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.
Single nucleotide polymorphisms (SNPs) have become the marker of choice for genome-wide association studies. In order to provide the best genome coverage for the analysis of performance and ...production traits, a large number of relatively evenly distributed SNPs are needed. Gene-associated SNPs may fulfill these requirements of large numbers and genome wide distribution. In addition, gene-associated SNPs could themselves be causative SNPs for traits. The objective of this project was to identify large numbers of gene-associated SNPs using high-throughput next generation sequencing.
Transcriptome sequencing was conducted for channel catfish and blue catfish using Illumina next generation sequencing technology. Approximately 220 million reads (15.6 Gb) for channel catfish and 280 million reads (19.6 Gb) for blue catfish were obtained by sequencing gene transcripts derived from various tissues of multiple individuals from a diverse genetic background. A total of over 35 billion base pairs of expressed short read sequences were generated. Over two million putative SNPs were identified from channel catfish and almost 2.5 million putative SNPs were identified from blue catfish. Of these putative SNPs, a set of filtered SNPs were identified including 342,104 intra-specific SNPs for channel catfish, 366,269 intra-specific SNPs for blue catfish, and 420,727 inter-specific SNPs between channel catfish and blue catfish. These filtered SNPs are distributed within 16,562 unique genes in channel catfish and 17,423 unique genes in blue catfish.
For aquaculture species, transcriptome analysis of pooled RNA samples from multiple individuals using Illumina sequencing technology is both technically efficient and cost-effective for generating expressed sequences. Such an approach is most effective when coupled to existing EST resources generated using traditional sequencing approaches because the reference ESTs facilitate effective assembly of the expressed short reads. When multiple individuals with different genetic backgrounds are used, RNA-Seq is very effective for the identification of SNPs. The SNPs identified in this report will provide a much needed resource for genetic studies in catfish and will contribute to the development of a high-density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for genome association studies and whole genome-based selection in catfish.
Upon the completion of whole genome sequencing, thorough genome annotation that associates genome sequences with biological meanings is essential. Genome annotation depends on the availability of ...transcript information as well as orthology information. In teleost fish, genome annotation is seriously hindered by genome duplication. Because of gene duplications, one cannot establish orthologies simply by homology comparisons. Rather intense phylogenetic analysis or structural analysis of orthologies is required for the identification of genes. To conduct phylogenetic analysis and orthology analysis, full-length transcripts are essential. Generation of large numbers of full-length transcripts using traditional transcript sequencing is very difficult and extremely costly.
In this work, we took advantage of a doubled haploid catfish, which has two sets of identical chromosomes and in theory there should be no allelic variations. As such, transcript sequences generated from next-generation sequencing can be favorably assembled into full-length transcripts. Deep sequencing of the doubled haploid channel catfish transcriptome was performed using Illumina HiSeq 2000 platform, yielding over 300 million high-quality trimmed reads totaling 27 Gbp. Assembly of these reads generated 370,798 non-redundant transcript-derived contigs. Functional annotation of the assembly allowed identification of 25,144 unique protein-encoding genes. A total of 2,659 unique genes were identified as putative duplicated genes in the catfish genome because the assembly of the corresponding transcripts harbored PSVs or MSVs (in the form of pseudo-SNPs in the assembly). Of the 25,144 contigs with unique protein hits, around 20,000 contigs matched 50% length of reference proteins, and over 14,000 transcripts were identified as full-length with complete open reading frames. The characterization of consensus sequences surrounding start codon and the stop codon confirmed the correct assembly of the full-length transcripts.
The large set of transcripts assembled in this study is the most comprehensive set of genome resources ever developed from catfish, which will provide the much needed resources for functional genome research in catfish, serving as a reference transcriptome for genome annotation, analysis of gene duplication, gene family structures, and digital gene expression analysis. The putative set of duplicated genes provide a starting point for genome scale analysis of gene duplication in the catfish genome, and should be a valuable resource for comparative genome analysis, genome evolution, and genome function studies.
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.
