Genetic Sex Markers Research Articles

Exploitation and Application of a New Genetic Sex Marker Based on Intron Insertion Variation of erc2 Gene in Oplegnathus punctatus.

Spotted knifejaw (Oplegnathus punctatus), one of the most valuable mariculture species, grows with significant sexual dimorphism, with males growing significantly faster than females. O. punctatus not only has excellent growth characteristics and high food value, but also shows high economic value in aquaculture, which has become a hotspot in the field of aquaculture. The current insufficiency of sex marker identification in O. punctatus restricts the process of its unisexual breeding. Rapid identification of sex will help to study the mechanisms of sex determination and accelerate the development of sex-controlled breeding. With the completion of the sequencing of the male and female genomes of O. punctatus, the efficient and precise development of genetic sex markers has been made possible. In this study, we used genome-wide information combined with molecular biology techniques from marker sequences to further establish a rapid method for DNA insertion variant detection in the intron of O. punctatus erc2 gene, which can be used to rapidly, accurately, and efficiently identify whether DNA insertion occurs in the intron of O. punctatus erc2 gene to be detected, and to identify the sex of O. punctatus to be detected. It could also be distinguished by agarose gel electrophoresis, which would shorten the time for accurate identification and improves the detection efficiency. Homozygous comparison of male and female individuals showed that the length of the DNA fragment of the erc2 gene was 239bp on chromosome X1 and 1173bp on chromosome Y. It can therefore be inferred that a 934bp insertion fragment exists on the Y chromosome. The PCR amplification results showed that two DNA fragments of 1173bp and 239bp could be amplified in male O. punctatus, and the 1173bp fragment was a marker fragment specific to the variant intron erc2 gene, while only a single DNA fragment of 239bp was amplified in female O. punctatus. It has important significance and application value in the study of neurotransmitter transmission and environmental adaptability of female and male fish based on erc2 gene, as well as the identification of male and female sex, the preparation of high male fry, and family breeding.

A new sex-specific genetic marker (fshr 1834G>T) for flathead grey mullet, Mugil cephalus, in Queensland, Australia

In this study, genetic sex marker candidates from northern hemisphere Mugil cephalus were tested to see if they could be used to sex a population originating from south-east Queensland, Australia. As such, a region of the follicle stimulating hormone receptor (fshr) gene was sequenced but did not contain previously published single nucleotide polymorphisms (SNPs). However, further screening of the sequenced fshr region revealed a promising sex marker candidate for Queensland M. cephalus, fshr 1834 G>T, which was accurate in 100% of fish tested (excluding intersex fish, which had the female genotype). While all females tested were homozygous G/G, males presented as either G/T (common) or T/T (lower frequency). Subsequently, a real-time high-resolution melt was developed to facilitate rapid and accurate genotyping of M. cephalus based on the fshr 1834 G>T SNP. Initial results suggest that fshr 1834 G>T is a useful SNP that can reduce the need for more invasive sampling techniques such as gonadal biopsy, provide information relating to the sex of captive stock prior to gonadal maturation, and may prove useful in wild population surveys and stock assessment.

Sex biased expression of Fem-1 in larval stages suggests its function in early sex differentiation of Litopeneaus vannamei

The Pacific white shrimp Litopenaeus vannamei (L. vannamei) is an economically important species worldwide. As females exhibit a faster growth rate than males, it is crucial to understand the mechanism of sex determination and differentiation for the purpose of mono-sex breeding in shrimp. The feminization-1 gene (Fem-1) serves as a sex-determining gene in Caenorhabditis elegans, and the Fem-1 family is implicated in sex development in several crustaceans. In this study, three Fem-1 family members, namely LvFem-1a, LvFem-1b, and LvFem-1c, were identified and characterized in L. vannamei. Based on genetic sex marker, individuals were separated by sex during the early developmental stages. The expression profiles of these genes were analyzed in female or male larvae. Significant differences between females and males were observed during the early stages of development. In Zoea III or earlier stages, the expressions levels in males were significantly higher than those in females. Intriguingly, there was a shift towards higher expression in females during the mysis and post-larval stages. These findings indicate that the Zoea III stage may be crucial for sex differentiation in shrimp. In addition, the analysis of gene expression across various ovarian stages and tissues was conducted. Results showed that all three genes were highly expressed in the ovary, with particularly elevated levels in ovarian development stage II. These findings suggest that the Fem-1 genes play a critical role in early ovary development. This study is the first research focusing on the sex-specific expression of sex differentiation genes in shrimp. The obtained results provide new insights into to the role of the Fem-1 gene family in sex differentiation and gonadal development.

