Generation of transgenic fish cell line with α-lactalbumin nanocarriers co-delivering Tol2 transposase mRNA and plasmids.

Fish cells, such as grass carp (Ctenopharyngodon idella) kidney (CIK) cells, are harder to transfect than mammalian cells. There is a need for an efficient gene delivery system for fish cells. Here, we used CIK cell line as a model to develop a strategy to enhance RNA and plasmid DNA transfection efficiency using a nanocarrier generated from α-lactalbumin (α-NC). α-NC absorbed nucleic acid cargo efficiently and exhibited low cytotoxicity. Plasmid transfection was more efficient with α-NC than with liposomal transfection reagents. We used α-NC to co-transfect Tol2 transposase mRNA and a plasmid containing Cas9 and GFP, generating a stable transgenic CIK cell line. Genome and RNA sequencing revealed that the Cas9 and GFP fragments were successfully inserted into the genome of CIK cells and efficiently transcribed. In this study, we established an efficient transfection system for fish cells using α-NC, simplifying the process of generating stable transgenic fish cell lines.

DNA Methylation and Chromatin Accessibility Impact Subgenome Expression Dominance in the Common Carp (Cyprinus carpio).

DNA methylation and chromatin accessibility play important roles in gene expression, but their function in subgenome expression dominance remains largely unknown. We conducted comprehensive analyses of the transcriptome, DNA methylation, and chromatin accessibility in liver and muscle tissues of allotetraploid common carp, aiming to reveal the function of epigenetic modifications in subgenome expression dominance. A noteworthy overlap in differential expressed genes (DEGs) as well as their functions was observed across the two subgenomes. In the promoter and gene body, the DNA methylation level of the B subgenome was significantly different than that of the A subgenome. Nevertheless, differences in DNA methylation did not align with changes in homoeologous biased expression across liver and muscle tissues. Moreover, the B subgenome exhibited a higher prevalence of open chromatin regions and greater chromatin accessibility, in comparison to the A subgenome. The expression levels of genes located proximally to open chromatin regions were significantly higher than others. Genes with higher chromatin accessibility in the B subgenome exhibited significantly elevated expression levels compared to the A subgenome. Contrastingly, genes without accessibility exhibited similar expression levels in both subgenomes. This study contributes to understanding the regulation of subgenome expression dominance in allotetraploid common carp.

Rapid and Non-Invasive Assessment of Texture Profile Analysis of Common Carp (Cyprinus carpio L.) Using Hyperspectral Imaging and Machine Learning.

Hyperspectral imaging (HSI) has been applied to assess the texture profile analysis (TPA) of processed meat. However, whether the texture profiles of live fish muscle could be assessed using HSI has not been determined. In this study, we evaluated the texture profile of four muscle regions of live common carp by scanning the corresponding skin regions using HSI. We collected skin hyperspectral information from four regions of 387 scaled and live common carp. Eight texture indicators of the muscle corresponding to each skin region were measured. With the skin HSI of live common carp, six machine learning (ML) models were used to predict the muscle texture indicators. Backpropagation artificial neural network (BP-ANN), partial least-square regression (PLSR), and least-square support vector machine (LS-SVM) were identified as the optimal models for predicting the texture parameters of the dorsal (coefficients of determination for prediction (rp) ranged from 0.9191 to 0.9847, and the root-mean-square error for prediction ranged from 0.1070 to 0.3165), pectoral (rp ranged from 0.9033 to 0.9574, and RMSEP ranged from 0.2285 to 0.3930), abdominal (rp ranged from 0.9070 to 0.9776, and RMSEP ranged from 0.1649 to 0.3601), and gluteal (rp ranged from 0.8726 to 0.9768, and RMSEP ranged from 0.1804 to 0.3938) regions. The optimal ML models and skin HSI data were employed to generate visual prediction maps of TPA values in common carp muscles. These results demonstrated that skin HSI and the optimal models can be used to rapidly and accurately determine the texture qualities of different muscle regions in common carp.

Fads2b Plays a Dominant Role in ∆6/∆5 Desaturation Activities Compared with Fads2a in Common Carp (Cyprinus carpio).

