Genome-Wide DNA Methylation and RNA Analysis Reveal Potential Mechanism of Resistance to Streptococcus agalactiae in GIFT Strain of Nile Tilapia (Oreochromis niloticus ).

Streptococcus agalactiae is an important pathogenic bacterium causing great economic loss in Nile tilapia (Oreochromis niloticus) culture. Resistant and susceptible groups sharing the same genome showed significantly different resistance to S. agalactiae in the genetically improved farmed tilapia strain of Nile tilapia. The resistance mechanism is unclear. We determined genome-wide DNA methylation profiles in spleen of resistant and susceptible O. niloticus at 5 h postinfection with S. agalactiae using whole-genome bisulfite sequencing. The methylation status was higher in the spleen samples from resistant fish than in the susceptible group. A total of 10,177 differentially methylated regions were identified in the two groups, including 3725 differentially methylated genes (DMGs) (3129 hyper-DMGs and 596 hypo-DMGs). The RNA sequencing showed 2374 differentially expressed genes (DEGs), including 1483 upregulated and 891 downregulated. Integrated analysis showed 337 overlapping DEGs and DMGs and 82 overlapping DEGs and differentially methylated region promoters. By integrating promoter DNA methylation with gene expression, we revealed four immune-related genes (Arnt2, Nhr38, Pcdh10, and Ccdc158) as key factors in epigenetic mechanisms contributing to pathogen resistance. Our study provided systematic methylome maps to explore the epigenetic mechanism and reveal the methylation loci of pathogen resistance and identified methylation-regulated genes that are potentially involved in defense against pathogens.

A high-density genetic map construction and sex-related loci identification in Chinese Giant salamander.

BACKGROUND: The Chinese giant salamander Andrias davidianus is an important amphibian species in China because of its increasing economic value, protection status and special evolutionary position from aquatic to terrestrial animal. Its large genome presents challenges to genetic research. Genetic linkage mapping is an important tool for genome assembly and determination of phenotype-related loci. RESULTS: In this study, we constructed a high-density genetic linkage map using ddRAD sequencing technology to obtain SNP genotyping data of members from an full-sib family which sex had been determined. A total of 10,896 markers were grouped and oriented into 30 linkage groups, representing 30 chromosomes of A. davidianus. The genetic length of LGs ranged from 17.61 cM (LG30) to 280.81 cM (LG1), with a mean inter-locus distance ranging from 0.11(LG3) to 0.48 cM (LG26). The total genetic map length was 2643.10 cM with an average inter-locus distance of 0.24 cM. Three sex-related loci and four sex-related markers were found on LG6 and LG23, respectively. CONCLUSION: We constructed the first High-density genetic linkage map and identified three sex-related loci in the Chinese giant salamander. Current results are expected to be a useful tool for future genomic studies aiming at the marker-assisted breeding of the species.

Characterization and function of the T-box 1 gene in Chinese giant salamander Andrias davidianus.

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx1 was knocked down by tbx1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh, cyp26a, dmrt1, and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.

Genome-wide RAD sequencing to identify a sex-specific marker in Chinese giant salamander Andrias davidianus.

BACKGROUND: Chinese giant salamander Andrias davidianus is an endangered species. The success of artificial breeding provides a useful way to protect this species. However, the method to identify the sex and mechanism of sex determination were unclear which hinder the improvement of the artificial breeding. Detection of a sex specific marker provides an effective approach to identify genetic sex and investigate the sex determination mechanism. RESULTS: We used restriction-site-associated DNA (RAD) sequencing to isolate a sex-specific genetic marker in A. davidianus to expand knowledge of the sex determination mechanism. Four male and four female specimens were subjected to RAD sequencing, which generated 934,072,989 reads containing approximately 134.4 Gb of sequences. The first round of comparison of the assembled sequence against the opposite sex raw reads revealed 19,097 female and 17,994 male unmatched sequences. Subsequently, 19,097 female sequences were subjected to a BLAST search against male genomic data, which revealed 308 sequences unmapped to the male genome. One hundred of these were randomly selected and validated by PCR in five male and five female specimens, and four putative sex-specific sequences were produced. Further validation was performed by PCR in another 24 females and 24 males, and all female individuals exhibited the expected specific bands, while the males did not. To apply the sex-specific marker, three specimens reversed from genetic female to physiological male were found in a group exposed to elevated temperature, and 13 individuals reversed from genetic male to physiological female were obtained in a 17ß-estradiol exposed group. CONCLUSION: This is the first report of a sex-specific marker in A. davidianus and may have potential for elucidation of its sex determination mechanism and, hence, its conservation.

