CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccßA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, ß-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccßA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

Response of cecropin transgenesis to challenge with Edwardsiella ictaluri in channel catfish Ictalurus punctatus.

Constructs bearing the cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (ß-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F3 channel catfish family transgenic for cecropin transgene driven by the CMV promoter, and one F1 channel catfish family transgenic for cecropin transgene driven by the common carp ß-actin promoter were produced. F3 and F1 individuals exhibited enhanced disease resistance when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). Inheritance of the transgene by the F1 and F3 generation was 15% and 60%, respectively. Growth rates of the cecropin transgenic and non-transgenic full siblings (controls) channel catfish were not different (P > 0.05). All transgenic fish showed significant resistance to infection by ESC at day 3 and day 4 post exposure (P = 0.005). No correlation was detected between body weight and time to death for all genetic groups (P = 0.34). Results of our study confirmed that genetic enhancement of E. ictaluri resistance can be achieved by cecropin transgenesis in channel catfish. In addition to survival rate, improving survival time is essential because the extension of survival time gives a better chance to apply treatments to stop the bacterial infection.

Transcriptome Analysis Reveals Key Gene Expression Changes in Blue Catfish Sperm in Response to Cryopreservation.

The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality.

Identification of an Epigenetically Marked Locus within the Sex Determination Region of Channel Catfish.

Channel catfish has an XY sex determination system. However, the X and Y chromosomes harbor an identical gene content of 950 genes each. In this study, we conducted comparative analyses of methylome and transcriptome of genetic males and genetic females before gonadal differentiation to provide insights into the mechanisms of sex determination. Differentially methylated CpG sites (DMCs) were predominantly identified on the sex chromosome, most notably within the sex determination region (SDR), although the overall methylation profiles across the entire genome were similar between genetic males and females. The drastic differences in methylation were located within the SDR at nucleotide position 14.0-20.3 Mb of the sex chromosome, making this region an epigenetically marked locus within the sex determination region. Most of the differentially methylated CpG sites were hypermethylated in females and hypomethylated in males, suggesting potential involvement of methylation modification in sex determination in channel catfish. Along with the differential methylation in the SDR, a number of differentially expressed genes within the SDR were also identified between genetic males and females, making them potential candidate genes for sex determination and differentiation in channel catfish.

Direct and pleiotropic effects of the Masou Salmon Delta-5 Desaturase transgene in F1 channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is the primary culture species in the US along with its hybrid made with male blue catfish, I. furcatus. In an effort to improve the nutritional value of channel catfish, the masou salmon Δ5-desaturase like gene (D5D) driven by the common carp beta-actin promoter (ßactin) was inserted into channel catfish. The objectives of this study were to determine the effectiveness of ßactin-D5D for improving n-3 fatty acid production in F1 transgenic channel catfish, as well as examine pleiotropic effects on growth, proximate analysis, disease resistance, and other performance traits. Transgenic F1 channel catfish showed a 33% increase in the relative proportion of n-3 fatty acids coupled with a 15% decrease in n-6 fatty acids and a 17% decrease in n-9 fatty acids when compared to non-transgenic full-siblings (P < 0.01, P < 0.01, P < 0.01). However, while the relative proportion of n-3 fatty acids was achieved, the total amount of fatty acids in the transgenic fish decreased resulting in a reduction of all fatty acids. Insertion of the ßactin-D5D transgene into channel catfish also had large effects on the body composition, and growth of channel catfish. Transgenic channel catfish grew faster, were more disease resistant, had higher protein and moisture percentage, but lower fat percentage than full-sib controls. There were sex effects as performance changes were more dramatic and significant in males. The ßactin-D5D transgenic channel catfish were also more uniform in their fatty acid composition, growth and other traits.

Chemokine C-C motif ligand 33 is a key regulator of teleost fish barbel development.

