Half-Smooth Tongue Sole Research Articles

Construction of a Genetic Linkage Map and Mapping of a Female-Specific DNA Marker in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

The half-smooth tongue sole (Cynoglossus semilaevis, hereafter, "tongue sole") is a marine flatfish with great commercial importance for fisheries and aquaculture in China. It has also been a promising model for the study of sex determination mechanisms in fish. Here, we report the construction of a genetic linkage map for the tongue sole, based on 137 markers including 103 AFLP markers, 33 microsatellite markers, and one female-specific DNA marker. Twenty-six linkage groups (LGs) were found. The total map length was 934.6 cM (Kosambi), with an average spacing of 8.4 cM, covering 64.4% of the estimated genome size. Furthermore, a female-specific SCAR marker, CseF-382, was mapped on LG5. This study represents the first genetic linkage map in the tongue sole. This map has great potential in the identification of quantitative traits loci and sex-related genes and marker-assisted selection in the tongue sole. Meanwhile, the new set of polymorphic microsatellite markers developed in this study is not only useful for genetic mapping but also of critical importance for studies on genetic diversity and broodstock management in tongue sole.

Molecular Cloning and Expression Analysis of FTZ-F1 in the Half-smooth Tongue-sole,<I>Cynoglossus semilaevis</I>

To investigate the expression characteristics of sex related gene of FTZ-F1 in the half-smooth tongue-sole (Cynoglossus semilaevis), the homologue FTZ-F1 (hsFTZ-F1) full-length cDNA was isolated from the testis by homologous cloning, and the cDNA included the open reading frame and a 66 bp 5'-UTR, along with a 1 619 bp 3'-UTR, encoding a predicted 485 amino acid protein. Sequence, tissue distribution and phylogenic analyses of the FTZ-F1 showed that the hsFTZ-F1 belonged to SF-1/Ad4BP group. The hsFTZ-F1 transcripts were highly abundant in the gonads, kidneys, brain and head-kidneys, but weakly in other tissues. However, the expression level in the brain and head-kidney of female was highly abundant than in the male. The hsFTZ-F1 expression was highly abundant in the embryo than in the larvae, which suggested that the hsFTZ-F1 may be involved in the organogenesis in the tongue sole.

Molecular cloning, characterization and expression analysis of gonadal P450 aromatase in the half-smooth tongue-sole, Cynoglossus semilaevis

Cytochrome P450 aromatase (P450arom) is an enzyme responsible for the conversion of androgen to estrogen. The P450arom plays an important role in gonadal differentiation and sex determination in teleosts. To study the role of gonadal cytochrome P450 aromatase (P450aromA) in sex differentiation, the cDNA was isolated from the ovary of the half-smooth tongue-sole, Cynoglossus semilaevis. The deduced amino acid sequence exhibited high homology with P450arom of other teleosts. Alignment and phylogenetic analysis of the P450arom showed that the tongue sole P450aromA belonged to the gonadal P450arom subfamily. The 5'-flanking region of the P450aromA gene contains two Ad4 binding motifs and a half site of the palindrome sequence of the estrogen-responsive element (the ERE half). P450aromA transcripts were highly abundant in the ovary, less abundant in the testis, but not present in the brain, liver, spleen, kidney, muscle, skin, heart, head kidney, gill, intestine, or eyes by RT-PCR. The P450aromA mRNA increased with gonad development, but was less abundant in the testis compare to the ovary in all phases. Highly abundant P450aromA was detected in the female ovary, but was less abundant in the testis in neo-males obtained by methyltestosterone or temperature treatment. These results imply that P450aromA may be involved in gonadal development and sex differentiation in the tongue sole.

Fosmid Library Construction and Initial Analysis of End Sequences in Female Half-smooth Tongue Sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis: Pleuronectiformes) is a commercially important cultured marine flatfish in China and forms an important fishery resource, but the research of its genome is underdeveloped. In this study, we constructed a female C. semilaevis fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 49,920 clones with an average insert size of about 39 kb, amounting to 3.23 genome equivalents. Fosmid stability assays indicate that female C. semilaevis DNA was stable during propagation in the fosmid system. Library screening with eight microsatellite markers yielded between two and five positive clones, and none of those tested was absent from the library. End-sequencing of both 5' and 3' ends of 1,152 individual clones generated 2,247 sequences after trimming, with an average sequence length of 855 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 259 (11.53%) and 287 (12.77%) significant hits (E < e (-5)), respectively. Repetitive sequences analysis resulted in 5.23% of base pairs masked using both the Fugu and Danio databases, repetitive elements were composed of retroelements, DNA transposons, satellites, simple repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for large-scale genome sequencing, physical mapping, and positional cloning, and provide a better understanding of female C. semilaevis genome.

CDNA cloning, tissues, embryos and larvae expression analysis of Sox10 in half-smooth tongue-sole, Cynoglossus semilaevis

A half-smooth tongue-sole, Cynoglossus semilaevis Sox10 (Accession no.: EU070763) was isolated from brain of tongue sole by using homologous cloning and RACE method. The complete cDNA of the tongue sole Sox10 contains a 35 bp 5′UTR, a 1338 bp open reading frame (ORF) encoding 445 amino acids and a 1155 bp 3′UTR. A condensed phylogenetic tree was constructed based on the amino acid sequences of tongue sole Sox10 and other well-defined vertebrate Sox. The overall topology of the tree showed the tongue sole Sox10 clusters with all Sox10. Alignment of amino acid residues of the tongue sole Sox10 gene with those from other vertebrate indicated high level conservation of amino acid sequence. The RT-PCR analysis demonstrated that the tongue sole Sox10 was highly expressed in brain, gills, skin and eyes, intermediately in spleen, heart, head–kidney and muscles, weakly expressed in kidneys and intestine and no expression in liver and gonad. The Sox10 was also expressed weakly in germ cell and zygote. We cannot detect the expression of the Sox10 in 8-cells stage. However it resumed expression weakly from blastula stage to middle of gastrula. And it expressed highly from neurula stage to 25 dah (day after hatching). It suggested that the Sox10 was involved in the development of embryos and larvae in tongue sole.

Molecular Cloning, Characterization and Expression Analysis of Brain P450arom in Half-smooth Tongue-sole, &lt;I&gt;Cynoglossus semilaevis&lt;/I&gt;Gunther

s:The 2184 bp full-length brain P450 aromatase (termed P450aromB) cDNA encoding a protein of 498 amino acid residues was isolated from the brain of the half-smooth tongue-sole (Cynoglossus semilaevis) by using homologous cloning to clarify the role in sex differentiation. Sequence and phylogenetic analyses showed that the tongue sole P450aromB belonged to brain P450arom subfamilies. The putative P450aromB amino acid residues shared 48.3%- 66.1% sequence affinity with other fish brain P450arom, and shared 34.2%-49.9% sequence affinity with ovarian P450arom of other fish. The RT-PCR analysis revealed that P450aromB mRNA was expressed at a high proportion in the brain and gills but less in the gonads and skin, but not in other tissues. However, the P450aromB decreased in the brain of sex-reversed males, treatment by Methyltestosterone or at high temperatures. These results suggested that the P450aromB is involved in the course of gonad development and sex determination in tongue soles.

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.