Identification of critical sex-biased genes in Andrias davidianus by de novo transcriptome.

The Chinese giant salamander Andrias davidianus is a protected amphibian with high nutritional and economic value. Understanding its sex determination mechanism is important for improving culture techniques and sex control in breeding. However, little information on the characterization of critical genes involved in sex is available. Herein, sequencing of ovary and test produced 40,783,222 and 46,128,902 raw reads, respectively, which were jointly assembled into 80,497 unigenes. Of these, 36,609 unigenes were annotated, of which 8907 were female-biased and 10,385 were male-biased. Several sex-related pathways were observed, including the Wnt signaling pathway. After elevated temperature and estrogen exposure, neomale and neofemale specimens were identified by a female-specific marker for the first time. RT-qPCR analysis showed the expression profile of ten selected sex-biased genes to be exhibited consistently in male and neomale and in female and neofemale, with the exception of the Amh and TfIIIa genes. Results suggested that these genes may play important roles in A. davidianus sex determination and gonad development. This provides a basis for further investigation of the molecular mechanisms of sex determination in amphibians.

Characterization and expression of galectin-3 after Streptococcus agalactiae and Aeromonas hydrophila challenge in GIFT strain Nile tilapia (Oreochromis niloticus).

In mammals, Galectin-3 has been revealed to be widely expressed in immune cells and played important role in immune reactions. However, Galectin-3 is frequently less reported in teleost. In the present study, a molecular characterization and expression analysis of galectin-3 were conducted in GIFT strain Nile tilapia. The full-length cDNA is 1034 bp with 690 bp of protein coding sequences. The result of qRT-PCR showed that the mRNA of galectin-3 was widely expressed in various tissues (heart, liver, spleen, gill, kidney, brain, intestine, skin, muscle, and ovary), and the higher expression was observed in immune-related tissues (liver and spleen). The time-course expression analysis revealed that galectin-3 was significantly up-regulated in intestine (5 h, 50 h, and 7 d), liver (5 h, 50 h, and 7 d), spleen (5 and 50 h), head-kidney (5 and 50 h), gill (5 h and 7 d) after Streptococcus agalactiae challenge, and significantly up-regulated in intestine (18, 24, 36, 72, and 96 h), liver (6, 18, 24, 96 h, and 6 d), spleen (18, 24, 36, 72, and 96 h), head-kidney (6, 12, 18, 24, 36, 72, and 96 h), and gill (12, 18, 24, and 36 h) after Aeromonas hydrophila challenge. Taken together, these data suggest that galectin-3 plays a role in immune responses in Nile tilapia after bacterial challenge.

Molecular characterization of manganese superoxide dismutase (MnSOD) from sterlet Acipenser ruthenus and its responses to Aeromonas hydrophila challenge and hypoxia stress.

A novel gene encoding the mitochondrial manganese superoxide dismutase from sterlet Acipenser ruthenus (Ar-MnSOD) was cloned. The full-length cDNA of MnSOD was of 1040 bp with a 672 bp open reading frame encoding 224 amino acids and the deduced amino acid sequence was located in mitochondria. Sequence comparison analysis showed that Ar-MnSOD was highly similar to MnSODs of invertebrates and vertebrates, especially those of freshwater Cyprinidae fishes and mammals. Phylogenetic analysis revealed that Ar-MnSOD was distant from MnSODs of other fishes and belonged to the family of mitochondrial MnSODs (mMnSOD). Consistently, Ar-MnSOD was located in mitochondria. The 3D structure of Ar-MnSOD was predicted and the overall structure was similar to that of MnSODs of humans and the bay scallop Argopecten irradians. In addition, mRNA of Ar-MnSOD was detected to extensively express in all tissues, with the highest level in brain and liver. Spleen and head kidney inoculation of Aeromonas hydrophila led to a significant up-regulation of Ar-MnSOD transcript levels. Also, hypoxia induced a transient increase in transcription of Ar-MnSOD in the gills, but not in the heart and brain, suggesting metabolic depression in these vital organs. The results also implied the anti-hypoxia properties of Ar-MnSOD in the related tissues and proved that Ar-MnSOD was involved in the stress response and (anti) oxidative processes triggered by hypoxia. The results indicated that Ar-MnSOD is induced upon A. hydrophila infection and hypoxia, consistent with its role in host immune and stress-induced anti-oxidative responses.

