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.

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.

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CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7 % for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25 μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individuals were gene edited in every tissue evaluated. Apparently, the plasmids were eventually degraded without integration, as they were not detectable in mutated individuals using PCR. Carp pituitary extract failed to induce spawning and restoration of fertility, indicating the need for developing other hormone therapies to achieve reversal of sterility upon demand. This is the first sterilization achieved using ZFN technology in an aquaculture species and the first successful gene editing of channel catfish. Our results will help understand the roles of the LH gene, purposeful sterilization of teleost fishes, and is a step towards control of domestic, hybrid, exotic, invasive, and transgenic fishes.

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.

As an important transcription factor, SOX2 involves in embryogenesis, maintenance of stem cells and proliferation of primordial germ cell (PGC). However, little was known about its function in mature gonads. Herein, we investigated the SOX2 gene profiles in testis of scallop, Chlamys farreri. The level of C. farreri SOX2 (Cf-SOX2) mRNA increased gradually along with gonadal development and reached the peak at mature stage, and was located in all germ cells, including spermatogonia, spermatocytes, spermatids and spermatozoa. Knockdown of Cf-SOX2 using RNAi leaded to a mass of germ cells lost, and only a few spermatogonia retained in the nearly empty testicular acini after 21 days. TUNEL assay showed that apoptosis occurred in spermatocytes. Furthermore, transcriptome profiles of the testes were compared between Cf-SOX2 knockdown and normal scallops, 131,340 unigenes were obtained and 2,067 differential expression genes (DEGs) were identified. GO and KEGG analysis showed that most DEGs were related to cell apoptosis (casp2, casp3, casp8), cell proliferation (samd9, crebzf, iqsec1) and spermatogenesis (htt, tusc3, zmynd10, nipbl, mfge8), and enriched in p53, TNF and apoptosis pathways. Our study revealed Cf-SOX2 is essential in spermatogenesis and testis development of C. farreri and provided important clues for better understanding of SOX2 regulatory mechanisms in bivalve testis.

The masou salmon Δ5-desaturase-like gene (D5D) driven by the common carp β-actin promoter was transferred into common carp (Cyprinus carpio) that were fed two diets. For P1 transgenic fish fed a commercial diet, Δ6-desaturase-like gene (D6D) and stearoyl-CoA desaturase (SCD) mRNA levels in muscle were up-regulated (P < 0.05) 12.7- and 17.9-fold, respectively, and the D6D mRNA level in the gonad of transgenic fish was up-regulated 6.9-fold (P < 0.05) compared to that of non-transgenic fish. In contrast, D6D and SCD mRNA levels in transgenic fish were dramatically down-regulated (P < 0.05), 50.2- and 16.7-fold in brain, and 5.4- and 2.4-fold in liver, respectively, in comparison with those of non-transgenic fish. When fed a specially formulated diet, D6D and SCD mRNA levels in muscle of transgenic fish were up-regulated (P < 0.05) 41.5- and 8.9-fold, respectively, and in liver 6.0- and 3.3-fold, respectively, compared to those of non-transgenic fish. In contrast, D6D and SCD mRNA levels in the gonad of transgenic fish were down-regulated (P < 0.05) 5.5- and 12.4-fold, respectively, and D6D and SCD mRNA levels in the brain were down-regulated 14.9- and 1.4-fold (P < 0.05), respectively, compared to those of non-transgenic fish. The transgenic common carp fed the commercial diet had 1.07-fold EPA, 1.12-fold DPA, 1.07-fold DHA, and 1.07-fold higher observed total omega-3 fatty acid levels than non-transgenic common carp. Although these differences were not statistically different (P > 0.05), there were significantly (P < 0.10) higher omega-3 fatty acid levels when considering the differences for all of the individual omega-3 fatty acids. The genotype × diet interactions observed indicated that the potential of desaturase transgenesis cannot be realized without using a well-designed diet with the needed amount of substrates.