Sex Locus Research Articles

A panel of tetranucleotide STR markers as an alternative approach to forensic DNA identification of wolf and dog.

Commercial panels of microsatellite (STR) loci are focused on the use of DNA of the domestic dog (Canis lupus familiaris) and are often inapplicable for genotyping the DNA of the gray wolf (Canis lupus lupus). We propose a CPlex test system, including one hexa- and 12 tetranucleotide autosomal STR loci, as well as two sex loci, that is equally efficient in DNA identification of biological samples of the wolf and the dog. Analysis of molecular variance between samples revealed significant differentiation values (FST = 0.0784, p < 0.001), which allows to use the panel to differentiate wolf and dog samples. Population subdivision coefficients (θ-values) were calculated for each of the 13 STR loci of the developed test system. It was shown that the values of the genotype frequency for dogs and wolves, without and with considering the θ-value, differ by three orders of magnitude (for dogs 8.9 × 10-16 and 2.1 × 10-14 and for wolves 1.9 × 10-15 and 4.5 × 10-14, respectively). The use of population subdivision coefficients will allow to identify the most reliable results of an expert identification study and the power of exclusion provided by the STR loci of the CPlex test system makes it possible to achieve a reliable level of evidence in forensic DNA analysis of both wolves and dogs. The test system has been validated for use in forensic identification of the dog and wolf based on biological traces found at crime scenes, as well as for individual identification and establishing biological relationship of animals of these species.

Absence of chromosome axis protein recruitment prevents meiotic recombination chromosome-wide in the budding yeast Lachancea kluyveri

Meiotic recombination shows broad variations across species and along chromosomes and is often suppressed at and around genomic regions determining sexual compatibility such as mating type loci in fungi. Here, we show that the absence of Spo11-DSBs and meiotic recombination on Lakl0C-left, the chromosome arm containing the sex locus of the Lachancea kluyveri budding yeast, results from the absence of recruitment of the two chromosome axis proteins Red1 and Hop1, essential for proper Spo11-DSBs formation. Furthermore, cytological observation of spread pachytene meiotic chromosomes reveals that Lakl0C-left does not undergo synapsis. However, we show that the behavior of Lakl0C-left is independent of its particularly early replication timing and is not accompanied by any peculiar chromosome structure as detectable by Hi-C in this yet poorly studied yeast. Finally, we observed an accumulation of heterozygous mutations on Lakl0C-left and a sexual dimorphism of the haploid meiotic offspring, supporting a direct effect of this absence of meiotic recombination on L. kluyveri genome evolution and fitness. Because suppression of meiotic recombination on sex chromosomes is widely observed across eukaryotes, the mechanism for recombination suppression described here may apply to other species, with the potential to impact sex chromosome evolution.

A maternal-to-zygotic-transition gene block on the zebrafish sex chromosome.

Wild zebrafish (Danio rerio) have a ZZ/ZW chromosomal sex-determination system with the major sex locus on the right arm of chromosome-4 (Chr4R) near the largest heterochromatic block in the genome, suggesting that Chr4R transcriptomics might differ from the rest of the genome. To test this hypothesis, we conducted an RNA-seq analysis of adult ZW ovaries and ZZ testes in the Nadia strain and identified 4 regions of Chr4 with different gene expression profiles. Unique in the genome, protein-coding genes in a 41.7 Mb section (Region-2) were expressed in testis but silent in ovary. The AB lab strain, which lacks sex chromosomes, verified this result, showing that testis-biased gene expression in Region-2 depends on gonad biology, not on sex-determining mechanism. RNA-seq analyses in female and male brains and livers validated reduced transcripts from Region-2 in somatic cells, but without sex specificity. Region-2 corresponds to the heterochromatic portion of Chr4R and its content of genes and repetitive elements distinguishes it from the rest of the genome. Region-2 lacks protein-coding genes with human orthologs; has zinc finger genes expressed early in zygotic genome activation; has maternal 5S rRNA genes, maternal spliceosome genes, a concentration of tRNA genes, and a distinct set of repetitive elements. The colocalization of (1) genes silenced in ovaries but not in testes that are (2) expressed in embryos briefly at the onset of zygotic genome activation; (3) maternal-specific genes for translation machinery; (4) maternal-specific spliceosome components; and (5) adjacent genes encoding miR-430, which mediates maternal transcript degradation, suggest that this is a maternal-to-zygotic-transition gene regulatory block.