Catadioptric freeform optical system design for LED off-axis road illumination applications.

The aim of this paper is to develop a new composite structure of catadioptric optical system containing both freeform refractive surface and freeform total internal reflective (TIR) surface for LED road illumination applications. The role of freeform refractive part is to generate the shifted general rectangular illumination pattern to optimally match the shape of the road surface. The application of TIR mechanism is aimed to control the stray light in the sidewalk direction of the road luminaire and maximize the efficient energy efficiency. In this paper, we use the "double pole" ray mapping technique to design the refractive optical surface and the θ-φ coordinate ray mapping technique to derive the freeform TIR surface. The simulation shows that the novel catadioptric design has relatively high collection efficiency, thus high average illuminance level inside the effective illumination area. This lens also has good control of stray light on the backside of the road luminaire.

Identification and expression of forkhead box genes in the Chinese giant salamander Andrias davidianus.

In the present study, 21 forkhead box (Fox) genes were identified in Andrias davidianus, including 13 full-length genes and eight partial sequences. Phylogenetic analysis showed that most were conserved in other investigated amphibians, whereas the Foxk1 gene was found exclusively in A. davidianus. Molecular evolution analysis indicated that most Fox genes underwent purifying selection, whereas two sites of the adFoxp4 gene showed positive selection and were located on the adFoxp4 protein surface. Expression profiles of all Fox genes identified were analysed in the hypothalamic-pituitary-gonad axis by reverse transcription-quantitative polymerase chain reaction. Eighteen genes exhibited sexually dimorphic expression (15 ovary-biased and three testis-biased genes), whereas two genes showed no difference between ovary and testis. Further investigation of 12 selected sexually dimorphic Fox genes showed changes in the expression profile of 11 genes in the ovary of larvae reared at high temperatures (28°C). The results of the present study provide information on Fox genes in an amphibian and suggest that they play key roles in sexual development and reproduction in A. davidianus.

Comparative transcriptome reveal the potential adaptive evolutionary genes in Andrias davidianus.

To search the evidence of molecular evolution mechanism for aquatic and cave habitat in Andrias davidianus, the evolution analysis was carried out among several species transcriptome data. The transcriptome data of Notophthalmus viridescens, Xenopus tropicalis, Cynops pyrrhogaster, Hynobius chinensis and A. davidianus were obtained from the Genbank and reassembled except Xenopus tropicalis. The BLAST search of transcriptome data obtained 1244 single-copy orthologous genes among five species. A phylogenetic tree showed A. davidianus to have the closest relationship to H. chinensis. Fourteen positively selected genes were detected in A. davidianus and N. vridescens group and fifteen in A. davidianus and H. chinensis group. Five genes were shared in the both groups which involved in the immune system, suggesting that A. davidianus adaptation to an aquatic and cave environment required rapid evolution of the immune system compared to N. viridescens and H. chinensis.

Epigenetic modification and inheritance in sexual reversal of fish.

