Mechanism Of Sex Determination Research Articles

Structural Variation Analysis in the samd3/elf3 Intergenic Region of the Barred knifejaw (Oplegnathus fasciatus) and the Development of Molecular Marker for Efficient Sex Identification.

The fish species Oplegnathus fasciatus exhibits an X1X1X2X2/X1X2Y sex determination mechanism. This species holds considerable economic value and displays pronounced sexual dimorphism in growth. Therefore, the development of a rapid and accurate method for sex identification is critical to enhancing breeding efficiency and maximizing production value. Using third-generation PacBio whole-genome sequencing, we identified a homologous region in the samd3/elf3 intergenic region of the X and Y chromosomes of O. fasciatus. Analysis of the whole-genome sequence revealed a large DNA insertion marker fragment within this region. Using specifically designed primers, two bands of 390bp and 1008bp were successfully amplified in males, whereas only a single 390bp band was detected in females. This marker can be easily distinguished by agarose gel electrophoresis, greatly enhancing the efficiency and accuracy of sex identification. This study not only expands the molecular marker system for sex identification of O. fasciatus but also offers a valuable methodological reference for sex identification in other economically important fish species. These findings have significant implications for germplasm improvement and efficient selection in aquaculture.

Dose-dependent role of AMH and AMHR2 signaling in male differentiation and regulation of sex determination in Spotted knifejaw (Oplegnathus punctatus) with X1X1X2X2/X1X2Y chromosome system

Sex determination mechanisms vary significantly across different chromosomal systems and evolutionary contexts. Nonetheless, the regulatory framework governing the multi-sex chromosome system (X1X1X2X2/X1X2Y) remains enigmatic. Through an examination of sex-related genes (dmrt1, hsd11b2, amh, sox9a, sox9b, foxl2, cyp19a), hormonal influences (E2, 11-KT), and histological analyses of gonadal development, we demonstrate that the critical period for sexual differentiation occurs between 35 to 60 days post-hatching (dph). Our multi-omics analysis identified amhr2 as a candidate sex-determining gene, revealing that the males possess three distinct amhr2 transcripts (amhr2ay, amhr2by, amhr2cy), whereas females express only one (amhr2a). In situ hybridization assays demonstrated that amhr2 is predominantly localized to primary spermatocyte and Sertoli cells of male testes. Notably, the specific mRNA expression of amhr2 is significantly enriched in amhr2cy, whose extracellular domain exhibits the highest binding affinity for Amh protein, with sexual expression differences manifesting as early as 5 dph. The outcomes of amhr2 interference (RNAi) experiments indicate that amhr2 knockdown leads to a reduction in the expression of male-related gene (dmrt1, amh, sox9a, sox9b), androgen synthesis genes (hsd11b2, cyp11a), and female-related genes (wnt4, foxl2, cyp19a, cyp19b). Conversely, overexpression of amhr2 yielded contrasting results. Our research supports the role of amhr2 as a pivotal candidate sex-determining gene. Furthermore, the dosage effect of amhr2, reflected in transcript abundance, mRNA expression levels, and binding efficacy, serves as a fundamental mechanism driving male differentiation and regulatory processes in Spotted knifejaw.

Reference genome provide insights into sex determination of silver aworana (Osteoglossum bicirrhosum)

BackgroundSilver arowana (Osteoglossum bicirrhosum) is a basal fish species with sexual monomorphism, while its sex determination mechanism has been poorly understood, posing a significant challenge to its captive breeding efforts.ResultsWe constructed two high-quality chromosome-level genome assemblies for both female and male silver arowana, with scaffold N50 values over 10 Mb. Combining re-sequencing data of 109 individuals, we identified a female-specific region, which was localized in a non-coding region, i.e., around 26-kb upstream of foxl2 gene (encoding forkhead box L2). Its strong interaction with the neighboring foxl2 on the same chromosome suggests foxl2 as a candidate sex-related gene in silver arowana. We subsequently propose a complex gene network in the sex determination process of silver arowana, with foxl2 acting as the central contributor. Transcriptome sequencing of gonads support our hypothesis that the regulation of foxl2 can be influenced by the spatial proximity of the female-specific fragment, thereby promoting ovarian function or inhibiting testicular function to stimulate gonadal differentiation. Furthermore, we found the sex chromosomes to be homomorphic with a potentially recent origin, as a linkage disequilibrium analysis proved minor recombination suppression.ConclusionsThese results taken together serve as a crucial foundation for conducting extensive investigations on the evolution and differentiation of sex-determining mechanisms, as well as the emergence and development of sex chromosomes in various fishes.

