Sex-determining Gene Research Articles

Generation of Pigs that Produce Single Sex Progeny.

In livestock industry, one sex is usually preferred over the other due to its impact on the production (e.g., milk from cows, eggs from laying hens, or meat from bulls). Boar taint, to which most of the consumers are susceptible, is a major challenge for the pork industry in the light of the enacted ban of castration without anesthesia from 2021 in Germany. Consequently, a shift towards an increased female ratio would be of great benefit for the pork production. We recently described that a CRISPR/Cas9-mediated knockout of the porcine SRY gene by intracytoplasmic microinjection or SCNT resulted in genetically male pigs with a female phenotype. This sex reversal study in pigs revealed a pivotal role of the SRY gene in male sex determination and might pave the way for the generation of boars that produce only female offspring.

Flower development and sex determination in horticultural crops

Horticultural crops are extremely valuable due to their high nutritional value, and fruits, in particular, provide indispensable vitamins and minerals. Fruit yield of edible crops is closely related to the number of flowers, which are often unisexual. The mechanism of sex differentiation in plants with unisexuality is complex, and research investigating this mechanism is in great demand. Sex determinants were first discovered in <italic>Cucurbitaceae</italic> (e.g., cucumber, melon, watermelon), and in recent years, with the rapid development of deep sequencing technologies and genomics, they have also been deciphered in some dioecious plants (e.g., persimmon, kiwifruit, asparagus). This has deepened our understanding of the evolution and diversification of sexual reproductive systems. This review summarizes recent research investigating flower sex-determination genes and their working networks, focusing on horticultural crops. Perspectives on future research in flower sex differentiation are also discussed.

Roles of anti-Müllerian hormone and its duplicates in sex determination and germ cell proliferation of Nile tilapia.

Duplicates of amh are crucial for fish sex determination and differentiation. In Nile tilapia, unlike in other teleosts, amh is located on X chromosome. The Y chromosome amh (amhΔ-y) is mutated with 5 bp insertion and 233 bp deletion in the coding sequence, and tandem duplicate of amh on Y chromosome (amhy) has been identified as the sex determiner. However, the expression of amh, amhΔ-y, and amhy, their roles in germ cell proliferation and the molecular mechanism of how amhy determines sex is still unclear. In this study, expression and functions of each duplicate were analyzed. Sex reversal occurred only when amhy was mutated as revealed by single, double, and triple mutation of the 3 duplicates in XY fish. Homozygous mutation of amhy in YY fish also resulted in sex reversal. Earlier and higher expression of amhy/Amhy was observed in XY gonads compared with amh/Amh during sex determination. Amhy could inhibit the transcription of cyp19a1a through Amhr2/Smads signaling. Loss of cyp19a1a rescued the sex reversal phenotype in XY fish with amhy mutation. Interestingly, mutation of both amh and amhy in XY fish or homozygous mutation of amhy in YY fish resulted in infertile females with significantly increased germ cell proliferation. Taken together, these results indicated that up-regulation of amhy during the critical period of sex determination makes it the sex-determining gene, and it functions through repressing cyp19a1a expression via Amhr2/Smads signaling pathway. Amh retained its function in controlling germ cell proliferation as reported in other teleosts, while amhΔ-y was nonfunctionalized.

CmLHP1 proteins play a key role in plant development and sex determination in melon (Cucumis melo).

In monoecious melon (Cucumis melo), sex is determined by the differential expression of sex determination genes (SDGs) and adoption of sex-specific transcriptional programs. Histone modifications such as H3K27me3 have been previously shown to be a hallmark associated to unisexual flower development in melon; yet, no genetic approaches have been conducted for elucidating the roles of H3K27me3 writers, readers, and erasers in this process. Here we show that melon homologs to Arabidopsis LHP1, CmLHP1A and B, redundantly control several aspects of plant development, including sex expression. Cmlhp1ab double mutants displayed an overall loss and redistribution of H3K27me3, leading to a deregulation of genes involved in hormone responses, plant architecture, and flower development. Consequently, double mutants display pleiotropic phenotypes and, interestingly, a general increase of the male:female ratio. We associated this phenomenon with a general deregulation of some hormonal response genes and a local activation of male-promoting SDGs and MADS-box transcription factors. Altogether, these results reveal a novel function for CmLHP1 proteins in maintenance of monoecy and provide novel insights into the polycomb-mediated epigenomic regulation of sex lability in plants.