Environmental sex determination (ESD) occurs in divergent, phylogenetically unrelated taxa, and in some species, co-occurs with genetic sex determination (GSD) mechanisms. Although epigenetic regulation in response to environmental effects has long been proposed to be associated with ESD, a systemic analysis on epigenetic regulation of ESD is still lacking. Using half-smooth tongue sole (Cynoglossus semilaevis) as a model-a marine fish that has both ZW chromosomal GSD and temperature-dependent ESD-we investigated the role of DNA methylation in transition from GSD to ESD. Comparative analysis of the gonadal DNA methylomes of pseudomale, female, and normal male fish revealed that genes in the sex determination pathways are the major targets of substantial methylation modification during sexual reversal. Methylation modification in pseudomales is globally inherited in their ZW offspring, which can naturally develop into pseudomales without temperature incubation. Transcriptome analysis revealed that dosage compensation occurs in a restricted, methylated cytosine enriched Z chromosomal region in pseudomale testes, achieving equal expression level in normal male testes. In contrast, female-specific W chromosomal genes are suppressed in pseudomales by methylation regulation. We conclude that epigenetic regulation plays multiple crucial roles in sexual reversal of tongue sole fish. We also offer the first clues on the mechanisms behind gene dosage balancing in an organism that undergoes sexual reversal. Finally, we suggest a causal link between the bias sex chromosome assortment in the offspring of a pseudomale family and the transgenerational epigenetic inheritance of sexual reversal in tongue sole fish.

Characterization of a low-density lipoprotein receptor, Lrp13, in Chinese tongue sole (Cynoglossus semilaevis) and medaka (Oryzias latipes).

As an important economic marine species cultured in China, Chinese tongue sole (Cynoglossus semilaevis) has interested us due to its sexual dimorphism and ZW/ZZ sex determination system. In a previous study, dmrt1 was identified as a dosage-dependent male-determining gene. In the present study, a female-specific expressed gene, cse0440, initially annotated as lrp1b-like, was identified from chromosome W of C. semilaevis. In view of the differences between cse0440 and lrp1b in terms of expression pattern, a phylogenetic analysis containing 85 LRP proteins was constructed and provided an evidence to re-annotate cse0440 as cseLRP13. In addition, two orthologues of cseLRP13 were separately identified from W and Z chromosomes: cseLRP13-W and cseLRP13-Z. The subsequent multiple sequence alignment and syntenic arrangements of LRP13 in C. semilaevis, Japanese medaka (Oryzias latipes), large yellow croaker (Larimichthys crocea), striped bass (Morone saxatilis), white perch (Morone americana) and Fugu rubripes (Takifugu rubripes) further supported this re-annotation. RT-PCR and in situ hybridization revealed that cselrp13 was exclusively expressed in the oocytes and follicles of ovaries. These results suggested that lrp13 may play important roles in female reproduction. In future, with the advancement of micromanipulation in flatfish, the detailed function of two lrp13 orthologues in C. semilaevis will be elucidated.

Sexually Dimorphic Expression of Foxl2 and Ftz-F1 in Chinese Giant Salamander Andrias Davidianus.

Foxl2 and FTZ-F1 play a crucial role in the regulation of gonad development in fish and mammals, but studies of their function in amphibians are scarce. We isolated the full length of Foxl2 (adFoxl2) and Ftz-F1 (adFtz-f1) cDNA from the Chinese giant salamander Andrias davidianus and quantified its expression in various tissues and developing gonads. The adFoxl2 gene encodes 301aa including a conserved forkhead box, and the adFtz-f1 gene encodes 467aa containing an Ftz-F1 box. The amino acid sequences showed high homology with other amphibians. adFoxl2 expression was high in ovary, whereas adFtz-f1 was higher in testis, moderate in pituitary, ovary, and kidney; and low in the remaining tested tissues. Expression of adFoxl2 gradually increased from 1Y to 5Y in ovary, whereas adFtz-f1 expression gradually decreased in testis. In addition, adFoxl2 and adFtz-f1 were detected in granulosa cell in ovary and in spermatocytes in testis. The adFoxl2 transcription was inhibited in brain and ovary after treatment with methyltestosterone and with letrozole, whereas adFtz-f1 expression was upregulated. High-temperature suppressed the expression of adFxl2 in ovary and enhanced the transcription of adFtz-f1. These results suggest that adFoxl2 functioned in ovary differentiation, whereas adFtz-f1 played a role in testis development, which lays a foundation for study of the sex differentiation mechanism in A. davidianus.

Cloning, expression of, and evidence of positive selection for, the prolactin receptor gene in Chinese giant salamander (Andrias davidianus).

