galactose. The protein also stimulates hemocytes to phagocytose invading bacterial pathogens. ScGal is an important immune factor in innate immunity, and a small protein with multiple important functions.
Subject(s)
Bacteria/immunology , Bivalvia/genetics , Bivalvia/immunology , Galectins/genetics , Hemocytes/immunology , Phagocytosis/immunology , Agglutination/immunology , Animals , Galactose/metabolism , Gills/metabolism , Hemolymph/metabolism , Immunity, Innate/genetics , Lipopolysaccharides/metabolism , Peptidoglycan/metabolism , Phagocytosis/genetics , Teichoic Acids/metabolismABSTRACT
Mucosal immune system is one of the most vital components in the innate immunity and constitutes the first line of host defense against bacterial infections, especially for the teleost, which live in the pathogen-rich aquatic environment. Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, play multiple roles at physiological and pathological states. In this regard, we sought here to identify Cathepsin A in turbot (SmCTSA), characterize its mucosal expression patterns following Vibrio anguillarum and Streptococcus iniae infections in mucosal tissues, and explore its binding ability with three microbial ligands for the first time. The SmCTSA was 2631 bp long containing a 1422 bp open reading frame (ORF) that encoded 473 amino acids. Phylogenetic analysis revealed that SmCTSA showed the closest relationship to half-smooth tongue sole (Cynoglossus semilaevis). In addition, SmCTSA was ubiquitously expressed in all examined healthy tissues, with high expression levels in head kidney (HK) and intestine, while the lowest expression level in blood. Moreover, SmCTSA was significantly differentially expressed at least two timepoints in each mucosal tissue, suggesting its potential important roles in innate immune responses of turbot. Finally, in vitro assays showed that recombinant SmCTSA bound Lipopolysaccharide (LPS) with high affinity, and lipoteichoic acid (LTA) and peptidoglycan (PGN) with relatively low affinity. This study provides valuable data for understanding the roles of ctsa in the host defense against bacterial infections.
Subject(s)
Cathepsin A/metabolism , Fish Diseases/immunology , Flatfishes/immunology , Immunity, Mucosal , Mucous Membrane/immunology , Animals , Binding Sites , Cathepsin A/genetics , Fish Diseases/microbiology , Gene Expression , Gene Expression Regulation , Immunity, Innate , Ligands , Lipopolysaccharides/metabolism , Mucous Membrane/microbiology , Phylogeny , RNA, Messenger/metabolism , Seafood/microbiology , Streptococcal Infections/immunology , Streptococcus iniae , Vibrio , Vibrio Infections/immunologyABSTRACT
Keloids result from abnormal proliferative scar formation with scar tissue expanding beyond the margin of the original wound and are mostly found in individuals of sub-Saharan African descent. The etiology of keloids has not been resolved but previous studies suggest that keloids are a genetically heterogeneous disorder. Although possible candidate genes have been suggested by genome-wide association studies using common variants, by upregulation in keloids or their involvement in syndromes that include keloid formation, rare coding variants that contribute to susceptibility in non-syndromic keloid formation have not been previously identified. Through analysis of whole-genome data we mapped a locus to chromosome 8p23.3-p21.3 with a statistically significant maximum multipoint LOD score of 4.48. This finding was followed up using exome sequencing and led to the identification of a c.1202T>C (p.(Leu401Pro)) variant in the N-acylsphingosine amidohydrolase (ASAH1) gene that co-segregates with the keloid phenotype in a large Yoruba family. ASAH1 is an acid ceramidase known to be involved in tumor formation by controlling the ratio of ceramide and sphingosine. ASAH1 is also involved in cell proliferation and inflammation, and may affect the development of keloids via multiple mechanisms. Functional studies need to clarify the role of the ASAH1 variant in wound healing.
