Yanliang Jiang

Genome sequence and genetic diversity of the common carp, Cyprinus carpio

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.

Generation of genome-scale gene-associated SNPs in catfish for the construction of a high-density SNP array

BackgroundSingle 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.ResultsTranscriptome 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.ConclusionsFor 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.

Transcriptomic signatures of attachment, NF-κB suppression and IFN stimulation in the catfish gill following columnaris bacterial infection.

Outbreaks of columnaris disease (Flavobacterium columnare) are common in wild and cultured freshwater fish worldwide. Disease occurrences, particularly those caused by virulent genomovar II isolates, in aquaculture species such as channel catfish can be devastating. In contrast to other important aquaculture pathogens, little is known about host immune responses to columnaris. Adhesion of F. columnare to gill tissue has been correlated in some previous studies to virulence and host susceptibility. Here, therefore, we conducted the first transcriptomic profiling of host responses to columnaris following an experimental challenge. We utilized Illumina-based RNA-seq expression profiling to examine transcript profiles at three timepoints (4h, 24h, and 48h) in catfish gill after bath immersion infection. Enrichment and pathway analyses of the differentially expressed genes revealed several central signatures following infection. These included the dramatic upregulation of a rhamnose-binding lectin, with putative roles in bacterial attachment and aggregation, suppression of NF-κB signalling via IκBs, BCL-3, TAX1BP1, and olfactomedin 4, and strong induction of IFN-inducible responses including iNOS2b, IFI44, and VHSV genes. Fifteen differentially expressed genes with varying expression profiles by RNA-seq, were validated by QPCR (correlation coefficients 0.85-0.94, p-value <0.001). Our results highlight several putative immune pathways and individual candidate genes deserving of further investigation in the context of development of therapeutic regimens and laying the foundation for selection of resistant catfish lines against columnaris.

Construction of Genetic Linkage Maps and Comparative Genome Analysis of Catfish Using Gene-associated Markers

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.

Efficient assembly and annotation of the transcriptome of catfish by RNA-Seq analysis of a doubled haploid homozygote.

BackgroundUpon 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.ResultsIn 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.ConclusionsThe 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.

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

BackgroundThrough the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energys Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification.ResultsA total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35% of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis.ConclusionsThis project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

RNA-Seq reveals expression signatures of genes involved in oxygen transport, protein synthesis, folding, and degradation in response to heat stress in catfish

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.

Development of the catfish 250K SNP array for genome-wide association studies.

BackgroundQuantitative 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.ResultsIn 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.ConclusionsThis 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.

Bulk segregant RNA-seq reveals expression and positional candidate genes and allele-specific expression for disease resistance against enteric septicemia of catfish

BackgroundThe application of RNA-seq has accelerated gene expression profiling and identification of gene-associated SNPs in many species. However, the integrated studies of gene expression along with SNP mapping have been lacking. Coupling of RNA-seq with bulked segregant analysis (BSA) should allow correlation of expression patterns and associated SNPs with the phenotypes.ResultsIn this study, we demonstrated the use of bulked segregant RNA-seq (BSR-Seq) for the analysis of differentially expressed genes and associated SNPs with disease resistance against enteric septicemia of catfish (ESC). A total of 1,255 differentially expressed genes were found between resistant and susceptible fish. In addition, 56,419 SNPs residing on 4,304 unique genes were identified as significant SNPs between susceptible and resistant fish. Detailed analysis of these significant SNPs allowed differentiation of significant SNPs caused by genetic segregation and those caused by allele-specific expression. Mapping of the significant SNPs, along with analysis of differentially expressed genes, allowed identification of candidate genes underlining disease resistance against ESC disease.ConclusionsThis study demonstrated the use of BSR-Seq for the identification of genes involved in disease resistance against ESC through expression profiling and mapping of significantly associated SNPs. BSR-Seq is applicable to analysis of genes underlining various performance and production traits without significant investment in the development of large genotyping platforms such as SNP arrays.

Identification and Characterization of Full-Length cDNAs in Channel Catfish (Ictalurus punctatus) and Blue Catfish (Ictalurus furcatus)

Background Genome annotation projects, gene functional studies, and phylogenetic analyses for a given organism all greatly benefit from access to a validated full-length cDNA resource. While increasingly common in model species, full-length cDNA resources in aquaculture species are scarce. Methodology and Principal Findings Through in silico analysis of catfish (Ictalurus spp.) ESTs, a total of 10,037 channel catfish and 7,382 blue catfish cDNA clones were identified as potentially encoding full-length cDNAs. Of this set, a total of 1,169 channel catfish and 933 blue catfish full-length cDNA clones were selected for re-sequencing to provide additional coverage and ensure sequence accuracy. A total of 1,745 unique gene transcripts were identified from the full-length cDNA set, including 1,064 gene transcripts from channel catfish and 681gene transcripts from blue catfish, with 416 transcripts shared between the two closely related species. Full-length sequence characteristics (ortholog conservation, UTR length, Kozak sequence, and conserved motifs) of the channel and blue catfish were examined in detail. Comparison of gene ontology composition between full-length cDNAs and all catfish ESTs revealed that the full-length cDNA set is representative of the gene diversity encoded in the catfish transcriptome. Conclusions This study describes the first catfish full-length cDNA set constructed from several cDNA libraries. The catfish full-length cDNA sequences, and data gleaned from sequence characteristics analysis, will be a valuable resource for ongoing catfish whole-genome sequencing and future gene-based studies of function and evolution in teleost fishes.