Occurrence of Intersex in the Marine Mussel Perumytilus purpuratus (Mollusca: Bivalvia): Does Gonadal Parasitism Play a Role?

Intersexuality is a reproductive phenomenon that occurs in some gonochoric species and refers to the simultaneous presence of both male and female gametes within the same individual. Although this phenomenon has been reported in various invertebrate species, many aspects remain poorly understood, especially in marine mussels. However, it has been suggested that the prevalence of parasites within populations could induce the occurrence of intersex animals. We studied intersexuality in the marine mollusk Perumytilus purpuratus, a key species of the rocky intertidal zone on the southeastern coast of the Pacific Ocean. A total of 6472 mussels from eight locations in northern and southern Chile were analyzed. We estimated the size of the specimens, the sex ratio of the population, and the prevalence of parasites. Additionally, we examined the germ cells of intersex mussels. The results showed that the male-to-female sex ratio (1:1) was maintained in the populations. Intersex mussels were found in six of the eight locations, representing 0.19% of the mussels analyzed. However, no parasites were found in the intersex animals. Additionally, the abundance of intersex individuals was not correlated with parasitism levels in the population. In intersex mussels, the gonadal tissue was compartmentalized, with male and female germ cells remaining separate. It is concluded that intersexuality in Perumytilus purpuratus is a low-frequency reproductive phenomenon, likely resulting from an alteration in the sex determination mechanism. Intersexuality offers a valuable opportunity to explore the biological aspects of sex determination in mussels. Therefore, further research in this area should be pursued.

Insights into Solea senegalensis Reproduction Through Gonadal Tissue Methylation Analysis and Transcriptomic Integration.

Fish exhibit diverse mechanisms of sex differentiation and determination, shaped by both external and internal influences, often regulated by distinct DNA methylation patterns responding to environmental changes. In S. senegalensis aquaculture, reproductive issues in captivity pose significant challenges, particularly the lack of fertilization capabilities in captive-bred males, hindering genetic improvement measures. This study analyzed the methylation patterns and transcriptomic profiles in gonadal tissue DNA from groups differing in rearing conditions and sexual maturity stages. RRBS (Reduced Representation Bisulfite Sequencing) was employed to detect notable methylation variations across groups, while RNA was extracted and sequenced for differential expression analysis. Our findings suggest that DNA methylation significantly regulates gene expression, acting as a mechanism that can both repress and enhance gene expression depending on the genomic context. The complexity of this epigenetic mechanism is evident from the varying levels of methylation and correlation rates observed in different CpGs neighboring specific genes linked to reproduction. Differential methylation comparisons revealed the highest number of differently methylated CpGs between maturation stages, followed by rearing conditions, and lastly between sexes. These findings underscore the crucial role of methylation in regulating gene expression and its potential role in sex differentiation, highlighting the complex interplay between epigenetic modifications and gene expression.

Gonadal expression profiles reveal the underlying mechanisms of temperature effects on sex determination in the large-scale loach (Paramisgurnus dabryanus)