A universal panel of STR loci for the study of polymorphism of the species Canis lupus and forensic identification of dog and wolf.

Commercial panels of microsatellite (STR) loci are intended for DNA analysis of the domestic dog (Canis lupus familiaris) and, therefore, when genotyping the Grey wolf (Canis lupus lupus), most markers reveal significant deviations from the Hardy-Weinberg equilibrium and have a low informative value, which complicates their use in a forensic examination. The aim of this study was to select STR markers that equally effectively reflect population polymorphism in the wolf and the dog, and to create a universal panel for the identification of individuals in forensic science. Based on the study of polymorphisms of 34 STR loci, a CPlex panel of 15 autosomal loci and two sex loci was developed, which is equally suitable for identifying wolfs and dogs. Analysis of molecular variance (AMOVA) between samples revealed significant differentiation values (FST = 0.0828, p < 0.05), which allows the panel to be used for differentiating between wolf and dog samples. For the first time in the forensic examination of objects of animal origin in the Republic of Belarus, population subdivision coefficients (θ-values) were calculated for each of the 15 STR loci of the test system being reported. It was shown that the values of the genotype frequency, when averaged over all studied animals without and with considering the θ-value, differ by three orders of magnitude (3.39 · 10-17 and 4.71 · 10-14, respectively). The use of population subdivision coefficients will provide the researcher with the most relevant results of an expert identification study. The test system was validated in accordance with the protocol of the Scientific Working Group on DNA Analysis Methods. A computational tool was developed to automate the analysis of genetic data on the wolf and dog in the forensic examination; two guides were approved for practicing forensic experts. This methodology is being successfully used in expert practice in investigating cases of illegal hunting, animal abuse and other offenses in the Republic of Belarus.

Assessing the role of individual foxes in environmental contamination with Echinococcus multilocularis through faecal samples

Key parasite transmission parameters are difficult to obtain from elusive wild animals. For Echinococcus multilocularis, the causative agent of alveolar echinococcosis (AE), the red fox is responsible for most of the environmental contamination in Europe. The identification of individual spreaders of E. multilocularis environmental contamination is crucial to improving our understanding of the ecology of parasite transmission in areas of high endemicity and optimising the effectiveness of prevention and control measures in the field. Genetic faecal sampling appears to be a feasible method to gain information about the faecal deposition of individual animals. We conducted a 4 year faecal sampling study in a village that is highly endemic for E. multilocularis, to assess the feasibility of individual identification and sexing of foxes to describe individual infection patterns. Individual fox identification from faecal samples was performed by obtaining reliable genotypes from 14 microsatellites and one sex locus, coupled with the detection of E. multilocularis DNA, first using captive foxes and then by environmental sampling. From a collection of 386 fox stools collected between 2017 and 2020, tested for the presence of E. multilocularis DNA, 180 were selected and 124 samples were successfully genotyped (68.9%). In total, 45 unique individual foxes were identified and 26 associated with at least one sample which tested positive for E. multilocularis (Em(+)). Estimation of the population size showed the fox population to be between 29 and 34 individuals for a given year and 67 individuals over 4 years. One-third of infected individuals (9/26 Em(+) foxes) deposited 2/3 of the faeces which tested positive for E. multilocularis (36/60 Em(+) stools). Genetic investigation showed a significantly higher average number of multiple stools for females than males, suggesting that the two sexes potentially defecated unequally in the studied area. Three partially overlapping clusters of fox faeces were found, with one cluster concentrating 2/3 of the total E. multilocularis-positive faeces. Based on these findings, we estimated that 12.5 million E. multilocularis eggs were produced during the study period, emphasizing the high contamination level of the environment and the risk of exposure faced by the parasite hosts.

