Genetic risk involved in stock enhancement of fish having environmental sex determination

Environmental sex determination is reported in various fish species, including some fishes subject to stock enhancement. We studied the influence of stock enhancement on the sex-determining system of fish with both genotypic and environmental sex determination. We constructed and analyzed recurrence formula models for the dynamics of gene frequency in the male heterogametic (XX female and XY male) sex-determining system of the Japanese flounder (Paralichthys olivaceus). In this species, an XX individual can develop as a phenotypic male (sex-reversed male) depending on the conditions experienced by a juvenile. We show that the release of sex-reversed males may result in the extinction of the Y gene that determines sex. We also studied how the risk depends on the kind of hatchery broodstock used (e.g., fish collected from the wild or fish from a lineage established in a hatchery), sex-reversal rates in the hatchery and in the wild, and the relative contribution of released fish to reproduction in the wild. We propose some ways to reduce the risk. Received: October 10, 2001 / Accepted: December 17, 2001     

Genetic and Physical Mapping of Sex-Linked AFLP Markers in Nile Tilapia (Oreochromis niloticus)

Identification of the sex-determining genes of the Nile tilapia (Oreochromis niloticus) has important implications for commercial aquaculture. We previously identified an XX/XY sex-determining locus in this species within a 10-cM interval between markers GM201 and UNH995 on linkage group one (LG1). In order to refine this region, we developed new AFLP markers using bulked segregant analysis of the mapping families. We identified three AFLP markers that showed a sex-specific pattern of segregation. All three mapped near, but just outside, the previously identified sex-determining region on LG1. Hybridization of BAC clones containing these markers to chromosome spreads confirmed that the XX/XY sex-determining locus is on one of the small chromosomes in O. niloticus.     

Isolation and Physical Mapping of Sex-Linked AFLP Markers in Nile Tilapia (Oreochromis niloticus L.)

Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.     

Accumulation of Y-specific satellite DNAs during the evolution of <Emphasis Type="Italic">Rumex acetosa</Emphasis> sex chromosomes

The study of the molecular structure of young heteromorphic sex chromosomes of plants has shed light on the evolutionary forces that control the differentiation of the X and Y during the earlier stages of their evolution. We have used the model plant Rumex acetosa, a dioecious species with multiple sex chromosomes, 2n = 12 + XX female and 2n = 12 + XY₁Y₂ male, to analyse the significance of repetitive DNA accumulation during the differentiation of the Y. A bulk segregant analysis (BSA) approach allowed us to identify and isolate random amplified polymorphic DNA (RAPD) markers linked to the sex chromosomes. From a total of 86 RAPD markers in the parents, 6 markers were found to be linked to the Ys and 1 to the X. Two of the Y-linked markers represent two AT-rich satellite DNAs (satDNAs), named RAYSII and RAYSIII, that share about 80% homology, as well as with RAYSI, another satDNA of R. acetosa. Fluorescent in situ hybridisation demonstrated that RAYSII is specific for Y₁, whilst RAYSIII is located in different clusters along Y₁ and Y₂. The two satDNAs were only detected in the genome of the dioecious species with XX/XY₁Y₂ multiple sex chromosome systems in the subgenus Acetosa, but were absent from other dioecious species with an XX/XY system of the subgenera Acetosa or Acetosella, as well as in gynodioecious or hermaphrodite species of the subgenera Acetosa, Rumex and Platypodium. Phylogenetic analysis with different cloned monomers of RAYSII and RAYSIII from both R. acetosa and R. papillaris indicate that these two satDNAs are completely separated from each other, and from RAYSI, in both species. The three Y-specific satDNAs, however, evolved from an ancestral satDNA with repeating units of 120 bp, through intermediate satDNAs of 360 bp. The data therefore support the idea that Y-chromosome differentiation and heterochromatinisation in the Rumex species having a multiple sex chromosome system have occurred by different amplification events from a common ancestral satDNA. Since dioecious species with multiple XX/XY₁Y₂ sex chromosome systems of the section Acetosa appear to have evolved from dioecious species with an XX/XY system, the amplification of tandemly repetitive elements in the Ys of the section Acetosa is a recent evolutionary process that has contributed to an increase in the size and differentiation of the already non-recombining Y chromosomes.     

