Lisui Bao

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.

Construction of a high-density, high-resolution genetic map and its integration with BAC-based physical map in channel catfish

Construction of genetic linkage map is essential for genetic and genomic studies. Recent advances in sequencing and genotyping technologies made it possible to generate high-density and high-resolution genetic linkage maps, especially for the organisms lacking extensive genomic resources. In the present work, we constructed a high-density and high-resolution genetic map for channel catfish with three large resource families genotyped using the catfish 250K single-nucleotide polymorphism (SNP) array. A total of 54,342 SNPs were placed on the linkage map, which to our knowledge had the highest marker density among aquaculture species. The estimated genetic size was 3,505.4 cM with a resolution of 0.22 cM for sex-averaged genetic map. The sex-specific linkage maps spanned a total of 4,495.1 cM in females and 2,593.7 cM in males, presenting a ratio of 1.7 : 1 between female and male in recombination fraction. After integration with the previously established physical map, over 87% of physical map contigs were anchored to the linkage groups that covered a physical length of 867 Mb, accounting for ∼90% of the catfish genome. The integrated map provides a valuable tool for validating and improving the catfish whole-genome assembly and facilitates fine-scale QTL mapping and positional cloning of genes responsible for economically important traits.

Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries. Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

Identification and analysis of genome-wide SNPs provide insight into signatures of selection and domestication in channel catfish (Ictalurus punctatus)

Domestication and selection for important performance traits can impact the genome, which is most often reflected by reduced heterozygosity in and surrounding genes related to traits affected by selection. In this study, analysis of the genomic impact caused by domestication and artificial selection was conducted by investigating the signatures of selection using single nucleotide polymorphisms (SNPs) in channel catfish (Ictalurus punctatus). A total of 8.4 million candidate SNPs were identified by using next generation sequencing. On average, the channel catfish genome harbors one SNP per 116 bp. Approximately 6.6 million, 5.3 million, 4.9 million, 7.1 million and 6.7 million SNPs were detected in the Marion, Thompson, USDA103, Hatchery strain, and wild population, respectively. The allele frequencies of 407,861 SNPs differed significantly between the domestic and wild populations. With these SNPs, 23 genomic regions with putative selective sweeps were identified that included 11 genes. Although the function for the majority of the genes remain unknown in catfish, several genes with known function related to aquaculture performance traits were included in the regions with selective sweeps. These included hypoxia-inducible factor 1β· HIFιβ ¨ and the transporter gene ATP-binding cassette sub-family B member 5 (ABCB5). HIF1β· is important for response to hypoxia and tolerance to low oxygen levels is a critical aquaculture trait. The large numbers of SNPs identified from this study are valuable for the development of high-density SNP arrays for genetic and genomic studies of performance traits in catfish.

High-density interspecific genetic linkage mapping provides insights into genomic incompatibility between channel catfish and blue catfish

Catfish is the leading aquaculture species in the United States. The interspecific hybrid catfish produced by mating female channel catfish with male blue catfish outperform both of their parent species in a number of traits. However, mass production of the hybrids has been difficult because of reproductive isolation. Investigations of genome structure and organization of the hybrids provide insights into the genetic basis for maintenance of species divergence in the face of gene flow, thereby helping develop strategies for introgression and efficient production of the hybrids for aquaculture. In this study, we constructed a high-density genetic linkage map using the hybrid catfish system with the catfish 250K SNP array. A total of 26,238 SNPs were mapped to 29 linkage groups, with 12,776 unique marker positions. The linkage map spans approximately 3240 cM with an average intermarker distance of 0.25 cM. A fraction of markers (986 of 12,776) exhibited significant deviation from the expected Mendelian ratio of segregation, and they were clustered in major genomic blocks across 15 LGs, most notably LG9 and LG15. The distorted markers exhibited significant bias for maternal alleles among the backcross progenies, suggesting strong selection against the blue catfish alleles. The clustering of distorted markers within genomic blocks should lend insights into speciation as marked by incompatibilities between the two species. Such findings should also have profound implications for understanding the genomic evolution of closely related species as well as the introgression of hybrid production programs in aquaculture.

Claudin multigene family in channel catfish and their expression profiles in response to bacterial infection and hypoxia as revealed by meta-analysis of RNA-Seq datasets

Claudins are one of the major groups of transmembrane proteins that play crucial roles in tight junctions. In addition to their function in the regulation of paracellular permeability, claudins are also involved in a number of biological processes related to pathogen infection, embryonic development, organ development and hypoxia response. Despite its importance, analyses of claudin genes in channel catfish have not been systematically performed. In this study, a total of 52 claudin genes were identified and characterized in channel catfish. Phylogenetic analyses were conducted to determine their identities and identify a number of lineage-specific claudin gene duplications in channel catfish. Expression profiles of catfish claudin genes in response to enteric septicemia of catfish (ESC) disease and hypoxia stress were determined by analyzing existing RNA-Seq datasets. Claudin genes were significantly down-regulated in the intestine at 3h post-infection, indicating that pathogens may disrupt the mucosal barrier by suppressing the expression of claudin genes. A total of six claudin genes were significantly regulated in the gill after hypoxia stress. Among them, the expressions of cldn-11b and cldn-10d were dramatically altered when comparing hypoxia tolerant fish with intolerant fish, though their specific roles involved in response to hypoxia stress remained unknown.

