Master Sex-determination Research Articles

Multi-genome comparisons reveal gain-and-loss evolution of anti-Mullerian hormone receptor type 2 as a candidate master sex-determining gene in Percidae

BackgroundThe Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii, and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems.ResultsWe explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex-determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplicates (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19–27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been likely lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome 18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variations (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex-determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in the testis than in the ovary.ConclusionsTogether, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Diversity and Convergence of Sex-Determination Mechanisms in Teleost Fish.

Sexual reproduction is prevalent across diverse taxa. However, sex-determination mechanisms are so diverse that even closely related species often differ in sex-determination systems. Teleost fish is a taxonomic group with frequent turnovers of sex-determining mechanisms and thus provides us with great opportunities to investigate the molecular and evolutionary mechanisms underlying the turnover of sex-determining systems. Here, we compile recent studies on the diversity of sex-determination mechanisms in fish. We demonstrate that genes in the TGF-β signaling pathway are frequently used for master sex-determining (MSD) genes. MSD genes arise via two main mechanisms, duplication-and-transposition and allelic mutations, with a few exceptions. We also demonstrate that temperature influences sex determination in many fish species, even those with sex chromosomes, with higher temperatures inducing differentiation into males in most cases. Finally, we review theoretical models for the turnover of sex-determining mechanisms and discuss what questions remain elusive.

Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci.

Homomorphic sex chromosomes and their turnover are common in teleosts. We investigated the evolution of nascent sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, focusing on their under-studied repetitive landscape. We combined bioinformatic analyses of the repeatome with molecular cytogenetic techniques, including comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, ribosomal RNA genes (rDNA) and bacterial artificial chromosomes (BACs), and immunostaining of SYCP3 and MLH1 proteins to mark lateral elements of synaptonemal complexes and recombination sites, respectively. Both species share the same heteromorphic XY sex chromosome system, which thus evolved prior to their divergence. This was corroborated by sequence analysis of a putative master sex determining (MSD) gene gdf6Y in both species. Based on their divergence, differentiation of the XY sex chromosome pair started approximately 2 million years ago. In all populations, the gdf6Y gene mapped within a region rich in satellite DNA on the Y chromosome long arms. Despite their heteromorphism, X and Y chromosomes mostly pair regularly in meiosis, implying synaptic adjustment. In N. kadleci, Y-linked paracentric inversions like those previously reported in N. furzeri were detected. An inversion involving the MSD gene may suppress occasional recombination in the region, which we otherwise evidenced in the N. furzeri population MZCS-121 of the Limpopo clade lacking this inversion. Y chromosome centromeric repeats were reduced compared with the X chromosome and autosomes, which points to a role of relaxed meiotic drive in shaping the Y chromosome repeat landscape. We speculate that the recombination rate between sex chromosomes was reduced due to heterochiasmy. The observed differences between the repeat accumulations on the X and Y chromosomes probably result from high repeat turnover and may not relate closely to the divergence inferred from earlier SNP analyses.

Characterization of the sex determining region of channel catfish (Ictalurus punctatus) and development of a sex-genotyping test

Channel catfish is an important species for aquaculture that exhibits a sexually dimorphic growth in favor of males. Genetic sexing and development of sex markers are crucial for the early identification of sex and of particular genotypes (YY males) for the production of all-male population in channel catfish aquaculture. In this study, we sequenced genomic DNA from pools of males and pools of females to better characterize the sex determining region (SDR) of channel catfish and to develop sex-specific markers for genetic sexing. Performing comparative analyses on male and female pooled genomic reads, we identified a large SDR (∼8.3 Mb) in the middle of channel catfish linkage group 4 (LG04). This non-recombining SDR contains a high-density of male-specific (Y chromosome) fixed single nucleotide polymorphisms (SNPs) along with ∼ 185 kb male-specific insertions or deletions. This SDR contains 95 annotated protein-encoding genes, including the recently reported putative channel catfish master sex determining (MSD) gene, breast cancer anti-estrogen resistance protein 1 (bcar1), located at one edge of the SDR. No sex-specific SNPs and/or indels were found in the coding sequence of bcar1, but one male-specific SNP was identified in its first intron. Based on this genomic information, we developed a PCR-based sex-specific genetic test. Genotyping results confirmed strong linkage between phenotypic sexes and the identified SDR in channel catfish. Our results confirm, using a Pool-Seq approach, that channel catfish is male heterogametic (XX-XY) with a large SDR on the LG04 sex chromosome. Furthermore, our genotyping primers can be used to identify XX, XY, and YY fish that will facilitate future research on sex determination and aquaculture applications in channel catfish.