Highly unsaturated fatty acids (HUFAs) are essential for mammalian health, development and growth. However, most mammals, including humans, are incapable of synthesizing n-6 and n-3 HUFAs. Fish can convert C18 unsaturated fatty acids into n-6 and n-3 HUFAs via fatty acid desaturase (Fads), in which Fads2 is a key enzyme in HUFA biosynthesis. The allo-tetraploid common carp theoretically encode two duplicated fads2 genes. The expression patterns and desaturase functions of these two homologous genes are still unknown. In this study, the full length of the fads2a and fads2b were identified in common carp (Cyprinus carpio). Expression analyses indicate that both genes were mainly expressed in the liver and the expression of fads2b is higher than fads2a at different developmental stages in carp embryos. Heterogenous expression and 3D docking analyses suggested that Fads2b demonstrated stronger ∆6 and ∆5 desaturase activities than Fads2a. The core promotor regions of fads2a and fads2b were characterized and found to have different potential transcriptional binding sites. These results revealed the same desaturase functions, but different activities of two homologues of fasd2 genes in common carp. The data showed that fads2b played a more important role in HUFA synthesis through both expression and functional analyses.

The Promoter SNPs Were Associated with Both the Contents of Poly-Unsaturated Fatty Acids (PUFAs) and the Expressions of PUFA-Related Genes in Common Carp.

The allo-tetraploid common carp encodes two duplicated fads2 genes (fads2a and fads2b) and two duplicated elovl5 genes (elovl5a and elovl5b). The coding SNPs (cSNPs) of these genes were reported to be significantly associated with the PUFA contents. Whether the promoter SNPs (pSNPs) were associated with the PUFA contents has not been reported yet. In this study, after sequencing the promoters of these four genes, we identified six pSNPs associated with the contents of PUFAs in common carp, including one elovl5a pSNP, one elovl5b pSNP, and four fads2b pSNPs. The pSNPs were predicted in the locations of transcriptional factor binding sites. Together with previously identified cSNPs in fads2b and elovl5b, the pSNPs and cSNPs of these two genes had the joint effects on the PUFA contents with higher explained percentage of phenotypic variation of the PUFA contents than single gene. The expression levels of both fads2a and fads2b were significantly positively correlated with the contents of six PUFAs. The fads2b pSNPs corresponding to higher fads2b expression levels were associated with higher PUFA contents. The pSNPs and cSNPs will be useful for the future selection breeding of common carp with higher PUFA contents.

Dominant Elongase Activity of Elovl5a but Higher Expression of Elovl5b in Common Carp (Cyprinus carpio).

Most diploid freshwater and marine fish encode one elovl5 elongase, having substrate specificity and activities towards C18, C20 and C22 polyunsaturated fatty acids (PUFAs). The allo-tetraploid common carp is hypothesized to encode two duplicated elovl5 genes. How these two elovl5 genes adapt to coordinate the PUFA biosynthesis through elongase function and expression divergence requires elucidation. In this study, we obtained the full-length cDNA sequences of two elovl5 genes in common carp, named as elovl5a and elovl5b. Functional characterization showed that both enzymes had elongase activity towards C18, C20 and C22 PUFAs. Especially, the activities of these two enzymes towards C22 PUFAs ranged from 3.87% to 8.24%, higher than those in most freshwater and marine fish. The Elovl5a had higher elongase activities than Elovl5b towards seven substrates. The spatial-temporal expression showed that both genes co-transcribed in all tissues and development stages. However, the expression levels of elovl5b were significantly higher than those of elovl5a in all examined conditions, suggesting that elovl5b would be the dominantly expressed gene. These two genes had different potential transcriptional binding sites. These results revealed the complicated roles of elovl5 on PUFA synthesis in common carp. The data also increased the knowledge of co-ordination between two homoeologs of the polyploid fish through function and expression divergence.

Association Analysis between Genetic Variants of elovl5a and elovl5b and Poly-Unsaturated Fatty Acids in Common Carp (Cyprinus carpio).

The allo-tetraploid common carp, one widely cultured food fish, is able to produce poly-unsaturated fatty acids (PUFAs). The genetic markers on the PUFA contents for breeding was limited. The polymorphisms in elovl5a and elovl5b, the rate-limiting enzymes in the PUFA biosynthesis, have not been investigated yet. Herein, we identified one coding SNP (cSNP) in elovl5a associated with the content of one PUFA and two cSNPs in elovl5b with the contents of eight PUFAs. The heterozygous genotypes in these three loci were associated with higher contents than the homozygotes. Together with previously identified two associated cSNPs in fads2b, we found the joint effect of these four cSNPs in fads2b and elovl5b on the PUFA contents with the increased explained percentages of PUFA contents. The genotype combinations of more heterozygotes were associated with higher PUFA contents than the other combinations. Using ten genomic selection programs with all cSNPs in fads2b and elovl5b, we obtained the high and positive correlations between the phenotypes and the estimated breeding values of eight PUFAs. These results suggested that elovl5b might be the major gene corresponding to common carp PUFA contents compared with elovl5a. The cSNP combinations in fads2b and elovl5b and the optimal genomic selection program will be used in the future selection breeding to improve the PUFA contents of common carp.

