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.

Complement system is one of the most important defense systems of innate immunity, which plays a crucial role in disease defense responses in channel catfish. However, inappropriate and excessive complement activation could lead to potential damage to the host cells. Therefore the complement system is controlled by a set of complement regulatory proteins to allow normal defensive functions, but prevent hazardous complement activation to host tissues. In this study, we identified nine complement regulatory protein genes from the channel catfish genome. Phylogenetic and syntenic analyses were conducted to determine their orthology relationships, supporting their correct annotation and potential functional inferences. The expression profiles of the complement regulatory protein genes were determined in channel catfish healthy tissues and after infection with the two main bacterial pathogens, Edwardsiella ictaluri and Flavobacterium columnare. The vast majority of complement regulatory protein genes were significantly regulated after bacterial infections, but interestingly were generally up-regulated after E. ictaluri infection while mostly down-regulated after F. columnare infection, suggesting a pathogen-specific pattern of regulation. Collectively, these findings suggested that complement regulatory protein genes may play complex roles in the host immune responses to bacterial pathogens 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.

Columnaris causes severe mortalities among many different wild and cultured freshwater fish species, but understanding of host resistance is lacking. Catfish, the primary aquaculture species in the United States, serves as a great model for the analysis of host resistance against columnaris disease. Channel catfish in general is highly resistant to the disease while blue catfish is highly susceptible. F2 generation of hybrids can be produced where phenotypes and genotypes are segregating, providing a useful system for QTL analysis. To identify genes associated with columnaris resistance, we performed a genome-wide association study (GWAS) using the catfish 250 K SNP array with 340 backcross progenies derived from crossing female channel catfish (Ictalurus punctatus) with male F1 hybrid catfish (female channel catfish I. punctatus × male blue catfish I. furcatus).

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.

MicroRNAs (miRNAs) are a class of endogenous non-coding small RNA with average length of 22 nucleotides, participating in the post-transcriptional regulation of gene expression. In this study, we report the identification and characterization of miRNAs in the tube foot of sea cucumber (Apostichopus japonicus) by next generation sequencing with Illumina HiSeq 2000 platform. Through the bioinformatic analysis, we identified 260 conserved miRNAs and six novel miRNAs from the tube foot small RNA transcriptome. Quantitative realtime PCR (qRT-PCR) was performed to characterize the specific expression in the tube foot. The results indicated that four miRNAs, including miR-29a, miR-29b, miR-2005 and miR-278-3p, were significantly up-regulated in the tube foot. The target genes of the four specifically expressed miRNAs were predicted in silico and validated by performing qRT-PCR. Gene ontology (GO) and KEGG pathway analyses with the target genes of these four miRNAs were conducted to further understand the regulatory function in the tube foot. This is the first study to profile the miRNA transcriptome of the tube foot in sea cucumber. This work will provide valuable genomic resources to understand the mechanisms of gene regulation in the tube foot, and will be useful to assist the molecular breeding in sea cucumber.

Epigenetic modulation is an important mechanism of miRNA dysregulation in cervical cancer. In this study, we firstly studied how this mechanism contributes to miR-375 downregulation in cervical cancer cells. Then, we further studied the association between miR-375 and MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in epithelial mesenchymal transition (EMT) of the cancer cells. HPV-16 positive SiHa and CaSki cells were used as in vitro model. Our data showed that HPV-16 E6 positively modulated DNMT1 expression in both SiHa and CaSki cells. Knockdown of DNMT1 partly restored miR-375 levels in the cells. The following methylation-specific PCR (MSP) assay and qRT-PCR analysis showed that methylation was common in the promoter region of miR-375 in both SiHa and CaSki cells and demethylation partly restored miR-375 levels in the cells. Therefore, we infer that miR-375 is downregulated partly due to promoter hypermethylation mediated by DNMT1 in HPV-16 positive cervical cancer cells. Our bioinformatics analysis showed that MALAT1 has three putative binding sites with miR-375 and the following dual luciferase assay confirmed two of them. QRT-PCR analysis showed that miR-375 overexpression significantly reduced MALAT1 expression, while MALAT1 overexpression reversely suppressed miR-375 levels. Therefore, we infer that there is a reciprocal regulation between miR-375 and MALAT1 in the cells. In SiHa cells, miR-375 overexpression or MALAT1 siRNA partly restored E-cadherin expression, significantly reduced N-cadherin and also reduced invasion capacity of SiHa cells. Therefore, these results suggest that miR-375 and MALAT1 form a functional axis modulating EMT in cervical cancer.