Identification of a candidate sex determination gene in Culaea inconstans suggests convergent recruitment of an Amh duplicate in two lineages of stickleback.

Sex chromosomes vary greatly in their age and levels of differentiation across the tree of life. This variation is largely due to the rates of sex chromosome turnover in different lineages; however, we still lack an explanation for why sex chromosomes are so conserved in some lineages (e.g. mammals, birds) but so labile in others (e.g. teleosts, amphibians). To identify general mechanisms driving transitions in sex determination systems or forces which favour their conservation, we first require empirical data on sex chromosome systems from multiple lineages. Stickleback fishes are a valuable model lineage for the study of sex chromosome evolution due to variation in sex chromosome systems between closely-related species. Here, we identify the sex chromosome and a strong candidate for the master sex determination gene in the brook stickleback, Culaea inconstans. Using whole-genome sequencing of wild-caught samples and a lab cross, we identify AmhY, a male specific duplication of the gene Amh, as the candidate master sex determination gene. AmhY resides on Chromosome 20 in C. inconstans and is likely a recent duplication, as both AmhY and the sex-linked region of Chromosome 20 show little sequence divergence. Importantly, this duplicate AmhY represents the second independent duplication and recruitment of Amh as the sex determination gene in stickleback and the eighth example known across teleosts. We discuss this convergence in the context of sex chromosome turnovers and the role that the Amh/AmhrII pathway, which is crucial for sex determination, may play in the evolution of sex chromosomes in teleosts.

Generation of a chromosome-level genome assembly for Pacific halibut (Hippoglossus stenolepis) and characterization of its sex-determining genomic region.

The Pacific halibut (Hippoglossus stenolepis) is a key species in the North Pacific Ocean and Bering Sea ecosystems, where it also supports important fisheries. However, the lack of genomic resources limits our understanding of evolutionary, environmental and anthropogenic forces affecting key life history characteristics of Pacific halibut and prevents the application of genomic tools in fisheries management and conservation efforts. In the present study, we report on the first generation of a high‐quality chromosome‐level assembly of the Pacific halibut genome, with an estimated size of 602 Mb, 24 chromosome‐length scaffolds that contain 99.8% of the assembly and a N50 scaffold length of 27.3 Mb. In the first application of this important resource, we conducted genome‐wide analyses of sex‐specific genetic variation by pool sequencing and characterized a potential sex‐determining region in chromosome 9 with a high density of female‐specific SNPs. Within this region, we identified the bmpr1ba gene as a potential candidate for master sex‐determining (MSD) gene. bmpr1ba is a member of the TGF‐β family that in teleosts has provided the largest number of MSD genes, including a paralogue of this gene in Atlantic herring. The genome assembly constitutes an essential resource for future studies on Pacific halibut population structure and dynamics, evolutionary history and responses to environmental and anthropogenic influences. Furthermore, the genomic location of the sex‐determining region in Pacific halibut has been identified and a putative candidate MSD gene has been proposed, providing further support for the rapid evolution of sex‐determining mechanisms in teleost fish.