Identification of sex locus and a male-specific marker in blunt-snout bream (Megalobrama amblycephala) using a whole genome resequencing method

Fish sex is often associated with its economic traits, such as growth and body color. The mechanism of fish sex determination is always a hot spot for scientific research. Fish is one of the most diverse groups of vertebrates, and its sex determination mechanism is diverse and complex. Blunt-snout bream (BSB), belonging to Cyprinidae and the genus Megalobrama, is an important economic fish in China. Ordinary consumers are more inclined to buy male fish that does not carry eggs, hence the preparation of this all-male group of BSB meets the needs of the industry. In this study, we applied a whole genome resequencing approach to search sex-linked markers and better characterized sex determination in this fish. Our results confirmed that BSB uses a male heterogametic (XX/XY) system. Individuals from two populations including 10 males and 10 females were sequenced using a whole genomic sequencing method. A large potential sex differentiated region with approximately 6 Mb at the beginning of linkage group (LG) 9 of female BSB genome was identified using genome comparative analysis. This region contained a high density of male-biased genetic polymorphisms and harbored 275 genes, whereas reported sex-related genes didn't exist in this region. With the help of male genome, some male-biased sequences were identified using genome-wide associated analysis, and subsequently a male-specific marker was developed. Our results exhibited that BSB had a considerably large sex locus on LG9, which was likely sex chromosome of this species. Moreover, we developed a male-specific marker for BSB. Our study not only is of great practical significance to the development of sex-controlled breeding technology, but also is of theoretical significance to further explore the sex determination mechanism in fish.

Investigation of powdery &amp; downy mildew segregation in Bogazkere hybrids of Turkish wine grape

Powdery mildew (Erysiphe necator syn. Uncinula necator) and downy mildew (Plasmapora viticola) are the most harmful diseases to grapevine production in Turkey. The aim of this study was to generate hybrids between the susceptible Turkish wine grape variety ‘Bogazkere’ and powdery and downy mildew resistant microvine breeding lines and evaluate their resistance to these important diseases. Marker Assisted Selection (MAS) was used to determine the inheritance of powdery and downy mildew resistance and flower sex loci of the 36 F1 progeny plants obtained by cross-pollination at genotypic level. Based on the PCR results, phenotyping and leaf bioassay were performed to validate initial MAS for 11 and 9 representative progeny with four resistant and susceptible controls. As a result, two tall powdery and downy mildew-resistant vines, three powdery mildew-resistant microvines and two downy mildew-resistant microvines were obtained. The MAS results were also validated by leaf bioassays, which confirmed that resistance genes segregated to the F1 progeny are functional and provide resistance. To the best of our knowledge, this research provides the first photographic validation of the powdery and downy mildew resistance of the Turkish variety ‘Bogazkere’. Our findings could potentially provide the wine industry with a resistant wine grape cultivar and an addition to the wine grape germplasm, which could contribute to future breeding programs. Most importantly, the success of the resistance genes segregated into the candidate progeny could help significantly reduce the use of chemicals against grapevine pathogens.

Combining two genetic sexing strains allows sorting of non-transgenic males for Aedes genetic control

Chemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01–0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors.

A Robust Test System for DNA Identification of Raccoon Dogs

To establish a system for DNA identification of biological samples of the raccoon dog (Nyctereutes procyonoides), we carried out a study of polymorphism of 39 microsatellite loci and three sex loci specific to various species of the Canidae family. This resulted in the development of the NPlex test system, which includes 14 autosomal STRs and two sex loci. The test system has been validated for use in forensic identification of racoon dogs based on biological traces (such as traces of blood and secretions, fragments of muscle tissue or bones, dermis and hair, etc.) found at crime scenes, as well as for establishing biological relationship of animals of this species. Validation efforts were performed in accordance with the protocol of the Scientific Working Group on DNA Analysis Methods.

