Sex-linked DNA Markers Research Articles

Screening and characterization of sex-linked DNA markers in Mozambique tilapia (Oreochromis mossambicus)

The Mozambique tilapia (Oreochromis mossambicus) is an important aquaculture fish. It shows sexual dimorphism in growth, with males growing much faster than females. Thus, production of all-male fingerlings of Mozambique tilapia would improve aquaculture efficiency. However, the lack of accurate sex-specific markers has hampered the mono-sex breeding and the study of the molecular mechanisms of sex determination in this species. Recently, we performed genome sequencing and re-sequencing of female and male Mozambique tilapia and identified the sex chromosome and sex determining region. In the present study, two sex-linked SNP markers were identified and validated. Male specific contigs were assembled from the unmapped reads after aligning male re-sequencing data against the published XX genome. Based on these male-specific contigs, three sex-linked DNA markers, Marker-1, Marker-2 and Marker-3, were developed and correctly identified the genetic sex of 16 fish. The universality of these three male-specific markers was then tested in a panel of 144 individuals, which showed a discrepancy of 5.5% (8/144) between genotypic sex and phenotypic sex. In conclusion, we developed three sex-linked markers to identify different sex genotypes in Mozambique tilapia. This study lays a foundation for the establishment of MAS-GMT (marker-assisted selection of genetically male tilapias) technique and sex-controlled breeding of Mozambique tilapia, as well as for the identification of the sex determining gene.

A high-density linkage map and sex-linked markers for the Amazon Tambaqui Colossoma macropomum

BackgroundTambaqui (Colossoma macropomum, Cuvier, 1818) is the most economically important native freshwater fish species in Brazil. It can reach a total length of over 1 m and a weight of over 40 kg. The species displays a clear sex dimorphism in growth performance, with females reaching larger sizes at harvest. In aquaculture, the production of monosex populations in selective breeding programmes has been therefore identified as a key priority.ResultsIn the present study, a genetic linkage map was generated by double digest restriction-site associated DNA (ddRAD) sequencing from 248 individuals sampled from two F1 families. The map was constructed using 14,805 informative SNPs and spanned 27 linkage groups. From this, the tambaqui draft genome was improved, by ordering the scaffolds into chromosomes, and sex-linked markers were identified. A total of 235 markers on linkage group 26 showed a significant association with the phenotypic sex, supporting an XX/XY sex determination system in the species. The four most informative sex-linked markers were validated on another 206 sexed individuals, demonstrating an accuracy in predicting sex ranging from 90.0 to 96.7%.ConclusionsThe genetic mapping and novel sex-linked DNA markers identified and validated offer new tools for rapid progeny sexing, thus supporting the development of monosex female production in the industry while also supporting breeding programmes of the species.

Effects of synthetic sex steroid hormone exposures on gonadal sex differentiation and dynamics of a male-related gene, Gonadal soma-derived factor (Gsdf) and an estrogen up-regulated gene, Choriogenine-H (ChgH) gene expression in the euryhaline Javafish medaka, Oryzias javanicus, based on genetic sexes

To clarify the basal aspects of sexual development in Javafish medaka, Oryzias javanicus (ZZ/ZW), a model marine species for ecotoxicity testing, we examined the details of gonadal sex differentiation and exogenous sex hormone-dependent sex reversals using genetic sex-linked DNA markers. Sex differences in germ cell numbers were observed at 5 days post hatching (dph), in which there was a significant increase in the germ cells of ZW. In ZW, diplotene oocytes and the ovarian cavity appeared at approximately 10, and 30 dph, respectively. In ZZ, spermatogonial proliferation was observed at approximately 20 dph. A ZZ-dominant expression of Gonadal soma-derived factor (Gsdf) mRNA was detected before hatching. The exposure of embryos to 17α-ethinylestradiol (EE2; 0.1, 1, 10 ng/mL) did not cause sex reversals in most cases. However, EE2 exposures led to significant Choriogenin-H (ChgH) mRNA expression, an estrogen up-regulated gene, in all fry; these exposures did not suppress Gsdf expression in ZZ fry. The exposure of embryos to 17α-methyltestosterone (MT; 0.1, 1, 10 ng/mL) caused sex reversals but only at low frequencies in ZW and ZZ fish. Although the 10 ng/mL MT exposure was accompanied by induction of significant Gsdf expression in ZW fry, induction of ChgH expression was also observed in several fry. Together, the present study indicates for the first time that male-dominant sexual dimorphic expression of Gsdf precedes the first morphological sex difference, i.e., the sex difference in germ cell number, and results strongly suggest that exogenous sex hormone-dependent sex reversal is not induced easily in O. javanicus.