The Rho family GTPases are a group of small monomeric G proteins, which are molecular switches in signaling pathways. They have been known to regulate a diverse range of cellular processes including actin cytoskeleton rearrangement and microtubule dynamics. In particular, their participations in immune responses are also significant. However, little information of the Rho GTPases is available in teleost including channel catfish, an economically important species and one of the best teleost models forimmunological research. In this study, Rho GTPase genes were identified from channel catfish and well annotated by phylogenetic and syntenic analyses. Their expression profiles were determined in channel catfish healthy tissues and infected tissues. Altogether seven Rho GTPase genes were significantly regulated after bacterial infection, with six genes in the gill after Flavobacterium columnare challenge and two genes in the intestine in response to Edwardsiella ictaluri. All the differentially expressed genes were up-regulated soon after bacterial infection. Different expression patterns between the two experiments were observed, which may be attributed to tissue-specific regulation or pathogen-specific regulation. These results suggested that Rho GTPases play important roles in immune responses to bacterial pathogens, setting a foundation for future investigation on Rho GTPases.

The current study was conducted to assess the effects of microinjection of different dosages of guide RNA (gRNA)/Cas9 protein on the mutation rate, embryo survival, embryonic development, hatchability and early fry survival in channel catfish, Ictalurus punctatus. Guide RNAs targeting two of the channel catfish immune-related genes, toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) and rhamnose binding lectin (RBL) genes, were designed and prepared. Three dosages of gRNA/Cas9 protein (low, 2.5 ng gRNA/7.5 ng Cas9, medium, 5 ng gRNA/15 ng Cas9 and high, 7.5 ng gRNA/22.5 ng Cas9) were microinjected into the yolk of one-cell embryos. Mutation rate increased with higher dosages (p < 0.05). Higher dosages increased the mutation frequency in individual embryos where biallelic mutations were detected. For both genes, microinjection procedures increased the embryo mortality (p < 0.05). Increasing the dosage of gRNA/Cas9 protein increased the embryo mortality and reduced the hatching percent (p < 0.05). Embryonic development was delayed when gRNAs targeting RBL gene were injected. Means of fry survival time were similar for different dosages (p > 0.05). The current results lay the foundations for designing gene editing experiments in channel catfish and can be used as a guide for other fish species.

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.

Fast growth is one of the most desired traits for all food animals, which affects the profitability of animal production. The Pacific oyster, Crassostrea gigas, is an important aquaculture shellfish around the world with the largest annual production. Growth of the Pacific oyster has been greatly improved by artificial selection breeding, but molecular mechanisms underlying growth remains poorly understood, which limited the molecular integrative breeding of fast growth with other superior traits. In this study, comparative transcriptome analyses between the fast-growing selectively bred Pacific oyster and unselected wild Pacific oysters were conducted by RNA-Seq. A total of 1,303 protein-coding genes differentially expressed between fast-growing oysters and wild controls were identified, of which 888 genes were expressed at higher levels in the fast-growing oysters. Functional analysis of the differentially expressed genes (DEGs) indicated that genes involved in microtubule motor activity and biosynthesis of nucleotides and proteins are potentially important for growth in the oyster. Positive selection analysis of genes at the transcriptome level showed that a significant number of ribosomal protein genes had undergone positive selection during the artificial selection breeding process. These results also indicated the importance of protein biosynthesis and metabolism for the growth of oysters. The alternative splicing (AS) of genes was also compared between the two groups of oysters. A total of 3,230 differential alternative splicing events (DAS) were identified, involved in 1,818 genes. These DAS genes were associated with specific functional pathways related to growth, such as "long-term potentiation," "salivary secretion," and "phosphatidylinositol signaling system." The findings of this study will be valuable resources for future investigation to unravel molecular mechanisms underlying growth regulation in the oyster and other marine invertebrates and to provide solid support for breeding application to integrate fast growth with other superior traits in the Pacific oyster.