Identification of sex determination locus in sea cucumber Apostichopus japonicus using genome-wide association study

BackgroundSex determination mechanisms are complicated and diverse across taxonomic categories. Sea cucumber Apostichopus japonicus is a benthic echinoderm, which is the closest group of invertebrates to chordate, and important economic and ecologically aquaculture species in China. A. japonicus is dioecious, and no phenotypic differences between males and females can be detected before sexual maturation. Identification of sex determination locus will broaden knowledge about sex-determination mechanism in echinoderms, which allows for the identification of sex-linked markers and increases the efficiency of sea cucumber breeding industry.ResultsHere, we integrated assembly of a novel chromosome-level genome and resequencing of female and male populations to investigate the sex determination mechanisms of A. japonicus. We built a chromosome-level genome assembly AJH1.0 using Hi-C technology. The assembly AJH1.0 consists of 23 chromosomes ranging from 22.4 to 60.4 Mb. To identify the sex-determination locus of A. japonicus, we conducted genome-wide association study (GWAS) and analyses of distribution characteristics of sex-specific SNPs and fixation index FST. The GWAS analysis showed that multiple sex-associated loci were located on several chromosomes, including chromosome 4 (24.8%), followed by chromosome 9 (10.7%), chromosome 17 (10.4%), and chromosome 18 (14.1%). Furthermore, analyzing the homozygous and heterozygous genotypes of plenty of sex-specific SNPs in females and males confirmed that A. japonicus might have a XX/XY sex determination system. As a physical region of 10 Mb on chromosome 4 included the highest number of sex-specific SNPs and higher FST values, this region was considered as the candidate sex determination region (SDR) in A. japonicus.ConclusionsIn the present study, we integrated genome-wide association study and analyses of sex-specific variations to investigate sex determination mechanisms. This will bring novel insights into gene regulation during primitive gonadogenesis and differentiation and identification of master sex determination gene in sea cucumber. In the sea cucumber industry, investigation of molecular mechanisms of sex determination will be helpful for artificial fertilization and precise breeding.

An ancient truncated duplication of the anti-Müllerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family.

The evolution of sex determination (SD) in teleosts is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified. Pangasiids are economically important catfishes in South Asian countries, but little is known about their SD system. Here, we generated novel genomic resources for 12Pangasiids and characterized their SD system. Based on a Pangasianodon hypophthalmus chromosome-scale genome assembly, we identified an anti-Müllerian hormone receptor type Ⅱ gene (amhr2) duplication, which was further characterized as being sex-linked in males and expressed only in testes. These results point to a Y chromosome male-specific duplication (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Reference-guided assembly of 11 additional Pangasiids, along with sex-linkage studies, revealed that this truncated amhr2by duplication is a male-specific conserved gene in Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication/insertion event at the root of the Siluroidei radiation that is dated to ~100million years ago. Together these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiids, a finding that highlights the recurrent use of the transforming growth factor β pathway, which is often used for the recruitment of teleost master SD genes, and provides another empirical case towards firther understanding of dynamics of SD systems.

Transposon-induced epigenetic silencing in the X chromosome as a novel form of dmrt1 expression regulation during sex determination in the fighting fish

BackgroundFishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens.ResultsWe conducted association mapping on four commercial and two wild populations of B. splendens. In three of the four commercial populations, the master sex determining (MSD) locus was found to be located in a region of ~ 80 kb on LG2 which harbours five protein coding genes, including dmrt1, a gene involved in male sex determination in different animal taxa. In these fish, dmrt1 shows a male-biased gonadal expression from undifferentiated stages to adult organs and the knockout of this gene resulted in ovarian development in XY genotypes. Genome sequencing of XX and YY genotypes identified a transposon, drbx1, inserted into the fourth intron of the X-linked dmrt1 allele. Methylation assays revealed that epigenetic changes induced by drbx1 spread out to the promoter region of dmrt1. In addition, drbx1 being inserted between two closely linked cis-regulatory elements reduced their enhancer activities. Thus, epigenetic changes, induced by drbx1, contribute to the reduced expression of the X-linked dmrt1 allele, leading to female development. This represents a previously undescribed solution in animals relying on dmrt1 function for sex determination. Differentiation between the X and Y chromosomes is limited to a small region of ~ 200 kb surrounding the MSD gene. Recombination suppression spread slightly out of the SD locus. However, this mechanism was not found in the fourth commercial stock we studied, or in the two wild populations analysed, suggesting that it originated recently during domestication.ConclusionsTaken together, our data provide novel insights into the role of epigenetic regulation of dmrt1 in sex determination and turnover of SD systems and suggest that fighting fish are a suitable model to study the initial stages of sex chromosome evolution.