Screening and characterization of sex-linked DNA markers and marker-assisted selection in blue tilapia (Oreochromis aureus)

The blue tilapia (Oreochromis aureus) is an important cultured fish worldwide, and exhibits sexual size dimorphism in favor of males, and therefore, mono-sex breeding is of great value for aquaculture. However, the lack of accurate sex-linked markers for blue tilapia has hampered the mono-sex culture in aquaculture, as well as study of sex determination in this species. In this study, WW super-females were bred and selected. Then, structure variations between WW and ZZ in sex determining region were identified from blue tilapia by comparative analysis of ZZ/ZW genome re-sequencing data from 5 different strains and ZZ/WW reference genome sequences. Based on these structure variations, 30 pairs of primers were designed to screen sex-linked markers. After two rounds of PCR screening, three markers, named as Marker-1, Marker-2 and Marker-3, were obtained to identify the genetic sex of blue tilapia. Marker-2 and Marker-3 were combined together for multiplex PCR amplification to distinguish ZZ, ZW and WW fish to make up for the lack of a co-dominant marker. In conclusion, three sex-linked markers were isolated and used for identification of different genotypes and establishment of MAS-GMT (marker-assisted selection of genetically male tilapias) technique in blue tilapia. It is also of great significance for isolation of sex determining gene, elucidation of the evolution of sex chromosomes and the transition of sex determination system.

Screening and characterization of sex-linked DNA markers and marker-assisted selection in the Southern catfish (Silurus meridionalis)

The Southern catfish (Silurus meridionalis), which exhibits a sexual dimorphism with female growth faster than male, is an economically important fish in China. However, the lack of a rapid and accurate sex specific marker has hampered the application of sex control in the breeding process, as well as the investigations of sex-determination in this species. In this study, by comparative analysis of next generation sequencing (NGS) and re-sequencing data from male and female samples, 38 candidate Y chromosome-specific sequences were screened out, their sex-specificity were validated in Southern catfish through PCR amplification. Finally, 8 sex-linked DNA markers were obtained to distinguish the genetic sex of Southern catfish. Importantly, one of those markers (Marker-8) could serve as a highly specific and robust co-dominant marker for genetic sex identification of XX, XY and YY individuals. Subsequently, a marker-assisted selection (MAS) technique was developed for the mass production of XX all-females for aquaculture and YY supermales for basic research especially in sex determination. In conclusion, our current data suggest an XY sex determination system in the Southern catfish. Sex-linked DNA markers obtained in this study will have potential applications in the isolation of sex determine gene, elucidation of the sex determination mechanism, and accelerate the development of sex control breeding in Southern catfish.

Efficacy of gene-based markers associated with sex expression in papaya

The papaya (Carica papaya L.) is a polygamous plant, has three types of sex forms, viz. male, female and hermaphrodite. Efforts were made to identify sex expression and validation of gene-linked molecular markers associated with it. Molecular markers (ISSR and SCAR) were employed to distinguish male, female and hermaphrodite sex forms in seedlings and well differentiated adult plant types. The important markers which have been found linked with sex forms in papaya were used for validation in population of six papaya genotypes. Total 10 sex linked DNA markers (9 SCAR and 1 ISSR) were employed for validation of sexes in 6 papaya genotypes (dioceious and gynodioecious). Out of 10 only five (T12, W11, SCAR SDSP, C09/20 and GACA4) were amplified for the sex expression. However, five SCAR (SCARp, SCARpm, T1, STS 807 and STS 831) marker did not get amplified with the all sex genotypes. Of the two marker systems tested, SCAR markers were most consistent. Markers, namely, T12, CFW+CRV and W11 were most informative to predict cent per cent sex forms. These markers can be used by the breeders and commercial papaya growers to identify desired sex types at seedling stage in nursery for establishing a productive plantation.

Identification of sex-linked DNA markers by AFLP in Portunus trituberculatus.

To investigate the mechanism of sex determination of crabs, amplified fragment-length polymorphism DNA analysis was applied to identify sex-specific markers for Portunus trituberculatus. Eleven pairs of primers were used to amplify DNA isolated from male and female crabs. A total of 481 and 499 fragments were amplified for females and males, respectively, and the ratios of polymorphic fragments were 20.59-65.52% and 27.27-62.22%, respectively. Distribution difference values between female and male groups for 10 polymorphisms were greater than 40%; Cluster analysis revealed that Nei's genetic distances for 57 polymorphisms clustered according to sex. The results indicate that a difference at the DNA level exists between female and male crabs and provide data for further study on sex determination.

