Xubo Wang

Detection of Alternative Splice and Gene Duplication by RNA Sequencing in Japanese Flounder, Paralichthys olivaceus

Japanese flounder (Paralichthys olivaceus) is one of the economic important fish in China. Sexual dimorphism, especially the different growth rates and body sizes between two sexes, makes this fish a good model to investigate mechanisms responsible for such dimorphism for both fundamental questions in evolution and applied topics in aquaculture. However, the lack of “omics” data has hindered the process. The recent advent of RNA-sequencing technology provides a robust tool to further study characteristics of genomes of nonmodel species. Here, we performed de novo transcriptome sequencing for a double haploid Japanese flounder individual using Illumina sequencing. A single lane of paired-end sequencing produced more than 27 million reads. These reads were assembled into 107,318 nonredundant transcripts, half of which (51,563; 48.1%) were annotated by blastx to public protein database. A total of 1051 genes that had potential alternative splicings were detected by Chrysalis implemented in Trinity software. Four of 10 randomly picked genes were verified truly containing alternative splicing by cloning and Sanger sequencing. Notably, using a doubled haploid Japanese flounder individual allow us to analyze gene duplicates. In total, 3940 “single-nucleotide polymorphisms” were detected form 1859 genes, which may have happened gene duplicates. This study lays the foundation for structural and functional genomics studies in Japanese flounder.

Isolation and characterization of 64 novel microsatellite markers from a fosmid library of female half‐smooth tongue sole (Cynoglossus semilaevis)

Microsatellite markers were developed from a fosmid library of female half‐smooth tongue sole, Cynoglossus semilaevis. Three hundred eighty‐four clones were randomly selected to sequence (double strand reading), and 168 sequences in 143 clones were found to contain microsatellites. Of the 101 primer pairs designed, 64 gave polymorphic polymerase chain reaction products. Based on characterization with 36 individuals, the number of alleles ranged from two to nine. The values of observed and expected heterozygosities varied from 0.06 to 1.00 and from 0.05 to 0.94, respectively. These markers have the potential as tools for population structure evaluation, ecological analyses and linkage map construction.

Sequencing and characterization of the transcriptome of half-smooth tongue sole (Cynoglossus semilaevis)

BackgroundHalf-smooth tongue sole (Cynoglossus semilaevis) is a valuable fish for aquaculture in China. This fish exhibits sexual dimorphism, particularly different growth rates and body sizes between two genders. Thus, C. semilaevis is a good model that can be used to investigate mechanisms responsible for such dimorphism, this model can also be utilized to answer fundamental questions in evolution and applied fields of aquaculture. Hence, advances in second-generation sequencing technology, such as 454 pyrosequencing, could provide a robust tool to study the genome characteristics of non-model species.ResultsIn this study, C. semilaevis was subjected to de novo transcriptome sequencing and characterization. A total of 749,954 reads were generated using a single 454 sequencing run in a full PicoTiter plate. These reads were then assembled into 62,632 contigs with a 10-fold average sequencing coverage. A total of 26,589 sequences were successfully annotated based on sequence similarities; among these sequences, 3,451 transcripts exhibited gene ontology terms and 2,362 showed enzyme commissions associated with 186 pathways from Kyoto Encyclopedia of Gene and Genomes pathways. A search of repetitive elements was performed, and 1,898 transposable elements were identified. Approximately 7,800 simple-sequence repeats and 21,234 single-nucleotide polymorphisms were also detected.ConclusionsOur data provided an integrated and comprehensive transcriptome resource for C. semilaevis. These data could be used for further research in population genetics, gene function, and tissue-specific gene expressions.