Barbels are important sensory organs in teleosts, reptiles, and amphibians. The majority of ∼4,000 catfish species, such as the channel catfish (Ictalurus punctatus), possess abundant whisker-like barbels. However, barbel-less catfish, such as the bottlenose catfish (Ageneiosus marmoratus), do exist. Barbeled catfish and barbel-less catfish are ideal natural models for determination of the genomic basis for barbel development. In this work, we generated and annotated the genome sequences of the bottlenose catfish, conducted comparative and subtractive analyses using genome and transcriptome datasets, and identified differentially expressed genes during barbel regeneration. Here, we report that chemokine C-C motif ligand 33 (ccl33), as a key regulator of barbel development and regeneration. It is present in barbeled fish but absent in barbel-less fish. The ccl33 genes are differentially expressed during barbel regeneration in a timing concordant with the timing of barbel regeneration. Knockout of ccl33 genes in the zebrafish (Danio rerio) resulted in various phenotypes, including complete loss of barbels, reduced barbel sizes, and curly barbels, suggesting that ccl33 is a key regulator of barbel development. Expression analysis indicated that paralogs of the ccl33 gene have both shared and specific expression patterns, most notably expressed highly in various parts of the head, such as the eye, brain, and mouth areas, supporting its role for barbel development.

Effectiveness of Cathelicidin Antimicrobial Peptide against Ictalurid Catfish Bacterial Pathogens.

One of the major goals in aquaculture is to protect fish against infectious diseases as disease outbreaks could lead to economic losses if not controlled. Antimicrobial peptides (AMPs), a class of highly conserved peptides known to possess direct antimicrobial activities against invading pathogens, were evaluated for their ability to protect Channel Catfish Ictalurus punctatus and hybrid catfish (female Channel Catfish × male Blue Catfish I. furcatus) against infection caused by the fish pathogen Aeromonas hydrophila ML09-119. To identify effective peptides, the minimum inhibitory concentrations against bacterial pathogens Edwardsiella ictaluri S97-773, Edwardsiella piscicida E22-10, A. hydrophila ML09-119, Aeromonas veronii 03X03876, and Flavobacterium columnare GL-001 were determined in vitro. In general and overall, cathelicidins derived from alligator and sea snake exhibited more potent and rapid antimicrobial activities against the tested catfish pathogens as compared to cecropin and pleurocidin AMPs and ampicillin, the antibiotic control. When the peptides (2.5 µg of peptide/g of fish) were injected into fish and simultaneously challenged with A. hydrophila through immersion, increased survival rates in Channel Catfish and hybrid catfish were observed in both cathelicidin (alligator and sea snake) treatments as compared to other peptides and the infected control (P < 0.001) with alligator cathelicidin being the overall best treatment. Bacterial numbers in the kidney and liver of Channel Catfish and hybrid catfish also decreased (P < 0.05) for cathelicidin-injected groups at 24 and 48 h after challenge infection. These results show the potential of cathelicidin to protect catfish against bacterial infections and suggest that an approach overexpressing the peptide in transgenic fish, which is the long-term goal of this research program, may provide a method of decreasing bacterial disease problems in catfish as delivering the peptides via individual injection or feeding would not be economically feasible.

Development of a spermatogonia cryopreservation protocol for blue catfish, Ictalurus furcatus.

Sustainability of channel catfish, Ictalurus punctatus ♀ × blue catfish, Ictalurus furcatus ♂ hybrid aquaculture relies on new innovative technologies to maximize fry output. Transplanting spermatogonial stem cells (SSCs) from blue catfish into channel catfish hosts has the potential to greatly increase gamete availability and improve hybrid catfish fry outputs. Cryopreservation would make these cells readily accessible for xenogenesis, but a freezing protocol for blue catfish testicular tissues has not yet been fully developed. Therefore, the objectives of this experiment were to identify the best permeating [dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol] and non-permeating (lactose or trehalose with egg yolk or BSA) cryoprotectants, their optimal concentrations, and the best freezing rates (-0.5, -1.0, -5.0, -10 °C/min until -80 °C) that yield the highest number of viable type A spermatogonia cells. Results showed that all of these factors had significant impacts on post-thaw cell production and viability. DMSO was the most efficient permeating cryoprotectant at a concentration of 1.0 M. The optimal concentration of each cryoprotectant depended on the specific cryoprotectant due to interactions between the two factors. Of the non-permeating cryoprotectants, 0.2 M lactose with egg yolk consistently improved type A spermatogonia production and viability beyond that of the 1.0 M DMSO control. The overall best freezing rate was consistent at -1 °C/min, but similar results were obtained using -0.5 °C/min. Overall, we recommend cryopreserving blue catfish testicular tissues in 1.0 M DMSO with 0.2 M lactose and egg yolk at a rate of either -0.5 or -1 °C/min to achieve the best cryopreservation outcomes. Continued development of cryopreservation protocols for blue catfish and other species will make spermatogonia available for xenogenic applications and genetic improvement programs.