Conversion of a male-specific RAPD marker into an STS marker in Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is an economically important plant. This species is dioecious, and male plants are considered to be more desirable than females due to their higher yields. To reduce the time required for asparagus breeding, molecular marker techniques have been employed to identify sex-linked DNA markers. In the present study, we converted the male-specific random amplified polymorphic DNA marker T35R54-1600 into a sequence tagged site marker. We cloned a male-specific DNA fragment amplified with the T35R54 primer and determined the sequence of the fragment. The size of T35R54-1600 is 1,586 bp, and this fragment is not homologous to known sex-linked BAC sequences, indicating that this fragment is a new sex-linked region. Within this fragment, we designed the primer pair ‘MSSTS710’ to amplify a 710 bp region. This marker could be used to identify the sex of eight cultivars of A. officinalis: ‘Mary Washington 500W’, ‘UC157’, ‘Harumachi Green’, ‘Super Welcome’, ‘F4’, ‘Pacific 2000’, ‘F2’ and ‘Backlim’. We also analyzed the applicability of this marker to two dioecious Asparagus species, A. schoberioides and A. kiusianus, which are cross-compatible with A. officinalis. Although male-specific DNA fragments of two dioecious Asparagus species, A. schoberioides and A. kiusianus, were generated using the existing male-specific marker Asp1T7sp, no amplicon was obtained using the MSSTS710 marker. Since MSSTS710 can be employed for sex identification only in A. officinalis and not in closely related Asparagus species, the DNA region around the MSSTS710 marker must be variable among Asparagus species.

Different origins of ZZ/ZW sex chromosomes in closely related medaka fishes, Oryzias javanicus and O. hubbsi

Although the sex-determining gene DMY has been identified on the Y chromosome in the medaka, Oryzias latipes, this gene is absent in most Oryzias species. Recent comparative studies have demonstrated that, in the javanicus species group, Oryzias dancena and Oryzias minutillus have an XX/XY sex determination system, while Oryzias hubbsi has a ZZ/ZW system. Furthermore, sex chromosomes were not homologous in these species. Here, we investigated the sex determination mechanism in Oryzias javanicus, another species in the javanicus group. Linkage analysis of isolated sex-linked DNA markers showed that this species has a ZZ/ZW sex determination system. The sex-linkage map showed a conserved synteny to the linkage group 16 of O. latipes, suggesting that the sex chromosomes in O. javanicus are not homologous to those in any other Oryzias species. Fluorescence in-situ hybridization analysis confirmed that the ZW sex chromosomes of O. javanicus and O. hubbsi are not homologous, and showed that O. javanicus has the morphologically heteromorphic sex chromosomes, in which the W chromosome has 4,6-diamino-2-phenylindole-positive heterochromatin at the centromere. These findings suggest the repeated evolution of new sex chromosomes from autosomes in Oryzias, probably through the emergence of new sex-determining genes.

Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of Gracilaria changii (Rhodophyta)

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males.

Evolution of ZZ/ZW and XX/XY sex-determination systems in the closely related medaka species, Oryzias hubbsi and O. dancena

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.

Evolution of Different Y Chromosomes in Two Medaka Species,Oryzias dancenaandO. latipes

Although the sex-determining gene DMY has been identified on the Y chromosome in the medaka (Oryzias latipes), this gene is absent in most Oryzias species, suggesting that closely related species have different sex-determining genes. Here, we investigated the sex-determination mechanism in O. dancena, which does not possess the DMY gene. Since heteromorphic sex chromosomes have not been reported in this species, a progeny test of sex-reversed individuals produced by hormone treatment was performed. Sex-reversed males yielded all-female progeny, indicating that O. dancena has an XX/XY sex-determination system. To uncover the cryptic sex chromosomes, sex-linked DNA markers were screened using expressed sequence tags (ESTs) established in O. latipes. Linkage analysis of isolated sex-linked ESTs showed a conserved synteny between the sex chromosomes in O. dancena and an autosome in O. latipes. Fluorescence in situ hybridization (FISH) analysis of these markers confirmed that sex chromosomes of these species are not homologous. These findings strongly suggest an independent origin of sex chromosomes in O. dancena and O. latipes. Further analysis of the sex-determining region in O. dancena should provide crucial insights into the evolution of sex-determination mechanisms in vertebrates.