Diversity of reptile sex chromosome evolution revealed by cytogenetic and linked-read sequencing.

Reptile sex determination is attracting much attention because the great diversity of sex-determination and dosage compensation mechanisms permits us to approach fundamental questions about mechanisms of sex chromosome turnover. Recent studies have made significant progress in better understanding diversity and conservation of reptile sex chromosomes, with however no reptile master sex determination genes identified. Here we describe an integrated genomics and cytogenetics pipeline, combining probes generated from the microdissected sex chromosomes with transcriptome and genome sequencing to explore the sex chromosome diversity in non-model Australian reptiles. We tested our pipeline on a turtle, two species of geckos, and a monitor lizard. Genes identified on sex chromosomes were compared to the chicken genome to identify homologous regions among the four species. We identified candidate sex determining genes within these regions, including conserved vertebrate sex-determining genes pdgfa, pdgfra amh and wt1, and demonstrated their testis or ovary-specific expression. All four species showed gene-by-gene rather than chromosome-wide dosage compensation. Our results imply that reptile sex chromosomes originated by independent acquisition of sex-determining genes on different autosomes, as well as translocations between different ancestral macro- and microchromosomes. We discuss the evolutionary drivers of the slow differentiation and turnover of reptile sex chromosomes.

Sex Chromosomes and Master Sex-Determining Genes in Turtles and Other Reptiles.

Among tetrapods, the well differentiated heteromorphic sex chromosomes of birds and mammals have been highly investigated and their master sex-determining (MSD) gene, Dmrt1 and SRY, respectively, have been identified. The homomorphic sex chromosomes of reptiles have been the least studied, but the gap with birds and mammals has begun to fill. This review describes our current knowledge of reptilian sex chromosomes at the cytogenetic and molecular level. Most of it arose recently from various studies comparing male to female gene content. This includes restriction site-associated DNA sequencing (RAD-Seq) experiments in several male and female samples, RNA sequencing and identification of Z- or X-linked genes by male/female comparative transcriptome coverage, and male/female transcriptomic or transcriptome/genome substraction approaches allowing the identification of Y- or W-linked transcripts. A few putative master sex-determining (MSD) genes have been proposed, but none has been demonstrated yet. Lastly, future directions in the field of reptilian sex chromosomes and their MSD gene studies are considered.

A single intronic single nucleotide polymorphism in splicing site of steroidogenic enzyme hsd17b1 is associated with phenotypic sex in oyster pompano, Trachinotus anak.

Teleosts show varied master sex determining (MSD) genes and sex determination (SD) mechanisms, with frequent turnovers of sex chromosomes. Tracing the origins of MSD genes and turnovers of sex chromosomes in a taxonomic group is of particular interest in evolutionary biology. Oyster pompano (Trachinotus anak), a marine fish, belongs to the family Carangidae, in which 17b-hydroxysteroid dehydrogenase 1 (hsd17b1) has repeatedly evolved to an MSD gene. Whole-genome resequencing identified a single nucleotide polymorphism (SNP) at chromosome 24 to be strictly associated with phenotypic sex, with females being the heterozygous sex. This SNP is located in a splicing site at the first exon/intron boundary of hsd17b1. The Z-linked SNP results in malfunction of all spliced isoforms, whereas the W-linked isoforms were predicted to have open reading frames that are conserved among vertebrates, suggesting that hsd17b1 is a female-determining gene. The differential alternative splicing patterns of ZZ and ZW genotypes were consistently observed both in undifferentiated stages and differentiated gonads. We observed elevated recombination around the SD locus and no differentiation between Z and W chromosomes. The extreme diversity of mutational mechanisms that hsd17b1 evolves to an MSD gene highlights frequent in situ turnovers between sex chromosomes in the Carangidae.

A supernumerary “B-sex” chromosome drives male sex determination in the Pachón cavefish, Astyanax mexicanus

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now.1,2 B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species3,4 that have often been considered as selfish genetic elements that behave as genome parasites.5,6 The observation that in some species Bs can be either restricted or predominant in one sex7-14 raised the interesting hypothesis that Bs could play a role in sex determination.15 The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a "B-sex" chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.