Prediction of the Sex-Associated Genomic Region in Tunas (Thunnus Fishes).

Fish species have a variety of sex determination systems. Tunas (genus Thunnus) have an XY genetic sex determination system. However, the Y chromosome or responsible locus has not yet been identified in males. In a previous study, a female genome of Pacific bluefin tuna (T. orientalis) was sequenced, and candidates for sex-associated DNA polymorphisms were identified by a genome-wide association study using resequencing data. In the present study, we sequenced a male genome of Pacific bluefin tuna by long-read and linked-read sequencing technologies and explored male-specific loci through a comparison with the female genome. As a result, we found a unique region carrying the male-specific haplotype, where a homolog of estrogen sulfotransferase gene was predicted to be encoded. The genome-wide mapping of previously resequenced data indicated that, among the functionally annotated genes, only this gene, named sult1st6y, was paternally inherited in the males of Pacific bluefin tuna. We reviewed the RNA-seq data of southern bluefin tuna (T. maccoyii) in the public database and found that sult1st6y of southern bluefin tuna was expressed in all male testes, but absent or suppressed in the female ovary. Since estrogen sulfotransferase is responsible for the inactivation of estrogens, it is reasonable to assume that the expression of sult1st6y in gonad cells may inhibit female development, thereby inducing the individuals to become males. Thus, our results raise a promising hypothesis that sult1st6y is the sex determination gene in Thunnus fishes or at least functions at a crucial point in the sex-differentiation cascade.

A chromosome-level genome of Portunus trituberculatus provides insights into its evolution, salinity adaptation and sex determination.

Portunus trituberculatus (Crustacea: Decapoda: Brachyura), commonly known as the swimming crab, is of major ecological importance, as well as being important to the fisheries industry. P. trituberculatus is also an important farmed species in China due to its rapid growth rate and high economic value. Here, we report the genome sequence of the swimming crab, which was assembled at the chromosome scale, covering ~1.2Gb, with 79.99% of the scaffold sequences assembled into 53 chromosomes. The contig and scaffold N50values were 108.7kb and 15.6Mb, respectively, with 19,981 protein-coding genes. Based on comparative genomic analyses of crabs and shrimps, the C2H2 zinc finger protein family was found to be the only gene family expanded in crab genomes, suggesting it was closely related to the evolution of crabs. The combination of transcriptome and bulked segregant analysis provided insights into the genetic basis of salinity adaptation and rapid growth in P. trituberculatus. In addition, the specific region of the Y chromosome was located for the first time in the genome of P. trituberculatus, and three genes were preliminarily identified as candidate genes for sex determination in this region. Decoding the swimming crab genome not only provides a valuable genomic resource for further biological and evolutionary studies, but is also useful for molecular breeding of swimming crabs.

A Chromosome-Level Genome Assembly of Mozambique Tilapia (Oreochromis mossambicus) Reveals the Structure of Sex Determining Regions.

