Comparative transcriptome analysis reveals immunoregulation mechanism of lncRNA-mRNA in gill and skin of large yellow croaker (Larimichthys crocea) in response to Cryptocaryon irritans infection.

BACKGROUND: Cryptocaryonosis caused by Cryptocaryon irritans is one of the major diseases of large yellow croaker (Larimichthys crocea), which lead to massive economic losses annually to the aquaculture industry of L. crocea. Although there have been some studies on the pathogenesis for cryptocaryonosis, little is known about the innate defense mechanism of different immune organs of large yellow croaker. RESULTS: In order to analyze the roles of long non-coding RNAs and genes specifically expressed between immune organs during the infection of C. irritans, in this study, by comparing transcriptome data from different tissues of L. crocea, we identified tissue-specific transcripts in the gills and skin, including 507 DE lncRNAs and 1592 DEGs identified in the gills, and 110 DE lncRNAs and 1160 DEGs identified in the skin. Furthermore, we constructed transcriptome co-expression profiles of L. crocea gill and skin, including 7,503 long noncoding RNAs (lncRNAs) and 23,172 protein-coding genes. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DEGs and the target genes of the DE lncRNAs in the gill were specifically enriched in several pathways related to immune such as HIF-1 signaling pathway. The target genes of DE lncRNAs and DEGs in the skin are specifically enriched in the complement and coagulation cascade pathways. Protein-protein interaction (PPI) network analysis identified 3 hub genes including NFKBIA, TNFAIP3 and CEBPB, and 5 important DE lncRNAs including MSTRG.24134.4, MSTRG.3038.5, MSTRG.27019.3, MSTRG.26559.1, and MSTRG.10983.1. The expression patterns of 6 randomly selected differentially expressed immune-related genes were validated using the quantitative real-time PCR method. CONCLUSIONS: In short, our study is helpful to explore the potential interplay between lncRNAs and protein coding genes in different tissues of L. crocea post C. irritans and the molecular mechanism of pathogenesis for cryptocaryonosis. HIGHLIGHTS: Skin and gills are important sources of pro-inflammatory molecules, and their gene expression patterns are tissue-specific after C. irritans infection. 15 DEGs and 5 DE lncRNAs were identified as hub regulatory elements after C. irritans infection The HIF-1 signaling pathway and the complement and coagulation cascade pathway may be key tissue-specific regulatory pathways in gills and skin, respectively.

Transcriptome analysis reveals the temporal gene expression patterns in skin of large yellow croaker (Larimichthys crocea) in response to Cryptocaryon irritans infection.

Large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. In the past decades, cryptocaryonosis caused by Cryptocryon irritans has led to huge economic losses, posing great threat to the healthy and sustainable development of L. crocea mariculture industry. As the largest immunologically active mucosal organ in fish, skin provides the first defense line against external pathogens. To better understand the gene expression dynamics, the large yellow croakers were artificially infected with C. irritans and their skin tissues were collected at 0 h, 24 h, 48 h, 72 h and 96 h post infection. The total RNA in the skin tissues were extracted and the transcriptome were sequenced. After sequencing, a total of 1,131, 311, 140 million high quality RNA-seq reads were collected. A set of 215, 473, 968, 1055 differentially expressed genes were identified at 24 h, 48 h, 72 h and 96 h post infection respectively. Further analysis clustered these DEGs into six profiles and 75 hub genes for six profiles were identified. Among these hub genes, 18 immune related genes including TLR5, TOPK, NFKBIZ, MAPK14A were identified post C. irritans infection. Cytokine-cytokine receptor interaction was the only pathway that significantly enriched at four timepoints post infection. This study provides an in-depth understanding of skin transcriptome variance of large yellow croaker after C. irritans infection, which would be helpful for further understanding of the molecular mechanism of L. crocea in response to C. irritans infection.

The adaptive evolution of Leuciscus waleckii in Lake Dali Nur and convergent evolution of Cypriniformes fishes inhabiting extremely alkaline environments.