Cathelicidins enhance protection of channel catfish, Ictalurus punctatus, and channel catfish ♀ × blue catfish, Ictalurus furcatus ♂ hybrid catfish against Edwarsiella ictaluri infection.

Cathelicidins are a class of antimicrobial peptides (AMPs) known to possess rapid and direct antimicrobial activities against a variety of microorganisms. Recently identified cathelicidins derived from alligator and sea snake were found to be more effective in inhibiting microbial growth than other AMPs previously characterized. The ability of these two cathelicidins along with the peptides, cecropin and pleurocidin, to protect channel catfish (Ictalurus punctatus, Rafinesque) and hybrid catfish (I. punctatus ♀ × blue catfish, Ictalurus furcatus, Valenciennes ♂) against Edwardsiella ictaluri, one of the most prevalent pathogens affecting commercial catfish industry, was investigated. Cathelicidin-injected fish (50 µg ml-1  fish-1 ) that were simultaneously challenged with E. ictaluri through bath immersion at a concentration of ~1 × 106 CFU/ml had increased survival rates compared with other peptide treatments and the infected control. Bacterial numbers were also reduced in the liver and kidney of channel catfish and hybrid catfish in the cathelicidin treatments 24 hr post-infection. After 8 days of challenge, serum was collected to determine immune-related parameters such as bactericidal activity, lysozyme, serum protein, albumin and globulin. These immune-related parameters were significantly elevated in fish injected with the two cathelicidins as compared to other peptide treatments. These results indicate that cathelicidins derived from alligator and sea snake can stimulate immunity and enhance the resistance to E. ictaluri infection in channel catfish and hybrid catfish.

Analysis of specific mRNA gene expression profiles as markers of egg and embryo quality for hybrid catfish aquaculture.

Despite best efforts to optimize reproduction, egg incubation, and larval performance in captivity, inconsistencies in hatchery fish production are still created by high variations in egg quality from individual females. In some hatchery species, egg quality and generation of viable embryos are correlated to abundances of specific mRNAs. Channel catfish females show considerable extremes in egg quality, causing inconsistencies in channel catfish, Ictalurus punctatus, female × blue catfish, Ictalurus furcatus, male hybrid fry production. The objectives of this study were to examine relative transcripts linked to egg and embryo quality and determine expression between low-hatch and high-hatch egg batches through early development (0, 24, 48, and 96 h post-fertilization; HPF). RNA was extracted from eggs/embryos of nine females (n = 4 high-quality, n = 5 low-quality) and Real-Time PCR was used to quantify relative gene expression. The transcripts assessed in this study perform critical cellular functions, including tubulin ß (tubb), cathepsin D (ctsd), cathepsin Z (ctsz), cathepsin B (ctsb), cyclin B (ccnb1), exportin-1 (xpo1), ring finger protein 213 (rnf213), glucocorticoid receptor-1 (GR-1), and heat shock protein 70 (hsp70). Relative gene expression of all transcripts except GR-1 and hsp70 were up-regulated in the high-hatch group and peaked at 48 HPF (neurulation stage), indicating the importance of these gene products at this threshold to normally progress until hatch. Due to lack of expression during earlier stages, maternally derived mRNAs for these genes do not seem to impact early embryonic development. Using mRNA markers as a selection mechanism for hatchery broodstock may lead to more high-hatch egg batches by reducing problems associated with poor egg quality.

The Y chromosome sequence of the channel catfish suggests novel sex determination mechanisms in teleost fish.