Prolactin receptor (PRLR) is a protein associated with reproduction in mammals and with osmoregulation in fish. In this study, the complete length of Chinese giant salamander Andrias davidianus prolactin receptor (AD-prlr) was cloned. Andrias davidianus prlr expression was high in the kidney, pituitary, and ovary and low in other examined tissues. The AD-prlr levels were higher in ovary than in testis, and increased in ovaries with age from 1 to 6 years. To determine effect of exogenous androgen and aromatase inhibitor on AD-prlr expression, methyltestosterone (MT) and letrozole (LE) were injected, resulting in decreased AD-prlr in both brain and ovary, with MT repressing prlr transcription more rapidly than did LE. The molecular evolution of prlr was assessed, and found to have undergone a complex evolution process. The obranch-site test detected four positively selected sites in ancestral lineages prior to the separation of mammals and birds. Fourteen sites underwent positive selection in ancestral lineages of birds and six were positively selected in amphibians. The site model showed that 16, 7, and 30 sites underwent positive selection in extant mammals, amphibians, and birds, respectively. The positively selected sites in amphibians were located outside the transmembrane domain, with four in the extracellular and three in the intracellular domain, indicating that the transmembrane region might be conserved and essential for protein function. Our findings provide a basis for further studies of AD-prlr function and molecular evolution in Chinese giant salamander. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 707-719, 2015. © 2015 Wiley Periodicals, Inc.

Expression and purification of half-smooth tongue sole (Cynoglossus semilaevis) CSDAZL protein.

The csdazl gene is a sex related gene of half-smooth tongue sole (Cynoglossus semilaevis). Our research group have cloned full length cDNA of csdazl, and studied its expression pattern. To get the further information of csdazl, we constructed a prokaryotic expression plasmid, pET-32a-CSDAZL, expressed in Escherichia coli BL21 and purified the fusion protein by His Trap. In order to detect the biological activity of the fusion protein, we injected the protein with liposome into fish, and detected other sex-related genes' mRNA expression. The results showed that the expression levels of half-smooth tongue sole female-related genes Cyp19a and Foxl2 significantly decreased between 6 and 24 h; however, both genes' expressions returned to their normal levels 72 h after injection, indicating that recombinant CSDAZL protein could down-regulated the expression of female-related genes, Foxl2 and Cyp19a genes, implying that the fusion protein has biological activity and csdazl plays a role in sex differentiation by regulating sex related genes' expression.

Regulatory mechanism of LncRNAs in gonadal differentiation of hermaphroditic fish, Monopterus albus.

BACKGROUND: Monopterus albus is a hermaphroditic fish with sex reversal from ovaries to testes via the ovotestes in the process of gonadal development, but the molecular mechanism of the sex reversal was unknown. METHODS: We produced transcriptomes containing mRNAs and lncRNAs in the crucial stages of the gonad, including the ovary, ovotestis and testis. The expression of the crucial lncRNAs and their target genes was detected using qRT‒PCR and in situ hybridization. The methylation level and activity of the lncRNA promoter were analysed by applying bisulfite sequencing PCR and dual-luciferase reporter assays, respectively. RESULTS: This effort revealed that gonadal development was a dynamic expression change. Regulatory networks of lncRNAs and their target genes were constructed through integrated analysis of lncRNA and mRNA data. The expression and DNA methylation of the lncRNAs MSTRG.38036 and MSTRG.12998 and their target genes Psmß8 and Ptk2ß were detected in developing gonads and sex reversal gonads. The results showed that lncRNAs and their target genes exhibited consistent expression profiles and that the DNA methylation levels were negatively regulated lncRNA expression. Furthermore, we found that Ptk2ß probably regulates cyp19a1 expression via the Ptk2ß/EGFR/STAT3 pathway to reprogram sex differentiation. CONCLUSIONS: This study provides novel insight from lncRNA to explore the potential molecular mechanism by which DNA methylation regulates lncRNA expression to facilitate target gene transcription to reprogram sex differentiation in M. albus, which will also enrich the sex differentiation mechanism of teleosts.