Leuciscus waleckii is widely distributed in Northeast Asia and has high economic value. The population in Lake Dali Nur can adapt to extremely alkaline-saline water with bicarbonate over 50 mmol/L (pH 9.6), thus providing an exceptional model for exploring the mechanisms of adaptive evolution under extreme alkaline environments. Here, we assembled a high-quality chromosome-level reference genome for L. waleckii from Lake Dali Nur. Based on the resequencing of 85 individuals from divergent populations, the historical population size of L.waleckii in Lake Dali Nur dramatically expanded in a thousand years approximately 13,000 years ago, and experienced a cliff recession in the process of adapting to the alkaline environment of Lake Dali Nur approximately 6,000 years ago. Genome scans between freshwater and alkaline populations further revealed the significant selective sweep regions from Lake Dali Nur, which harbour a set of candidate genes involved in hypoxia tolerance, ion transport, acid-base regulation and nitrogen metabolism. 5 alkali population-specific nonsynonymous mutations were identified in CA15 gene copies. In addition, two sites with convergent amino acid mutation were detected in the RHCG-a gene among several alkali environment adapted Cypriniformes fish. Our findings provide comprehensive insight into the genomic mechanisms of L. waleckii and reveal their adaptative evolution under extreme alkaline environments.

Genome-Wide Association and Expression Analysis Revealed the Candidate Variants and Molecular Underpinnings of Cold-Stress Response in Large Yellow Croaker.

Large yellow croaker (Larimichthys crocea) is one of the most economically important fish in China. Recently, global climate change has caused more and more intense and extreme low temperature weathers, resulting in huge losses to the large yellow croaker industry. Therefore, it is essential to understand the mechanisms of low-temperature tolerance in large yellow croaker. Here, we conducted an integrative analysis of genome-wide association study (GWAS) and transcriptome analysis to identify candidate variants and reveal the molecular underpinning of cold-stress response in large yellow croaker. A total of 8 significant single nucleotide polymorphisms (SNPs) loci on 6 chromosomes were identified in the GWAS analysis, and 5764 (gill) and 3588 (liver) differentially expressed genes (DEGs) were detected in cold-stressed large yellow croaker, respectively. Further comparative and functional analysis of the candidate genes and DEGs highlighted the importance of pathways/genes related to immune response, cellular stress response, lipid transport, and metabolism in the cold-stress response of large yellow croaker. Our results provide insights into the cold tolerance of large yellow croaker and contribute to genomic-based selection for low-temperature-resistant large yellow croaker.

Genome-Wide Association Analysis Reveals the Genetic Architecture of Parasite (Cryptocaryon irritans) Resistance in Large Yellow Croaker (Larimichthys crocea).

Large yellow croaker is an important marine culture species in China. Recently, the large yellow croaker industry is threatened by various disease problems, especially for the white spot disease, which is caused by parasite Cryptocaryon irritans. In the current study, we conducted a genome-wide association study (GWAS) for C. irritans resistance in two large yellow croaker populations (n = 264 and n = 480, respectively). We identified 15 QTL with explained genetic variance ranging from 1 to 8% in the two populations. One QTL on chromosome 23 was shared by the two populations, and three QTL had been reported in the previous study. We identified a lot of biological pathways associated with C. irritans resistance, such as hormone transport, response to bacterium, apoptotic process, acute inflammatory response to antigenic stimulus, and NF-kappa B signaling pathway. The genes casp8 and traf6 involved in regulatory network for apoptosis and inflammation were identified to be candidate genes for C. irritans resistance. Our results showed the complex polygenic architecture of resistance of large yellow croaker against C. irritans. These results would be helpful for the researches of the molecular mechanism of C. irritans resistance and genome-assisted breeding of large yellow croaker.

Genome-Wide Association Study Identifies Genomic Loci of Sex Determination and Gonadosomatic Index Traits in Large Yellow Croaker (Larimichthys crocea).

