Whole-genome resequencing reveals recent signatures of selection in five populations of largemouth bass ( Micropterus salmoides).

Largemouth bass ( Micropterus salmoides) is an economically important fish species in North America, Europe, and China. Various genetic improvement programs and domestication processes have modified its genome sequence through selective pressure, leaving nucleotide signals that can be detected at the genomic level. In this study, we sequenced 149 largemouth bass fish, including protospecies (imported from the US) and improved breeds (four domestic breeding populations from China). We detected genomic regions harboring certain genes associated with improved traits, which may be useful molecular markers for practical domestication, breeding, and selection. Subsequent analyses of genetic diversity and population structure revealed that the improved breeds have undergone more rigorous genetic changes. Through selective signal analysis, we identified hundreds of putative selective sweep regions in each largemouth bass line. Interestingly, we predicted 103 putative candidate genes potentially subjected to selection, including several associated with growth (p sst1 and grb10), early development ( klf9, sp4, and sp8), and immune traits ( pkn2, sept2, bcl6, and ripk2). These candidate genes represent potential genomic landmarks that could be used to improve important traits of biological and commercial interest. In summary, this study provides a genome-wide map of genetic variations and selection footprints in largemouth bass, which may benefit genetic studies and accelerate genetic improvement of this economically important fish.

Correlation analysis of mandarin fish (Siniperca chuatsi) growth hormone gene polymorphisms and growth traits.

Screening of trait-associated molecular markers can be used to enhance the efficiency of selective breeding. Previously, we produced the first high-density genetic linkage map for the mandarin fish (Siniperca chuatsi) and identified 11 quantitative-trait loci significantly associated with growth, of which one is located within the growth hormone (GH) gene. To investigate the GH gene polymorphisms and their correlation with growth, the complete sequence was cloned and 32 single-nucleotide polymorphisms (SNPs) and one simple-sequence repeat (SSR) were identified. Of which, eight SNPs (G1-G8) and the SSR (GH-AG)were selected for genotyping and correlation analysis with growth traits in a random population. The results showed that the four novel polymorphicloci (G1, G2, G3 and GH-AG) were significantly correlated with growth traits of mandarin fish (P < 0.05). Of these, G1, G3 and GH-AG showed highly significant correlations with multiple growth traits (P < 0.01) and the combined SNP analysis showed that G1-G3 formed four effective diplotypes (D1-D4), among which D1 was highly significantly greater than D4 (P < 0.01) for some important growth traits. In conclusion, our results show that the four polymorphic loci G1-G3 and GH-AG within the mandarin fish GH gene are significantly correlated with growth traits and could be used as candidate molecular markers for selective breedingof superior varieties of mandarin fish.

Β-defensin in Nile tilapia (Oreochromis niloticus): Sequence, tissue expression, and anti-bacterial activity of synthetic peptides.

Beta-defensins (ß-defensins) are small cationic amphiphilic peptides that are widely distributed in plants, insects, and vertebrates, and are important for their antimicrobial properties. In this study, the ß-defensin (Onß-defensin) gene of the Nile tilapia (Oreochromis niloticus) was cloned from spleen tissue. Onß-defensin has a genomic DNA sequence of 674 bp and produces a cDNA of 454 bp. Sequence alignments showed that Onß-defensin contains three exons and two introns. Sequence analysis of the cDNA identified an open reading frame of 201 bp, encoding 66 amino acids. Bioinformatic analysis showed that Onß-defensin encodes a cytoplasmic protein molecule containing a signal peptide. The deduced amino acid sequence of this peptide contains six conserved cysteine residues and two conserved glycine residues, and shows 81.82% and 78.33% sequence similarities with ß-defensin-1 of fugu (Takifugu rubripes) and rainbow trout (Oncorhynchus mykiss), respectively. Real-time quantitative PCR showed that the level of Onß-defensin expression was highest in the skin (307.1-fold), followed by the spleen (77.3-fold), kidney (17.8-fold), and muscle (16.5-fold) compared to controls. By contrast, low levels of expression were found in the liver, heart, intestine, stomach, and gill (<3.0-fold). Artificial infection of tilapia with Streptococcus agalactiae (group B streptococcus [GBS] strain) resulted in a significantly upregulated expression of Onß-defensin in the skin, muscle, kidney, and gill. In vitro antimicrobial experiments showed that a synthetic Onß-defensin polypeptide had a certain degree of inhibitory effect on the growth of Escherichia coli DH5α and S. agalactiae. The results indicate that Onß-defensin plays a role in immune responses that suppress or kill pathogens.