Monopterus albus is a hermaphroditic fish that undergoes sex reversal from female to male via intersex during the process of the gonadal differentiation which was an ideal model for epigenetic modification research. After laying eggs, the female M.albus reversal to the intersex. So that the female have a shorter stage and smaller body size which cause low egg production. In the present study, we produced the transcriptomes which contain mRNA and lncRNA in the crucial stage of the gonad including ovary, ovotestis and testis. This effort reveals that gonadal development was a dynamic expression changes. Regulatory networks of lncRNAs and its target genes were constructed though integrated analysis of lncRNA and mRNA data. We found DNA methylation was negatively associated with lncRNA (MSTRG.38036 and MSTRG.12998) expression in developing gonads. Additionally, 17α-methyltestosterone inhibit the expression of lncRNA and increase methylation. Furthermore, we found that Ptk2ß probably regulates cyp19a1 expression via the Ptk2ß/EGFR/STAT3 pathway to reprogram sex differentiation. The present study on the gonadal differentiation of M. albus provides novel insights from lncRNA to explore potential molecular mechanism. In the future, function of the lncRNA will be further studied and the gene editing technology will be applied to cultivate the female with high fecundity to improve the yield of fish fry.

Chromosome-Scale, Haplotype-Resolved Genome Assembly of Non-Sex-Reversal Females of Swamp Eel Using High-Fidelity Long Reads and Hi-C Data.

The Asian swamp eel (Monopterus albus) is an excellent model species for studying sex change and chromosome evolution. M. albus is also widely reared in East Asia and South-East Asia because of its great nutritional value. The low fecundity of this species (about 300 eggs per fish) greatly hinders fries production and breeding programs. Interestingly, about 3-5% of the eels could remain as females for 3 years and lay more than 3,000 eggs per fish, which are referred to as non-sex-reversal (NSR) females. Here, we presented a new chromosome-level genome assembly of such NSR females using Illumina, HiFi, and Hi-C sequencing technologies. The new assembly (Mal.V2_NSR) is 838.39 Mb in length, and the N50 of the contigs is 49.8 Mb. Compared with the previous assembly obtained using the continuous long-read sequencing technology (Mal.V1_CLR), we found a remarkable increase of continuity in the new assembly Mal.V2_NSR with a 20-times longer contig N50. Chromosomes 2 and 12 were assembled into a single contig, respectively. Meanwhile, two highly contiguous haplotype assemblies were also obtained, with contig N50 being 14.54 and 12.13 Mb, respectively. BUSCO and Merqury analyses indicate completeness and high accuracy of these three assemblies. A comparative genomic analysis revealed substantial structural variations (SVs) between Mal.V2_NSR and Mal.V1_CLR and two phased haplotype assemblies, as well as whole chromosome fusion events when compared with the zig-zag eel. Additionally, our newly obtained assembly provides a genomic view of sex-related genes and a complete landscape of the MHC genes. Therefore, these high-quality genome assemblies would provide great help for future breeding works of the swamp eel, and it is a valuable new reference for genetic and genomic studies of this species.

Integrated chromatin accessibility and DNA methylation analysis to reveal the critical epigenetic modification and regulatory mechanism in gonadal differentiation of the sequentially hermaphroditic fish, Monopterus albus.

BACKGROUND: Monopterus albus is a hermaphroditic and economically farmed fish that undergoes sex reversal from ovary to testis via ovotestis during gonadal development. The epigenetic changes that are associated with gonadal development in this species remain unclear. METHODS: We produced DNA methylome, transcriptome, and chromatin accessibility maps of the key stages of gonad development: ovary, ovotestis, and testis. The expression of the key candidate genes was detected using qRT-PCR and in situ hybridization and the methylation levels were analysed using bisulphite sequencing PCR. Promoter activity and regulation were assessed using dual-luciferase reporter assays. RESULTS: Gonadal development exhibits highly dynamic transcriptomic, DNA methylation, and chromatin accessibility changes. We found that DNA methylation status, especially of the transcription start site, was significantly negatively correlated with gene expression while chromatin accessibility exhibited no correlation with gene expression during gonadal development. The epigenetic signatures revealed many novel regulatory elements and genes involved in sex reversal, which were validated. DNA methylation detection and site mutation of plastin-2 promoter, as a candidate gene, revealed that DNA methylation could impact the binding of transcription factor dmrt1 and foxl2 through methylation and demethylation to regulate plastin-2 expression during gonadal development. CONCLUSIONS: These data provide novel insights into epigenetic modification and help elucidate the potential molecular mechanism by which dynamic modification of DNA methylation plays a crucial role in gonadal development.