Larimichthys crocea is one of the traditional marine culture fishes in China, widely distributed in South China Sea, East Sea, and southern Yellow Sea. Sex dimorphism is evident in this species that females present a substantial growth strength than males, suggesting breeding females could obtain more economic benefits in L. crocea aquaculture industry. With the continuous expansion of aquaculture industry, both identifying sex-associated genome region and understanding the genetic basis underlying gonad differentiation and development matter to not only sex control aquaculture but also breeding industry. Thus, genome-wide association analysis (GWAS) of sex determination was conducted with a random breeding population of 905 individuals (including 463 females and 442 males) by ddRAD sequencing. For sex determination, 21 significant single nucleotide polymorphisms (SNPs) in chromosome (Chr) 22 were identified. Surrounding these SNPs, we founded 14 candidate genes, including dmrt1, dmrt3, and piwil2, fam102a, and odf2. The sex-associated region was narrowed down further to 2.4 Mb on Chr22 through Fst scanning and insertion-deletion (InDel) analysis. Besides, 3 SNPs in the supposed sex-determining region on Chr22 were identified as highly associated with gonad differentiation through GWAS on gonadosomatic index (GSI) in 350 males and 231 females. Because of the significant difference of GSI between females and males of L. crocea, GWAS on GSI of different genders was also conducted independently. Finally, we identified a SNP in Chr18 showing genome-wide significant association with male GSI (MGSI) and three genes axl, cyp2a10, and cyp2g1 involved in the gonadal development regulation process of aromatase. Overall, this study explored the genetic basis of sex determination mechanism and provided novel insights into gonad differentiation and development, offering solid genetic support for sex control breeding, marker-assisted selection, and marine resources conservation.

Genome-Wide Association Study of Body Shape-Related Traits in Large Yellow Croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) is one of the most important cultured marine fish on the southeast coast of China. Its body shape is important for the aquaculture industry since it affects the behavior such as swimming, ingesting, and evading, as well as customer preference. Due to the greater consumer demand of small head, slender body large yellow croaker, selecting and breeding of slender individuals with the assistance of genetic markers will benefit the industry quickly. In this study, several traits were employed to represent body shape, including body depth/body length (BD/BL), body thickness/body length (BT/BL), caudal peduncle depth/caudal peduncle length (CPDLR), tail length/body length (TL/BL), and body area/head area (BA/HA). Genome-wide association study was conducted with a panmictic population of 280 individuals to identify SNP and genes potentially associated with body shape. A set of 20 SNPs on 12 chromosomes were identified to be significantly associated with body shape-related traits. Besides, 5 SNPs were identified to be suggestive associated with CPDLR and BT/BL. Surrounding these SNPs, we found some body shape-related candidate genes, including fabp1, acrv1, bcor, mstn, bambi, and neo1, which involved in lipid metabolism, TGF-ß signaling, and BMP pathway and other important regulatory pathways. These results will be useful for the understanding of the genetic basis of body shape formation and helpful for body shape controlling of large yellow croaker by using marker-assisted selection.

Construction of a High-Density Genetic Linkage Map and QTL Mapping for Growth-Related Traits in Takifugu bimaculatus.

