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.

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.

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.

Optical meta-waveguides for integrated photonics and beyond.

The growing maturity of nanofabrication has ushered massive sophisticated optical structures available on a photonic chip. The integration of subwavelength-structured metasurfaces and metamaterials on the canonical building block of optical waveguides is gradually reshaping the landscape of photonic integrated circuits, giving rise to numerous meta-waveguides with unprecedented strength in controlling guided electromagnetic waves. Here, we review recent advances in meta-structured waveguides that synergize various functional subwavelength photonic architectures with diverse waveguide platforms, such as dielectric or plasmonic waveguides and optical fibers. Foundational results and representative applications are comprehensively summarized. Brief physical models with explicit design tutorials, either physical intuition-based design methods or computer algorithms-based inverse designs, are cataloged as well. We highlight how meta-optics can infuse new degrees of freedom to waveguide-based devices and systems, by enhancing light-matter interaction strength to drastically boost device performance, or offering a versatile designer media for manipulating light in nanoscale to enable novel functionalities. We further discuss current challenges and outline emerging opportunities of this vibrant field for various applications in photonic integrated circuits, biomedical sensing, artificial intelligence and beyond.

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.

Crucial role of dead end gene for primordial germ cell survival in rice field eel (Monopterus albus).

The dead end gene has been identified as a essential factor for Primordial germ cells (PGCs) migration and survival in many species, but its role in Monopterus albus is unclear. In order to clarify the function of dead end gene in M.albus PGCs migration and survival, we first characterized the expression profile of M.albus dead end (Madnd) in developing embryos and various tissues. qRT-PCR revealed that Madnd transcripts were exclusively detected in gonad, including ovary, testis and ovotestis.Embryos injected with a Madnd morpholino (Madnd-MO) exhibited down-regulation of the vasa gene. Furthermore, the GFP signal show the PGCs migration in control group were injected with GFP-nanos3 3'-UTR mRNA for visualization, as described in a previous study, yet it was disappeared after embryos injected with Madnd-MO.Finally, we characterized the genomics sequence of the Madnd gene and designed five gRNAs for genome editing. Three gRNAs were selected for microinjection according to the results of in vitro tests. gRNAd1 was used for microinjection with the Cas9 protein and was confirmed to be effective. Our analysis in this study suggested that Madnd play a key role in PGCs migration and survival in M. albus. These data provide the basis for the production of fast-growing and reproductively M.albus sterile.

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers in swamp eel (Monopterus albus).

OBJECTIVES: Swamp eel is one model species for sexual reversion and an aquaculture fish in China. One local strain with deep yellow and big spots of Monopterus albus has been selected for consecutive selective breeding. The objectives of this study were characterizing the Simple Sequence Repeats (SSRs) of M. albus in the assembled genome obtained recently, and developing polymorphic SSRs for future breeding programs. METHODS: The genome wide SSRs were mined by using MISA software, and their types and genomic distribution patterns were investigated. Based on the available flanking sequences, primer pairs were batched developed, and Polymorphic SSRs were identified by using Polymorphic SSR Retrieval tool. The obtained polymorphic SSRs were validated by using e-PCR and capillary electrophoresis, then they were used to investigate genetic diversity of one breeding population. RESULTS: A total of 364,802 SSRs were identified in assembled M. albus genome. The total length, density and frequency of SSRs were 8,204,641 bp, 10,259 bp/Mb, and 456.16 loci/Mb, respectively. Mononucleotide repeats were predominant among SSRs (33.33%), and AC and AAT repeats were the most abundant di- and tri-nucleotide repeats motifs. A total of 287,189 primer pairs were designed, and a high-density physical map was constructed (359.11 markers per Mb). A total of 871 polymorphic SSRs were identified, and 38 SSRs of 101 randomly selected ones were validated by using e-PCR and capillary electrophoresis. Using these 38 polymorphic SSRs, 201 alleles were detected and genetic diversity level (Na, PIC, HO, and He) was evaluated. CONCLUSIONS: The genome-wide SSRs and newly developed SSR markers will provide a useful tool for genetic mapping, diversity analysis studies in swamp eel in the future. The high level of genetic diversity (Na = 5.29, PIC = 0.5068, HO = 0.4665, He = 0.5525) but excess of homozygotes (FIS = 0.155) in one breeding population provide baseline information for future breeding program.

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.

A high-quality de novo genome assembly of one swamp eel (Monopterus albus) strain with PacBio and Hi-C sequencing data.

The swamp eel (Monopterus albus) is one economically important fish in China and South-Eastern Asia and a good model species to study sex inversion. There are different genetic lineages and multiple local strains of swamp eel in China, and one local strain of M. albus with deep yellow and big spots has been selected for consecutive selective breeding due to superiority in growth rate and fecundity. A high-quality reference genome of the swamp eel would be a very useful resource for future selective breeding program. In the present study, we applied PacBio single-molecule sequencing technique (SMRT) and the high-throughput chromosome conformation capture (Hi-C) technologies to assemble the M. albus genome. A 799 Mb genome was obtained with the contig N50 length of 2.4 Mb and scaffold N50 length of 67.24 Mb, indicating 110-fold and ∼31.87-fold improvement compared to the earlier released assembly (∼22.24 Kb and 2.11 Mb, respectively). Aided with Hi-C data, a total of 750 contigs were reliably assembled into 12 chromosomes. Using 22,373 protein-coding genes annotated here, the phylogenetic relationships of the swamp eel with other teleosts showed that swamp eel separated from the common ancestor of Zig-zag eel ∼49.9 million years ago, and 769 gene families were found expanded, which are mainly enriched in the immune system, sensory system, and transport and catabolism. This highly accurate, chromosome-level reference genome of M. albus obtained in this work will be used for the development of genome-scale selective breeding.