The sex determination mechanism in large-scale loach (Paramisgurnus dabryanus) follows a ZZ/ZW system, with sexual differentiation regulated by both genotypic factors and temperature effects (GSD+TSD), where elevated temperatures result in a higher proportion of males. Currently, research on the sex determination mechanisms in large-scale loach is limited, and the specific gene expression profiles and the role of temperature in influencing sex remain largely unknown. This study investigated the impact of temperature on the sex ratio in cultured populations of the large-scale loach, and then identified a female-specific genetic marker by whole genome sequencing, facilitating the distinguishing of females, males, and pseudo-males within this population. Transcriptomic analysis was subsequently performed on these groups, and the data revealed a similar expression pattern between pseudo-males and true-males. The research combined differential expression analysis with WGCNA to construct a regulatory network of nine sex differentiation-related genes (SDG) (map3k4, trpv4, hsd17b12a, wt1, ar, dmrt1, bcar1, sox9a, cyp17a1), indicating that sex differentiation in large-scale loach is probably driven by the regulation of male-related genes. The transcriptomic analysis suggested that temperature significantly modified the expression of SDG in the ovaries, while in the testes, it predominantly affects metabolism-related pathways. We established a temperature-sensitive gene network in females, based on the correlation between gene expression and temperature, as well as the number of co-regulated genes in female data. We propose that, with increasing temperature, wt1 serves as a central regulator, leading to the down-regulation of foxl2a, cyp19a1a, and the cholesterol biosynthesis-related gene sqlea, ultimately resulting in the development of pseudo-males.

Highlighting the Importance of Correct Sex Identification in Chondrichthyan Genomic Studies, Using the White Shark as an Example

The increased availability of reference genome assemblies of sharks and rays has contributed greatly to our understanding of their biology, including their sex-determination mechanisms. However, several publicly available genome assemblies of sharks and rays appear to be missing information about the sex of the source individuals. This can confound the investigation into genetic sex-determining elements and hinder the discovery of sex-specific patterns. Herein, we highlight the importance of clear and accurate sex identification in sharks and rays for future genome assemblies, using an example of a white shark (Carcharodon carcharias) genome, in which the phenotypically assigned sex conflicts with the genetic information. This genome assembly was reported to be sourced from a juvenile female (BioSample: SAMN01915239). We analyzed the assembly by mapping its available genome sequences to the current white shark reference genome assembly and compared the read coverage to sequences collected from other samples. Evidence suggests that this specimen is genetically male, which contradicts its assignment based on phenotype. Therefore, we urge researchers to provide as much accurate information (e.g., sex, sampling localities, and life history) as possible when publishing genome assemblies for sharks and rays (or for any other organism).

MOLECULAR CLONING AND QUANTITATIVE mRNA EXPRESSION OF sox9 GENE IN GONADAL DEVELOPMENTAL PERIOD OF HYPOATHERINA TSURUGAE

Sox9 is a transcription factor of high mobility group (HMG) box family DNA binding domain. It plays a crucial role in gonadogenesis during embryonic developmental period. 1454 bp of sox9 mRNA transcript of Hypoatherina tsurugae (D. S. Jordan & Starks) was cloned and sequenced. It consists of an open reading frame (ORF) of 1436 bp, that encodes a 479 aa protein, found to be identical to the HMG box of other fish species. A phylogenetic tree was constructed by comparing the mRNA sequence of 50 different fishes across various taxa available in the NCBI database and using as outgroup Acipenser sinensis. The tree shows a high homology of sox9 from H. tsurugae with that from Maelanotaenia boesemani, the two forming a single clade. The expression of sox9 was studied in amhy+ (male) individuals. It begins from baseline at 0 wah (week after hatching) and is expressed in an increasing fashion. In amhy- (female) individuals it is highly expressed at initial stage (0 wah) and the expression reaches its peak at 2 wah then declines, indicating the low expression needed for differentiation of the female sex organs. The histological sections of gonads were studied in different stages of biweekly collected larvae during the sex determination/differentiation period and it showed that differentiation of gonads male/female is decided at 6 wah. In this stage the primary oocytes are clearly recognized and it correlates with the expression of sox9 genes. These finding add to the knowledge for a better understanding of molecular mechanisms of sex determination and differentiation period in fishes.