Subject(s)
Acid Ceramidase/genetics , Keloid/genetics , Mutation, Missense , Adult , Female , Humans , Keloid/diagnosis , Male , PedigreeABSTRACT
Gnathodiaphyseal dysplasia (GDD; MIM#166260) is an autosomal dominant syndrome with characteristic cemento-osseous lesions of jawbones, bone fragility, and diaphyseal sclerosis of tubular bones. To date, only five mutations in the proposed calcium-activated chloride channel ANO5/TMEM16E gene have been identified. In this study, we describe two families and two singular patients with three new mutations. One Caucasian family with seven affected members exhibited frequent bone fractures and florid osseous dysplasia (p.Cys356Tyr), while one Chinese family with two affected members suffered from cementoma and purulent osteomyelitis (p.Cys360Tyr). In addition, two different novel mutations (p.Gly518Glu and p.Arg215Gly) were identified in sporadic patients without family history. In vitro studies overexpressing GDD mutations (p.Cys356Tyr and p.Cys360Tyr) showed significantly reduced ANO5 protein. It appears that all GDD mutations known so far locate in an extracellular domain following the first transmembrane domain or in the 4th putative transmembrane domain. Both wild-type and mutant ANO5 protein localize to the endoplasmic reticulum. After Ano5 gene knock-down with shRNA in MC3T3-E1 osteoblast precursors we saw elevated expression of osteoblast-related genes such as Col1a1, osteocalcin, osterix and Runx2 as well as increased mineral nodule formation in differentiating cells. Our data suggest that ANO5 plays a role in osteoblast differentiation.
Subject(s)
Anoctamins/genetics , Mutation, Missense , Osteogenesis Imperfecta/genetics , Osteogenesis Imperfecta/pathology , Adolescent , Adult , Aged , Asian People , Child , Child, Preschool , Family Health , Female , Humans , Male , White PeopleABSTRACT
Catfish represent 12% of teleost or 6.3% of all vertebrate species, and are of enormous economic value. Here we report a high-quality reference genome sequence of channel catfish (Ictalurus punctatus), the major aquaculture species in the US. The reference genome sequence was validated by genetic mapping of 54,000 SNPs, and annotated with 26,661 predicted protein-coding genes. Through comparative analysis of genomes and transcriptomes of scaled and scaleless fish and scale regeneration experiments, we address the genomic basis for the most striking physical characteristic of catfish, the evolutionary loss of scales and provide evidence that lack of secretory calcium-binding phosphoproteins accounts for the evolutionary loss of scales in catfish. The channel catfish reference genome sequence, along with two additional genome sequences and transcriptomes of scaled catfishes, provide crucial resources for evolutionary and biological studies. This work also demonstrates the power of comparative subtraction of candidate genes for traits of structural significance.
Subject(s)
Animal Scales/metabolism , Biological Evolution , Fish Proteins/genetics , Genome , Ictaluridae/genetics , Phylogeny , Animal Scales/anatomy & histology , Animals , Base Sequence , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Chromosome Mapping , Fish Proteins/metabolism , Gene Expression Regulation , Gene Ontology , Ictaluridae/classification , Molecular Sequence Annotation , Open Reading Frames , Phosphoproteins/genetics , Phosphoproteins/metabolism , Polymorphism, Single Nucleotide , Sequence AlignmentABSTRACT
The razor clam Sinonovacula constricta is an important commercial species. The deficiency of developmental transcriptomic data is becoming the bottleneck of further researches on the mechanisms underlying settlement and metamorphosis in early development. In this study, de novo transcriptome sequencing was performed for S. constricta at different early developmental stages by using Illumina HiSeq 2000 paired-end (PE) sequencing technology. A total of 112,209,077 PE clean reads were generated. De novo assembly generated 249,795 contigs with an average length of 585 bp. Gene annotation resulted in the identification of 22,870 unigene hits against the NCBI database. Eight unique sequences related to metamorphosis were identified and analyzed using real-time PCR. The razor clam reference transcriptome would provide useful information on early developmental and metamorphosis mechanisms and could be used in the genetic breeding of shellfish.
