Genome-wide identification and differentially expression analysis of lncRNAs in tilapia.

BACKGROUND: Long noncoding RNAs (LncRNAs) play important roles in fundamental biological processes. However, knowledge about the genome-wide distribution and stress-related expression of lncRNAs in tilapia is still limited. RESULTS: Genome-wide identification of lncRNAs in the tilapia genome was carried out in this study using bioinformatics tools. 103 RNAseq datasets that generated in our laboratory or collected from NCBI database were analyzed. In total, 72,276 high-confidence lncRNAs were identified. The averaged positive correlation coefficient (r_mean = 0.286) between overlapped lncRNA and mRNA pairs showed significant differences with the values for all lncRNA-mRNA pairs (r_mean = 0.176, z statistics = - 2.45, p value = 0.00071) and mRNA-mRNA pairs (r_mean = 0.186, z statistics = - 2.23, p value = 0.0129). Weighted correlation network analysis of the lncRNA and mRNA datasets from 12 tissues identified 21 modules and many interesting mRNA genes that clustered with lncRNAs. Overrepresentation test indicated that these mRNAs enriched in many biological processes, such as meiosis (p = 0.00164), DNA replication (p = 0.00246), metabolic process (p = 0.000838) and in molecular function, e.g., helicase activity (p = 0.000102) and catalytic activity (p = 0.0000612). Differential expression (DE) analysis identified 99 stress-related lncRNA genes and 1955 tissue-specific DE lncRNA genes. MiRNA-lncRNA interaction analysis detected 72,267 lncRNAs containing motifs with sequence complementary to 458 miRNAs. CONCLUSIONS: This study provides an invaluable resource for further studies on molecular bases of lncRNAs in tilapia genomes. Further function analysis of the lncRNAs will help to elucidate their roles in regulating stress-related adaptation in tilapia.

Genetic mechanism of body size variation in groupers: Insights from phylotranscriptomics.

Animal body size variation is of particular interest in evolutionary biology, but the genetic basis remains largely unknown. Previous studies have shown the presence of two parallel evolutionary genetic clusters within the fish genus Epinephelus with evident divergence in body size, providing an excellent opportunity to investigate the genetic basis of body size variation in vertebrates. Herein, we performed phylotranscriptomic analysis and reconstructed the phylogeny of 13 epinephelids originating from the South China Sea. Two genetic clades with an estimated divergence time of approximately 15.4 million years ago were correlated with large and small body size, respectively. A total of 180 rapidly evolving genes and two positively selected genes were identified between the two groups. Functional enrichment analyses of these candidate genes revealed distinct enrichment categories between the two groups. These pathways and genes may play important roles in body size variation in groupers through complex regulatory networks. Based on our results, we speculate that the ancestors of the two divergent groups of groupers may have adapted to different environments through habitat selection, leading to genetic variations in metabolic patterns, organ development, and lifespan, resulting in body size divergence between the two locally adapted populations. These findings provide important insights into the genetic mechanisms underlying body size variation in groupers and species differentiation.

Genome-Wide Characterization of Alternative Splicing Events and Their Responses to Cold Stress in Tilapia.

Alternative splicing (AS) is an important post-transcriptional regulatory mechanism for cells to generate transcript variability and proteome diversity. No systematic investigation of AS events among different tissues in response to stressors is available for tilapia currently. In this study, AS among different tissues was identified and the cold stress-related AS events were explored in a Nile tilapia (Oreochromis niloticus) line based on 42 RNA-seq datasets using a bioinformatics pipeline. 14,796 (82.76%; SD = 2,840) of the expression genes showed AS events. The two most abundant AS types were alternative transcription start site (TSS) and terminal site (TTS) in tilapia. Testis, brain and kidney possess the most abundant AS gene number, while the blood, muscle and liver possess the least number in each tissue. Furthermore, 208 differentially alternative splicing (DAS) genes in heart and 483 DAS in brain in response to cold stress. The number of AS types for alternative exon end, exon skipping and retention of single intron increased significantly under cold stress. GO enrichment and pathway overrepresentation analysis indicated that many DAS genes, e.g., genes in circadian clock pathway, may influence expression of downstream genes under cold stress. Our study revealed that AS exists extensively in tilapia and plays an important role in cold adaption.