A Novel Look at Dosage-Sensitive Sex Locus Xp21.2 in a Case of 46,XY Partial Gonadal Dysgenesis without NR0B1 Duplication

A region of 160 kb at Xp21.2 has been defined as dosage-sensitive sex reversal (DSS) and includes the NR0B1 gene, considered to be the candidate gene involved in XY gonadal dysgenesis if overexpressed. We describe a girl with 46,XY partial gonadal dysgenesis carrying a 297 kb duplication at Xp21.2 upstream of NR0B1 initially detected by chromosomal microarray analysis. Fine mapping of the breakpoints by whole-genome sequencing showed a tandem duplication of TASL (CXorf21), GK and partially TAB3, upstream of NR0B1. This is the first description of an Xp21.2 duplication upstream of NR0B1 associated with 46,XY partial gonadal dysgenesis.

Development of a 135K SNP genotyping array for Actinidia arguta and its applications for genetic mapping and QTL analysis in kiwifruit.

Kiwifruit (Actinidia spp) is a woody, perennial and deciduous vine. In this genus, there are multiple ploidy levels but the main cultivated cultivars are polyploid. Despite the availability of many genomic resources in kiwifruit, SNP genotyping is still a challenge given these different levels of polyploidy. Recent advances in SNP array technologies have offered a high-throughput genotyping platform for genome-wide DNA polymorphisms. In this study, we developed a high-density SNP genotyping array to facilitate genetic studies and breeding applications in kiwifruit. SNP discovery was performed by genome-wide DNA sequencing of 40 kiwifruit genotypes. The identified SNPs were stringently filtered for sequence quality, predicted conversion performance and distribution over the available Actinidia chinensis genome. A total of 134 729 unique SNPs were put on the array. The array was evaluated by genotyping 400 kiwifruit individuals. We performed a multidimensional scaling analysis to assess the diversity of kiwifruit germplasm, showing that the array was effective to distinguish kiwifruit accessions. Using a tetraploid F1 population, we constructed an integrated linkage map covering 3060.9cM across 29 linkage groups and performed QTL analysis for the sex locus that has been identified on Linkage Group 3 (LG3) in Actinidia arguta. Finally, our dataset presented evidence of tetrasomic inheritance with partial preferential pairing in A. arguta. In conclusion, we developed and evaluated a 135K SNP genotyping array for kiwifruit. It has the advantage of a comprehensive design that can be an effective tool in genetic studies and breeding applications in this high-value crop.

Characterization of sex locus and sex-specific sequences in the mandarin fishes

Sex determination of teleost fish switched frequently. Sex markers are especially practical in identifying early sex and helpful in generating mono-sex population in aquatic species. And sex determination in mandarin fishes is still unclear. In this study, we applied a pool-sequencing approach to better characterize sex-specific markers and sex determination in mandarin fishes. Using genome comparative analysis, we identified significant sex differentiated region (~5.0 Mb) and sex chromosome (LG2) in Siniperca chuatsi. Similar sex-differentiated regions were also identified on LG4 in other mandarin fishes including Siniperca knerii, Siniperca scherzeri, and Coreoperca whiteheadi. These non-recombining regions contained high-density of male-biased genetic polymorphisms accompanied with male-biased fragments (~ 23 kb in total). Meanwhile, sequence synteny analysis showed that whole genomes, including sex chromosomes and even sex-differentiated regions among these species were in good synteny. In addition, the sex-differentiated region harbored 124 genes without “usual suspects” sex determining genes. Based on this genetic information, we successfully developed male-specific genetic markers for mandarin fishes. And genotyping results showed phenotypic sexes were strongly linked to its genetic sexes in one Siniperca chuatsi population. Our results exhibited that sex-differentiated regions and sex chromosomes were relatively conserved in the four mandarin fishes. All the four mandarin fishes were identified to utilize a male heterogametic (XX-XY) sex determination system. Besides, we also developed sex-linked genetic markers. Our study will facilitate future research on sex determination and aquaculture applications in mandarin fishes.

Development of a microsatellite panel for identification of biological samples of moose (Alces alces) in forensic research

For forensic analysis of evidence from illegal hunting sites of moose, a panel of 15 microsatellite (4 tetranucleotide and 11 dinucleotide) loci and 1 sex locus (amelogenin) was proposed. The cumulative power of discrimination of individual based on the common genetic database of moose (for a panel of 15 microsatellite loci) is more than 99.999 999999 999 %. The genetic and forensic indicators of a microsatellite panel were evaluated on a population sample of moose on a national scale (383 samples) for the first time in Europe and forensic methodology of the DNA identification of the moose (Alces alces) introduced into the national legal system.