Genetic sex determination improves Canadian Atlantic salmon (Salmo salar) population assessments

AbstractEstimating egg deposition for Atlantic salmon population assessments is made difficult by their lack of sexual dimorphism prior to the autumn spawning season. We quantified the effect of sex misclassification from subjective examination of external morphology on egg deposition estimates in four Atlantic salmon populations across multiple years. Sex classification of Canadian salmon using the genetic sex marker (sdY) was accurate (>97%), whereas sex classification based on subjective examination of external morphology was inaccurate, with misclassification rates dependent on sea age, life history, and sampling season. Sex misclassification led to annual egg deposition estimates that ranged from −36% to +56%. We found that sex could not be discriminated based on measures of external morphology.

Development of genetic sex markers of zig-zag eel (Mastacembelus armatus) by a NGS method

Identifying sex-specific marker with universal applicability is important for the study of unisexual reproduction and sex determination mechanism. In this study, two genetic sex markers of zig-zag eel were identified using a NGS method. Twelve potential male-specific sequences were first obtained by whole genome re-sequencing analysis of three males and three females. After PCR amplification and agarose gel electrophoresis for validation, two male-specific markers were obtained. Moreover, these markers were further verified in different populations in the Pearl River basin, and the accuracy of M2 markers was 100%. In addition, these markers were used to identify the genetic sex of different periods of embryonic development and hatching of juveniles at different time points in the cultured population, and it was found that male to female switching was common in the cultured population. Besides, we also found some embryos with YY genotype. These universal sex-specific markers will lay a good foundation for understanding of sex determination mechanism and development of all-male breeding of zig-zag eel.

Photosynthetic and antioxidant activity in the sex-related heat stress response of the dioecious species Rumex thyrsiflorus Fingerh.

Rumex thyrsiflorus Fingerh. is a dioecious species with heteromorphic sex chromosomes. Due to sexual dimorphism and the content of bioactive constituents used in pharmacology, this species is an interesting object of study. A complex analysis of selected physiological and biochemical aspects of the sex-related response to heat stress in vitro and in vivo was carried out. The experiment included in vitro regenerated plants and plants obtained from seeds. Regenerants were obtained from hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.27 μM thidiazuron (TDZ). The sex of the plants was determined by molecular analysis based on genetic sex markers. Analysis of the main photosynthetic parameters indicated that in vitro regenerated plants showed a decrease in photosystem II (PSII) activity when directly exposed to a stressor. However, in contrast to the seed-derived plants, they adapted efficiently to the recovery conditions within 1 week after the stress was terminated. Furthermore, in vitro regenerated male and female plants acclimatised well to field conditions and showed greater stress tolerance based on better efficiency of the photosynthetic apparatus and the highest chlorophyll a/b ratio. In case of plants derived from seeds, male plants were less sensitive to heat stress and showed greater stability of PSII at high temperatures compared to female plants. The results showed that the response to high-temperature stress depends on the sex and the origin of the plants, i. e. in vitro regenerated plants versus plants obtained from seeds. Even short-term heat stress resulted in differences in photosynthetic efficiency. Biochemical analysis of antioxidant activity in response to heat stress, carried out for the first time in Rumex thyrsiflorus, has allowed the identification of the following forms of superoxide dismutase (SOD): manganese (MnSOD), iron (FeSOD) and two copper-zinc isoforms (Cu/ZnSOD I and Cu/ZnSOD II).

Whole-Genome Inter-Sex Variation in Russian Sturgeon (Acipenser gueldenstaedtii).

The Russian sturgeon (Acipenser gueldenstaedtii, AG) is an endangered fish species increasingly raised on fish farms for black caviar. Understanding the process of sex determination in AG is, therefore, of scientific and commercial importance. AG lacks sexual dimorphism until sexual maturation and has a predominantly octoploid genome without a definite sex chromosome. A conserved short female-specific genomic sequence was recently described, leading to the development of a genetic sex marker. However, no biological function has been reported for this sequence. Thus, the mechanism of sex determination and the overall inter-sex genomic variation in AG are still unknown. To comprehensively analyze the inter-sex genomic variation and assess the overall inter-species variation between AG and A. ruthenus (AR, sterlet), a related tetraploid sturgeon species, we performed whole-genome sequencing on DNA from 10 fish-farm-raised adult AG (5 males and 5 females). We produced a partially assembled, ~2390 MBp draft genome for AG. We validated in AG the female-specific region previously described in AR. We identified ~2.8 million loci (SNP/indels) varying between the species, but only ~7400 sex-associated loci in AG. We mapped the sex-associated AG loci to the AR genome and identified 15 peaks of sex-associated variation (10 kb segments with 30 or more sex-associated variants), 1 of which matched the previously reported sex-variable region. Finally, we identified 14 known and predicted genes in proximity to these peaks. Our analysis suggests that one or more of these genes may have functional roles in sex determination and/or sexual differentiation in sturgeons. Further functional studies are required to elucidate these roles.