Evolution of ZZ/ZW and XX/XY sex-determination systems in the closely related medaka species, <Emphasis Type="Italic">Oryzias hubbsi</Emphasis> and <Emphasis Type="Italic">O. dancena</Emphasis>

A DM-domain gene on the Y chromosome was identified as the sex-determining gene in the medaka, Oryzias latipes, and named DMY (also known as dmrt1bY). However, this gene is absent in most Oryzias fishes, suggesting that closely related species have another sex-determining gene. In fact, it has been demonstrated that the Y chromosome in O. dancena is not homologous to that in O. latipes, whereas both species have an XX/XY sex-determination system. Through a progeny test of sex-reversed fish and a linkage analysis of isolated sex-linked DNA markers, we show that O. hubbsi, which is one of the most closely related species to O. dancena, has a ZZ/ZW system. In addition, genetic and fluorescence in situ hybridization mapping of the sex-linked markers revealed that sex chromosomes in O. hubbsi and O. dancena are not homologous, indicating different origins of these ZW and XY sex chromosomes. Furthermore, we found that O. hubbsi has morphologically heteromorphic sex chromosomes, in which the W chromosome has 4,6-diamidino-2-phenylindole (DAPI)-positive heterochromatin blocks and is larger than the Z chromosome, although such differentiated sex chromosomes have not been observed in other Oryzias species. These findings suggest that a variety of sex-determining mechanisms and sex chromosomes have evolved in Oryzias.     

Mapping and Validation of the Major Sex-Determining Region in Nile Tilapia (Oreochromis niloticus L.) Using RAD Sequencing

Sex in Oreochromis niloticus (Nile tilapia) is principally determined by an XX/XY locus but other genetic and environmental factors also influence sex ratio. Restriction Associated DNA (RAD) sequencing was used in two families derived from crossing XY males with females from an isogenic clonal line, in order to identify Single Nucleotide Polymorphisms (SNPs) and map the sex-determining region(s). We constructed a linkage map with 3,802 SNPs, which corresponded to 3,280 informative markers, and identified a major sex-determining region on linkage group 1, explaining nearly 96% of the phenotypic variance. This sex-determining region was mapped in a 2 cM interval, corresponding to approximately 1.2 Mb in the O. niloticus draft genome. In order to validate this, a diverse family (4 families; 96 individuals in total) and population (40 broodstock individuals) test panel were genotyped for five of the SNPs showing the highest association with phenotypic sex. From the expanded data set, SNPs Oni23063 and Oni28137 showed the highest association, which persisted both in the case of family and population data. Across the entire dataset all females were found to be homozygous for these two SNPs. Males were heterozygous, with the exception of five individuals in the population and two in the family dataset. These fish possessed the homozygous genotype expected of females. Progeny sex ratios (over 95% females) from two of the males with the “female” genotype indicated that they were neomales (XX males). Sex reversal induced by elevated temperature during sexual differentiation also resulted in phenotypic males with the “female” genotype. This study narrows down the region containing the main sex-determining locus, and provides genetic markers tightly linked to this locus, with an association that persisted across the population. These markers will be of use in refining the production of genetically male O. niloticus for aquaculture.     

Identification of the sex-determining locus in the Thai medaka, Oryzias minutillus

A sex-determining gene, DMY, which is comparable to the SRY gene in mammals, has been identified in the medaka, Oryzias latipes. Although Oryzias curvinotus, a closely related species to O. latipes also has DMY, this gene has not been found in other Oryzias fishes. It has recently been demonstrated that the sex chromosomes of Oryzias dancena and Oryzias hubbsi differ from those of O. latipes and these species have XX/XY and ZZ/ZW systems, respectively. This may suggest that Oryzias species have evolved different sex-determining genes on different sex chromosomes. In the present study, we investigated the sex determination mechanism in Oryzias minutillus, which is closely related to O. dancena and O. hubbsi. Linkage analysis using 14 isolated sex-linked DNA markers showed that this species has an XX/XY sex determination system. These sex-linked markers were located on linkage group 8 of O. latipes, suggesting that the sex chromosomes of O. minutillus are homologous to the autosomes of other Oryzias species. Furthermore, fluorescence in situ hybridization using a tightly sex-linked marker demonstrated that the XY sex chromosomes of O. minutillus and O. dancena were not homologous. These findings provide additional evidence for independent origins of sex chromosomes and sex-determining genes in these closely related species.     