Takifugu bimaculatus is a euryhaline species, distributed ranging from the southern Yellow Sea to the South China Sea. Their tolerance to a wide range of salinity and temperature, coupled with a desirable firm texture, makes T. bimaculatus a strong candidate for Takifugu aquaculture in subtropics areas. Due to the increasing demand in markets and emerging of the Takifugu aquaculture industry, close attention has been paid to improvement on the T. bimaculatus production. In aquaculture, the great effort has been put into marker-assisted selective breeding, and efficient improvement was realized. However, few genetic resources on T. bimaculatus are provided so far. Aiming at understanding the genetic basis underlying important economic growth traits, facilitating genetic improvement and enriching the genetic resource in T. bimaculatus, we constructed the first genetic linkage map for T. bimaculatus via double digestion restriction-site association DNA sequencing and conducted quantitative traits locus (QTL) mapping for growth-related traits. The map comprised 1976 single nucleotide polymorphism markers distributed on 22 linkage groups (LG), with a total genetic distance of 2039.74 cM. Based on the linkage map, a chromosome-level assembly was constructed whereby we carried out comparative genomics analysis, verifying the high accuracy on contigs ordering of the linkage map. On the other hand, 18 QTLs associated with growth traits were detected on LG6, LG7, LG8, LG10, LG20, and LG21 with phenotypical variance ranging from 15.1 to 56.4%. Candidate genes participating in cartilage development, fat accumulation, and other growth-related regulation activities were identified from these QTLs, including col11a1, foxa2, and thrap3. The linkage map provided a solid foundation for chromosomes assembly and refinement. QTLs reported here unraveled the genomic architecture of some growth traits, which will advance the investigation of aquaculture breeding efforts in T. bimaculatus.

Identification and Expression Analysis of Long Non-coding RNA in Large Yellow Croaker (Larimichthys crocea) in Response to Cryptocaryon irritans Infection.

Large-scale transcription studies have revealed numerous lncRNAs (long non-coding RNAs). lncRNAs have been proposed to participate in the regulation of a diverse range of biological processes, including transcriptional regulation. Although lncRNAs have attracted increasing attention, the studies in large yellow croaker (Larimichthys crocea) are still rare, and they lack systematic analysis. In this study, 101 RNA-seq datasets varied in ages, sexes, and tissues were retrieved from the NCBI database to generate a comprehensive catalog of large yellow croaker transcriptome database. A set of 14,599 high-confidence lncRNAs from 13,673 loci were identified and characterized. Furthermore, RNA-seq datasets obtained from the infection of C. irritans were employed to investigate the differential expression pattern of lncRNAs and analyze potential biological functions. A total of 77 differentially expressed lncRNAs targeting to 567 protein-coding genes were identified by using expression analysis. Several immune genes, including TLR5, CD2AP, and MMP9, were highlighted. With GO enrichment and KEGG pathway analysis, the immune-related terms or pathways were enriched. This study created a comprehensive dataset of lncRNAs for large yellow croaker, which would be helpful for the researches of functional roles of lncRNAs in large yellow croaker.

The sequencing and de novo assembly of the Larimichthys crocea genome using PacBio and Hi-C technologies.

Larimichthys crocea is an endemic marine fish in East Asia that belongs to Sciaenidae in Perciformes. L. crocea has now been recognized as an "iconic" marine fish species in China because not only is it a popular food fish in China, it is a representative victim of overfishing and still provides high value fish products supported by the modern large-scale mariculture industry. Here, we report a chromosome-level reference genome of L. crocea generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The genome sequences were assembled into 1,591 contigs with a total length of 723.86 Mb and a contig N50 length of 2.83 Mb. After chromosome-level scaffolding, 24 scaffolds were constructed with a total length of 668.67 Mb (92.48% of the total length). Genome annotation identified 23,657 protein-coding genes and 7262 ncRNAs. This highly accurate, chromosome-level reference genome of L. crocea provides an essential genome resource to support the development of genome-scale selective breeding and restocking strategies of L. crocea.

Constructing a High-Density Genetic Linkage Map for Large Yellow Croaker (Larimichthys crocea) and Mapping Resistance Trait Against Ciliate Parasite Cryptocaryon irritans.