Subject(s)
Bivalvia/genetics , Gene Expression Regulation, Developmental , Larva/genetics , Molecular Sequence Annotation , Transcriptome , Animals , Bivalvia/classification , Bivalvia/growth & development , Databases, Genetic , Gene Expression Profiling , Gene Ontology , High-Throughput Nucleotide Sequencing , Larva/growth & development , Metamorphosis, Biological/genetics , Phylogeny , Real-Time Polymerase Chain ReactionABSTRACT
BACKGROUND: Columnaris causes severe mortalities among many different wild and cultured freshwater fish species, but understanding of host resistance is lacking. Catfish, the primary aquaculture species in the United States, serves as a great model for the analysis of host resistance against columnaris disease. Channel catfish in general is highly resistant to the disease while blue catfish is highly susceptible. F2 generation of hybrids can be produced where phenotypes and genotypes are segregating, providing a useful system for QTL analysis. To identify genes associated with columnaris resistance, we performed a genome-wide association study (GWAS) using the catfish 250 K SNP array with 340 backcross progenies derived from crossing female channel catfish (Ictalurus punctatus) with male F1 hybrid catfish (female channel catfish I. punctatus × male blue catfish I. furcatus). RESULTS: A genomic region on linkage group 7 was found to be significantly associated with columnaris resistance. Within this region, five have known functions in immunity, including pik3r3b, cyld-like, adcyap1r1, adcyap1r1-like, and mast2. In addition, 3 additional suggestively associated QTL regions were identified on linkage groups 7, 12, and 14. The resistant genotypes on the QTLs of linkage groups 7 and 12 were found to be homozygous with both alleles being derived from channel catfish. The paralogs of the candidate genes in the suggestively associated QTL of linkage group 12 were found on the QTLs of linkage group 7. Many candidate genes on the four associated regions are involved in PI3K pathway that is known to be required by many bacteria for efficient entry into the host. CONCLUSION: The GWAS revealed four QTLs associated with columnaris resistance in catfish. Strikingly, the candidate genes may be arranged as functional hubs; the candidate genes within the associated QTLs on linkage groups 7 and 12 are not only co-localized, but also functionally related, with many of them being involved in the PI3K signal transduction pathway, suggesting its importance for columnaris resistance.
Subject(s)
Catfishes/genetics , Disease Resistance/genetics , Genome-Wide Association Study , Quantitative Trait Loci , Animals , Female , Genetic Association Studies , Genetic Linkage , Genomics , Linkage Disequilibrium , Male , Mortality , Polymorphism, Single NucleotideABSTRACT
The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.
Subject(s)
Carps/genetics , Evolution, Molecular , Genetic Variation , Genome/genetics , Animals , Base Sequence , Chromosome Mapping , Gene Expression Profiling , Genome Components/genetics , Molecular Sequence Data , Sequence Analysis, DNA , Skin/metabolism , Species SpecificityABSTRACT
DNA isolated from saliva has become an attractive alternative to the use of blood-derived DNA in genetic studies and is now extensively used in many applications. This review addresses advantages of saliva DNA for large population-based studies and points out caveats for use in certain techniques. We compiled data comparing saliva-derived genomic DNA to blood-derived DNA with regards to quality, quantity and convenience. Special attention is given to the usefulness of saliva-derived DNA for PCR-based methods and genome-wide analyses.
ABSTRACT
Calanus sinicus Brodsky (Copepoda, Crustacea) is a dominant zooplanktonic species widely distributed in the margin seas of the Northwest Pacific Ocean. In this study, we utilized an RNA-Seq-based approach to develop molecular resources for C. sinicus. Adult samples were sequenced using the Illumina HiSeq 2000 platform. The sequencing data generated 69,751 contigs from 58.9 million filtered reads. The assembled contigs had an average length of 928.8 bp. Gene annotation allowed the identification of 43,417 unigene hits against the NCBI database. Gene ontology (GO) and KEGG pathway mapping analysis revealed various functional genes related to diverse biological functions and processes. Transcripts potentially involved in stress response and lipid metabolism were identified among these genes. Furthermore, 4,871 microsatellites and 110,137 single nucleotide polymorphisms (SNPs) were identified in the C. sinicus transcriptome sequences. SNP validation by the melting temperature (T m )-shift method suggested that 16 primer pairs amplified target products and showed biallelic polymorphism among 30 individuals. The present work demonstrates the power of Illumina-based RNA-Seq for the rapid development of molecular resources in nonmodel species. The validated SNP set from our study is currently being utilized in an ongoing ecological analysis to support a future study of C. sinicus population genetics.
