Different sex determination systems in two closely related Eurasian minnow (Phoxinus) species.

Relationships between Vertebrate ZW and XY Sex Chromosome Systems

fshr: a fish sex-determining locus shows variable incomplete penetrance across flathead grey mullet populations.

The genetic basis of sex determination is typically conserved within species if not within broader lineages. For example, within the stickleback family (Gasterosteidae), AmhY has been identified as a master sex-determination (MSD) gene in multiple species across two genera. By contrast, the existence of within-species variability in the genetic basis of sex determination is not frequently observed but provides an opportunity to understand the evolution and turnover of sex determination systems. In this study, we investigated the consistency with which AmhY is involved in sex determination across 610 individuals from five brook stickleback (Culaea inconstans) populations. We designed a PCR-restriction enzyme assay to identify the presence of AmhY in each individual and recorded sexual morphology in each individual in the field at the time of capture. We found that the genetic sex (presence/absence of AmhY) did not match the field-determined phenotypic sex in up to 44% of individuals within a population. This variation in the genetic basis of sex determination in brook stickleback suggests that the mechanism of sex determination in this species is likely more complex than thought when AmhY was first implicated and may still be evolving. Such within-species variation provides an opportunity to further investigate how and why transitions in sex-determination mechanisms occur.

Since the molecular mechanisms underlying sex determination in Procambarus clarkii are still unclear, it is important to investigate the genetic basis of sex determination in crustaceans. Currently, the molecular mechanisms of sex determination and the gender-specific markers in this species remain poorly understood. In this study, a total of 14,046,984 SNPs and 2,160,652 InDels were identified through genome-wide resequencing of 89 individuals (45 females and 44 males). Further analysis confirmed that the candidate chromosome was Chr38, the sex determination system was identified as XY, and the sex determination region was located at Chr38: 6,000,000-21,100,000 bp. A pair of sex-specific molecular markers has been identified based on a 21 bp female-specific insertion within the candidate sex-determining region. Additionally, SOAT, NPC1, PTGS2, FANCD1, and VAlRS were identified as candidate sex-determining genes through the screening of candidate genes and RT-qPCR validation analysis. These findings provide a robust foundation for investigating sex-determining mechanisms in crustaceans. Through the integration of genome-wide association studies (GWAS), selection signals, and transcriptome analysis, we identified, for the first time, genes associated with sex determination, growth, and immunity. These genes represent promising candidates for further functional studies and genetic improvement in Procambarus clarkii.

The objectives of this project were to: 1) Identify genetic markers linked to sex-determining genes in various experimental and commercial stocks of O. niloticusand O. aureus, as well as red tilapias; 2) Develop additional markers tightly linked to these sex determiners, and develop practical, non-destructive genetic tests for identifying genotypic sex in young tilapia; A third aim, to map sex modifier loci, was removed during budget negotiations at the start of the project. Background to the topic. A major obstacle to profitable farming of tilapia is the tendency of females to reproduce at a small size during the production cycle, diverting feed and other resources to a large population of small, unmarketable fish. Several approaches for producing all-male fingerlings have been tried, including interspecific hybridization, hormonal masculinization, and the use of YY-supermale broodstock. Each method has disadvantages that could be overcome with a better understanding of the genetic basis of sex determination in tilapia. The lack of sex-linked markers has been a major impediment in research and development of efficient monosex populations for tilapia culture. Major conclusions, solutions, achievements. We identified DNA markers linked to sex determining genes in six closely related species of tilapiine fishes. The mode of sex determination differed among species. In Oreochromis karongaeand Tilapia mariaethe sex-determining locus is on linkage group (LG) 3 and the female is heterogametic (WZ-ZZ system). In O. niloticusand T. zilliithe sex-determining locus is on LG1 and the male is heterogametic (XX-XY system). We have nearly identified the series of BAC clones that completely span the region. A more complex pattern was observed in O. aureus and O. mossambicus, in which markers on both LG1 and LG3 were associated with sex. We found evidence for sex-linked lethal effects on LG1, as well as interactions between loci in the two linkage groups. Comparison of genetic and physical maps demonstrated a broad region of recombination suppression harboring the sex-determining locus on LG3. We also mapped 29 genes that are considered putative regulators of sex determination. Amhand Dmrta2 mapped to separate QTL for sex determination on LG23. The other 27 genes mapped to various linkage groups, but none of them mapped to QTL for sex determination, so they were excluded as candidates for sex determination in these tilapia species. Implications, both scientific and agricultural. Phylogenetic analysis suggests that at least two transitions in the mode of sex determination have occurred in the evolution of tilapia species. This variation makes tilapias an excellent model system for studying the evolution of sex chromosomes in vertebrates. The genetic markers we have identified on LG1 in O. niloticusaccurately diagnose the phenotypic sex and are being used to develop monosex populations of tilapia, and eliminate the tedious steps of progeny testing to verify the genetic sex of broodstock animals.