MicroRNAs (miRNAs) regulate post-transcription gene expression by targeting genes and play crucial roles in diverse biological processes involving body color formation. However, miRNAs and miRNA-targets underlying shell color polymorphism remain largely unknown in mollusca. Using four shell colors full-sib families of the Pacific oyster Crassostrea gigas, we systematically identified miRNAs and miRNA-targets in the mantles, which organ could produce white, golden, black or partially pigmented shell. RNA sequencing and analysis identified a total of 53 known miRNA and 91 novel miRNAs, 47 of which were detected to differentially express among six pairwise groups. By integrating miRNA and mRNA expression profiles, a total of 870 genes were predicted as targets of differentially expressed miRNAs, mainly involving in biomineralization and pigmentation through functional enrichment. Furthermore, a total of four miRNAs and their target mRNAs were predicted to involve in synthesis of melanin, carotenoid or tetrapyrrole. Of them, lgi-miR-317 and its targets peroxidase and lncRNA TCONS_00951105 are implicated in acting as the competing endogenous RNA to regulate melanogenesis. Our studies revealed the systematic characterization of miRNAs profiles expressed in oyster mantle, which might facilitate understanding the intricate molecular regulation of shell color polymorphism and provide new insights into breeding research in oyster.

Transcriptome complexity plays crucial roles in regulating the biological functions of eukaryotes. Except for functional genes, alternative splicing and fusion transcripts produce a vast expansion of transcriptome diversity. In this study, we applied PacBio single-molecule long-read sequencing technology to unveil the whole transcriptome landscape of Lateolabrax maculatus. We obtained 28,809 high-quality non-redundant transcripts, including 18,280 novel isoforms covering 8,961 annotated gene loci within the current reference genome and 3,172 novel isoforms. A total of 10,249 AS events were detected, and intron retention was the predominant AS event. In addition, 1,359 alternative polyadenylation events, 3,112 lncRNAs, 29,609 SSRs, 365 fusion transcripts, and 1,194 transcription factors were identified in this study. Furthermore, we performed RNA-Seq analysis combined with Iso-Seq results to investigate salinity regulation mechanism at the transcripts level. A total of 518 transcripts were differentially expressed, which were further divided into 8 functional groups. Notably, transcripts from the same genes exhibited similar or opposite expression patterns. Our study provides a comprehensive view of the transcriptome complexity in L. maculatus, which significantly improves current gene models. Moreover, the diversity of the expression patterns of transcripts may enhance the understanding of salinity regulatory mechanism in L. maculatus and other euryhaline teleosts.

The diverse modes of sexual reproduction in Bivalvia make it an excellent clade to understand the evolution of sex and sex determination. The cosmopolitan Pacific oyster Crassostrea gigas is an ideal model for bivalve sex determination studies because of its complicated sexuality, including dioecy, sex change and rare hermaphroditism. A major barrier to C. gigas sex determination study has been the lack of information on the type of sex determination. To identify its sex-determining system, sex observation by following the same individual in two consecutive years was conducted on 760 oysters from distinct populations. Stable sexuality and sex reversal in both directions were observed, which provides a case against the protandry of C. gigas. Restriction site-associated DNA sequencing (RAD-seq) based on 26 samples with unchanged and converted sexualities was carried out for identifying sex-linked marker. One SNP Cgsl-40 was proved to be sex-related, but sex-biased heterozygosity varied between populations for RAD-seq and validation, showing no evidence for sex chromosomes or single-locus models for C. gigas primary sex determination. Information obtained in our study provides novel insight into sex determination mechanism in C. gigas.

Insecticides, especially pyrethroids, are the most important in the insect pest control and preventing insect vector-borne human diseases. However, insect pests, including mosquitoes, have developed resistance in the insecticides that used against them. Cytochrome P450s are associated with insecticide resistance through overexpression and detoxification mechanisms in insect species. In this study, we utilized a powerful tool, the RNAi technique, to determine the roles of key P450 genes overexpressed in permethrin resistant mosquitoes that confer insecticide resistance to unravel the molecular basis of resistance mechanisms in the mosquito Culex quinquefasciatus. The results showed that knockdown of 8 key P450 genes using RNAi techniques significantly decreased resistance to permethrin in resistant mosquitoes. In silico modeling and docking analysis further revealed the potential metabolic function of overexpressed P450 genes in the development of insecticide resistance in mosquitoes. These findings not only highlighted the functional importance of these P450 genes in insecticide resistance, but also revealed that overexpression of multiple P450 genes was responsible for the high levels of insecticide resistance in a mosquito population of Culex quinquefasciatus.