Characterization of Pisrt1/Foxl2 in Ellobius lutescens and exclusion as sex-determining genes

The rodent Ellobius lutescens is an exceptional mammal which determines male sex constitutively without the SRY gene and, therefore, may serve as an animal model for human 46,XX female-to-male sex reversal. It was suggested that other factors of the network of sex-determining genes determine maleness in these animals. However, some sex-determining genes like SOX9 and SF1 have already been excluded by segregation analysis as primary sex-determining factors in E. lutescens. In this work, we have cloned and characterized two genes of the PIS (polled intersex syndrome) gene interval, which were reported as candidates in female-to-male sex reversal in hornless goats recently. The genes Foxl2 and Pisrt1 from that interval were identified in E. lutescens DNA and mapped to Chromosome 8. We have excluded linkage of Foxl2 and Pisrt1 loci with the sex of the animals. Hence, the involvement of this gene region in sex determination may be specific for goats and is not a general mechanism of XX sex reversal or XX male sex determination.The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession number AY623815.     

Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations

The medaka, Oryzias latipes, has an XX/XY sex determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a prime candidate for the sex-determining gene. Furthermore, the crucial role of DMY during male development was established by studying two wild-derived XY female mutants. In this study, to find new DMY and sex-determination related gene mutations, we conducted a broad survey of the genotypic sex (DMY-negative or DMY-positive) of wild fish. We examined 2274 wild-caught fish from 40 localities throughout Japan, and 730 fish from 69 wild stocks from Japan, Korea, China, and Taiwan. The phenotypic sex type agreed with the genotypic sex of most fish, while 26 DMY-positive (XY) females and 15 DMY-negative (XX) males were found from 13 and 8 localities, respectively. Sixteen XY sex-reversals from 11 localities were mated with XY males of inbred strains, and the genotypic and phenotypic sexes of the F(1) progeny were analyzed. All these XY sex-reversals produced XY females in the F(1) generation, and all F(1) XY females had the maternal Y chromosome. These results show that DMY is a common sex-determining gene in wild populations of O. latipes and that all XY sex-reversals investigated had a DMY or DMY-linked gene mutation.     

Heterochromosomes in Mytilicola intestinalis Steuer (Copepoda)

The male and female diploid complements in Mytilicola are formed both by 22 chromosomes. Two different somatic metaphase figures are found: with two minute-size chromosomes and with three minute-size chromosomes. The study of meiosis has shown that the female sex is heterogametic. The chromosome complement is 10_II+XX in the male and 10_II+XY in the female. A bivalent formed by two different-length homologues, is to be seen in the female meiosis at pachytene; it shows, sometimes, a light positive hetero-pycnosis.     