First genetic assessment of brackish water polychaete Tylorrhynchus heterochaetus: mitochondrial COI sequences reveal strong genetic differentiation and population expansion in samples collected from southeast China and north Vietnam.

Tylorrhynchus heterochaetus is a widespread benthic polychaete worm found in coastal brackish waters of the west Pacific. It has high ecological and economic value as a biomarker of water quality and as a high-quality feed in aquaculture and fisheries and is considered a delicacy in some areas of Asia. However, it has experienced a marked reduction in recent years due to overexploitation as well as changes in the environment and climate. Here, to comprehensively understand its genetic background and thus provide insights for better conservation and utilization of this species, we assessed the genetic variability and demographic history of T. heterochaetus individuals sampled from eight locations along the coasts of southeast China and north Vietnam based on mitochondrial cytochrome c oxidase I ( COI) sequences. We observed high haplotype diversity ( Hd), with an average of 0.926, but relatively low nucleotide diversity ( π), with a mean of 0.032 across all samples. A total of 94 polymorphic sites and 85 haplotypes were identified among 320 individuals. The pairwise genetic distances among haplotypes ranged from 0.001 to 0.067, with the high intraspecific divergence possibly reflecting geographic isolation and gene pool fragmentation. Significant genetic structures were revealed among the studied locations; specifically, the eight locations could be treated as six genetically different populations based on pairwise Φ ST results (0.026-0.951, P<0.01). A significant pattern of isolation-by-distance was detected between the genetic and geographic distances ( r=0.873, P=0.001). Three geographic lineages were defined based on phylogenetic tree and network analyses of COI haplotypes. AMOVA results indicated that genetic variations mainly occurred among the three lineages (89.96%). Tests of neutrality and mismatch distribution suggested that T. heterochaetus underwent recent population expansion. These results provide the first report on the genetic status of T. heterochaetus and will be valuable for the management of genetic resources and better understanding of the ecology and evolution in this species.

Whole-genome sequencing of leopard coral grouper ( Plectropomus leopardus) and exploration of regulation mechanism of skin color and adaptive evolution.

Leopard coral groupers belong to the Plectropomus genus of the Epinephelidae family and are important fish for coral reef ecosystems and the marine aquaculture industry. To promote future research of this species, a high-quality chromosome-level genome was assembled using PacBio sequencing and Hi-C technology. A 787.06 Mb genome was assembled, with 99.7% (784.57 Mb) of bases anchored to 24 chromosomes. The leopard coral grouper genome size was smaller than that of other groupers, which may be related to its ancient status among grouper species. A total of 22 317 protein-coding genes were predicted. This high-quality genome of the leopard coral grouper is the first genomic resource for Plectropomus and should provide a pivotal genetic foundation for further research. Phylogenetic analysis of the leopard coral grouper and 12 other fish species showed that this fish is closely related to the brown-marbled grouper. Expanded genes in the leopard coral grouper genome were mainly associated with immune response and movement ability, which may be related to the adaptive evolution of this species to its habitat. In addition, we also identified differentially expressed genes (DEGs) associated with carotenoid metabolism between red and brown-colored leopard coral groupers. These genes may play roles in skin color decision by regulating carotenoid content in these groupers.

Differential Transcriptomic and Metabolomic Responses in the Liver of Nile Tilapia (Oreochromis niloticus) Exposed to Acute Ammonia.