Analysis of 52 Rab GTPases from channel catfish and their involvement in immune responses after bacterial infections

Rab genes, encoding a large family of monomeric G-proteins, contain over 60 members in the human genome. They have been recognized as crucial regulators for membrane trafficking including cargo sorting, vesicle formation, budding, motility, docking, fusion, secretory and endocytic pathway of host immune responses. However, little is known of the Rab gene family in teleost fish species. The development of full-length transcripts and whole genome sequences allow the identification and annotation of Rab GTPase gene family in catfish. In this study, a total of 52 Rab genes were identified from catfish cDNA and genome databases. Phylogenetic analysis assigned them into eleven subfamilies. Most Rab GTPases are conserved among vertebrates, though some of which are absent in fish genomes. Analysis of multiple RNA-seq datasets, along with real time PCR analysis revealed up-regulation of 10 Rab genes after bacterial infection. These included Rab3a, Rab4a, Rab4b, Rab5a, Rab5c, Rab7a, Rab9a, Rab11a, Rab11b, and Rab33a. Their up-regulation are temporally and spatially regulated in various tissues, but mostly induced at early stages after infection and in the gill and liver tissues, with the exception of Rab5c that is mostly up-regulated in the head kidney and trunk kidney. The complex pattern of their induced expression suggested both specific and cooperative actions of a these Rab genes in the acute immune responses to bacterial infection.

Development of a 690 K SNP array in catfish and its application for genetic mapping and validation of the reference genome sequence

Single nucleotide polymorphisms (SNPs) are capable of providing the highest level of genome coverage for genomic and genetic analysis because of their abundance and relatively even distribution in the genome. Such a capacity, however, cannot be achieved without an efficient genotyping platform such as SNP arrays. In this work, we developed a high-density SNP array with 690,662 unique SNPs (herein 690 K array) that were relatively evenly distributed across the entire genome, and covered 98.6% of the reference genome sequence. Here we also report linkage mapping using the 690 K array, which allowed mapping of over 250,000 SNPs on the linkage map, the highest marker density among all the constructed linkage maps. These markers were mapped to 29 linkage groups (LGs) with 30,591 unique marker positions. This linkage map anchored 1,602 scaffolds of the reference genome sequence to LGs, accounting for over 97% of the total genome assembly. A total of 1,007 previously unmapped scaffolds were placed to LGs, allowing validation and in few instances correction of the reference genome sequence assembly. This linkage map should serve as a valuable resource for various genetic and genomic analyses, especially for GWAS and QTL mapping for genes associated with economically important traits.

Generation of physical map contig-specific sequences useful for whole genome sequence scaffolding.

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.

Suppression of adipocyte differentiation and lipid accumulation by stearidonic acid (SDA) in 3T3-L1 cells

BackgroundIncreased consumption of omega-3 (ω-3) fatty acids found in cold-water fish and fish oil has been reported to protect against obesity. A potential mechanism may be through reduction in adipocyte differentiation. Stearidonic acid (SDA), a plant-based ω-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids; however, its role in adipocyte differentiation is unknown. This study was designed to evaluate the effects of SDA on adipocyte differentiation in 3T3-L1 cells.Methods3T3-L1 preadipocytes were differentiated in the presence of SDA or vehicle-control. Cell viability assay was conducted to determine potential toxicity of SDA. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) quantification in differentiated 3T3-L1 adipocytes. Adipocyte differentiation was evaluated by adipogenic transcription factors and lipid accumulation gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Fatty acid analysis was conducted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).Results3T3-L1 cells treated with SDA were viable at concentrations used for all studies. SDA treatment reduced lipid accumulation in 3T3-L1 adipocytes. This anti-adipogenic effect by SDA was a result of down-regulation of mRNA levels of the adipogenic transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBPα, C/EBPβ), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol-regulatory element binding protein-1c (SREBP-1c). SDA treatment resulted in decreased expression of the lipid accumulation genes adipocyte fatty-acid binding protein (AP2), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD-1), lipoprotein lipase (LPL), glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK). The transcriptional activity of PPARγ was found to be decreased with SDA treatment. SDA treatment led to significant EPA enrichment in 3T3-L1 adipocytes compared to vehicle-control.ConclusionThese results demonstrated that SDA can suppress adipocyte differentiation and lipid accumulation in 3T3-L1 cells through down-regulation of adipogenic transcription factors and genes associated with lipid accumulation. This study suggests the use of SDA as a dietary treatment for obesity.