Bulked Segregant Analysis and Association Analysis Identified the Polymorphisms Related to the Intermuscular Bones in Common Carp (Cyprinus carpio).

The allotetraploid common carp is one of the most important freshwater food fish. However, the IBs found in allotetraploid common carp increase the difficulty in fish meat processing and consumption. Although candidate genes associated with the total IB number have been identified, the SNPs associated with the numbers of the total IBs and different forms of IBs have not yet been identified, hindering the breeding of IB-reduced common carp. Herein, the numbers of different types of IBs in three common carp strains were measured. Using whole-genome resequencing and bulked segregant analysis in three pairs of IB-more and IB-less groups, we identified the consensus nonsynonymous SNPs in three strains of common carp. Screening the flanking regions of these SNPs led to the detection of other SNPs. Association study detected 21 SNPs significantly associated with the number of total IBs, epineural-IBs, and ten detailed types of IBs. We observed the joint effects of multiple SNPs on each associated IB number with an improved explained percentage of phenotypic variation. The resulting dataset provides a resource to understand the molecular mechanisms of IB development in different common carp strains. These SNPs are potential markers for future selection to generate IB-reduced common carp.

Parallel subgenome structure and divergent expression evolution of allo-tetraploid common carp and goldfish.

How two subgenomes in allo-tetraploids adapt to coexistence and coordinate through structure and expression evolution requires extensive studies. In the present study, we report an improved genome assembly of allo-tetraploid common carp, an updated genome annotation of allo-tetraploid goldfish and the chromosome-scale assemblies of a progenitor-like diploid Puntius tetrazona and an outgroup diploid Paracanthobrama guichenoti. Parallel subgenome structure evolution in the allo-tetraploids was featured with equivalent chromosome components, higher protein identities, similar transposon divergence and contents, homoeologous exchanges, better synteny level, strong sequence compensation and symmetric purifying selection. Furthermore, we observed subgenome expression divergence processes in the allo-tetraploids, including inter-/intrasubgenome trans-splicing events, expression dominance, decreased expression levels, dosage compensation, stronger expression correlation, dynamic functionalization and balancing of differential expression. The potential disorders introduced by different progenitors in the allo-tetraploids were hypothesized to be alleviated by increasing structural homogeneity and performing versatile expression processes. Resequencing three common carp strains revealed two major ecotypes and uncovered candidate genes relevant to growth and survival rate.

Association between the Polymorphisms of fads2a and fads2b and Poly-Unsaturated Fatty Acids in Common Carp (Cyprinus carpio).

Fatty acid desaturase 2 (fads2) is one of the rate-limiting enzymes in PUFAs biosynthesis. Compared with the diploid fish encoding one fads2, the allo-tetraploid common carp, one most important food fish, encodes two fads2 genes (fads2a and fads2b). The associations between the contents of different PUFAs and the polymorphisms of fads2a and fads2b have not been studied. The contents of 12 PUFAs in common carp individuals were measured, and the polymorphisms in the coding sequences of fads2a and fads2b were screened. We identified five coding single nucleotide polymorphisms (cSNPs) in fads2a and eleven cSNPs in fads2b. Using the mixed linear model and analysis of variance, a synonymous fads2a cSNP was significantly associated with the content of C20:3n-6. One non-synonymous fads2b cSNP (fads2b.751) and one synonymous fads2b cSNP (fads2b.1197) were associated with the contents of seven PUFAs and the contents of six PUFAs, respectively. The heterozygous genotypes in both loci were associated with higher contents than the homozygous genotypes. The fads2b.751 genotype explained more phenotype variation than the fads2b.1197 genotype. These two SNPs were distributed in one haplotype block and associated with the contents of five common PUFAs. These results suggested that fads2b might be the major gene responding to common carp PUFA contents and that fads.751 might be the main effect SNP. These cSNPs would be potential markers for future selection to improve the PUFA contents in common carp.