Subject(s)
Copepoda/genetics , Ecological and Environmental Phenomena , Transcriptome/genetics , Animals , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation , Gene Ontology , Genetic Loci , Genetic Markers , Microsatellite Repeats/genetics , Molecular Sequence Annotation , Nucleic Acid Denaturation , Polymorphism, Single Nucleotide/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNAABSTRACT
BACKGROUND: Quantitative traits, such as disease resistance, are most often controlled by a set of genes involving a complex array of regulation. The dissection of genetic basis of quantitative traits requires large numbers of genetic markers with good genome coverage. The application of next-generation sequencing technologies has allowed discovery of over eight million SNPs in catfish, but the challenge remains as to how to efficiently and economically use such SNP resources for genetic analysis. RESULTS: In this work, we developed a catfish 250K SNP array using Affymetrix Axiom genotyping technology. The SNPs were obtained from multiple sources including gene-associated SNPs, anonymous genomic SNPs, and inter-specific SNPs. A set of 640K high-quality SNPs obtained following specific requirements of array design were submitted. A panel of 250,113 SNPs was finalized for inclusion on the array. The performance evaluated by genotyping individuals from wild populations and backcross families suggested the good utility of the catfish 250K SNP array. CONCLUSIONS: This is the first high-density SNP array for catfish. The array should be a valuable resource for genome-wide association studies (GWAS), fine QTL mapping, high-density linkage map construction, haplotype analysis, and whole genome-based selection.
Subject(s)
Catfishes/genetics , Genomics/methods , Oligonucleotide Array Sequence Analysis/methods , Polymorphism, Single Nucleotide , Animals , Chromosome Mapping , Fish Proteins/genetics , Genetic Association Studies/methods , Genotype , Haplotypes , High-Throughput Nucleotide Sequencing , Phenotype , Quantitative Trait Loci/genetics , Reproducibility of ResultsABSTRACT
Along with the rapid advances of the nextgen sequencing technologies, more and more species are added to the list of organisms whose whole genomes are sequenced. However, the assembled draft genome of many organisms consists of numerous small contigs, due to the short length of the reads generated by nextgen sequencing platforms. In order to improve the assembly and bring the genome contigs together, more genome resources are needed. In this study, we developed a strategy to generate a valuable genome resource, physical map contig-specific sequences, which are randomly distributed genome sequences in each physical contig. Two-dimensional tagging method was used to create specific tags for 1,824 physical contigs, in which the cost was dramatically reduced. A total of 94,111,841 100-bp reads and 315,277 assembled contigs are identified containing physical map contig-specific tags. The physical map contig-specific sequences along with the currently available BAC end sequences were then used to anchor the catfish draft genome contigs. A total of 156,457 genome contigs (~79% of whole genome sequencing assembly) were anchored and grouped into 1,824 pools, in which 16,680 unique genes were annotated. The physical map contig-specific sequences are valuable resources to link physical map, genetic linkage map and draft whole genome sequences, consequently have the capability to improve the whole genome sequences assembly and scaffolding, and improve the genome-wide comparative analysis as well. The strategy developed in this study could also be adopted in other species whose whole genome assembly is still facing a challenge.
Subject(s)
Contig Mapping/methods , Chromosomes, Artificial, Bacterial/genetics , GenomicsABSTRACT
BACKGROUND: Comparative mapping is a powerful tool to study evolution of genomes. It allows transfer of genome information from the well-studied model species to non-model species. Catfish is an economically important aquaculture species in United States. A large amount of genome resources have been developed from catfish including genetic linkage maps, physical maps, BAC end sequences (BES), integrated linkage and physical maps using BES-derived markers, physical map contig-specific sequences, and draft genome sequences. Application of such genome resources should allow comparative analysis at the genome scale with several other model fish species. RESULTS: In this study, we conducted whole genome comparative analysis between channel catfish and four model fish species with fully sequenced genomes, zebrafish, medaka, stickleback and Tetraodon. A total of 517 Mb draft genome sequences of catfish were anchored to its genetic linkage map, which accounted for 62% of the total draft genome sequences. Based on the location of homologous genes, homologous chromosomes were determined among catfish and the four model fish species. A large number of conserved syntenic blocks were identified. Analysis of the syntenic relationships between catfish and the four model fishes supported that the catfish genome is most similar to the genome of zebrafish. CONCLUSION: The organization of the catfish genome is similar to that of the four teleost species, zebrafish, medaka, stickleback, and Tetraodon such that homologous chromosomes can be identified. Within each chromosome, extended syntenic blocks were evident, but the conserved syntenies at the chromosome level involve extensive inter-chromosomal and intra-chromosomal rearrangements. This whole genome comparative map should facilitate the whole genome assembly and annotation in catfish, and will be useful for genomic studies of various other fish species.