Isolation, polymorphism and expression study of two distinct major histocompatibility complex class II B genes from half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex (MHC) class II antigens are important in vertebrate immune system, which present peptides to CD4+ T cells. In the present study, cDNAs encoding MHC class II B gene were isolated from the cDNA library of half‐smooth tongue sole (Cynoglossus semilaevis), and the full length cDNA sequences were got by rapid amplification of cDNA ends polymerase chain reaction. The polymorphism of its open reading frame, 3′ untranslated region and intron 1 was studied. Nineteen class II B alleles were identified from nine individuals and clustered into two groups, designated as Cyse‐DAB and Cyse‐DBB. The deduced amino acid sequences among Cyse‐DAB and Cyse‐DBB alleles shared identities from 94.0% to 99.6% and 92.4% to 99.6%, respectively, while the identities between Cyse‐DAB and Cyse‐DBB genes varied from 85.1% to 92.0%. Three Cyse‐DAB alleles and one Cyse‐DBB allele were observed in each of two individuals, and three Cyse‐DBB alleles and one Cyse‐DAB allele in another individual, which indicated at least two loci existed in each gene. Two different 3′ UTR sequences were identified and one belonged to Cyse‐DAB, the other belonged to Cyse‐DBB. Both five Cyse‐DAB and five Cyse‐DBB intron 1 sequences were identified from genomic sequences, among which two sequences of each gene were identified in a single individual, which suggested the existence of at least two Cyse‐DAB and two Cyse‐DBB loci. Both the two genes’‐specific tissue and developmental stage expression were studied by reverse‐transcription polymerase chain reaction, which showed that the two genes had similar expression patterns in tissue study with high expression in spleen and kidney, low expression in liver, gill and ovary, moderate expression in brain, heart, intestine and testis. While in developmental analysis, Cyse‐DBB had higher expression than Cyse‐DAB in early developmental stages, which indicated that the two genes might have different functions in those stages. Therefore, in half‐smooth tongue sole, two different MHC class II B genes exist and could differentiate from each other whether by sequence analysis or by developmental stage expression study.

A microsatellite genetic linkage map of half smooth tongue sole (Cynoglossus semilaevis).

Half smooth tongue sole, Cynoglossus semilaevis (Pleuronectiformes, Cynoglossidae), is an important aquaculture species throughout coastal areas of China which has a high nutritional and economic value. Genetic linkage map is an important tool for accelerating aquatic breeding process through marker assisted selection (MAS) and quantitative trait locus (QTL). Here, 325 polymorphic microsatellite markers were explored and sex-specific genetic linkage map were constructed using these markers. The female map contained 193 markers located at 21 linkage groups, with a total length of 1041cM and an average resolution of 6.8cM; the male map contained 195 markers located at 21 linkage groups, with a total length of 1154cM and an average resolution of 7.2cM, they covered approximately 76.72% and 78.12% genomes, respectively. The recombination ratio of female/male was about 1:1.02 estimated in the sex-specific frame map. All developed microsatellite markers and this linkage map could serve as the foundation for further study in high density linkage map construction.

Molecular characterization and functional analysis of the GATA4 in tongue sole (Cynoglossus semilaevis)

The GATA family of transcription factors is characterized by two zinc finger domains and is involved in different cellular processes. GATA4 is a highly conserved transcription factor that regulates embryonic morphogenesis and cellular differentiation. GATA4 in vertebrates regulates its target genes to influence genital ridge differentiation. In this study, the GATA4 from tongue sole (Cynoglossus semilaevis) was characterized to understand the function of this transcription factor in sex differentiation. The full-length cDNA of C. semilaevis GATA4 comprised 2031bp, encoding a predicted polypeptide consisting of 402 amino acids with two conserved zinc finger domains. Phylogenetic, gene structure, and synteny analyses showed that C. semilaevis GATA4 was homologous to tetrapod GATA4. The mRNA transcript of C. semilaevis GATA4 exhibited high expression in the heart, liver, and gonad. GATA4 expression is dimorphic in the male and female gonads. Embryonic development expression profiles revealed the possible involvement of C. semilaevis GATA4 in morphogenesis. In situ hybridization results showed strong GATA4 signals in the spermatogonia and spermatocytes of the testis and in the oogonia, primary oocytes, and secondary oocytes of the ovary. The expression of C. semilaevis GATA4 in the male, pseudomale, and female gonads showed significantly different methylation levels of the two CpG sites (-2738 and -2647) among the three genders. Basing on these results, we speculated that GATA4 plays a potential role in sex differentiation. This study lays the groundwork for further sex control breeding techniques in C. semilaevis.

Molecular Cloning, Promoter Analysis and Expression Profiles of the sox3 Gene in Japanese Flounder, Paralichthys olivaceus

Sox3, which belongs to the SoxB1 subgroup, plays major roles in neural and gonadal development. In the present study, Japanese flounder Paralichthys olivaceus sox3 gene (Posox3) and its promoter sequence were isolated and characterized. The deduced PoSox3 protein contained 298 amino acids with a characteristic HMG-box domain. Alignment and phylogenetic analyses indicated that PoSox3 shares highly identical sequence with Sox3 homologues from different species. The promoter region of Posox3 has many potential transcription factor (TF) binding sites. The expression profiles of Posox3 in different developmental stages and diverse adult tissues were analyzed by quantitative real-time RT-PCR (qRT-PCR). Posox3 mRNA was maternally inherited, and maintained at a considerably high expression level between the blastula stage and the hatching stage during embryonic development. Posox3 was abundantly expressed in the adult brain and showed sexually dimorphic expression pattern. In situ hybridization (ISH) was carried out to investigate the cellular distribution of Posox3 in the ovary, and results showed the uniform distribution of Posox3 throughout the cytoplasm of oogonia and stage I–III oocytes. These results indicate that Posox3 has potentially vital roles in embryonic and neural development and may be involved in the oogenesis process. Our work provides a fundamental understanding of the structure and potential functions of Sox3 in Paralichthys olivaceus.