The Mozambique tilapia (Oreochromis mossambicus) is a fascinating taxon for evolutionary and ecological research. It is an important food fish and one of the most widely distributed tilapias. Because males grow faster than females, genetically male tilapia are preferred in aquaculture. However, studies of sex determination and sex control in O. mossambicus have been hindered by the limited characterization of the genome. To address this gap, we assembled a high-quality genome of O. mossambicus, using a combination of high coverage of Illumina and Nanopore reads, coupled with Hi-C and RNA-Seq data. Our genome assembly spans 1,007 Mb with a scaffold N50 of 11.38 Mb. We successfully anchored and oriented 98.6% of the genome on 22 linkage groups (LGs). Based on re-sequencing data for male and female fishes from three families, O. mossambicus segregates both an XY system on LG14 and a ZW system on LG3. The sex-patterned SNPs shared by two XY families narrowed the sex determining regions to ∼3 Mb on LG14. The shared sex-patterned SNPs included two deleterious missense mutations in ahnak and rhbdd1, indicating the possible roles of these two genes in sex determination. This annotated chromosome-level genome assembly and identification of sex determining regions represents a valuable resource to help understand the evolution of genetic sex determination in tilapias.

Sexual diversification of splicing regulation during embryonic development in honeybees (Apis mellifera), A haplodiploid system.

The honeybee is a haplodiploid organism in which sexual development is determined by the complementary sex determiner (csd) gene and realized by sex-specific splicing processes involving the feminizer (fem) gene. We used high throughput transcriptome sequencing (RNA-Seq) to characterize the transcriptional differences between the sexes caused by the fertilization and sex determination processes in honeybee (Apis mellifera) embryos. We identified 758, 372 and 43 differentially expressed genes (DEGs) and 58, 176 and 233 differentially spliced genes (DSGs) in 10-15-h-old, 25-40-h-old and 55-70-h-old female and male embryos, respectively. The early difference in male and female embryos in response to the fertilization and non-fertilization processes resulted mainly in differential expression of genes (758 DEGs vs. 58 DSGs). In the latest sampled embryonic stage, the transcriptional differences between the sexes were dominated by alternative splicing of transcripts (43 DEGs vs. 233 DSGs). Interestingly, differentially spliced transcripts that encode RNA-binding properties were overrepresented in 55-70-h-old embryos, indicating a more diverse regulation via alternative splicing than previous work on the sex determination pathway suggested. These stage- and sex-specific transcriptome data from honeybee embryos provide a comprehensive resource for examining the roles of fertilization and sex determination in developmental programming in a haplodiploid system.

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 duplicated copy of id2b is an unusual sex-determining candidate gene on the Y chromosome of arapaima (Arapaima gigas)

Arapaima gigas is one of the largest freshwater fish species of high ecological and economic importance. Overfishing and habitat destruction are severe threats to the remaining wild populations. By incorporating a chromosomal Hi-C contact map, we improved the arapaima genome assembly to chromosome-level, revealing an unexpected high degree of chromosome rearrangements during evolution of the bonytongues (Osteoglossiformes). Combining this new assembly with pool-sequencing of male and female genomes, we identified id2bbY, a duplicated copy of the inhibitor of DNA binding 2b (id2b) gene on the Y chromosome as candidate male sex-determining gene. A PCR-test for id2bbY was developed, demonstrating that this gene is a reliable male-specific marker for genotyping. Expression analyses showed that this gene is expressed in juvenile male gonads. Its paralog, id2ba, exhibits a male-biased expression in immature gonads. Transcriptome analyses and protein structure predictions confirm id2bbY as a prime candidate for the master sex-determiner. Acting through the TGFβ signaling pathway, id2bbY from arapaima would provide the first evidence for a link of this family of transcriptional regulators to sex determination. Our study broadens our current understanding about the evolution of sex determination genetic networks and provide a tool for improving arapaima aquaculture for commercial and conservation purposes.