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.

Deep Transcriptomic Analysis Reveals the Dynamic Developmental Progression during Early Development of Channel Catfish (Ictalurus punctatus).

The transition from fertilized egg to larva in fish is accompanied with various biological processes. We selected seven early developmental stages in channel catfish, Ictalurus punctatus, for transcriptome analysis, and covered 22,635 genes with 590 million high-quality RNA-sequencing (seq) reads. Differential expression analysis between neighboring developmental timepoints revealed significantly enriched biological categories associated with growth, development and morphogenesis, which was most evident at 2 vs. 5 days post fertilization (dpf) and 5 vs. 6 dpf. A gene co-expression network was constructed using the Weighted Gene Co-expression Network Analysis (WGCNA) approach and four critical modules were identified. Among candidate hub genes, GDF10, FOXA2, HCEA and SYCE3 were involved in head formation, egg development and the transverse central element of synaptonemal complexes. CK1, OAZ2, DARS1 and UBE2V2 were mainly associated with regulation of cell cycle, growth, brain development, differentiation and proliferation of enterocytes. IFI44L and ZIP10 were critical for the regulation of immune activity and ion transport. Additionally, TCK1 and TGFB1 were related to phosphate transport and regulating cell proliferation. All these genes play vital roles in embryogenesis and regulation of early development. These results serve as a rich dataset for functional genomic studies. Our work reveals new insights of the underlying mechanisms in channel catfish early development.

Comparative transcriptome analysis of the swimbladder reveals expression signatures in response to low oxygen stress in channel catfish, Ictalurus punctatus.

Channel catfish is the leading aquaculture species in the US, and one of the reasons for its application in aquaculture is its relatively high tolerance against hypoxia. However, hypoxia can still cause huge economic losses to the catfish industry. Studies on hypoxia tolerance, therefore, are important for aquaculture. Fish swimbladder has been considered as an accessory respiration organ surrounded by a dense capillary countercurrent exchange system. In this regard, we conducted RNA-Seq analysis with swimbladder samples of catfish under hypoxic and normal conditions to determine if swimbladder was responsive to low oxygen treatment and to reveal genes, their expression patterns, and pathways involved in hypoxia responses in catfish. A total of 155 differentially expressed genes (DEGs) were identified from swimbladder of adult catfish, whereas a total of 2,127 DEGs were identified from swimbladder of fingerling catfish under hypoxic condition as compared with untreated controls. Subsequent pathway analysis revealed that many DEGs under hypoxia were involved in HIF signaling pathway ( nos2, eno2, camk2d2, prkcb, cdkn1a, eno1, and tfrc), MAPK signaling pathway (voltage-dependent calcium channel subunit genes), PI3K/Akt/mTOR signaling pathway ( itga6, g6pc, and cdkn1a), Ras signaling pathway ( efna3 and ksr2), and signaling by VEGF ( fn1, wasf3, and hspb1) in catfish swimbladder. This study provided insights into regulation of gene expression and their involved gene pathways in catfish swimbladder in response to low oxygen stresses.

Comparative transcriptome analysis reveals conserved branching morphogenesis related genes involved in chamber formation of catfish swimbladder.

The swimbladder is an internal gas-filled organ in teleosts. Its major function is to regulate buoyancy. The swimbladder exhibits great variation in size, shape, and number of compartments or chambers among teleosts. However, genomic control of swimbladder variation is unknown. Channel catfish ( Ictalurus punctatus), blue catfish ( Ictalurus furcatus), and their F1 hybrids of female channel catfish × male blue catfish (C × B hybrid catfish) provide a good model in which to investigate the swimbladder morphology, because channel catfish possess a single-chambered swimbladder, whereas blue catfish possess a bichambered swimbladder; C × B hybrid catfish possess a bichambered swimbladder but with a significantly reduced posterior chamber. Here we determined the transcriptional profiles of swimbladder from channel catfish, blue catfish, and C × B hybrid catfish. We examined their transcriptomes at both the fingerling and adult stages. Through comparative transcriptome analysis, ~4,000 differentially expressed genes (DEGs) were identified. Among these DEGs, members of the Wnt signaling pathway ( wnt1, wnt2, nfatc1, rac2), Hedgehog signaling pathway ( shh), and growth factors ( fgf10, igf-1) were identified. As these genes were known to be important for branching morphogenesis of mammalian lung and of mammary glands, their association with budding of the posterior chamber primordium and progressive development of bichambered swimbladder in fish suggest that these branching morphogenesis-related genes and their functions in branching are evolutionarily conserved across a broad spectrum of species.