Chromosome-level genome assembly of a stored-product psocid, Liposcelis tricolor (Psocodea: Liposcelididae)

Liposcelis tricolor (Psocoptera: Liposcelididae) is a significant pest affecting stored products globally. However, due to the lack of a detailed genomic reference, the mechanisms of sex determination, stress resistance, and potential control methods for this booklouse remain poorly understood. In this study, the chromosome-level genome of L. tricolor was assembled by employing Illumina, Nanopore, and Hi-C sequencing technologies. The final genome size was determined to be 229.33 Mb, anchored to 9 pseudo-chromosomes. BUSCO analysis showed that 99.2% of complete BUSCOs were identified, suggesting the high completeness of the genome. A total of 91.49 Mb of repetitive sequences, accounting for 38.84% of the total genome, were annotated, and 15,647 protein-coding genes were predicted, with 88.17% functionally annotated. Additionally, we identified 25 typical sex-determining genes based on the genomic data. This high-quality genome assembly provides a crucial foundation for advancing our comprehension of the molecular biology, genetics, and potential control strategies for psocid L. tricolor.

Transient receptor potential a1b regulates primordial germ cell numbers and sex differentiation in developing zebrafish.

Temperature is a leading environmental factor determining the sex ratio of some animal populations, such as fish, amphibians, and reptiles. However, the underlying mechanism by which temperature affects gender is still poorly understood. Transient receptor potential a1b (Trpa1b) belongs to the ion channel family of transient receptor potentials and exhibits dual thermosensitivity to heat and cold. In this study, we have unveiled a novel function of the trpa1b gene. Zebrafish generated through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with Trpa1b-null manifest a male-biased sex ratio. The quantity of primordial germ cells (PGCs) in zebrafish is closely linked to gender determination and gonadal development. Yet the role of the trpa1b gene in zebrafish reproductive development remains unexplored in the literature. Our investigation revealed a significant reduction in PGCs in Trpa1b mutant zebrafish compared to their wild-type counterparts 24-h postfertilization (hpf). Transcriptome sequencing of tissues near the reproductive crest of embryos at 1.25 days postfertilization (dpf) revealed differential changes in PGC-related marker genes and genes related to sperm cell development and differentiation. The relative expression of ddx4 and sycp3 genes was significantly downregulated, whereas amh was significantly upregulated at 20 dpf in trpa1b-/- zebrafish. The results of this study provide valuable insights and references for studying the molecular mechanism of sex determination in zebrafish. Undoubtedly, these results will further enhance our understanding of gender differentiation and gonadal development in fish and other vertebrates.

Genome Insights and Identification of Sex Determination Region and Sex Markers in Argyrosomus japonicus.

Argyrosomus japonicus, a member of the Sciaenidae family, is widely distributed across the sea areas near China, Japan, Australia, and South Africa. The aim of this study is to provide a high-quality genome with new technology and to understand the sex determination mechanism of this species. We generated a high-quality chromosome-level genome for Argyrosomus japonicus using PacBio HiFi and Hi-C sequencing technologies. To map the sex determination region, we employed re-sequencing data from 38 A. japonicus and conducted genome-wide association studies (GWASs) on sex phenotypes. Utilizing Hifiasm, we assembled a 708.8 Mb genome with a contig N50 length of 30 Mb. Based on Hi-C data, these contigs were organized into 24 chromosomes. The completeness of the assembly was assessed to be 99% using BUSCO, and over 98% according to Merqury. We identified a total of 174.57 Mb of repetitive elements and annotated 24,726 protein-coding genes in the genome. We mapped a 2.8 Mb sex determination region on chromosome 9, within which we found two sex-linked markers. Furthermore, we confirmed that the XX-XY sex determination system is adopted in A. japonicus. The findings of this study provide significant insights into genetic breeding, genome evolution research, and sex control breeding in A. japonicus.

Characterization of the sex determining region and development of a molecular sex identification method in a Salangid fish