The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. In this study, we subjected channel catfish (
) to ...a 10-week feeding trial to assess how different dietary amounts of frass inclusion would affect both systemic and mucosal tissue gene expression, especially in regard to growth and immune-related genes. Fish were divided in quadruplicate aquaria, and five experimental diets comprising 0, 50, 100, 200, and 300 g of frass per kilogram of feed were fed twice daily. At the end of the trial, liver, head kidney, gill, and intestine samples were collected for gene expression analyses. First, liver and intestine samples from fish fed with a no frass inclusion diet (control), low-frass (50 g/kg) inclusion diet, or a high-frass (300 g/kg) inclusion diet were subjected to Illumina RNA sequencing to determine global differential gene expression among diet groups. Differentially expressed genes (DEGs) included the upregulation of growth-related genes such as glucose-6-phosphatase and myostatin, as well as innate immune receptors and effector molecules such as toll-like receptor 5, apolipoprotein A1, C-type lectin, and lysozyme. Based on the initial screenings of low/high frass using RNA sequencing, a more thorough evaluation of immune gene expression of all tissues sampled, and all levels of frass inclusion, was further conducted. Using targeted quantitative PCR panels for both innate and adaptive immune genes from channel catfish, differential expression of genes was identified, which included innate receptors (TLR1, TLR5, TLR9, and TLR20A), proinflammatory cytokines (IL-1β type a, IL-1β type b, IL-17, IFN-γ, and TNFα), chemokines (CFC3 and CFD), and hepcidin in both systemic (liver and head kidney) and mucosal (gill and intestine) tissues. Overall, frass from black soldier fly larvae inclusion in formulated diets was found to alter global gene expression and activate innate and adaptive immunity in channel catfish, which has the potential to support disease resistance in this species in addition to demonstrated growth benefits.
► Complete set of NLR receptors from catfish. ► Phylogenetic analysis and comparative analysis of NLRs among teleosts and mammalian species. ► Expression analysis of representative NLR receptors. ► ...Expression analysis of representative NLR receptors after bacterial infection.
Innate immune system plays a significant role in all multicellular organisms. The key feature of the system is its ability to recognize and respond to invading microorganisms. Vertebrates including teleost fish have evolved an array of pathogen recognition receptors (PRRs) for detecting and responding to various pathogen-associated molecular patterns (PAMPs), including Toll-like receptors (TLRs), nucleotide-binding domain, leucine-rich repeat containing receptors (NLRs), and the retinoic acid inducible gene I (RIG-I) like receptors (RLRs). In this study, we identified 22 NLRs including six members of the NLR-A subfamily (NODs), two members of the NLR-B subfamily, 11 members of the NLR-C subfamily, and three genes that do not belong to any of these three subfamilies: Apaf1, CIITA, and NACHT-P1. Phylogenetic analysis indicated that orthologs of the mammalian NOD1, NOD2, NOD3, NOD4, and NOD5 were all identified in catfish. In addition, an additional truncated NOD3-like gene was also identified in catfish. While the identities of subfamily A NLRs could be established, the identities of the NLR-B and NLR-C subfamilies were inconclusive at present. Expression of representative NLR genes was analyzed using RT-PCR and qRT-PCR. In healthy catfish tissues, all the tested NLR genes were found to be ubiquitously expressed in all 11 tested catfish tissues. Analysis of expression of these representative NLR genes after bacterial infection with Edwardsiella ictaluri revealed a significant up-regulation of all tested genes in the spleen and liver, but a significant down-regulation in the intestine and head kidney, suggesting their involvement in the immune responses of catfish against the intracellular bacterial pathogen in a tissue-specific manner. The up-regulation and down-regulation of the tested genes exhibited an amazing similarity of expression profiles after infection, suggesting the co-regulation of these genes.
SNPs are abundant, codominantly inherited, and sequence-tagged markers. They are highly adaptable to large-scale automated genotyping, and therefore, are most suitable for association studies and ...applicable to comparative genome analysis. However, discovery of SNPs requires genome sequencing efforts through whole genome sequencing or deep sequencing of reduced representation libraries. Such genome resources are not yet available for many species including catfish. A large resource of ESTs is to become available in catfish allowing identification of large number of SNPs, but reliability of EST-derived SNPs are relatively low because of sequencing errors. This project was designed to answer some of the questions relevant to quality assessment of EST-derived SNPs.
wo factors were found to be most significant for validation of EST-derived SNPs: the contig size (number of sequences in the contig) and the minor allele sequence frequency. The larger the contigs were, the greater the validation rate although the validation rate was reasonably high when the contigs contain four or more EST sequences with the minor allele sequence being represented at least twice in the contigs. Sequence quality surrounding the SNP under test is also crucially important. PCR extension appeared to be limited to a very short distance, prohibiting successful genotyping when an intron was present, a surprising finding.
Stringent quality assessment measures should be used when working with EST-derived SNPs. In particular, contigs containing four or more ESTs should be used and the minor allele sequence should be represented at least twice. Genotyping primers should be designed from a single exon, completely avoiding introns. Application of such quality assessment measures, along with large resources of ESTs, should provide effective means for SNP identification in species where genome sequence resources are lacking.