Comparative genome analysis of 52 fish species suggests differential associations of repetitive elements with their living aquatic environments.

BACKGROUND: Repetitive elements make up significant proportions of genomes. However, their roles in evolution remain largely unknown. To provide insights into the roles of repetitive elements in fish genomes, we conducted a comparative analysis of repetitive elements of 52 fish species in 22 orders in relation to their living aquatic environments. RESULTS: The proportions of repetitive elements in various genomes were found to be positively correlated with genome sizes, with a few exceptions. More importantly, there appeared to be specific enrichment between some repetitive element categories with species habitat. Specifically, class II transposons appear to be more abundant in freshwater bony fish than in marine bony fish when phylogenetic relationship is not considered. In contrast, marine bony fish harbor more tandem repeats than freshwater species. In addition, class I transposons appear to be more abundant in primitive species such as cartilaginous fish and lamprey than in bony fish. CONCLUSIONS: The enriched association of specific categories of repetitive elements with fish habitats suggests the importance of repetitive elements in genome evolution and their potential roles in fish adaptation to their living environments. However, due to the restriction of the limited sequenced species, further analysis needs to be done to alleviate the phylogenetic biases.

Genome sequence of walking catfish (Clarias batrachus) provides insights into terrestrial adaptation.

BACKGROUND: Walking catfish (Clarias batrachus) is a freshwater fish capable of air-breathing and locomotion on land. It usually inhabits various low-oxygen habitats, burrows inside the mudflat, and sometimes "walks" to search for suitable environments during summer. It has evolved accessory air-breathing organs for respiring air and corresponding mechanisms to survive in such challenging environments. Thereby, it serves as a great model for understanding adaptations to terrestrial life. RESULTS: Comparative genomics with channel catfish (Ictalurus punctatus) revealed specific adaptations of C. batrachus in DNA repair, enzyme activator activity, and small GTPase regulator activity. Comparative analysis with 11 non-air-breathing fish species suggested adaptive evolution in gene expression and nitrogenous waste metabolic processes. Further, myoglobin, olfactory receptor related to class A G protein-coupled receptor 1, and sulfotransferase 6b1 genes were found to be expanded in the air-breathing walking catfish genome, with 15, 15, and 12 copies, respectively, compared to non-air-breathing fishes that possess only 1-2 copies of these genes. Additionally, we sequenced and compared the transcriptomes of the gill and the air-breathing organ to characterize the mechanism of aerial respiration involved in elastic fiber formation, oxygen binding and transport, angiogenesis, ion homeostasis and acid-base balance. The hemoglobin genes were expressed dramatically higher in the air-breathing organ than in the gill of walking catfish. CONCLUSIONS: This study provides an important genomic resource for understanding the adaptive mechanisms of walking catfish to terrestrial environments. It is possible that the coupling of enhanced abilities for oxygen storage and oxygen transport through genomic expansion of myoglobin genes and transcriptomic up-regulation of hemoglobin and angiogenesis-related genes are important components of the molecular basis for adaptation of this aquatic species to terrestrial life.

GWAS analysis using interspecific backcross progenies reveals superior blue catfish alleles responsible for strong resistance against enteric septicemia of catfish.