Profile of Jennifer Marshall Graves

“Sometimes when you ask a functional question, you get an evolutionary answer,” says Jennifer Marshall Graves, a leader in the field of epigenetics and comparative genomics. Her team’s vast body of research on Australian fauna has helped the distinguished professor at Melbourne’s La Trobe University (LTU) uncover fundamental insights into mammalian sex chromosomes and sex-determining genes. Elected as an International Member of the National Academy of Sciences in 2019, Graves compared DNA sequences across widely divergent animals and demonstrates in her Inaugural Article (1) that bird and reptile microchromosomes, once thought to be insignificant, are ancient and highly conserved and represent the original building blocks of vertebrate genomes. Graves was born in Adelaide, Australia. Her father, the soil physicist T. J. Marshall, formulated the Marshall equation, which describes the movement of fluids in soil. Her mother, Ann Marshall, was a geologist and urban geographer whose activism concerning overdevelopment in Adelaide left a mark on the city. Graves recalls being “turned onto genetics” when high school biology teacher Doreen McCarthy used colorful images of budgerigars to demonstrate Mendel’s laws of genetics. Graves says, “I was hooked and enrolled in science at Adelaide University in 1960.” Shortly thereafter, during her mother’s sabbatical, she spent a semester at the University of California, Berkeley, where she studied cell biology taught by Daniel Mazia. Returning to Adelaide, she received the Sir Ronald A. Fisher Award for Genetics in 1962 and conducted honors research on X chromosome inactivation in kangaroo females with advisor David Hayman. At Adelaide, she earned both an undergraduate degree in genetics and physical chemistry and a Master’s degree in genetics. Jennifer Marshall Graves. Image credit: GAMMA/Micheline Pelletier. The Fisher Award, a £10 book token, was followed by a Fulbright Travel Grant, which enabled doctoral studies at Berkeley under Mazia and cell geneticist …

Sex determination gene transformer regulates the male-female difference in Drosophila fat storage via the adipokinetic hormone pathway.

Sex differences in whole-body fat storage exist in many species. For example, Drosophila females store more fat than males. Yet, the mechanisms underlying this sex difference in fat storage remain incompletely understood. Here, we identify a key role for sex determination gene transformer (tra) in regulating the male-female difference in fat storage. Normally, a functional Tra protein is present only in females, where it promotes female sexual development. We show that loss of Tra in females reduced whole-body fat storage, whereas gain of Tra in males augmented fat storage. Tra's role in promoting fat storage was largely due to its function in neurons, specifically the Adipokinetic hormone (Akh)-producing cells (APCs). Our analysis of Akh pathway regulation revealed a male bias in APC activity and Akh pathway function, where this sex-biased regulation influenced the sex difference in fat storage by limiting triglyceride accumulation in males. Importantly, Tra loss in females increased Akh pathway activity, and genetically manipulating the Akh pathway rescued Tra-dependent effects on fat storage. This identifies sex-specific regulation of Akh as one mechanism underlying the male-female difference in whole-body triglyceride levels, and provides important insight into the conserved mechanisms underlying sexual dimorphism in whole-body fat storage.

Identification of male-specific SNP markers and development of rapid PCR-based genetic sex identification method in channel catfish (Ictalurus punctatus)

Channel catfish (Ictalurus punctatus) is an important aquaculture species worldwide. Although studies of the sex determination mechanism of channel catfish began during the 1980s, the mechanism and sex-specific genes remain unclear. In this study, based on the results of sex-related quantitative trait locus (QTL) mapping and targeted next-generation sequencing (NGS) technique, 13 male-specific single nuclear polymorphisms (SNPs) were identified in the coding region of the zbtb38 gene of channel catfish by targeted next-generation sequencing. Among them, six substitutions resulted in changes in the amino acid coding in zbtb38 on both the X and Y chromosomes. It is speculated that zbtb38 on the Y chromosome has an important role in testicular differentiation and is an important candidate gene for sex determination in channel catfish. Subsequently, male-specific primers were designed according to male-specific nucleotides, which amplified an expected band from males but not from females. Thus, a rapid and effective method was successfully established to identify the genetic sex of channel catfish. Our study contributes to the study of the sex determination mechanisms and development of mono-sex populations in channel catfish.

Sex Determination and Differentiation in Teleost: Roles of Genetics, Environment, and Brain.