Subject(s)
Chromosomes/genetics , Evolution, Molecular , Ictaluridae/genetics , Synteny/genetics , Animals , Chromosome Mapping , Genome , Oryzias/genetics , Phylogeny , Smegmamorpha/genetics , Tetraodontiformes/genetics , Zebrafish/geneticsABSTRACT
BACKGROUND: The razor clam Sinonovacula constricta is a benthic intertidal bivalve species with important commercial value. Despite its economic importance, knowledge of its transcriptome is scarce. Next generation sequencing technologies offer rapid and efficient tools for generating large numbers of sequences, which can be used to characterize the transcriptome, to develop effective molecular markers and to identify genes associated with growth, a key breeding trait. RESULTS: Total RNA was isolated from the mantle, gill, liver, siphon, gonad and muscular foot tissues. High-throughput deep sequencing of S. constricta using 454 pyrosequencing technology yielded 859,313 high-quality reads with an average read length of 489 bp. Clustering and assembly of these reads produced 16,323 contigs and 131,346 singletons with average lengths of 1,376 bp and 458 bp, respectively. Based on transcriptome sequencing, 14,615 sequences had significant matches with known genes encoding 147,669 predicted proteins. Subsequently, previously unknown growth-related genes were identified. A total of 13,563 microsatellites (SSRs) and 13,634 high-confidence single nucleotide polymorphism loci (SNPs) were discovered, of which almost half were validated. CONCLUSION: De novo sequencing of the razor clam S. constricta transcriptome on the 454 GS FLX platform generated a large number of ESTs. Candidate growth factors and a large number of SSRs and SNPs were identified. These results will impact genetic studies of S. constricta.
Subject(s)
Bivalvia/genetics , Expressed Sequence Tags , Intercellular Signaling Peptides and Proteins/genetics , Microsatellite Repeats , Molecular Sequence Annotation , Transcriptome , Animals , Bivalvia/classification , Bivalvia/growth & development , Gills/chemistry , Gills/growth & development , Gills/metabolism , Gonads/chemistry , Gonads/growth & development , Gonads/metabolism , High-Throughput Nucleotide Sequencing , Intercellular Signaling Peptides and Proteins/metabolism , Liver/chemistry , Liver/growth & development , Liver/metabolism , Muscles/chemistry , Muscles/metabolism , Phylogeny , Polymorphism, Single Nucleotide , Sequence Analysis, DNA/methodsABSTRACT
Outbreak of Taura syndrome virus (TSV) is one of the major pathogens of the Pacific white shrimp Litopenaeus vannamei. Although selective breeding for improvement of TSV resistance in L. vannamei has been successfully developed and has led to a great benefit to the shrimp farming industry worldwide. The molecular mechanisms underlying the viral resistance in shrimp remain largely unknown. In the present study, we conducted the first transcriptomic profiling of host responses in hemolymph and hemocytes in order to identify the differentially expressed genes associated with resistance to TSV in L. vannamei. High-throughput RNA-Seq was employed, obtaining 193.6 and 171.2 million high-quality Illumina reads from TSV-resistant and susceptible L. vannamei lines respectively. A total of 61,937 contigs were generated with an average length of 546.26 bp. BLASTX-based gene annotation (E-value < 10(-5)) allowed the identification of 12,398 unique proteins against the NCBI non-redundant NR database. In addition, comparison of digital gene expression between resistant and susceptible strains revealed 1374 significantly differentially expressed contigs (representing 697 unigenes). Gene pathway analysis of the differentially expressed gene set highlighted several putative genes involved in the immune response activity including (1) pathogen/antigen recognition including immune regulator, adhesive protein and signal transducer; (2) coagulation; (3) proPO pathway cascade; (4) antioxidation; and (5) protease. The expression patterns of 22 differentially expressed genes involving immune response were validated by quantitative real-time RT-PCR (average correlation coefficients 0.94, p-value < 0.001). Our results provide valuable information on gene functions associated with resistance to TSV in L. vannamei.