Temperature-dependent effects of house fly proto-Y chromosomes on gene expression could be responsible for fitness differences that maintain polygenic sex determination.

Sex determination, the developmental process by which sexually dimorphic phenotypes are established, evolves fast. Evolutionary turnover in a sex determination pathway may occur via selection on alleles that are genetically linked to a new master sex determining locus on a newly formed proto-sex chromosome. Species with polygenic sex determination, in which master regulatory genes are found on multiple different proto-sex chromosomes, are informative models to study the evolution of sex determination and sex chromosomes. House flies are such a model system, with male determining loci possible on all six chromosomes and a female-determiner on one of the chromosomes as well. The two most common male-determining proto-Y chromosomes form latitudinal clines on multiple continents, suggesting that temperature variation is an important selection pressure responsible for maintaining polygenic sex determination in this species. Temperature-dependent fitness effects could be manifested through temperature-dependent gene expression differences across proto-Y chromosome genotypes. These gene expression differences may be the result of cis regulatory variants that affect the expression of genes on the proto-sex chromosomes, or trans effects of the proto-Y chromosomes on genes elswhere in the genome. We used RNA-seq to identify genes whose expression depends on proto-Y chromosome genotype and temperature in adult male house flies. We found no evidence for ecologically meaningful temperature-dependent expression differences of sex determining genes between male genotypes, but we were probably not sampling an appropriate developmental time-point to identify such effects. In contrast, we identified many other genes whose expression depends on the interaction between proto-Y chromosome genotype and temperature, including genes that encode proteins involved in reproduction, metabolism, lifespan, stress response, and immunity. Notably, genes with genotype-by-temperature interactions on expression were not enriched on the proto-sex chromosomes. Moreover, there was no evidence that temperature-dependent expression is driven by chromosome-wide cis-regulatory divergence between the proto-Y and proto-X alleles. Therefore, if temperature-dependent gene expression is responsible for differences in phenotypes and fitness of proto-Y genotypes across house fly populations, these effects are driven by a small number of temperature-dependent alleles on the proto-Y chromosomes that may have trans effects on the expression of genes on other chromosomes.

Evolution of master sex determiners: TGF-β signalling pathways at regulatory crossroads.

To date, more than 20 different vertebrate master sex-determining genes have been identified on different sex chromosomes of mammals, birds, frogs and fish. Interestingly, six of these genes are transcription factors (Dmrt1- or Sox3- related) and 13 others belong to the TGF-β signalling pathway (Amh, Amhr2, Bmpr1b, Gsdf and Gdf6). This pattern suggests that only a limited group of factors/signalling pathways are prone to become top regulators again and again. Although being clearly a subordinate member of the sex-regulatory network in mammals, the TGF-β signalling pathway made it to the top recurrently and independently. Facing this rolling wave of TGF-β signalling pathways, this review will decipher how the TGF-β signalling pathways cope with the canonical sex gene regulatory network and challenge the current evolutionary concepts accounting for the diversity of sex-determining mechanisms. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

A 180 Myr-old female-specific genome region in sturgeon reveals the oldest known vertebrate sex determining system with undifferentiated sex chromosomes.

Several hypotheses explain the prevalence of undifferentiated sex chromosomes in poikilothermic vertebrates. Turnovers change the master sex determination gene, the sex chromosome or the sex determination system (e.g. XY to WZ). Jumping master genes stay main triggers but translocate to other chromosomes. Occasional recombination (e.g. in sex-reversed females) prevents sex chromosome degeneration. Recent research has uncovered conserved heteromorphic or even homomorphic sex chromosomes in several clades of non-avian and non-mammalian vertebrates. Sex determination in sturgeons (Acipenseridae) has been a long-standing basic biological question, linked to economical demands by the caviar-producing aquaculture. Here, we report the discovery of a sex-specific sequence from sterlet (Acipenser ruthenus). Using chromosome-scale assemblies and pool-sequencing, we first identified an approximately 16 kb female-specific region. We developed a PCR-genotyping test, yielding female-specific products in six species, spanning the entire phylogeny with the most divergent extant lineages (A. sturio, A. oxyrinchus versus A. ruthenus, Huso huso), stemming from an ancient tetraploidization. Similar results were obtained in two octoploid species (A. gueldenstaedtii, A. baerii). Conservation of a female-specific sequence for a long period, representing 180 Myr of sturgeon evolution, and across at least one polyploidization event, raises many interesting biological questions. We discuss a conserved undifferentiated sex chromosome system with a ZZ/ZW-mode of sex determination and potential alternatives.This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.