Species and gender differentiation between and among domestic and wild animals using mitochondrial and sex-linked DNA markers

In many African countries accurate and reliable identification of poached wildlife products like carcasses or meat presents a big problem when morphological characters such as skin hair or bones are missing. We describe a molecular based approach that has a potential of serving as a forensic tool in game meat identification in Africa. A mitochondial DNA marker (mt700) and one restriction enzyme, Rsa1 were used in the PCR-RFLP species identification of game meat obtained from two National Parks in Tanzania. Species-specific reference DNA fragment patterns were obtained using fresh meat from ten wildlife and four domesticated species. All species except the zebra, produced unique monomorphic RFLP patterns. Collectively, these patterns demonstrate the potential ability of genetic techniques for discriminating between and among wildlife and domestic species. The reference PCR-RFLP fragments enabled species identification of about 79% of unknown meat samples. In addition, sex was also assigned to all of the samples following successful amplification of gender-specific, SRY and ZFY/X, chromosomal domains. Although the present study has been conducted on a limited range both in numbers and genetic diversity of wildlife species present in Africa, the results demonstrate the potential usefulness of the DNA approach in wildlife forensics in the continent.

Detection of ayu sex-linked DNA markers using homologous clones

ABSTRACT: In order to detect sex-linked DNA markers, linkage analysis was performed in each of three ayu half-sib families using 32 amplified fragment length polymorphism primer combinations, four microsatellite DNA primer sets and phenotypic sex (male). In each family the sex-determining locus appeared in a distal position on one of the linkage groups in which the D7-141* marker was strongly associated with male gender. The D7-141* marker was amplified in all of the male ayu (100%), and 8.7% of the female ayu. The D7-141* marker was detected in all male ayu at a level similar to that in the 40 wild individuals. These results suggest that the D7-141* marker is a male-specific marker linked with the sex-determining locus of the male in amphidromous ayu.

Y-chromosomal DNA markers for discrimination of chemical substance and effluent effects on sexual differentiation in salmon.

Chinook salmon alevins were exposed during their labile period for sex differentiation to different concentrations of bleached kraft mill effluent (BKME), primary sewage effluent, secondary sewage effluent (SE), 17ss-estradiol, testosterone, and nonylphenol. After exposure for 29 days post hatching (DPH), fish were allowed to grow until 103 and 179 DPH, at which time their genetic sex was determined using Y-chromosomal DNA markers and their gonadal sex was determined by histology. Independent of treatment, all fish identified as genetic females (XX) in these experiments possessed normal female gonads. Only the highest dose of some treatments affected the development of gonads in genetic XY males. At 103 DPH, some genetic males treated with 1 micro g estradiol/L, BKME 100%,and SE 30% developed as physiological females, presenting ovaries identical to genetic females in the control group. The physiological female condition in XY fish was also observed in these treatments groups at 179 DPH, which suggests that the effect is permanent, whereas in other groups the effect changed between sampling periods. Identification of the genetic sex of individual animals using sex-linked DNA markers provides a useful tool for investigating environmental factors influencing sex determination and differentiation.

Sex determination in the parasitic nematode Strongyloides ratti.

The parasitic nematode Strongyloides ratti reproduces by both parthenogenesis and sexual reproduction, but its genetics are poorly understood. Cytological evidence suggests that sex determination is an XX/XO system. To investigate this genetically, we isolated a number of sex-linked DNA markers. One of these markers, Sr-mvP1, was shown to be single copy and present at a higher dose in free-living females than in free-living males. The inheritance of two alleles of Sr-mvP1 by RFLP analysis was consistent with XX female and XO male genotypes. Analysis of the results of sexual reproduction demonstrated that all progeny inherit the single paternal X chromosome and one of the two maternal X chromosomes. Therefore, all stages of the S. ratti life cycle, with the exception of the free-living males, are XX and genetically female. These findings are considered in relation to previous analyses of S. ratti and to other known sex determination systems.

DNA sex identification in the three‐spined stickleback

Two sex linked DNA markers are isolated in the three‐spined sticklebackGasterosteus aculeatusand a sex identification test is developed.

DNA sex identification in the three-spined stickleback

Two sex linked DNA markers are isolated in the three-spined stickleback Gasterosteus aculeatus and a sex identification test is developed.