Sex locus and sex markers identification using whole genome pool-sequencing approach in the largemouth bass (Micropterus Salmoides L.)

The mechanism of sex determination in teleost evolves in an amazing speed contrary to mammals and birds. Different populations of the same species may use different sex determination systems. Largemouth bass is an aquaculture importance and recreational fish, which belongs to the family of Centrarchidae, genus Micropterus. However, molecular basis of genetic sex determination currently remains unclear. We applied a whole genome pool-sequencing approach to better characterize sex determination in this fish. Our results confirmed that sex determination system in largemouth bass is male heterogametic (XX/XY). In this study, males and females genomic DNA pools were sequenced using a whole genomic sequencing approach. Using genome comparative analysis, we identified a large sex differentiated region (~ 1.7 Mb) at the beginning of chromosome 7 on largemouth bass. This non-recombination region contained enriched male-biased fragments and high-density of male-biased genetic polymorphisms. Meanwhile, sequence synteny analysis between/within sex chromosomes showed that many chromosomal rearrangement events have taken place in this region. Based on this genetic information, we successfully developed male specific genetic markers. Additionally, 96 genes were located on that sex differentiated region, whereas “usual suspects” sex determining gene didn't exist in this region. It is implied, in largemouth bass, a new gene has evolved as the master sex determining gene. Our results exhibited that largemouth bass has a large sex locus on Chromosome 7, which is likely the largemouth bass sex chromosome. The presence of numerous chromosomal rearrangements and the absence of “usual suspects” sex determining gene in this sex differentiated region indicate recombination is suppressed on that region and a new master sex determining gene may be co-opted in largemouth bass. Besides, we also developed sex-linked genetic markers. Our study will facilitate future researches on sex determination and aquaculture applications in largemouth bass.

Development and validation of a novel 26-plex system for prenatal diagnosis with forensic markers.

Short tandem repeat (STR) loci are commonly used in forensic casework, such as personal identification and paternity testing. In recent years, STR has also been widely used for rapid, accurate and automated prenatal diagnosis, known as quantitative fluorescent PCR (QF-PCR). Despite their usefulness, the current systems often lack the power to detect mosaicism for Turner syndrome. In this study, we developed a novel 26-plex system that combined the 22 STRs in chromosome 21/18/13/X, 3 sex loci and 1 quality control marker (TAF9L). The system was generated to achieve greater diagnostic power of trisomy 21/18/13 and sex chromosome abnormalities. Studies of the sensitivity, specificity, stability and accuracy were performed according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. Compared with the results of the chromosomal microarray analysis (CMA)/copy number variation sequencing (CNV-seq), the detection ratio of non-mosaic chromosome abnormalities of this system in the identification of chromosome 21/18/13/X/Y aneuploidies reached 100%, and the rate of negative results was consistently 100% based on 203 prenatal diagnosis sample analyses. In addition, our results suggested that this panel was a useful tool for mosaicism for Turner syndrome cases. Interestingly, we found one case with large segment loss of chromosome X, which indicated that we should be alert to this situation when the STR genotype of the parent-child is inconsistent in forensic genetics. In summary, this study demonstrated that our system is an accurate, cost-effective and rapid approach for the detection of chromosome numerical abnormalities in prenatal diagnosis.