Coleoid cephalopods have distinctive neural and morphological characteristics compared to other invertebrates. Early studies reported massive genomic rearrangements occurred before the split of octopus and squid lineages (Proc Natl Acad Sci U S A 116:3030-5, 2019), which might be related to the neural innovations of their brain, yet the details remain elusive. Here we combine genomic and single-nucleus transcriptome analyses to investigate the octopod chromosome evolution and cerebral characteristics.

Papilla and skin are two important organs of the sea cucumber. Both tissues have ectodermic origin, but they are morphologically and functionally very different. In the present study, we performed comparative transcriptome analysis of the papilla and skin from the sea cucumber (Apostichopus japonicus) in order to identify and characterize gene expression profiles by using RNA-Seq technology. We generated 30.6 and 36.4 million clean reads from the papilla and skin and de novo assembled in 156,501 transcripts. The Gene Ontology (GO) analysis indicated that cell part, metabolic process and catalytic activity were the most abundant GO category in cell component, biological process and molecular funcation, respectively. Comparative transcriptome analysis between the papilla and skin allowed the identification of 1,059 differentially expressed genes, of which 739 genes were expressed at higher levels in papilla, while 320 were expressed at higher levels in skin. In addition, 236 differentially expressed unigenes were not annotated with any database, 160 of which were apparently expressed at higher levels in papilla, 76 were expressed at higher levels in skin. We identified a total of 288 papilla-specific genes, 171 skin-specific genes and 600 co-expressed genes. Also, 40 genes in papilla-specific were not annotated with any database, 2 in skin-specific. Development-related genes were also enriched, such as fibroblast growth factor, transforming growth factor-β, collagen-α2 and Integrin-α2, which may be related to the formation of the papilla and skin in sea cucumber. Further pathway analysis identified ten KEGG pathways that were differently enriched between the papilla and skin. The findings on expression profiles between two key organs of the sea cucumber should be valuable to reveal molecular mechanisms involved in the development of organs that are related but with morphological differences in the sea cucumber.

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.

Recent advances in next-generation sequencing technologies have drastically increased throughput and significantly reduced sequencing costs. However, the average read lengths in next-generation sequencing technologies are short as compared with that of traditional Sanger sequencing. The short sequence reads pose great challenges for de novo sequence assembly. As a pilot project for whole genome sequencing of the catfish genome, here we attempt to determine the proper sequence coverage, the proper software for assembly, and various parameters used for the assembly of a BAC physical map contig spanning approximately a million of base pairs.

The lectin pathway of the complement system is characterized by two groups of soluble pattern recognition molecules, mannose-binding lectins (MBLs) and ficolins. These molecules recognize and bind carbohydrates in pathogens and activate complement leading to opsonization, leukocyte activation, and direct pathogen killing. While MBLs have been reported in many fish species, ficolins do not appear to be present in the teleost lineage, despite their importance in invertebrate and higher vertebrate innate immunity. A protein with a similar fibrinogen-like domain, microfibrillar-associated protein 4, MFAP4, is present in fish, albeit with no described immune function. We examined whether MFAP4 genes in fish may potentially act as pathogen receptors in the absence of ficolin. We isolated and characterized five MFAP4 genes from channel catfish. Linkage mapping and phylogenetic analysis indicated that at least three of the catfish MFAP4 genes are tightly clustered on a single chromosome, suggesting that they may have arisen through tandem duplication. Divergent, duplicated families of MFAP4 genes are also present in other teleost species. Expression analysis of the catfish MFAP4 transcripts revealed unique patterns of homeostatic expression among the genes in gill, spleen, skin, liver, and muscle. Expression of the five MFAP4 transcripts showed significant changes in expression as soon as 4h after infection with either Edwardsiella ictaluri or Flavobacterium columnare with modulation of expression continuing up to 7 d following pathogen exposure. Several different tissues and gene-specific patterns were captured and transcript expression changes of >30-fold were observed over the course of the bacterial challenges. Our results suggest a novel role for MFAP4 in teleost immune responses.