Sex segregation ratio and gender expression in the genus Actinidia

The sex segregation ratio was checked in bi-parental families of Actinidia deliciosa (2n=6x=174) obtained by crossing four females (A12, Mo3, Br4, Hw1) with two males (T2, M1) and one fruiting male (M3h, subandroecious) according to a factorial mating design. The M3h fruiting male was also selfed. The sex ratio was checked in maternal families of A. kolomikta (2n=2x) and A. chinensis (2n=2x) as well as in A. deliciosa. Seedlings of both diploid species took 3–4 years to progress beyond juvenility, whereas a noticeable number of seedlings from biparental crosses of A. deliciosa involving A12 and Hw1 as seed parents were still non-flowering after seven growing seasons. Open-pollinated families of both diploid and hexaploid species as well as most families from biparental crosses showed a sex segregation ratio approaching 11. Subandroecious lines with different degrees of ovary and pistil development appeared in proportions of 0–4.2%, depending on the cross, but only 6 of the 2567 male vines checked were capable of setting fruit. No case of self-fertility or apomixis was detected among 1866 bagged female vines. Selfed M3h progenies gave only female and male phenotypes in a ratio of 1 female to 3 males. No off-type vines were found among these progenies. The same disomic sex segregation ratio seems to be operating at different ploidy levels in the genus Actinidia. Since selfed fruiting males produced both female and male individuals, the male sex appears to be the heterogametic one. Such evidence indicates that a monofactorial system based on one or more linked genes or on an X/Y chromosome set must be controlling sex expression. How a monofactorial sex-determining mechanism could operate in polyploids to give a 11 female: male ratio is discussed. Minor modifying gene(s) seem to be responsible for the feminization of males, and their expression appears enhanced by environmental conditions. Masculinizing gene(s) seem to be lacking in female genotypes.     

Cytogenetic variability in genus odontocheila (Coleoptera,Cicindelidae): karyotypes,C-banding,NORs and localisation of ribosomal genes of O. confusa and O. nodicornis

Two species of Odontocheila, O. confusa and O. nodicornis, from the Neotropical Region were studied regarding their karyotypes, localisation and activity of ribosomal genes and C-banding. The species, although belonging to the same genus, have quite distinct karyotypes. O. confusa has 10 pairs of autosomes and a single sex chromosome mechanism of the XY/XX type, thus a diploid value of 2n = 22 in males and females. One aneuploid male with a diploid number of 2n = 20 and one male with three B chromosomes were found in a total of eight males studied. O. nodicornis has 17 autosomal pairs and also a single chromosome system but of the X0/XX type, thus a diploid value of 2n = 35 in males and 2n = 36 in females. Fluorescence in situ hybridisation (FISH) revealed the presence of rDNA clusters in two autosomes in both species in mitotic and meiotic figures. Silver staining of male interphase nuclei confirmed the FISH results and showed that all rDNA genes were active. C-banding analysis revealed the presence of constitutive heterochromatin in the centromeres of all chromosomes in the two species plus two pairs in O. nodicornis with terminal positive C-bands. These results are discussed from the cytogenetic and evolutionary point of view.     

Genetics of dioecy and causal sex chromosomes in plants

Dioecy (separate male and female individuals) ensures outcrossing and is more prevalent in animals than in plants. Although it is common in bryophytes and gymnosperms, only 5% of angiosperms are dioecious. In dioecious higher plants, flowers borne on male and female individuals are, respectively deficient in functional gynoecium and roecium. Dioecy is inherited via three sex chromosome systems: XX/XY, XX/X0 and WZ/ZZ, such that XX or WZ is female and XY, X0 or ZZ are males. The XX/XY system generates the rarer XX/X0 and WZ/ZZ systems. An autosome pair begets XY chromosomes. A recessive loss-of-androecium mutation (ana) creates X chromosome and a dominant gynoecium-suppressing (GYS) mutation creates Y chromosome. The ana/ANA and gys/GYS loci are in the sex-determining region (SDR) of the XY pair. Accumulation of inversions, deleterious mutations and repeat elements, especially transposons, in the SDR of Y suppresses recombination between X and Y in SDR, making Y labile and increasingly degenerate and heteromorphic from X. Continued recombination between X and Y in their pseudoautosomal region located at the ends of chromosomal arms allows survival of the degenerated Y and of the species. Dioecy is presumably a component of the evolutionary cycle for the origin of new species. Inbred hermaphrodite species assume dioecy. Later they suffer degenerate-Y-led population regression. Cross-hybridization between such extinguishing species and heterologous species, followed by genome duplication of segregants from hybrids, give rise to new species.     