BackgroundThe short-snout icefish, Neosalanx brevirostris, a member of the Salangidae family, is an economically important fishery species in China. Understanding the mechanisms underlying sex determination in this species has crucial implications for conservation, ecology and evolution. Meanwhile, there is a shortage of rapid and cost-effective genetic methods for sex identification, which poses challenges in identifying the sex of immature individuals in sex determination mechanism studies and aquaculture breeding applications.ResultsBased on whole genome resequencing data, sex-specific loci and regions were found to be concentrated in a region on chromosome 2. All sex-specific loci exhibited excess heterozygosity in females and complete homozygosity in males. This sex determining region contains seven genes, including cytochrome P450 aromatase CYP19B, which is involved in steroidogenesis and is associated with 24 sex-specific loci and two W-deletions. A haploid female-specific sequence was identified as paralogous to a diploid sequence with a significant length difference, making it suitable for rapid and cost-effective genetic sex identification by traditional PCR and agarose gel electrophoresis, which were further validated in 24 females and 24 males with known phenotypic sexes.ConclusionsOur results confirm that N.brevirostris exhibits a female heterogametic sex determination system (ZZ/ZW), with chromosome 2 identified as the putative sex chromosome containing a relatively small sex determining region (~ 48 Kb). The gene CYP19B is proposed as a candidate sex determining gene. Moreover, the development of PCR based method enables genetic sex identification at any developmental stage, thereby facilitating further studies on sex determination mechanisms and advancing aquaculture breeding applications for this species.

Comparative Transcriptome Analysis of Sexual Differentiation in Male and Female Gonads of Nao-Zhou Stock Large Yellow Croaker (Larimichthys crocea).

The Nao-zhou stock large yellow croaker (Larimichthys crocea) is a unique economic seawater fish species in China and exhibits significant dimorphism in both male and female phenotypes. Cultivating all-female seedlings can significantly improve breeding efficiency. To accelerate the cultivation process of all female seedlings of this species, it is necessary to deeply understand the regulatory mechanisms of sexual differentiation and gonadal development. This study used Illumina high-throughput sequencing to sequence the transcriptome of the testes and ovaries of Nao-zhou stock large yellow croaker to identify genes and molecular functions related to sex determination. A total of 10,536 differentially expressed genes were identified between males and females, including 5682 upregulated and 4854 downregulated genes. Functional annotation screened out 70 important candidate genes related to sex, including 34 genes highly expressed in the testis (including dmrt1, foxm1, and amh) and 36 genes highly expressed in the ovary (including gdf9, hsd3b1, and sox19b). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis found that differentially expressed genes were significantly enriched in nine signaling pathways related to sex determination and gonadal development, including steroid hormone biosynthesis, MAPK signaling pathway, and the TGF-beta signaling pathway. By screening sex-related differentially expressed genes and mapping protein-protein interaction networks, hub genes such as dmrt1, amh, and cyp19a1a were found to be highly connected. The expression levels of 15 sex-related genes, including amh, dmrt1, dmrt2a, foxl1, and zp3b, were determined by qRT-PCR and RNA sequencing. This study screened for differentially expressed genes related to sex determination and differentiation of Nao-zhou stock large yellow croaker and revealed the signaling pathways involved in gonad development of male and female individuals. The results provide important data for future research on sex determination and differentiation mechanisms, thereby providing a scientific basis for the cultivation of all-female seedlings.

Cephalopod Sex Determination and its Ancient Evolutionary Origin.

Octopuses, squids, and cuttlefishes - the coleoid cephalopods - are a remarkable branch in the tree of life whose members exhibit a repertoire of sophisticated behaviors (Hanlon and Messenger, 2018). As a clade, coleoids harbor an incredible variety of novel traits including the most complex nervous system among invertebrates, derived camera-type eyes, and rapid adaptive camouflage abilities (Young, 1971; Hanlon, 2007). The burst of evolutionary novelty that distinguishes cephalopods is even more striking in a phylogenetic context; cephalopods are a deeply diverged lineage that last share a common ancestor with other extant molluscs in the Cambrian period, roughly 550 million years ago (Ponder and Lindberg, 2008; Huang et al., 2022). With recent advances in genome sequencing technologies, we have the capability to explore the genomic foundations of cephalopod novelties. Here, using PacBio long-read sequencing of genomic DNA and IsoSeq full-length mRNA sequencing, we provide a novel chromosome-scale reference genome and annotation for a female California two-spot octopus ( O. bimaculoides ). Our assembly reveals that the female octopus has just one sex chromosome, consistent with a ZO karyotype, while the male has two (ZZ), providing the first evidence of genetic sex determination in cephalopods. We use our assembly and annotation in combination with existing genomic information from other cephalopods to create the first whole genome alignments from this group and demonstrate that the sex chromosome is of an ancient origin, before the radiation of extant cephalopods approximately 480 million years ago (Huang et al., 2022), and has been conserved to the present day in all cephalopod genomes available.