Subject(s)
Animal Diseases/genetics , Crustacea/genetics , Crustacea/virology , RNA Virus Infections/genetics , RNA/genetics , Animal Diseases/immunology , Animal Diseases/virology , Animals , Crustacea/immunology , Gene Expression , Hemocytes/immunology , Hemocytes/virology , Hemolymph/immunology , Hemolymph/virology , RNA/immunology , RNA Virus Infections/immunology , RNA Virus Infections/virology , Transcriptome/genetics , Transcriptome/immunologyABSTRACT
BACKGROUND: Catfish has a male-heterogametic (XY) sex determination system, but genes involved in gonadogenesis, spermatogenesis, testicular determination, and sex determination are poorly understood. As a first step of understanding the transcriptome of the testis, here, we conducted RNA-Seq analysis using high throughput Illumina sequencing. METHODOLOGY/PRINCIPAL FINDINGS: A total of 269.6 million high quality reads were assembled into 193,462 contigs with a N50 length of 806 bp. Of these contigs, 67,923 contigs had hits to a set of 25,307 unigenes, including 167 unique genes that had not been previously identified in catfish. A meta-analysis of expressed genes in the testis and in the gynogen (double haploid female) allowed the identification of 5,450 genes that are preferentially expressed in the testis, providing a pool of putative male-biased genes. Gene ontology and annotation analysis suggested that many of these male-biased genes were involved in gonadogenesis, spermatogenesis, testicular determination, gametogenesis, gonad differentiation, and possibly sex determination. CONCLUSION/SIGNIFICANCE: We provide the first transcriptome-level analysis of the catfish testis. Our analysis would lay the basis for sequential follow-up studies of genes involved in sex determination and differentiation in catfish.
Subject(s)
Catfishes/genetics , High-Throughput Nucleotide Sequencing , Testis/metabolism , Transcriptome/genetics , Animals , Female , Gene Ontology , Genetic Linkage , Male , Molecular Sequence Annotation , Sex Characteristics , Sex Determination Processes , Spermatogenesis/geneticsABSTRACT
Cathepsin B, a lysosomal cysteine protease, has drawn much attention in vertebrates. However, very little is known about the functions of cathepsin B in bivalves. In this study, we identified the cathepsin B gene in the razor clam Sinonovacula constricta. The protein has a typical cysteine protease structure, comprising a 15-residue putative signal peptide, a 75-residue propeptide and a 249-residue mature domain. In the mature domain, there is an occluding loop, an oxyanion hole (Gln) and a catalytic triad (Cys, His and Asn). The cathepsin B gene is expressed in a wide range of tissues but appears to exhibit greatest level of expression in the liver. During the early developmental stages, the transcript could be detected widely. After the clam was infected with Vibrio anguillarum, the expression of the cathepsin B gene showed the most significant up-regulation in the liver and mantle tissues at 8h after infection. The fact that bacterial infection can induce the expression of the cathepsin B transcript suggests that cathepsin B could play an important role in the innate immunity of clams.
Subject(s)
Bivalvia/immunology , Cathepsin B/immunology , Immunity, Innate , Liver/immunology , Amino Acid Sequence , Animals , Bivalvia/enzymology , Bivalvia/virology , Cathepsin B/classification , Cathepsin B/genetics , Gene Expression Regulation , Liver/enzymology , Liver/virology , Molecular Sequence Data , Organ Specificity , Phylogeny , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Vibrio/immunologyABSTRACT
Temperature is one of the most prominent abiotic factors affecting ectotherms. Most fish species, as ectotherms, have extraordinary ability to deal with a wide range of temperature changes. While the molecular mechanism underlying temperature adaptation has long been of interest, it is still largely unexplored with fish. Understanding of the fundamental mechanisms conferring tolerance to temperature fluctuations is a topic of increasing interest as temperature may continue to rise as a result of global climate change. Catfish have a wide natural habitat and possess great plasticity in dealing with environmental variations in temperature. However, no studies have been conducted at the transcriptomic level to determine heat stress-induced gene expression. In the present study, we conducted an RNA-Seq analysis to identify heat stress-induced genes in catfish at the transcriptome level. Expression analysis identified a total of 2,260 differentially expressed genes with a cutoff of twofold change. qRT-PCR validation suggested the high reliability of the RNA-Seq results. Gene ontology, enrichment, and pathway analyses were conducted to gain insight into physiological and gene pathways. Specifically, genes involved in oxygen transport, protein folding and degradation, and metabolic process were highly induced, while general protein synthesis was dramatically repressed in response to the lethal temperature stress. This is the first RNA-Seq-based expression study in catfish in response to heat stress. The candidate genes identified should be valuable for further targeted studies on heat tolerance, thereby assisting the development of heat-tolerant catfish lines for aquaculture.