Transcriptome sequencing and analysis for the pigmentation of scale and skin in common carp (Cyprinus carpio).

BACKGROUND: Teleost scale not only provides a protective layer resisting penetration and pathogens but also participate in coloration. It is interesting to study the mechanism of teleost scale formation. Furthermore, whether there existed consensus genes between scale coloration and skin coloration has not been examined yet. METHODS AND RESULTS: We analyzed the transcriptome profiles of red scale, white scale, red skin, and white skin of common carp (Cyprinus carpio). Pair-wise comparison identified 3391 differentially expressed genes (DEGs) between scale and skin, respectively. The 1765 up-regulated genes (UEGs) in scale, as the down-regulated genes in skin, preferred mineralization and other scale development-related processes. The 1626 skin UEGs were enriched in the morphogenesis of skin and appendages. We also identified 195 UEGs in white scale and 223 UEGs in red scale. The white scale UEGs primarily participated in regulation of growth and cell migration. The UEGs in red scale preferred pigment cell differentiation and retinoid metabolic process. A total of 22 DEGs had consensus expression patterns in skin and scale of the same coloration. The expression levels of these DEGs clearly grouped skin and scale of the same coloration together with principle component analysis and correlation analysis. Eleven consensus DEGs were homologous to the orthologs of Poropuntius huangchuchieni, 82% of which were under strong purifying selection. Eight processes including lipid storage and lipid catabolism were shared in both scale pigmentation and skin pigmentation. CONCLUSIONS: We identified consensus DEGs and biological processes in scale and skin pigmentation. Our transcriptome analysis will contribute to further elucidation of mechanisms of teleost scale formation and coloration.

A chromosome-level genome of the mud crab (Scylla paramamosain estampador) provides insights into the evolution of chemical and light perception in this crustacean.

Mud crabs, found throughout the Indo-Pacific region, are coastal species that are important fisheries resources in many tropical and subtropical Asian countries. Here, we present a chromosome-level genome assembly of a mud crab (Scylla paramamosain). The genome is 1.55 Gb (contig N50 191 kb) in length and encodes 17,821 proteins. The heterozygosity of the assembled genome was estimated to be 0.47%. Effective population size analysis suggested that an initial large population size of this species was maintained until 200 thousand years ago. The contraction of cuticle protein and opsin genes compared with Litopenaeus vannamei is assumed to be correlated with shell hardness and light perception ability, respectively. Furthermore, the analysis of three chemoreceptor gene families, the odorant receptor (OR), gustatory receptor (GR) and ionotropic receptor (IR) families, suggested that the mud crab has no OR genes and shows a contraction of GR genes and expansion of IR genes. The numbers of the three gene families were similar to those in three other decapods but different from those in two nondecapods and insects. In addition, IRs were more diversified in decapods than in nondecapod crustaceans, and most of the expanded IRs in the mud crab genome were clustered with the antennal IR clades. These findings suggested that IRs might exhibit more diverse functions in decapods than in nondecapods, which may compensate for the smaller number of GR genes. Decoding the S. paramamosain genome not only provides insight into the genetic changes underpinning ecological traits but also provides valuable information for improving the breeding and aquaculture of this species.

Chromosome-level genome assembly of golden pompano (Trachinotus ovatus) in the family Carangidae.

Golden pompano (Trachinotus ovatus), a marine fish in the Carangidae family, has a wide geographical distribution and adapts to severe environmental rigours. It is also an economically valuable aquaculture fish. To understand the genetic mechanism of adaption to environmental rigours and improve the production in aquaculture, we assembled its genome. By combination of Illumina and Pacbio reads, the obtained genome sequence is 647.5 Mb with the contig N50 of 1.80 Mb and the scaffold N50 of 5.05 Mb. The assembly covers 98.9% of the estimated genome size (655 Mb). Based on Hi-C data, 99.4% of the assembled bases are anchored into 24 pseudo-chromosomes. The annotation includes 21,915 protein-coding genes, in which 95.7% of 2,586 BUSCO vertebrate conserved genes are complete. This genome is expected to contribute to the comparative analysis of the Carangidae family.

Correction to: P_RNA_scaffolder: a fast and accurate genome scaffolder using paired-end RNAsequencing reads.

Following the publication of this article.