Genetic and physical maps around the sex-determining <Emphasis Type="Italic">M</Emphasis>-locus of the dioecious plant asparagus

Asparagus officinalis L. is a dioecious plant. A region called the M-locus located on a pair of homomorphic sex chromosomes controls the sexual dimorphism in asparagus. The aim of this work was to clone the region determining sex in asparagus from its position in the genome. The structure of the region encompassing M should be investigated and compared to the sex-determining regions in other dioecious model species. To establish an improved basis for physical mapping, a high-resolution genetic map was enriched with AFLP markers closely linked to the target locus by carrying out a bulked segregant analysis. By screening a BAC library with AFLP- and STS-markers followed by chromosome walking, a physical map with eight contigs could be established. However, the gaps between the contigs could not be closed due to a plethora of repetitive elements. Surprisingly, two of the contigs on one side of the M-locus did not overlap although they have been established with two markers, which mapped in a distance as low as 0.25 cM flanking the sex locus. Thus, the clustering of the markers indicates a reduced recombination frequency within the M-region. On the opposite side of the M-locus, a contig was mapped in a distance of 0.38 cM. Four closely linked BAC clones were partially sequenced and 64 putative ORFs were identified. Interestingly, only 25% of the ORFs showed sequence similarity to known proteins and ESTs. In addition, an accumulation of repetitive sequences and a low gene density was revealed in the sex-determining region of asparagus. Molecular cytogenetic and sequence analysis of BACs flanking the M-locus indicate that the BACs contain highly repetitive sequences that localize to centromeric and pericentromeric locations on all asparagus chromosomes, which hindered the localization of the M-locus to the single pair of sex chromosomes. We speculate that dioecious Silene, papaya and Asparagus species may represent three stages in the evolution of XX, XY sex determination systems. Given that asparagus still rarely produces hermaphroditic flowers and has homomorphic sex chromosomes, this species may be an ideal system to further investigates early sex chromosome evolution and the origins of dioecy.     

黑斑蛙的减数分裂研究

本文研究了黑斑蛙的减数分裂,发现其性染色体所形成的性二价体主要呈末端与末端联接,浓缩期占79.6％,中期Ⅰ占75％,这进一步证明黑斑蛙确实存在XY型性别决定机制,这种XY型性染色体虽形态相同,但已发生了质的分化,可能是同型异质。黑斑蛙的性染色体并不形成性泡,少数二价体有中间交叉。     

Increase of cortisol levels after temperature stress activates dmrt1a causing female‐to‐male sex reversal and reduced germ cell number in medaka

In vertebrates, there is accumulating evidence that environmental factors as triggers for sex determination and genetic sex determination are not two opposing alternatives but that a continuum of mechanisms bridge those extremes. One prominent example is the model fish species Oryzias latipes which has a stable XX/XY genetic sex determination system, but still responds to environmental cues, where high temperatures lead to female‐to‐male sex reversal. However, the mechanisms behind are still unknown. We show that high temperatures increase primordial germ cells (PGC) numbers before they reach the genital ridge, which, in turn, regulates the germ cell proliferation. Complete ablation of PGCs led to XX males with germ cell less testis, whereas experimentally increased PGC numbers did not reverse XY genotypes to female. For the underlying molecular mechanism, we provide support for the explanation that activation of the dmrt1a gene by cortisol during early development of XX embryos enables this autosomal gene to take over the role of the male determining Y‐chromosomal dmrt1bY.     

Sex-determining mechanisms in animals

Biological mechanisms leading to the development of males and females are extremely varied. In the XX/XY system, the male has an unequal pair of chromosomes, while in the ZZ/ZW system, the unequal pair is in the female. Sex can also be determined by the temperature of incubation. Recent research has focused on the identification of sex-determining genes, culminating in the demonstration that the Sry gene on the Y chromosome of mice can induce male development in genetically female XX mouse embryos. Nevertheless, the occurrence of phenotypes in apparent contrast to the genotype suggests that the genetic male/female switch is not simple, and there may be common features linking different sex-determining mechanisms. There is increasing evidence that such a link may be provided by the induction of growth differences, and that the primary sex difference may result from the distinction between fast versus slow growth.     

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.