Rapid chromosome evolution and acquisition of thermosensitive stochastic sex determination in nematode androdioecious hermaphrodites

The factors contributing to evolution of androdioecy, the coexistence of hermaphrodites and males such as in Caenorhabditis elegans, remains poorly known. However, nematodes exhibit androdioecy in at last 13 genera with the predatory genus Pristionchus having seven independent transitions towards androdioecy. Nonetheless, associated genomic architecture and sex determination mechanisms are largely known from Caenorhabditis. Here, studying 47 Pristionchus species, we observed repeated chromosome evolution which abolished the ancestral XX/XO sex chromosome system. Two phylogenetically unrelated androdioecious Pristionchus species have no genomic differences between sexes and mating hermaphrodites with males resulted in hermaphroditic offspring only. We demonstrate that stochastic sex determination is influenced by temperature in P. mayeri and P. entomophagus, and CRISPR engineering indicated a conserved role of the transcription factor TRA-1 in P. mayeri. Chromosome-level genome assemblies and subsequent genomic analysis of related Pristionchus species revealed stochastic sex determination to be derived from XY sex chromosome systems through sex chromosome-autosome fusions. Thus, rapid karyotype evolution, sex chromosome evolution and evolvable sex determination mechanisms are general features of this genus, and represent a dynamic background against which androdioecy has evolved recurrently. Future studies might indicate that stochastic sex determination is more common than currently appreciated.

Identification of Genes and Long Non-Coding RNAs Putatively Related to Portunus trituberculatus Sex Determination and Differentiation Using Oxford Nanopore Technology Full-Length Transcriptome Sequencing.

The swimming crab (Portunus trituberculatus) is an economically important species in China, and its growth traits show obvious sexual dimorphism. Thus, it is important to study the mechanism of sex determination and differentiation in this species. Herein, we identified 2138 differentially expressed genes and 132 differentially expressed long non-coding RNAs (lncRNAs) using Oxford Nanopore Technology full-length transcriptome sequencing. We predicted 561 target genes of the differentially expressed lncRNAs according to their location and base pair complimentary principles. Furthermore, pathways related to sex determination, differentiation, and reproduction were enriched for lncRNAs according to gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses. This indicated that lncRNAs might play regulatory roles in these pathways. Our results could form the basis for future studies of sex determination and differentiation in P. trituberculatus.

Identification of sex-specific markers using genome re-sequencing in the blunt snout bream (Megalobrama amblycephala)

BackgroundThe blunt snout bream (Megalobrama amblycephala) is an important economic freshwater fish in China with tender flesh and high nutritional value. With the cultivation of superior new varieties and the expansion of breeding scale, it becomes imperative to employ sex-control technology to cultivate monosexual populations of M. amblycephala, thereby preventing the deterioration of desirable traits. The development of specific markers capable of accurately identifying the sex of M. amblycephala would facilitate the determination of the genetic sex of the breeding population before gonad maturation, thereby expediting the processes of sex-controlled breeding of M. amblycephala.ResultsA whole-genome re-sequencing was performed for 116 females and 141 males M. amblycephala collected from nine populations. Seven candidate male-specific sequences were identified through comparative analysis of male and female genomes, which were further compared with the sequencing data of 257 individuals, and finally three male-specific sequences were generated. These three sequences were further validated by PCR amplification in 32 males and 32 females to confirm their potential as male-specific molecular markers for M. amblycephala. One of these markers showed potential applicability in M. pellegrini as well, enabling males to be identified using this specific molecular marker.ConclusionsThe study provides a high-efficiency and cost-effective approach for the genetic sex identification in two species of Megalobrama. The developed markers in this study have great potential in facilitating sex-controlled breeding of M. amblycephala and M. pellegrini, while also contributing valuable insights into the underlying mechanisms of fish sex determination.

Genome Survey of Male Rana dybowskii to Further Understand the Sex Determination Mechanism.