Establishment of New Genetic Markers and Methods for Sex Determination of Mouse and Human Cells using Polymerase Chain Reactions and Crude DNA Samples.

Background: The currently available methods for sexing human or mouse cells have weaknesses. Therefore, it is necessary to establish new methods. Methods: We used bioinformatics approach to identify genes that have alleles on both the X and Y chromosomes of mouse and human genomes and have a region showing a significant difference between the X and Y alleles. We then used polymerase chain reactions (PCR) followed by visualization of the PCR amplicons in agarose gels to establish these genomic regions as genetic sex markers. Results: Our bioinformatics analyses identified eight mouse sex markers and 56 human sex markers that are new, i.e. are previously unreported. Six of the eight mouse markers and 14 of the 56 human markers were verified using PCR and ensuing visualization of the PCR amplicons in agarose gels. Most of the tested and untested sex markers possess significant differences in the molecular weight between the X- and Y-derived PCR amplicons and are thus much better than most, if not all, previously-reported genetic sex markers. We also established several simple and essentially cost-free methods for extraction of crude genomic DNA from cultured cells, blood samples, and tissues that could be used as template for PCR amplification. Conclusion: We have established new sex genetic markers and methods for extracting genomic DNA and for sexing human and mouse cells. Our work may also lend some methodological strategies to the identification of new genetic sex markers for other organismal species.

Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats.

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex‐ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex‐reversing stimuli more frequently, which may lead to higher sex‐reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR‐based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous oestrogenic pollutants in a common garden experiment. We found negligible sex‐reversal frequency in free‐living adults despite the presence of various endocrine‐disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg L−1 17α‐ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L−1 EE2 and a glyphosate‐based herbicide with 3 µg L−1 or 3 mg L−1 glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex‐reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species‐specific resistance to sex reversal.

Sex-Based Variation of Gene Expression in the Gonads and Fins of Russian Sturgeon (<i>Acipenser gueldenstaedtii</i>)

Russian sturgeon (Acipenser gueldenstaedtii) is a primitive freshwater fish and a source of black caviar. The genes involved in sexual determination and differentiation are still unknown and there are no molecular markers for sex identification in this species. Studying the variation of the sex-based differences in genomic sequences and in gene expression in the sturgeon may lead to markers of sex in early stages of development and advances in aquaculture, as well as provide novel insights about the evolution of reproduction, sex determination, and sexual differentiation mechanisms in vertebrates. Previous studies by our and other groups have identified differentially expressed genes in the gonads of adult female and male sturgeon. The current study aimed to test whether these genes were also differentially expressed in non-gonadal tissue, namely fins. We measured by qRT-PCR the mRNA levels of 29 known and novel sex-related genes in the gonads and fins of males (4 years old) and females (7 years old; sexual maturation is earlier in males than in females). Six genes (ATP6, IGFRM, LIA1A, S1A, NPL1A, GAPDH and SOX9) showed higher expression in female fins. However, only ATP6 mRNA levels differed in fins of males and females of the same age (4 years old). These findings underscore the impracticality of sex identification based on gene expression in non-gonadal tissue and the need for genetic sex markers in the Russian sturgeon.

Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use.

Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex-linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex-reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress-induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North-Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female-to-male sex-reversed adults had similar body mass as normal males. We recorded no events of male-to-female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human-induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex-reversed individuals surviving to adulthood may participate in breeding.

A newly developed genetic sex marker and its application to understanding chemically induced feminisation in roach (Rutilus rutilus).

Oestrogenic wastewater treatment works (WwTW) effluents discharged into UK rivers have been shown to affect sexual development, including inducing intersex, in wild roach (Rutilus rutilus). This can result in a reduced breeding capability with potential population level impacts. In the absence of a sex probe for roach it has not been possible to confirm whether intersex fish in the wild arise from genetic males or females, or whether sex reversal occurs in the wild, as this condition can be induced experimentally in controlled exposures to WwTW effluents and a steroidal oestrogen. Using restriction site-associated DNA sequencing (RAD-seq), we identified a candidate for a genetic sex marker and validated this marker as a sex probe through PCR analyses of samples from wild roach populations from nonpolluted rivers. We also applied the sex marker to samples from roach exposed experimentally to oestrogen and oestrogenic effluents to confirm suspected phenotypic sex reversal from males to females in some treatments, and also that sex-reversed males are able to breed as females. We then show, unequivocally, that intersex in wild roach populations results from feminisation of males, but find no strong evidence for complete sex reversal in wild roach at river sites contaminated with oestrogens. The discovered marker has utility for studies in roach on chemical effects, wild stock assessments, and reducing the number of fish used where only one sex is required for experimentation. Furthermore, we show that the marker can be applied nondestructively using a fin clip or skin swab, with animal welfare benefits.