Ammonia is toxic to aquatic animal. Currently, only limited works were reported on the responses of aquatic animals after ammonia exposure using "omics" technologies. Tilapia suffers from the stress of ammonia-nitrogen during intensive recirculating aquaculture. Optimizing ammonia stress tolerance has become an important issue in tilapia breeding. The molecular and biochemical mechanisms of ammonia-nitrogen toxicity have not been understood comprehensively in tilapia yet. In this study, using RNA-seq and gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer (GC-TOF-MS) techniques, we investigated differential expressed genes (DEGs) and metabolomes in the liver at 6 h post-challenges (6 hpc) and 24 h post-challenges (24 hpc) under high concentration of ammonia-nitrogen treatment. We detected 2258 DEGs at 6 hpc and 315 DEGs at 24 hpc. Functional enrichment analysis indicated that DEGs were significantly associated with cholesterol biosynthesis, steroid and lipid metabolism, energy conservation, and mitochondrial tissue organization. Metabolomic analysis detected 31 and 36 metabolites showing significant responses to ammonia-nitrogen stress at 6 and 24 hpc, respectively. D-(Glycerol 1-phosphate), fumaric acid, and L-malic acid were found significantly down-regulated at both 6 and 24 hpc. The integrative analysis of transcriptomics and metabolomics suggested considerable alterations and precise control of gene expression at both physiological and molecular levels in response to the stress of ammonia-nitrogen in tilapia.

Identifying a Long QTL Cluster Across chrLG18 Associated with Salt Tolerance in Tilapia Using GWAS and QTL-seq.

Understanding the genetic mechanism of osmoregulation is important for the improvement of salt tolerance in tilapia. In our previous study, we have identified a major quantitative trait locus (QTL) region located at 23.0 Mb of chrLG18 in a Nile tilapia line by QTL-seq. However, the conservation of these QTLs in other tilapia populations or species is not clear. In this study, we successfully investigated the QTLs associated with salt tolerance in a mass cross population from the GIFT line of Nile tilapia (Oreochromis niloticus) using a ddRAD-seq-based genome-wide association study (GWAS) and in a full-sib family from the Malaysia red tilapia strain (Oreochromis spp) using QTL-seq. Our study confirmed the major QTL interval that is located at nearly 23.0 Mb of chrLG18 in Nile tilapia and revealed a long QTL cluster across chrLG18 controlling for the salt-tolerant trait in both red tilapia and Nile tilapia. This is the first GWAS analysis on salt tolerance in tilapia. Our finding provides important insights into the genetic architecture of salinity tolerance in tilapia and supplies a basis for fine mapping QTLs, marker-assisted selection, and further detailed functional analysis of the underlying genes for salt tolerance in tilapia.

Identifying selectively important amino acid positions associated with alternative habitat environments in fish mitochondrial genomes.

Fish species inhabitating seawater (SW) or freshwater (FW) habitats have to develop genetic adaptations to alternative environment factors, especially salinity. Functional consequences of the protein variations associated with habitat environments in fish mitochondrial genomes have not yet received much attention. We analyzed 829 complete fish mitochondrial genomes and compared the amino acid differences of 13 mitochondrial protein families between FW and SW fish groups. We identified 47 specificity determining sites (SDS) that associated with FW or SW environments from 12 mitochondrial protein families. Thirty-two (68%) of the SDS sites are hydrophobic, 13 (28%) are neutral, and the remaining sites are acidic or basic. Seven of those SDS from ND1, ND2 and ND5 were scored as probably damaging to the protein structures. Furthermore, phylogenetic tree based Bayes Empirical Bayes analysis also detected 63 positive sites associated with alternative habitat environments across ten mtDNA proteins. These signatures could be important for studying mitochondrial genetic variation relevant to fish physiology and ecology.

Molecular cloning and functional analysis of polymeric immunoglobulin receptor gene in orange-spotted grouper (Epinephelus coioides).

As one of the most important mucosal effectors, polymeric immunoglobulin receptor (pIgR) mediates the transcytosis of polymeric immunoglobulins (pIgs) to protect the organisms. In this study, a full-length cDNA of pIgR was isolated from orange-spotted grouper (Epinephelus coioides), and the sequence analysis of deduced protein revealed the presence of only two Ig-like domains (ILDs), and the absence of the conserved Ig-binding site and complementary determining region (CDR). The grouper pIgR mRNA was detected in almost all the peripheral tissues examined, especially the mucosal tissues by RT-PCR. Additionally, recombinant grouper pIgR was stably expressed in the COS-7 cell line and identified as a 40-kDa transmembrane receptor. Furthermore, the association of recombinant pIgR and purified grouper pIgM was demonstrated. Taken together, the present study provided strong evidence that grouper pIgR was produced as a transmembrane protein, and probably involved in the pIgM transport.