Pesticide multiresidue analysis of peanuts using automated gel permeation chromatography clean-up/gas chromatography-mass spectrometry

A method based on automated gel permeation chromatography (GPC) and on-line quantitative concentration was developed for the determination of residues of 38 typically used pesticides in high-oil peanuts. Pesticides were extracted using acetonitrile into an oil fraction containing little of the peanut matrix. After changing the solvent to the mobile phase (1 : 1 cyclohexane : ethyl acetate), clean-up was carried out using GPC, the final collected solution being automatically concentrated to a fixed volume and transferred into vials for gas chromatography (GC) injection. The pesticides were analysed by gas chromatography-mass spectrometry (GC/MS) in the selected ion monitoring (SIM) mode. Average recoveries (spiked levels of 0.02, 0.05, and 0.1 µg g−1) were between 70 and 117%. The relative standard deviation of the method was 3.5–21% (n = 6). This approach gave comparable results with a previously published method.

Evolutionary Conservation of pou5f3 Genomic Organization and Its Dynamic Distribution during Embryogenesis and in Adult Gonads in Japanese Flounder Paralichthys olivaceus

Octamer-binding transcription factor 4 (Oct4) is a member of POU (Pit-Oct-Unc) transcription factor family Class V that plays a crucial role in maintaining the pluripotency and self-renewal of stem cells. Though it has been deeply investigated in mammals, its lower vertebrate homologue, especially in the marine fish, is poorly studied. In this study, we isolated the full-length sequence of Paralichthys olivaceus pou5f3 (Popou5f3), and we found that it is homologous to mammalian Oct4. We identified two transcript variants with different lengths of 3′-untranslated regions (UTRs) generated by alternative polyadenylation (APA). Quantitative real-time RT-PCR (qRT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) were implemented to characterize the spatial and temporal expression pattern of Popou5f3 during early development and in adult tissues. Our results show that Popou5f3 is maternally inherited, abundantly expressed at the blastula and early gastrula stages, then greatly diminishes at the end of gastrulation. It is hardly detectable from the heart-beating stage onward. We found that Popou5f3 expression is restricted to the adult gonads, and continuously expresses during oogenesis while its dynamics are downregulated during spermatogenesis. Additionally, numerous cis-regulatory elements (CRE) on both sides of the flanking regions show potential roles in regulating the expression of Popou5f3. Taken together, these findings could further our understanding of the functions and evolution of pou5f3 in lower vertebrates, and also provides fundamental information for stem cell tracing and genetic manipulation in Paralichthys olivaceus.

Molecular characterization and expression profiles of GATA6 in tongue sole (Cynoglossus semilaevis).

GATA-binding protein 6 (GATA6), a transcription factor of the GATA family, plays an important role in gonadal cell proliferation, differentiation, and endoderm development. In this study, the full-length coding sequence of tongue sole (Cynoglossus semilaevis) GATA6 was identified. The sequence consisted of 1494 nucleotides encoding a peptide of 497 amino acids, which included two conserved zinc finger domains. Phylogenetic, gene structure, and synteny analysis showed that C. semilaevis GATA6 was homologous to teleost and tetrapod GATA6. C. semilaevis GATA6 mRNA exhibited high expression in heart, intestine, liver, kidney, and gonad. Embryonic development expression profiles revealed that GATA6 is involved in morphogenesis because its expression increased at the blastula stage. The in situ hybridization results showed strong GATA6 signals in spermatogonia, spermatocytes, and Sertoli cells of the testis. The signals were also detected in the oogonia and oocytes of the ovary. The expression of C. semilaevis GATA6 was sexually dimorphic, and the methylation pattern in the promoter region varied among males, females, and pseudomales. These results suggested that GATA6 might influence the gonad development and reproduction of C. semilaevis. This study provides the groundwork for further development of breeding techniques in C. semilaevis.