Estrogen concentration and estrogen receptor-β expression in postmenopausal colon cancer considering patient/tumor background

A large number of studies have suggested an inhibitory role of estrogens against colorectal cancer (CRC), but persistent controversy exists. CRC characteristics are affected by sex, age, and tumor locus, suggesting the need for a systematic study considering these factors. The purpose of this study was to verify the difference in the pathobiological role of estrogens in CRC according to patient/tumor backgrounds. Surgical specimens from 116 postmenopausal women (≥ 70years/o, n = 74; < 70years/o, n = 42) were studied. Estrogen receptor-β (ER-β), the main ER in the colorectal epithelium, was immunohistochemically examined. The concentrations of estradiol (E2) and estrone (E1) were examined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These factors were compared according to the tissue type (cancerous or non-cancerous), patients' age, tumor backgrounds (locus, histology, pathological stage, status of mismatch repair protein = MMR), and clinical outcome. ER-β-positivity, higher E2 concentration, deficient-MMR, and medullary/mucinous histology (Med/Muc) were closely related to right-sided tumors in women who were aged ≥ 70years /o (R-Ca ≥ 70) and also closely related to each other. ER-β reduction compared with non-cancerous counterparts was observed only in left-sided tumors of patients < 70years /o (L-Ca < 70), non-Med/Muc, or proficient-MMR tumors. The present results suggest that estrogens do not suppress, but rather promote, R-Ca ≥ 70, Med/Muc, or deficient-MMR tumors, whereas estrogens suppress L-Ca < 70, non-Med/Muc, or proficient-MMR tumors, confirming the difference in pathobiological role of estrogens in postmenopausal colon cancer according to the patients' age and tumor background. This may at least partly explain the controversy regarding the association between estrogens and CRC.

Genetic mapping of sex and self-incompatibility determinants in the androdioecious plant Phillyrea angustifolia

The diversity of mating and sexual systems in angiosperms is spectacular, but the factors driving their evolution remain poorly understood. In plants of the Oleaceae family, an unusual self-incompatibility (SI) system has been discovered recently, whereby only two distinct homomorphic SI specificities segregate stably. To understand the role of this peculiar SI system in preventing or promoting the diversity of sexual phenotypes observed across the family, an essential first step is to characterize the genetic architecture of these two traits. Here, we developed a high-density genetic map of the androdioecious shrub P. angustifolia based on a F1 cross between a hermaphrodite and a male parent with distinct SI genotypes. Using a double restriction-site associated digestion (ddRAD) sequencing approach, we obtained reliable genotypes for 196 offspring and their two parents at 10,388 markers. The resulting map comprises 23 linkage groups totaling 1,855.13 cM on the sex-averaged map. We found strong signals of association for the sex and SI phenotypes, that were each associated with a unique set of markers on linkage group 12 and 18 respectively, demonstrating inheritance of these traits as single, independent, mendelian factors. The P. angustifolia linkage map shows robust synteny to the olive tree genome overall. Two of the six markers strictly associated with SI in P. angustifolia have strong similarity with a recently identified 741kb chromosomal region fully linked to the SI phenotype on chromosome 18 of the olive tree genome, providing strong cross-validation support. The SI locus stands out as being markedly rearranged, while the sex locus has remained relatively more collinear between the two species. This P. angustifolia linkage map will be a useful resource to investigate the various ways by which the sex and SI determination systems have co-evolved in the broader phylogenetic context of the Oleaceae family.

Lessons from an unusual vertebrate sex-determining gene.

So far, very few sex-determining genes have been identified in vertebrates and most of them, the so-called 'usual suspects', evolved from genes which fulfil essential functions during sexual development and are thus already tightly linked to the process that they now govern. The single exception to this 'usual suspects' rule in vertebrates so far is the conserved salmonid sex-determining gene, sdY (sexually dimorphic on the Y chromosome), that evolved from a gene known to be involved in regulation of the immune response. It is contained in a jumping sex locus that has been transposed or translocated into different ancestral autosomes during the evolution of salmonids. This special feature of sdY, i.e. being inserted in a 'jumping sex locus', could explain how salmonid sex chromosomes remain young and undifferentiated to escape degeneration. Recent knowledge on the mechanism of action of sdY demonstrates that it triggers its sex-determining action by deregulating oestrogen synthesis that is a conserved and crucial pathway for ovarian differentiation in vertebrates. This result suggests that sdY has evolved to cope with a pre-existing sex differentiation regulatory network. Therefore, 'limited options' for the emergence of new master sex-determining genes could be more constrained by their need to tightly interact with a conserved sex differentiation regulatory network rather than by being themselves 'usual suspects', already inside this sex regulatory network. 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.

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.