Allelic diversification after transposable element exaptation promoted gsdf as the master sex determining gene of sablefish.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor, beta receptor 1a (tgfbr1a) gene, gonadal somatic cell derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY. By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out, for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

A Supernumerary “B-Sex” Chromosome Drives Male Sex Determination in the Pachón Cavefish, <i>Astyanax mexicanus</i>

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now. B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species, that have often been considered as selfish genetic elements that behave as genome parasites. The observation that in some species Bs can be either restricted or predominant in one sex raised the interesting hypothesis that Bs could play a role in sex determination. The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachon cave, we show that Bs are strongly male-predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachon cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b gdf6b). Supporting its role as an MSD gene, we found that the Pachon cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachon cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachon cavefish B is a “B-sex” chromosome containing duplicated copies of the gdf6b gene acting as an MSD gene promoting male sex determination in this species.

Dose-dependent action of the RNA binding protein FOX-1 to relay X-chromosome number and determine C. elegans sex.

We demonstrate how RNA binding protein FOX-1 functions as a dose-dependent X-signal element to communicate X-chromosome number and thereby determine nematode sex. FOX-1, an RNA recognition motif protein, triggers hermaphrodite development in XX embryos by causing non-productive alternative pre-mRNA splicing of xol-1, the master sex-determination switch gene that triggers male development in XO embryos. RNA binding experiments together with genome editing demonstrate that FOX-1 binds to multiple GCAUG and GCACG motifs in a xol-1 intron, causing intron retention or partial exon deletion, thereby eliminating male-determining XOL-1 protein. Transforming all motifs to GCAUG or GCACG permits accurate alternative splicing, demonstrating efficacy of both motifs. Mutating subsets of both motifs partially alleviates non-productive splicing. Mutating all motifs blocks it, as does transforming them to low-affinity GCUUG motifs. Combining multiple high-affinity binding sites with the twofold change in FOX-1 concentration between XX and XO embryos achieves dose-sensitivity in splicing regulation to determine sex.

Assembly of the threespine stickleback Y chromosome reveals convergent signatures of sex chromosome evolution

BackgroundHeteromorphic sex chromosomes have evolved repeatedly across diverse species. Suppression of recombination between X and Y chromosomes leads to degeneration of the Y chromosome. The progression of degeneration is not well understood, as complete sequence assemblies of heteromorphic Y chromosomes have only been generated across a handful of taxa with highly degenerate sex chromosomes. Here, we describe the assembly of the threespine stickleback (Gasterosteus aculeatus) Y chromosome, which is less than 26 million years old and at an intermediate stage of degeneration. Our previous work identified that the non-recombining region between the X and the Y spans approximately 17.5 Mb on the X chromosome.ResultsWe combine long-read sequencing with a Hi-C-based proximity guided assembly to generate a 15.87 Mb assembly of the Y chromosome. Our assembly is concordant with cytogenetic maps and Sanger sequences of over 90 Y chromosome BAC clones. We find three evolutionary strata on the Y chromosome, consistent with the three inversions identified by our previous cytogenetic analyses. The threespine stickleback Y shows convergence with more degenerate sex chromosomes in the retention of haploinsufficient genes and the accumulation of genes with testis-biased expression, many of which are recent duplicates. However, we find no evidence for large amplicons identified in other sex chromosome systems. We also report an excellent candidate for the master sex-determination gene: a translocated copy of Amh (Amhy).ConclusionsTogether, our work shows that the evolutionary forces shaping sex chromosomes can cause relatively rapid changes in the overall genetic architecture of Y chromosomes.