The large yellow croaker (Larimichthys crocea) is the most economically important marine cage-farming fish in China in the past decade. However, the sustainable development of large yellow croaker aquaculture has been severely hampered by several diseases, of which, the white spot disease caused by ciliate protozoan parasite Cryptocaryon irritans ranks the most damaging disease in large yellow croaker cage farms. To better understand the genetic basis of parasite infection and disease resistance to C. irritans, it is vital to map the traits and localize the underlying candidate genes in L. crocea genome. Here, we constructed a high-density genetic linkage map using double-digest restriction-site associated DNA (ddRAD)-based high-throughput SNP genotyping data of a F1 mapping family, which had been challenged with C. irritans for resistant trait measure. A total of 5261 SNPs was grouped and oriented into 24 linkage groups (LGs), representing 24 chromosomes of L. crocea. The total genetic map length was 1885.67 cM with an average inter-locus distance of 0.36 cM. Quantitative trait loci (QTL) mapping identified seven significant QTLs in four LGs linked to C. irritans disease resistance. Candidate genes underlying disease resistance were identified from the reference genome, including ifnar1, ifngr2, ikbke, and CD112. Comparative genomic analysis between large yellow croaker and the four closely related species revealed high evolutionary conservation of chromosomes, though inter-chromosomal rearrangements do exist. Especially, the croaker genome structure was closer to the medaka genome than stickleback, indicating that the croaker genome might retain the teleost ancestral genome structure. The high-density genetic linkage map provides an important tool and resource for fine mapping, comparative genome analysis, and molecular selective breeding of large yellow croaker.

The sequence and de novo assembly of Takifugu bimaculatus genome using PacBio and Hi-C technologies.

Takifugu bimaculatus is a native teleost species of the southeast coast of China where it has been cultivated as an important edible fish in the last decade. Genetic breeding programs, which have been recently initiated for improving the aquaculture performance of T. bimaculatus, urgently require a high-quality reference genome to facilitate genome selection and related genetic studies. To address this need, we produced a chromosome-level reference genome of T. bimaculatus using the PacBio single molecule sequencing technique (SMRT) and High-through chromosome conformation capture (Hi-C) technologies. The genome was assembled into 2,193 contigs with a total length of 404.21 Mb and a contig N50 length of 1.31 Mb. After chromosome-level scaffolding, 22 chromosomes with a total length of 371.68 Mb were constructed. Moreover, a total of 21,117 protein-coding genes and 3,471 ncRNAs were annotated in the reference genome. The highly accurate, chromosome-level reference genome of T. bimaculatus provides an essential genome resource for not only the genome-scale selective breeding of T. bimaculatus but also the exploration of the evolutionary basis of the speciation and local adaptation of the Takifugu genus.

Genome-Wide Association Study of Growth and Body-Shape-Related Traits in Large Yellow Croaker (Larimichthys crocea) Using ddRAD Sequencing.

Large yellow croaker (Larimichthys crocea) is an economically important marine fish species of China. Due to overfishing and marine pollution, the wild stocks of this croaker have collapsed in the past decades. Meanwhile, the cultured croaker is facing the difficulties of reduced genetic diversity and low growth rate. To explore the molecular markers related to the growth traits of croaker and providing the related SNPs for the marker-assisted selection, we used double-digest restriction-site associated DNA (ddRAD) sequencing to dissect the genetic bases of growth traits in a cultured population and identify the SNPs that associated with important growth traits by GWAS. A total of 220 individuals were genotyped by ddRAD sequencing. After quality control, 27,227 SNPs were identified in 220 samples and used for GWAS analysis. We identified 13 genome-wide significant associated SNPs of growth traits on 8 chromosomes, and the beta P of these SNPs ranged from 0.01 to 0.86. Through the definition of candidate regions and gene annotation, candidate genes related to growth were identified, including important regulators such as fgf18, fgf1, nr3c1, cyp8b1, fabp2, cyp2r1, ppara, and ccm2l. We also identified SNPs and candidate genes that significantly associated with body shape, including bmp7, col1a1, col11a2, and col18a1, which are also economically important traits for large yellow croaker aquaculture. The results provided insights into the genetic basis of growth and body shape in large yellow croaker population and would provide reliable genetic markers for molecular marker-assisted selection in the future. Meanwhile, the result established a basis for our subsequent fine mapping and related gene study.