Infectious diseases pose significant threats to the catfish industry. Enteric septicemia of catfish (ESC) caused by Edwardsiella ictaluri is the most devastating disease for catfish aquaculture, causing huge economic losses annually. Channel catfish and blue catfish exhibit great contrast in resistance against ESC, with channel catfish being highly susceptible and blue catfish being highly resistant. As such, the interspecific backcross progenies provide an ideal system for the identification of quantitative trait locus (QTL). We previously reported one significant QTL on linkage group (LG) 1 using the third-generation backcrosses, but the number of founders used to make the second- and third-generation backcross progenies was very small. Although the third-generation backcross progenies provided a greater power for fine mapping than the first-generation backcrosses, some major QTL for disease resistance may have been missing due to the small numbers of founders used to produce the higher generation backcrosses. In this study, we performed a genome-wide association study using first-generation backcrosses with the catfish 690 K SNP arrays to identify additional ESC disease resistance QTL, especially those at the species level. Two genomic regions on LG1 and LG23 were determined to be significantly associated with ESC resistance as revealed by a mixed linear model and family-based association test. Examination of the resistance alleles indicated their origin from blue catfish, indicating that at least two major disease resistance loci exist among blue catfish populations. Upon further validation, markers linked with major ESC disease resistance QTL should be useful for marker-assisted introgression, allowing development of highly ESC resistant breeds of catfish.

Genome-wide association analysis of intra-specific QTL associated with the resistance for enteric septicemia of catfish.

Disease resistance is one of the most important traits for aquaculture industry. For catfish industry, enteric septicemia of catfish (ESC), caused by the bacterial pathogen Edwardsiella ictaluri, is the most severe disease, causing enormous economic losses every year. In this study, we used three channel catfish families with 900 individuals (300 fish per family) and the 690K catfish SNP array, and conducted a genome-wide association study to detect the quantitative trait loci (QTL) associated with ESC resistance. Three significant QTL, with two of located on LG1 and one on LG26, and three suggestive QTL located on LG1, LG3, and LG21, respectively, were identified to be associated with ESC resistance. With a well-assembled- and -annotated reference genome sequence, genes around the involved QTL regions were identified. Among these genes, 37 genes had known functions in immunity, which may be involved in ESC resistance. Notably, nlrc3 and nlrp12 identified here were also found in QTL regions of ESC resistance in the channel catfish × blue catfish interspecific hybrid system, suggesting this QTL was operating within both intra-specific channel catfish populations and interspecific hybrid backcross populations. Many of the genes of the Class I MHC pathway, for mediated antigen processing and presentation, were found in the QTL regions. The positional correlation found in this study and the expressional correlation found in previous studies indicated that Class I MHC pathway was significantly associated with ESC resistance. This study validated one QTL previously identified using the second and fourth generation of the interspecific hybrid backcross progenies, and identified five additional QTL among channel catfish families. Taken together, it appears that there are only a few major QTL for ESC disease resistance, making marker-assisted selection an effective approach for genetic improvements of ESC resistance.

Historical demography of common carp estimated from individuals collected from various parts of the world using the pairwise sequentially markovian coalescent approach.

The inference of historical demography of a species is helpful for understanding species' differentiation and its population dynamics. However, such inference has been previously difficult due to the lack of proper analytical methods and availability of genetic data. A recently developed method called Pairwise Sequentially Markovian Coalescent (PSMC) offers the capability for estimation of the trajectories of historical populations over considerable time periods using genomic sequences. In this study, we applied this approach to infer the historical demography of the common carp using samples collected from Europe, Asia and the Americas. Comparison between Asian and European common carp populations showed that the last glacial period starting 100 ka BP likely caused a significant decline in population size of the wild common carp in Europe, while it did not have much of an impact on its counterparts in Asia. This was probably caused by differences in glacial activities in East Asia and Europe, and suggesting a separation of the European and Asian clades before the last glacial maximum. The North American clade which is an invasive population shared a similar demographic history as those from Europe, consistent with the idea that the North American common carp probably had European ancestral origins. Our analysis represents the first reconstruction of the historical population demography of the common carp, which is important to elucidate the separation of European and Asian common carp clades during the Quaternary glaciation, as well as the dispersal of common carp across the world.

Testicular germ line cell identification, isolation, and transplantation in two North American catfish species.

Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 µm, diameter with distinct nucleus 7-8 µm diameter) and spermatogonia (12-15 µm, with distinct nucleus 6-7.5 µm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 µm) and type B spermatogonia (diameter 10-11 µm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.