Intercross population study reveals that co-mutation of mitfa genes in two subgenomes induces red skin color in common carp ( Cyprinus carpio wuyuanensis).

Common carp are among the oldest domesticated fish in the world. As such, there are many food and ornamental carp strains with abundant phenotypic variations due to natural and artificial selection. Hebao red carp (HB, Cyprinus carpio wuyuanensis), an indigenous strain in China, is renowned for its unique body morphology and reddish skin. To reveal the genetic basis underlying the distinct skin color of HB, we constructed an improved high-fidelity (HiFi) HB genome with good contiguity, completeness, and correctness. Genome structure comparison was conducted between HB and a representative wild strain, Yellow River carp (YR, C. carpio haematopterus), to identify structural variants and genes under positive selection. Signatures of artificial selection during domestication were identified in HB and YR populations, while phenotype mapping was performed in a segregating population generated by HB×YR crosses. Body color in HB was associated with regions with fixed mutations. The simultaneous mutation and superposition of a pair of homologous genes ( mitfa) in chromosomes A06 and B06 conferred the reddish color in domesticated HB. Transcriptome analysis of common carp with different alleles of the mitfa mutation confirmed that gene duplication can buffer the deleterious effects of mutation in allotetraploids. This study provides new insights into genotype-phenotype associations in allotetraploid species and lays a foundation for future breeding of common carp.

Cysestermerol A, a rare stilbene sestermer with significant hypoglycemic activities from Cynodon dactylon.

Cysestermerol A (1), a rare and new stilbene sestermer, was isolated from the whole herb of Cynodon dactylon. The planar and relative structures of 1 were elucidated based on HRESIMS, one- and two-dimensional NMR analyses, and its absolute configuration was further established by electronic circular dichroism calculations. Compound 1 obviously increased the glucose consumption in HepG2 cells equivalent to the positive control rosiglitazone and markedly inhibited the activity of α-glucosidase in vitro.

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.

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.

QTL Mapping for Red Blotches in Malaysia Red Tilapia (Oreochromis spp.).

Body color is an interesting economic trait in fish. Red tilapia with red blotches may decrease its commercial values. Conventional selection of pure red color lines is a time-consuming and labor-intensive process. To accelerate selection of pure lines through marker-assisted selection, in this study, double-digest restriction site-associated DNA sequencing (ddRAD-seq) technology was applied to genotype a full-sib mapping family of Malaysia red tilapia (Oreochromis spp.) (N = 192). Genome-wide significant quantitative trait locus (QTL)-controlling red blotches were mapped onto two chromosomes (chrLG5 and chrLG15) explaining 9.7% and 8.2% of phenotypic variances by a genome-wide association study (GWAS) and linkage-based QTL mapping. Six SNPs from the chromosome chrLG5 (four), chrLG15 (one), and unplaced supercontig GL831288-1 (one) were significantly associated to the red blotch trait in GWAS analysis. We developed nine microsatellite markers and validated significant correlations between genotypes and blotch data (p < 0.05). Our study laid a foundation for exploring a genetic mechanism of body colors and carrying out genetic improvement for color quality 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.

Marker-assisted selection of YY supermales from a genetically improved farmed tilapia-derived strain.

Genetically improved farmed tilapia (GIFT) and GIFT-derived strains account for the majority of farmed tilapia worldwide. As male tilapias grow much faster than females, they are often considered more desirable in the aquacultural industry. Sex reversal of females to males using the male sex hormone 17-α-methyltestosterone (MT) is generally used to induce phenotypic males during large-scale production of all male fingerlings. However, the widespread use of large quantities of sex reversal hormone in hatcheries may pose a health risk to workers and ecological threats to surrounding environments. Breeding procedures to produce genetically all-male tilapia with limited or no use of sex hormones are therefore urgently needed. In this study, by applying marker-assisted selection (MAS) for the selection of YY supermales from a GIFT-derived strain, we identified 24 XY pseudofemale and 431 YY supermale tilapias. Further performance evaluation on the progenies of the YY supermales resulted in male rates of 94.1%, 99.5% and 99.6%, respectively, in three populations, and a daily increase in body weight of 1.4 g at 3 months (n=997). Our study established a highly effective MAS procedure in the selection of YY supermales from a GIFT-derived strain. Furthermore, the development of MAS-selected YY supermales will help reduce the utilization of hormones for controlling sex in the tilapia aquaculture.

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.

Identifying a Major QTL Associated with Salinity Tolerance in Nile Tilapia Using QTL-Seq.

Selection of new lines with high salinity tolerance allows for economically feasible production of tilapias in brackish water areas. Mapping QTLs and identifying the markers linked to salinity-tolerant traits are the first steps in the improvement of the tolerance in tilapia through marker-assisted selection techniques. By using QTL-seq strategy and linkage-based analysis, two significant QTL intervals (chrLG4 and chrLG18) on salinity-tolerant traits were firstly identified in the Nile tilapia. Fine mapping with microsatellite and SNP markers suggested a major QTL region that located at 23.0 Mb of chrLG18 and explained 79% of phenotypic variation with a LOD value of 95. Expression analysis indicated that at least 10 genes (e.g., LACTB2, KINH, NCOA2, DIP2C, LARP4B, PEX5R, and KCNJ9) near or within the QTL interval were significantly differentially expressed in intestines, brains, or gills under 10, 15, or 20 ppt challenges. Our findings suggest that QTL-seq can be effectively utilized in QTL mapping of salinity-tolerant traits in fish. The identified major QTL is a promising locus to improve our knowledge on the genetic mechanism of salinity tolerance in tilapia.

Acute hypoxia stress induced abundant differential expression genes and alternative splicing events in heart of tilapia.