Simple SummaryThe fate of the gonad in teleost is influenced by various factors, including genetics and external factors. Several species-specific genes and environmental factors involved in sex determination and differentiation have been identified in teleost. In addition, these factors are species-specific. At the brain level, suppression of key molecule of the hypothalamus–gonadal axis affects sex determination. At the same time, pituitary hormones are required for regulating sex differentiation. However, the role of the brain during sex determination and differentiation remains elusive. In this review, we have gathered and discussed the findings on the role of prominent genes, environmental factors, and the brain in regulating sex determination and differentiation of teleost. The fish reproductive system is a complex biological system. Nonetheless, reproductive organ development is conserved, which starts with sex determination and then sex differentiation. The sex of a teleost is determined and differentiated from bipotential primordium by genetics, environmental factors, or both. These two processes are species-specific. There are several prominent genes and environmental factors involved during sex determination and differentiation. At the cellular level, most of the sex-determining genes suppress the female pathway. For environmental factors, there are temperature, density, hypoxia, pH, and social interaction. Once the sexual fate is determined, sex differentiation takes over the gonadal developmental process. Environmental factors involve activation and suppression of various male and female pathways depending on the sexual fate. Alongside these factors, the role of the brain during sex determination and differentiation remains elusive. Nonetheless, GnRH III knockout has promoted a male sex-biased population, which shows brain involvement during sex determination. During sex differentiation, LH and FSH might not affect the gonadal differentiation, but are required for regulating sex differentiation. This review discusses the role of prominent genes, environmental factors, and the brain in sex determination and differentiation across a few teleost species.

Application of Next Generation Semiconductor-Based Sequencing for the Identification of Apis mellifera Complementary Sex Determiner (csd) Alleles from Honey DNA.

Simple SummaryHoney contains traces of the DNA of the honey bees that produced it. This environmental DNA can therefore be used to investigate the genome of the honey bees. In this study, we used a next generation sequencing technology to analyze the variability of a key gene of Apis mellifera L., the complementary sex determiner (csd) gene, using honey environmental DNA as a source of honey bee DNA. This gene determines the sex of the bees. Two different alleles at this locus are needed to produce females whereas males have only one copy of this gene as they are haploid. In case two identical alleles are present in a diploid individual, the larvae are not vital and are discarded by the workers. Therefore, there is an advantage in maintaining a large csd diversity in honey bee populations. In light of the recent decline in honey bee populations, it is important to monitor the allele variability at this gene. The applied methodology provided a new strategy to disclose the genetic diversity at the csd gene at the population-wide level and identify most, if not all, csd alleles present in the colonies in a single analysis.The complementary sex determiner (csd) gene plays an essential role in the sex determination of Apis mellifera L. Females develop only if fertilized eggs have functional heterozygous genotypes at this gene whereas males, being haploids, are hemizygous. Two identical csd alleles produce non vital males. In light of the recent decline in honey bee populations, it is therefore important to monitor the allele variability at this gene. In this study, we tested the application of next generation semiconductor-based sequencing technology (Ion Torrent) coupled with environmental honey DNA as a source of honey bee genome information to retrieve massive sequencing data for the analysis of variability at the hypervariable region (HVR) of the csd gene. DNA was extracted from 12 honey samples collected from honeycombs directly retrieved from 12 different colonies. A specifically designed bioinformatic pipeline, applied to analyze a total of about 1.5 million reads, identified a total of 160 different csd alleles, 55% of which were novel. The average number of alleles per sample was compatible with the number of expected patrilines per colony, according to the mating behavior of the queens. Allele diversity at the csd could also provide information useful to reconstruct the history of the honey.