Potential antagonistic relationship of fgf9 and rspo1 genes in WNT4 pathway to regulate the sex differentiation in Chinese giant salamander (Andrias davidianus).

Farmed chinese giant salamander (Andrias davidianus) was an important distinctive economically amphibian that exhibited male-biased sexual size dimorphism. Fgf9 and rspo1 genes antagonize each other in Wnt4 signal pathway to regulate mammalian gonadal differentiation has been demonstrated. However, their expression profile and function in A. davidianus are unclear. In this study, we firstly characterized fgf9 and rspo1 genes expression in developing gonad. Results showed that fgf9 expression level was higher in testes than in ovaries and increased from 1 to 6 years while rspo1 expression was higher in ovaries than in testes. In situ hybridization assay showed that both fgf9 and rspo1 genes expressed at 62 dpf in undifferentiated gonad, and fgf9 gene was mainly expressed in spermatogonia and sertoli cells in testis while strong positive signal of rspo1 was detected in granular cell in ovary. During sex-reversal, fgf9 expression was significantly higher in reversed testes and normal testes than in ovaries, and opposite expression pattern was detected for rspo1. When FH535 was used to inhibit Wnt/ß-catenin pathway, expression of rspo1, wnt4 and ß-catenin was down-regulated. Conversely, expression of fgf9, dmrt1, ftz-f1 and cyp17 were up-regulated. Furthermore, when rspo1 and fgf9 were knocked down using RNAi technology, respectively. We observed that female biased genes were down regulated in ovary primordial cells after rspo1 was knocked down, while the opposite expression profile was observed in testis primordial cells after fgf9 was knocked down. These results suggested that fgf9 and rspo1 played an antagonistic role to regulate sex differentiation in the process of the gonadal development and provided a foundation for further functional characterizations. The data also provided basic information for genome editing breeding to improve the Chinese giant salamander farming industry.

The Complete Mitochondrial Genome of One Breeding Strain of Asian Swamp Eel (Monopterus albus, Zuiew 1793) Using PacBio and Illumina Sequencing Technologies and Phylogenetic Analysis in Synbranchiformes.

Asian swamp eel (Monopterus albus, Zuiew 1793) is a commercially important fish due to its nutritional value in Eastern and Southeastern Asia. One local strain of M. albus distributed in the Jianghan Plain of China has been subjected to a selection breeding program because of its preferred body color and superiority of growth and fecundity. Some members of the genus Monopterus have been reclassified into other genera recently. These classifications require further phylogenetic analyses. In this study, the complete mitochondrial genomes of the breeds of M. albus were decoded using both PacBio and Illumina sequencing technologies, then phylogenetic analyses were carried out, including sampling of M. albus at five different sites and 14 species of Synbranchiformes with complete mitochondrial genomes. The total length of the mitogenome is 16,621 bp, which is one nucleotide shorter than that of four mitogenomes of M. albus sampled from four provinces in China, as well as one with an unknown sampling site. The gene content, gene order, and overall base compositions are almost identical to the five reported ones. The results of maximum likelihood (ML) and Bayesian inference analyses of the complete mitochondrial genome and 13 protein-coding genes (PCGs) were consistent. The phylogenetic trees indicated that the selecting breed formed the deepest branch in the clade of all Asian swamp eels, confirmed the phylogenetic relationships of four genera of the family Synbranchidae, also providing systematic phylogenetic relationships for the order Synbranchiformes. The divergence time analyses showed that all Asian swamp eels diverged about 0.49 million years ago (MYA) and their common ancestor split from other species about 45.96 MYA in the middle of the Miocene epoch. Altogether, the complete mitogenome of this breed of M. albus would serve as an important dataset for germplasm identification and breeding programs for this species, in addition to providing great help in identifying the phylogenetic relationships of the order Synbranchiformes.