Hypoxia is one of the critical environmental stressors for fish in aquatic environments. Although accumulating evidences indicate that gene expression is regulated by hypoxia stress in fish, how genes undergoing differential gene expression and/or alternative splicing (AS) in response to hypoxia stress in heart are not well understood. Using RNA-seq, we surveyed and detected 289 differential expressed genes (DEG) and 103 genes that undergo differential usage of exons and splice junctions events (DUES) in heart of a hypoxia tolerant fish, Nile tilapia, Oreochromis niloticus following 12h hypoxic treatment. The spatio-temporal expression analysis validated the significant association of differential exon usages in two randomly selected DUES genes (fam162a and ndrg2) in 5 tissues (heart, liver, brain, gill and spleen) sampled at three time points (6h, 12h, and 24h) under acute hypoxia treatment. Functional analysis significantly associated the differential expressed genes with the categories related to energy conservation, protein synthesis and immune response. Different enrichment categories were found between the DEG and DUES dataset. The Isomerase activity, Oxidoreductase activity, Glycolysis and Oxidative stress process were significantly enriched for the DEG gene dataset, but the Structural constituent of ribosome and Structural molecule activity, Ribosomal protein and RNA binding protein were significantly enriched only for the DUES genes. Our comparative transcriptomic analysis reveals abundant stress responsive genes and their differential regulation function in the heart tissues of Nile tilapia under acute hypoxia stress. Our findings will facilitate future investigation on transcriptome complexity and AS regulation during hypoxia stress in fish.

Genome-Wide QTL Analysis Identified Significant Associations Between Hypoxia Tolerance and Mutations in the GPR132 and ABCG4 Genes in Nile Tilapia.

Exposure to hypoxia induces both acute and chronic stress responses, which plays an important role in health of cultured organisms including growth, reproduction, immunity, and other energy demanding activities. Application of advanced genomic technologies allows rapid identification of hypoxia trait-associated genes and precise selection of superior brood stocks with high tolerance in tilapia. By applying QTL-seq and double-digest restriction-site associated DNA sequencing (ddRAD-seq) techniques, we identified four genome-wide significant quantitative trait loci (QTLs) for hypoxia tolerance and many suggestive QTLs in Nile tilapia. These QTLs explained 6.6-14.7% of the phenotypic variance. Further analysis revealed that single nucleotide polymorphisms (SNPs) in exons of both GPR132 and ABCG4 genes located in genome-wide QTL intervals were significantly associated with hypoxia-tolerant traits. Expression analysis of both genes suggested that they were strong candidate genes involved into hypoxia tolerance in tilapia. Our findings suggest that both QTL-seq and ddRAD-seq techniques can be effectively utilized in QTL mapping of hypoxia traits in fish. Our data supply a basis for further marker-assisted selection of super lines with a high level of tolerance against low oxygen stress in the tilapia.

Two new stilbene trimers from Cynodon dactylon.

Many naturally occurring oligostilbenes have drawn considerable attention because of their intricate structures and diverse bioactivities. Two new stilbene trimers, cystibenetrimerol A (1) and cystibenetrimerol B (2) were isolated from the dried grass of Cynodon dactylon (L.) Pers. The planar structures and stereo configurations of them were elucidated by spectroscopic and spectrometric methods. The isolation and structures elucidation of two new stilbene trimers suggested the ordinary grass belonging to the family Poaceae may be a rich source of stilbene oligomers.

Characterization and functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in tilapia.

Hypoxia is a major cause of fish morbidity and mortality in the aquatic environment. Hypoxia-inducible factors are very important modulators in the transcriptional response to hypoxic stress. In this study, we characterized and conducted functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in Nile tilapia (Oreochromis niloticus). By cloning and Sanger sequencing, we obtained the full length cDNA sequences for HIF1α (2686bp) and HIF1αn (1308bp), respectively. The CDS of HIF1α includes 15 exons encoding 768 amino acid residues and the CDS of HIF1αn contains 8 exons encoding 354 amino acid residues. The complete CDS sequences of HIF1α and HIF1αn cloned from tilapia shared very high homology with known genes from other fishes. HIF1α show differentiated expression in different tissues (brain, heart, gill, spleen, liver) and at different hypoxia exposure times (6h, 12h, 24h). HIF1αn expression level under hypoxia is generally increased (6h, 12h, 24h) and shows extremely highly upregulation in brain tissue under hypoxia. A functional determination site analysis in the protein sequences between fish and land animals identified 21 amino acid sites in HIF1α and 2 sites in HIF1αn as significantly associated sites (α = 0.05). Phylogenetic tree-based positive selection analysis suggested 22 sites in HIF1α as positively selected sites with a p-value of at least 95% for fish lineages compared to the land animals. Our study could be important for clarifying the mechanism of fish adaptation to aquatic hypoxia environment.

Copy Number Variations in Tilapia Genomes.

Discovering the nature and pattern of genome variation is fundamental in understanding phenotypic diversity among populations. Although several millions of single nucleotide polymorphisms (SNPs) have been discovered in tilapia, the genome-wide characterization of larger structural variants, such as copy number variation (CNV) regions has not been carried out yet. We conducted a genome-wide scan for CNVs in 47 individuals from three tilapia populations. Based on 254 Gb of high-quality paired-end sequencing reads, we identified 4642 distinct high-confidence CNVs. These CNVs account for 1.9% (12.411 Mb) of the used Nile tilapia reference genome. A total of 1100 predicted CNVs were found overlapping with exon regions of protein genes. Further association analysis based on linear model regression found 85 CNVs ranging between 300 and 27,000 base pairs significantly associated to population types (R 2 > 0.9 and P > 0.001). Our study sheds first insights on genome-wide CNVs in tilapia. These CNVs among and within tilapia populations may have functional effects on phenotypes and specific adaptation to particular environments.