Two myostatin genes exhibit divergent and conserved functions in grass carp (Ctenopharyngodon idellus).

Myostatin (MSTN) is an important negative regulator of myogenesis, which inhibits myoblast proliferation and differentiation. Here, we report the isolation and characterization of two mstn genes in grass carp (Ctenopharyngodon idellus). Grass carp mstn-1 and mstn-2 cDNAs are highly divergent, sharing a relatively low amino acid sequence identity of 66%. In adult fish, both orthologs are expressed in numerous tissues and they are differentially regulated during a fasting/refeeding treatments. During embryogenesis, the mRNA levels of both mstn-1 and -2 were upregulated significantly at the beginning of somitogenesis, and maintained at high levels until hatching. Using in situ hybridization, grass carp mstn-1 mRNA was found to ubiquitously express at 12hpf, with strong signals in the notochord, and in the eyes, brain and tailbud at 24hpf, and in brain and notochord at 36hpf. In comparison, the mstn-2 mRNA can be detected in the eyes, brain and notochord at 24hpf, and in the notochord and hindbrain at 36hpf. Further overexpression of mstn-1 mRNA caused a strongly ventralized phenotype by inhibiting dorsal tissue development, while injection of mstn-2 mRNA resulted in obvious embryonic abnormalities in grass carp. These results provide some new insights into the functional conservation and divergence of mstn genes in teleost species.

Transcriptome profiling and digital gene expression analysis of Nile tilapia (Oreochromis niloticus) infected by Streptococcus agalactiae.

Tilapia is an important freshwater aquaculture species worldwide. In recent years, streptococcal diseases have severely threatened development of tilapia aquaculture, while effective prevention and control methods have not yet been established. In order to understand the immunological response of tilapia to infection by Streptococcus agalactiae (S. agalactiae), this study employed Solexa/Illumina RNA-seq and digital gene expression (DGE) technology to investigate changes in the tilapia transcriptome before and after S. agalactiae infection. We obtained 82,799 unigenes (mean size: 618 bp) using de novo assembly. Unigenes were annotated by comparing against databases including Nr, Swissprot, cluster of orthologous groups of proteins, Kyoto encyclopedia of genes and genomes, and gene ontology. Combined with DGE technology, transcriptomic changes in tilapia before and after bacteria challenging were examined. A total of 774 significantly up-regulated and 625 significantly down-regulated unigenes were identified, among which 293 were mapped to 181 signaling pathways including 17 immune-related pathways involving 65 differentially expressed genes. We observed a change in the expression of six genes in the Toll-like receptor signaling pathway, and this was subsequently confirmed via quantitative real-time PCR. This comparative study of the tilapia transcriptome before and after S. agalactiae infection identified important differentially-expressed immune-related genes and signaling pathways that will provide useful insights for further analysis of the mechanisms of tilapia defense against S. agalactiae infection.

Identification of a second follistatin gene in grass carp (Ctenopharyngodon idellus) and its regulatory function in myogenesis during embryogenesis.

Follistatin can antagonize the function of myostatin as a competitive binding protein and promote muscle growth in vivo. Here, we report the isolation and characterization of a second follistatin gene fst2 in grass carp (Ctenopharyngodon idellus). The grass carp fst2 cDNA was 1,376 bp in length, with an open reading frame (ORF) encoding 350 amino acid residues. A relatively low sequence identity of 78% was found between grass carp Fst2 and its paralog Fst1. Sequence and phylogenetic analyses suggest that the grass carp fst2 originated from fish-specific gene duplication. In adult fish, fst2 mRNA expression was observed in most tissues but was strongly expressed in the eyes, muscles, skin and ovary. Grass carp fst2 mRNA could be detected as early as 16 h post-fertilization (hpf), while fst1 mRNA was detected throughout embryogenesis. Using in situ hybridization, fst2 transcripts were detected in the anterior somites at 24 hpf and in the brain and posterior somites at 36 hpf. Meanwhile, fst1 mRNA was transcribed mainly in the optic vesicle and at the cephalic mesoderm at 12 hpf, in the eyes, cephalic mesoderm and at the lateral edge of most somites at 24 hpf, and mainly in the brain at 36 hpf. Furthermore, overexpression of fst2 mRNA markedly affected the formation of the embryonic midline and somite structures. Based on comparisons with fst1, our findings suggest that fst2 retained the ancestral functions of regulating muscle development and growth during embryogenesis in grass carp.