Rana dybowskii is one of the important aquaculture species in Northeast China. The fallopian tubes of female R. dybowskii are used to prepare oviductus ranae (an important traditional Chinese medicine). Therefore, R. dybowskii females have higher economical value than males. An increasing female R. dybowskii population can increase the benefits from R. dybowskii culture. However, the genome of amphibians is complex, making it difficult to investigate their sex determination mechanism. In this study, we analyzed the genome of male R. dybowskii using next-generation sequencing technology. A total of 200,046,452,400 bp of clean data were obtained, and the K-mer analysis indicated that the depth was 50×. The genome size of R. dybowskii was approximately 3585.05 M, with a heterozygosity rate, repeat sequence ratio, and genome GC content of 1.15%, 68.96%, and approximately 43.0%, respectively. In total, 270,785 contigs and 498 scaffolds were generated. The size of the contigs and scaffolds was 3,748,543,415 and 3,765,862,278 bp, respectively, with the N50 length of 31,988 and 336,385,783. The longest contig and scaffold were of the size 137,967,485 and 1,808,367,828 bp, respectively. The number of contigs and scaffolds > 10K nt was 99,620 and 451, respectively. Through annotation, 40,913 genes were obtained, including 156,609 CDS (i.e., 3.83 CDS per gene). Sequence alignment was performed with the assembled scaffolding genome in this study. Two and one fragment had high homology with two male-specific DNA molecular markers of R. dybowskii discovered previously (namely, MSM-222 and MSM-261, respectively). In addition, the Dmrt1 gene of R. dybowskii was obtained with a length of 18,893 bp by comparison and splicing. The forward primers amplifying MSM-222 and MSM-261 were located at 322-343 and 14,501-14,526 bp of Dmrt1, respectively. However, sequence alignment revealed that MSM-222 and MSM-261 were not located on Dmrt1, and only some homologous parts were observed. This indicated that in addition to Dmrt1, other important genes may play a crucial role in the sex determination mechanism of R. dybowskii. Our study provided a foundation for the subsequent high-quality genome construction and provided important genomic resources for future studies on R. dybowskii.

Molecular and genetic mechanisms of sex determination in poplar

The study of molecular and genetic mechanisms of sex determination in poplar is of interest not only in the fundamental aspect, but also in the applied aspect. In landscaping of large settlements, it is advisable to use male individuals of Populus genus due to their hypoallergenicity and increased resistance to environmental pollution, stress conditions and pathogens. However, sex determination in poplars is complicated by the complex genetic structure of the sex-determining region of the genome (SDR). In this review, the emergence, evolution, structure and function of the SDR in the genus Populus are discussed. Current insights into the structure and function of the key regulator of sex selection in poplars, the orthologous ARR16/ARR17 gene, and the possible role of other genes differentially expressed between male and female plants, including microRNAs, in this process are discussed in detail. The great diversity of species and the high complexity of SDR organization justify the need for further study of the molecular mechanisms of sex determination in poplars.

Identification of two effective sex-specific DNA markers in silver arowana (Osteoglossum bicirrhosum)

The silver arowana (Osteoglossum bicirrhosum), often referred to as a living fossil, presents significant challenges in sex identification based on morphological characteristics, with its sex determination mechanism remaining poorly understood. This issue poses considerable obstacles to its captive breeding efforts. Given the species' substantial value for both consumption and ornamental purposes, it is a critical candidate for aquaculture. Through genome sequencing, read mapping, and alignment analysis of five male and five female silver arowanas, along with one mixed pool of males, we successfully identified two sex-specific SNPs and confirmed a ZW model of sex determination in this ancient species. These two SNPs demonstrated remarkable accuracy, achieving 100 % success in genetic sex identification, as validated by Sanger sequencing of PCR products from 135 individuals. Both sex-specific SNPs are located approximately 26 kb upstream of the foxl2 gene, indicating a potentially pivotal role for the foxl2 gene in the sex-determination process of the silver arowana. Consequently, we have developed precise molecular markers for sex identification in the silver arowana, significantly enhancing reproductive efficiency and promoting further industrial development. This achievement lays a foundational basis for investigating the sex-determination mechanism of the silver arowana and other osteoglossid fish, thereby advancing aquaculture practices within this species.