Cottus rhenanus and Cottus perifretum have formed hybrid lineages and narrow hybrid zones that can be best explained through the action of natural selection. However, the underlying selective forces as well as their genomic targets are not well understood. This study identifies genomic regions in the parental species that cause hybrid incompatibilities and tests whether these manifest in a sex-specific manner to learn about processes that affect natural hybridization in Cottus. Interspecific F2 crosses were analyzed for 255 markers for genetic mapping and to detect transmission distortion as a sign for genetic incompatibilities. The Cottus map consists of 24 linkage groups with a total length of 1575.4 cM. A male heterogametic (XY) sex determination region was found on different linkage groups in the two parental species. Genetic incompatibilities were incomplete, varied among individuals and populations and were not associated with the heterogametic sex. The variance between populations and individuals makes it unlikely that there are species-specific incompatibility loci that could affect the gene pool of natural hybrids in a simple and predictable way. Conserved synteny with sequenced fish genomes permits to genetically study the Cottus genome through the transfer of genomic information from the model fish species. Homology relationships of candidate genomic regions in Cottus indicate that sex determination is not based on the same genomic regions found in other fish species. This suggests a fast evolutionary turnover of the genetic basis of sex determination that, together with the small size of the heterogametic regions, may contribute to the absence of fitness effects related to the Haldane's rule.

In this chapter the different categories of homomorphic and heteromorphic sex chromosomes, types of sex-determining mechanisms, known sex-linked genes, and data about sex-determining genes in the Amphibia have been compiled. Thorough cytogenetic analyses have shown that both XY/XX and ZW/ZZ sex chromosomes exist in the order Anura and Urodela. In some species quite unusual systems of sex determination have evolved (e.g. 0W-females/00-males or the co-existence of XY/XX and ZW/ZZ sex chromosomes within the same species). In the third order of the Amphibia, the Gymnophiona (or Apoda) there is still no information regarding any aspect of sex determination. Whereas most species of Anura and Urodela present undifferentiated, homomorphic sex chromosomes, there is also a considerable number of species in which an increasing structural complexity of the Y and W chromosomes exists. In various cases, the morphological differentiation of the sex chromosomes occurred as a result of quantitative and/or qualitative changes to the repetitive DNA sequences in the constitutive heterochromatin of the Y and W chromosomes. The greater the structural differences between the sex chromosomes, the lesser the extent of pairing in meiosis. No dosage compensation of the sex-linked genes in the somatic cells of the homogametic (XX or ZZ) individuals have been detected. The genes located to date on the amphibian sex chromosomes lead to the conclusion that there is no common ancestral or conserved sex-linkage group. In all amphibians, genetic sex determination (GSD) seems to operate, although environmental factors may influence sex determination and differentiation. Despite the accumulated evidence that GSD is operating in Anura and Urodela, there is little substantial information about how it functions. Although several DNA sequences homologous to the mammalian ZFY, SRY and SOX genes have been detected in the Anura or Urodela, none of these genes is an appropriate candidate to explain sex determination in these vertebrates.

Sex-determining regions have been identified in the Nile tilapia on linkage groups (LG) 1, 20 and 23, depending on the domesticated strains used. Sex determining studies on wild populations of this species are scarce. Previous work on two wild populations, from Lake Volta (Ghana) and from Lake Koka (Ethiopia), found the sex-determining region on LG23. These populations have a Y-specific tandem duplication containing two copies of the Anti-Müllerian Hormone amh gene (named amhY and amhΔY). Here, we performed a whole-genome short-reads analysis using male and female pools on a third wild population from Lake Hora (Ethiopia). We found no association of sex with LG23, and no duplication of the amh gene. Furthermore, we found no evidence of sex linkage on LG1 or on any other LGs. Long read whole genome sequencing of a male from each population confirmed the absence of a duplicated region on LG23 in the Lake Hora male. In contrast, long reads established the structure of the Y haplotype in Koka and Kpandu males and the order of the genes in the duplicated region. Phylogenies constructed on the nuclear and mitochondrial genomes, showed a closer relationship between the two Ethiopian populations compared to the Ghanaian population, implying an absence of the LG23Y sex-determination region in Lake Hora males. Our study supports the hypothesis that the amh region is not the sex-determining region in Hora males. The absence of the Y amh duplication in the Lake Hora population reflects a rapid change in sex determination within Nile tilapia populations. The genetic basis of sex determination in the Lake Hora population remains unknown.