Nanomaterial-induced mesenchymal stem cell differentiation into osteoblast for counteracting bone resorption in the osteoporotic rats

The current approach was designed to unearth the therapeutic potential of osteoblasts infusion, yielded from cultivating rat mesenchymal stem cells of bone marrow source in osteogenic differentiation media supplied with either hydroxyapatite nanoparticles (HA-NPs), chitosan/hydroxyapatite nanomaterials (C/HA-NPs), or chitosan nanoparticles, in the osteoporotic rats. The successful migration of the osteoblasts to the diseased bones of rats in C/HA-NPs and HA-NPs groups was evidenced by PCR screening of the Y-linked sex-determining gene (SRY) in the femoral bone tissue. Serum bone biomarker levels and gene expression patterns of cathepsin K, receptor activator of nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) were assessed. Additionally, histological examination of the femoral bone tissues of rats was performed. The current outcomes revealed that osteoblast implantation, resulted from C/HA-NPs or HA-NPs group, significantly lessened bone sialoprotein level. In Addition, it yielded a significant decline in the gene expression patterns of cathepsin K, RANKL, and RANKL/OPG proportion as well as up-regulation in BMP-2 and Runx-2 gene expression levels as opposed to the untreated ovariectomized animals. Moreover, it could restrain bone resorption and refine bone histoarchitecture. Conclusively, this study sheds light on the therapeutic significance of osteoblasts transplantation in alleviating the intensity of the bone remodeling cycle, consequently representing a hopeful therapeutic approach for primary osteoporosis.

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.

Sex-Biased Gene Expression and Isoform Profile of Brine Shrimp Artemia franciscana by Transcriptome Analysis.

The brine shrimp Artemia has a ZW sex determination system with ZW chromosomes in females and ZZ chromosomes in males. Artemia has been considered a promising model organism for ZW sex-determining systems, but the genes involved in sex determination and differentiation of Artemia have not yet been identified. Here, we conducted transcriptome sequencing of female and male A. franciscana using PacBio Iso-Seq and Illumina RNA-Seq techniques to identify candidate sex determination genes. Among the 42,566 transcripts obtained from Iso-Seq, 23,514 were analyzed. Of these, 2065 (8.8%) were female specific, 2513 (10.7%) were male specific, and 18,936 (80.5%) were co-expressed in females and males. Based on GO enrichment analysis and expression values, we found 10 female-biased and 29 male-biased expressed genes, including DMRT1 and Sad genes showing male-biased expression. Our results showed that DMRT1 has three isoforms with five exons, while Sad has seven isoforms with 2-11 exons. The Sad gene is involved in ecdysteroid signaling related to molting and metamorphosis in arthropods. Further studies on ecdysteroid biosynthetic genes are needed to improve our understanding of Artemia sex determination. This study will provide a valuable resource for sex determination and differentiation studies on Artemia and other crustaceans with ZW systems.

DNA methylation is involved in sexual differentiation and sex chromosome evolution in the dioecious plant garden asparagus

DNA methylation is a crucial regulatory mechanism in many biological processes. However, limited studies have dissected the contribution of DNA methylation to sexual differentiation in dioecious plants. In this study, we investigated the variances in methylation and transcriptional patterns of male and female flowers of garden asparagus. Compared with male flowers, female flowers at the same stages showed higher levels of DNA methylation. Both male and female flowers gained DNA methylation globally from the premeiotic to meiotic stages. Detailed analysis revealed that the increased DNA methylation was largely due to increased CHH methylation. Correlation analysis of differentially expressed genes and differentially methylated regions suggested that DNA methylation might not have contributed to the expression variation of the sex-determining genes SOFF and TDF1 but probably played important roles in sexual differentiation and flower development of garden asparagus. The upregulated genes AoMS1, AoLAP3, AoAMS, and AoLAP5 with varied methylated CHH regions might have been involved in sexual differentiation and flower development of garden asparagus. Plant hormone signaling genes and transcription factor genes also participated in sexual differentiation and flower development with potential epigenetic regulation. In addition, the CG and CHG methylation levels in the Y chromosome were notably higher than those in the X chromosome, implying that DNA methylation might have been involved in Y chromosome evolution. These data provide insights into the epigenetic modification of sexual differentiation and flower development and improve our understanding of sex chromosome evolution in garden asparagus.