As the global market for fisheries and aquaculture products expands, mislabeling of these products has become a growing concern in the food safety arena. Molecular species identification techniques ...hold the potential for rapid, accurate assessment of proper labeling. Here we developed and evaluated DNA barcodes for use in differentiating United States domestic and imported catfish species. First, we sequenced 651 base-pair barcodes from the cytochrome oxidase I (COI) gene from individuals of 9 species (and an Ictalurid hybrid) of domestic and imported catfish in accordance with standard DNA barcoding protocols. These included domestic Ictalurid catfish, and representative imported species from the families of Clariidae and Pangasiidae. Alignment of individual sequences from within a given species revealed highly consistent barcodes (98% similarity on average). These alignments allowed the development and analyses of consensus barcode sequences for each species and comparison with limited sequences in public databases (GenBank and Barcode of Life Data Systems). Validation tests carried out in blinded studies and with commercially purchased catfish samples (both frozen and fresh) revealed the reliability of DNA barcoding for differentiating between these catfish species. The developed protocols and consensus barcodes are valuable resources as increasing market and governmental scrutiny is placed on catfish and other fisheries and aquaculture products labeling in the United States.
► Three RLR genes, RIG-I, MDA5 and LGP2, were identified from channel catfish. ► The three genes showed close phylogenetic relation with the RLRs of other teleosts. ► The genes were found to be ...constitutively expressed in various tissues of catfish. ► Virus-infected catfish ovarian cells showed increased expression of all the genes. ► Bacterial infection also resulted in increased expression of the genes in liver.
Vertebrates including teleost fish have evolved an array of pathogen recognition receptors (PRRs) for detecting and responding to various pathogen-associated molecular patterns (PAMPs), including Toll-like receptors (TLRs), nucleotide-binding domain, leucine-rich repeat containing receptors (NLRs), and the retinoic acid inducible gene I (RIG-I) like receptors (RLRs). As a part of the series of studies targeted to characterize catfish PRRs, we described 22 NLR receptors in the sister contribution. Here in this study, we focused on cytosolic PRRs recognizing nucleotide pathogen-associated molecular patterns (PAMPs) of invading viruses, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors). Three RLRs with DExD/H domain containing RNA helicases, retinoic acid inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), were identified from channel catfish, Ictalurus punctatus. The catfish RIG-I encodes 937 amino acids that contains two CARDs, a DExDc, a HELICc and a RD domains. MDA5 encodes 1005 amino acids with all the domains identified for RIG-I. LGP2 encodes 677 amino acids that contain other domains but not the CARD domain at the N-terminus. Phylogenetic analyses of the three genes of catfish showed close clustering with their counterparts from other teleost fish. All the genes were found to be constitutively expressed in various tissues of catfish with minor variations. Channel catfish ovarian cells when infected with channel catfish virus showed significant increase in the transcript abundance of all the three genes. Further, RLR genes showed significant increases in expression in the liver tissue collected at different time-points after bacterial infection as well. The results indicate that the catfish RLRs may play important roles in antiviral and anti-bacterial immune responses.
A genetic linkage map of the channel catfish genome (N=29) was constructed using EST-based microsatellite and single nucleotide polymorphism (SNP) markers in an interspecific reference family. A ...total of 413 microsatellites and 125 SNP markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 allowed mapping of 331 markers (259 microsatellites and 72 SNPs) to 29 linkage groups. Each linkage group contained 3-18 markers. The largest linkage group contained 18 markers and spanned 131.2 cM, while the smallest linkage group contained 14 markers and spanned only 7.9 cM. The linkage map covered a genetic distance of 1811 cM with an average marker interval of 6.0 cM. Sex-specific maps were also constructed; the recombination rate for females was 1.6 times higher than that for males. Putative conserved syntenies between catfish and zebrafish, medaka, and Tetraodon were established, but the overall levels of genome rearrangements were high among the teleost genomes. This study represents a first-generation linkage map constructed by using EST-derived microsatellites and SNPs, laying a framework for large-scale comparative genome analysis in catfish. The conserved syntenies identified here between the catfish and the three model fish species should facilitate structural genome analysis and evolutionary studies, but more importantly should facilitate functional inference of catfish genes. Given that determination of gene functions is difficult in nonmodel species such as catfish, functional genome analysis will have to rely heavily on the establishment of orthologies from model species.
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