Chapter 10 - Sex Determination in Insects

Moths and butterflies (Lepidoptera) are the largest group of organisms with female heterogamety and the sex chromosome system WZ/ZZ (female/male) or exceptionally Z0/ZZ. However, the genetic basis of sex determination in Lepidoptera remained unknown for a long time until the sex-determining pathway was discovered in 2014 in the silkworm Bombyx mori. In this species, the dominant W chromosome carries a Feminizer (Fem) gene encoding a precursor of a Fem piRNA that promotes femaleness by downregulating the expression of a Z-linked gene, Masculinizer (Masc). In the W chromosome absence, Masc promotes male development and controls dosage compensation. In the 10 years since this discovery, significant progress has been made in understanding the molecular mechanisms of sex determination in Lepidoptera. Data from recent studies discussed in this review suggest a conserved role for Masc in male sex determination and dosage compensation in the clade Ditrysia, which comprises the majority of Lepidoptera. Although the primary sex-determining signals are not conserved, the presence of feminizing piRNAs of different origins in distantly related species suggests convergent evolution of a similar mechanism of female sex determination. A unique exception is zygosity-based sex determination in the butterfly Bicyclus anynana, where the primary signal is the state of the hypervariable Masc gene. In other species with a dispensable W chromosome, such as the silkmoth Samia cynthia, sex is determined by the Z:A ratio, but a molecular mechanism is not yet known. Overall, the available data suggest considerable diversity in the upstream molecular mechanisms of sex determination in Lepidoptera.

The Lamprey Gonad

10 - Sex Determination in Insects

Amaranthus tuberculatus and Amaranthus palmeri are agronomically important weed species, both with stable dioecious reproductive systems. An understanding of the genetic basis of sex determination may lead to new methods of managing these troublesome weeds. Previous research identified genomic sequences associated with maleness in each species. Male-specific sequences were used to identify genomic regions in both species that are believed to contain sex-determining genes, i.e. the male-specific Y (MSY) region. These regions were compared to understand if sex determination is controlled via the same physiological pathway and if dioecy evolved independently. A contiguously assembled candidate MSY region identified in Amaranthuspalmeri is approximately 1.3Mb with 121 predicted gene models. In Amaranthustuberculatus, several contigs, with combined length of 4.6Mb and with 147 gene models, were identified as belonging to the MSY region. Synteny was not detected between the two species' candidate MSY regions but they shared two predicted genes. With lists of candidate genes for sex determination containing fewer than 200 in each species, future research can address whether sex determination is controlled via similar physiological pathways and whether dioecy has indeed evolved independently in these species.

BackgroundSex chromosomes are in some species largely undifferentiated (homomorphic) with restricted sex determination regions. Homomorphic but different sex chromosomes are found in the closely related genera Populus and Salix indicating flexible sex determination systems, ideal for studies of processes involved in sex chromosome evolution. We have performed genome-wide association studies of sex and analysed sex chromosomes in a population of 265 wild collected Salix viminalis accessions and studied the sex determining locus.ResultsA total of 19,592 markers were used in association analyses using both Fisher’s exact tests and a single-marker mixed linear model, which resulted in 48 and 41 sex-associated (SA) markers respectively. Across all 48 SA markers, females were much more often heterozygous than males, which is expected if females were the heterogametic sex. The majority of the SA markers were, based on positions in the S. purpurea genome, located on chromosome 15, previously demonstrated to be the sex chromosome. Interestingly, when mapping the genotyping-by-sequencing sequence tag harbouring the two SA markers with the highest significance to the S. viminalis genomic scaffolds, five regions of very high similarity were found: three on a scaffold that represents a part of chromosome 15, one on a scaffold that represents a part of chromosome 9 and one on a scaffold not anchored to the genome. Based on segregation differences of the alleles at the two marker positions and on differences in PCR amplification between females and males we conclude that females had multiple copies of this DNA fragment (chromosome 9 and 15), whereas males only had one (chromosome 9). We therefore postulate that the female specific sequences have been copied from chromosome 9 and inserted on chromosome 15, subsequently developing into a hemizygous W chromosome linked region.ConclusionsOur results support that sex determination in S. viminalis is controlled by one locus on chromosome 15. The segregation patterns observed at the SA markers furthermore confirm that S. viminalis females are the heterogametic sex. We also identified a translocation from chromosome 9 to the W chromosome.

Sex determination pathways regulate male and female-specific development and differentiation and offer potential targets for genetic pest management methods. Insect sex determination pathways are comprised of primary signals, relay genes and terminal genes. Primary signals of coleopteran, dipteran, hymenopteran and lepidopteran species are highly diverse and regulate the sex-specific splicing of relay genes based on the primary signal dosage, amino acid composition or the interaction with paternally inherited genes. In coleopterans, hymenopterans and some dipterans, relay genes are Transformer orthologs from the serine-arginine protein family that regulate sex-specific splicing of the terminal genes. Alternative genes regulate the splicing of the terminal genes in dipterans that lack Transformer orthologs and lepidopterans. Doublesex and Fruitless orthologs are the terminal genes. Doublesex and Fruitless orthologs are highly conserved zinc-finger proteins that regulate the expression of downstream proteins influencing physical traits and courtship behaviours in a sex-specific manner. Genetic pest management methods can use different mechanisms to exploit or disrupt female-specific regions of different sex determination genes. Female-specific regions of sex determination genes can be exploited to produce a lethal gene only in females or disrupted to impede female development or fertility. Reducing the number of fertile females in pest populations creates a male-biased sex ratio and eventually leads to the local elimination of the pest population. Knowledge on the genetic basis of sex determination is important to enable these sex determination pathways to be exploited for genetic pest management.