Correction to: L_RNA_scaffolder: scaffolding genomes with transcripts.

Following the publication of this article [1], the authors reported that the link to the software described in the article is no longer valid.

LR_Gapcloser: a tiling path-based gap closer that uses long reads to complete genome assembly.

Background: Completing a genome is an important goal of genome assembly. However, many assemblies, including reference assemblies, are unfinished and have a number of gaps. Long reads obtained from third-generation sequencing (TGS) platforms can help close these gaps and improve assembly contiguity. However, current gap-closure approaches using long reads require extensive runtime and high memory usage. Thus, a fast and memory-efficient approach using long reads is needed to obtain complete genomes. Findings: We developed LR_Gapcloser to rapidly and efficiently close the gaps in genome assembly. This tool utilizes long reads generated from TGS sequencing platforms. Tested on de novo assembled gaps, repeat-derived gaps, and real gaps, LR_Gapcloser closed a higher number of gaps faster and with a lower error rate and a much lower memory usage than two existing, state-of-the art tools. This tool utilized raw reads to fill more gaps than when using error-corrected reads. It is applicable to gaps in the assemblies by different approaches and from large and complex genomes. After performing gap-closure using this tool, the contig N50 size of the human CHM1 genome was improved from 143 kb to 19 Mb, a 132-fold increase. We also closed the gaps in the Triticum urartu genome, a large genome rich in repeats; the contig N50 size was increased by 40%. Further, we evaluated the contiguity and correctness of six hybrid assembly strategies by combining the optimal TGS-based and next-generation sequencing-based assemblers with LR_Gapcloser. A proposed and optimal hybrid strategy generated a new human CHM1 genome assembly with marked contiguity. The contig N50 value was greater than 28 Mb, which is larger than previous non-reference assemblies of the diploid human genome. Conclusions: LR_Gapcloser is a fast and efficient tool that can be used to close gaps and improve the contiguity of genome assemblies. A proposed hybrid assembly including this tool promises reference-grade assemblies. The software is available at http://www.fishbrowser.org/software/LR_Gapcloser/.

P_RNA_scaffolder: a fast and accurate genome scaffolder using paired-end RNA-sequencing reads.

BACKGROUND: Obtaining complete gene structures is one major goal of genome assembly. Some gene regions are fragmented in low quality and high-quality assemblies. Therefore, new approaches are needed to recover gene regions. Genomes are widely transcribed, generating messenger and non-coding RNAs. These widespread transcripts can be used to scaffold genomes and complete transcribed regions. RESULTS: We present P_RNA_scaffolder, a fast and accurate tool using paired-end RNA-sequencing reads to scaffold genomes. This tool aims to improve the completeness of both protein-coding and non-coding genes. After this tool was applied to scaffolding human contigs, the structures of both protein-coding genes and circular RNAs were almost completely recovered and equivalent to those in a complete genome, especially for long proteins and long circular RNAs. Tested in various species, P_RNA_scaffolder exhibited higher speed and efficiency than the existing state-of-the-art scaffolders. This tool also improved the contiguity of genome assemblies generated by current mate-pair scaffolding and third-generation single-molecule sequencing assembly. CONCLUSIONS: The P_RNA_scaffolder can improve the contiguity of genome assembly and benefit gene prediction. This tool is available at http://www.fishbrowser.org/software/P_RNA_scaffolder .

Characterization and phylogenetic analysis of the mitochondrial genome of Schizothorax taliensis, a national-protected fish in China.

Schizothorax taliensis is a national key protected fish in China. The mitochondrial genome of S. taliensis is 16,578 bp in length and includes two ribosomal RNA genes, 22 tRNA genes, and 13 protein-coding genes. The phylogenetic analysis showed that S. taliensis belongs to Cyprinidae family and is closely related to other Schizothorax fish. This mitogenome will contribute to the further conservation and genetic studies of this endangered fish.

Whole genome sequencing yields the complete mitogenome of Pandalus borealis, an indicator species of the Arctic ecosystem.

Pandalus borealis is an important indicator species to study the state of the Arctic ecosystem. The mitochondrial genome of P. borealis is 15,956 bp in length and encodes 13 protein-coding genes. The phylogenetic tree of eleven shrimps revealed that P. borealis belonged to Pandalidae family and was closely related to C. crassicornis. This mitogenome will be of significance to study the Arctic ecosystem state and perform the resource protection of this species.