Goldfish transposase Tgf2 presumably from recent horizontal transfer is active.

Hobo/Activator/Tam3 (hAT) superfamily transposons occur in plants and animals and play a role in genomic evolution. Certain hAT transposons are active and have been developed as incisive genetic tools. Active vertebrate elements are rarely discovered; however, Tgf2 transposon was recently discovered in goldfish (Carassius auratus). Here, we found that the endogenous Tgf2 element can transpose in goldfish genome. Seven different goldfish mRNA transcripts, encoding three lengths of Tgf2 transposase, were identified. Tgf2 transposase mRNA was detected in goldfish embryos, mainly in epithelial cells; levels were high in ovaries and mature eggs and in all adult tissues tested. Endogenous Tgf2 transposase mRNA is active in mature eggs and can mediate high rates of transposition (>30%) when injected with donor plasmids harboring a Tgf2 cis-element. When donor plasmid was coinjected with capped Tgf2 transposase mRNA, the insertion rate reached >90% at 1 yr. Nonautonomous copies of the Tgf2 transposon with large-fragment deletions and low levels of point mutations were also detected in common goldfish. Phylogenetic analysis indicates the taxonomic distribution of Tgf2 in goldfish is not due to vertical inheritance. We propose that the goldfish Tgf2 transposon originated by recent horizontal transfer and maintains a highly native activity.

ENU-induced mutagenesis in grass carp (Ctenopharyngodon idellus) by treating mature sperm.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful approach for genetic improvement of plants, as well as for inducing functional mutants in animal models including mice and zebrafish. In the present study, mature sperm of grass carp (Ctenopharyngodon idellus) were treated with a range of ENU concentrations for 45 min, and then wild-type eggs were fertilized. The results indicated that the proportion of embryos with morphological abnormalities at segmentation stage or dead fry at hatching stage increased with increasing ENU dose up to 10 mM. Choosing a dose that was mutagenic, but provided adequate numbers of viable fry, an F1 population was generated from 1 mM ENU-treated sperm for screening purposes. The ENU-treated F1 population showed large variations in growth during the first year. A few bigger mutants with morphologically normal were generated, as compared to the controls. Analysis of DNA from 15 F1 ENU-treated individuals for mutations in partial coding regions of igf-2a, igf-2b, mstn-1, mstn-2, fst-1 and fst-2 loci revealed that most ENU-treated point mutations were GC to AT or AT to GC substitution, which led to nonsense, nonsynonymous and synonymous mutations. The average mutation rate at the examined loci was 0.41%. These results indicate that ENU treatment of mature sperm can efficiently induce point mutations in grass carp, which is a potentially useful approach for genetic improvement of these fish.

IGF binding protein 1 is correlated with hypoxia-induced growth reduce and developmental defects in grass carp (Ctenopharyngodon idellus) embryos.

The effects of hypoxia on embryonic development and the underlying cellular and molecular mechanisms are poorly understood in teleost fish, although the hypoxic effects on embryonic growth and development have been reported in the zebrafish model. Here, we found that hypoxia caused significant developmental delay and growth retardation during grass carp embryogenesis. Placing the embryos in hypoxic conditions at different developmental stages strongly induced the mRNA expression of insulin-like growth factor-binding protein 1 (IGFBP1), an inhibitory protein that binds to IGF and inhibits its subsequent actions in vivo. Further gain-of-function analysis results provided strong evidence to support the hypothesis that IGFBP1 plays an important role in mediating hypoxic-induced growth and developmental defects. Overexpression of IGFBP1 mRNA reduced the growth rate to a degree that was similar to hypoxia. Additionally, overexpression of IGFBP1 caused significant developmental defects in the formation of midline, somite and hindbrain structures during grass carp embryogenesis. Taken together, our studies suggest that IGFBP1 plays a key role in mediating these hypoxia-induced embryonic growth retardation and developmental delay in grass carp.