Genome of the tropical plant Marchantia inflexa: implications for sex chromosome evolution and dehydration tolerance

We present a draft genome assembly for the tropical liverwort, Marchantia inflexa, which adds to a growing body of genomic resources for bryophytes and provides an important perspective on the evolution and diversification of land plants. We specifically address questions related to sex chromosome evolution, sexual dimorphisms, and the genomic underpinnings of dehydration tolerance. This assembly leveraged the recently published genome of related liverwort, M. polymorpha, to improve scaffolding and annotation, aid in the identification of sex-linked sequences, and quantify patterns of sequence differentiation within Marchantia. We find that genes on sex chromosomes are under greater diversifying selection than autosomal and organellar genes. Interestingly, this is driven primarily by divergence of male-specific genes, while divergence of other sex-linked genes is similar to autosomal genes. Through analysis of sex-specific read coverage, we identify and validate genetic sex markers for M. inflexa, which will enable diagnosis of sex for non-reproductive individuals. To investigate dehydration tolerance, we capitalized on a difference between genetic lines, which allowed us to identify multiple dehydration associated genes two of which were sex-linked, suggesting that dehydration tolerance may be impacted by sex-specific genes.

Female versus male: Rumex thyrsiflorus Fingerh. under in vitro conditions. Does sex influence in vitro morphogenesis?

Rumex thyrsiflorus Fingerh. is a dioecious plant with polymorphic sex chromosomes (XX in females, XY1Y2 in males). This species is an interesting plant for study on the structure and function of sex chromosomes, the sex ratio, and is also a valuable source of bioactive constituents. A procedure for the micropropagation of R. thyrsiflorus from hypocotyl explants, which can be useful for analyses of sex-specific morphogenetic reactions under in vitro conditions, was developed. The molecular analysis, based on genetic sex markers, allowed the sex of explants to be determined in order to create male or female in vitro cultures. Regeneration primarily occurred via indirect adventitious shoot formation as well as via somatic embryogenesis, which was confirmed by histological and scanning electron microscope (SEM) analyses. The highest frequency of explants that revealed a morphogenetic response with the highest number of regenerated adventitious shoot buds (average 11.5 shoots per explant) was obtained on a Murashige and Skoog (MS) medium supplemented with 2.27 µM thidiazuron (TDZ). Molecular analysis revealed a female-biased sex ratio under these conditions. The relationship between the sex of the explants and their morphogenetic potential was studied. The efficiency of morphogenesis varied between the two sexes and depended on the ratio of auxin and cytokinin in the medium. The germination pattern of male and female seeds under in vitro conditions was also analysed. No differences in germination time between male and female seeds and in terms of male and female seedling length were observed, which indicated that there was no competition between the sexes during seed germination when grown under uniform growth conditions.

Production of a YY Male Brook Trout Broodstock for Potential Eradication of Undesired Brook Trout Populations

AbstractBrook Trout Salvelinus fontinalis introduced outside of their native range often negatively impact native aquatic fauna or provide marginal fisheries and are frequently targeted for manual or piscicide removal in lakes and streams. Unfortunately, complete eradication of exotic Brook Trout populations via these methods is rarely achieved; new approaches are needed. A potential alternative is a Trojan Y Chromosome (TYC) program in which hatchery‐produced genetically YY male fish would be regularly released into an undesired population over time, skewing the population towards 100% males, theoretically resulting in wild population extirpation. We developed two genetic sex markers for Brook Trout and employed juvenile sex reversal methods commonly used in commercial aquaculture to develop a YY broodstock that can produce offspring for possible future use as biological control agents. Our search for genetic sex markers proved successful, with genotypic sex determination for two assays matching the observed phenotype for 90 out of 90 individuals. In the first phase of the program, estradiol‐infused feed readily feminized genetic XY males into neofemales (FXY fish) at a high rate (99.6%; n = 224). Survival of progeny from such egg‐laying FXY fish averaged 88% to eye‐up and 91% from eye‐up to ponding, values similar to untreated Brook Trout reared at the same facility. In the second program phase, we cultured both sperm‐ and egg‐producing supermales (YY fish), a vital step towards development of TYC technology on a large aquaculture scale. Results showed that, in the hatchery, estradiol treatment does not reduce Brook Trout growth. This study demonstrates that hatchery production of a YY Brook Trout broodstock is feasible, modest in cost (less than US$10,000), and can be completed in 4 years. Although several hurdles remain before a full‐scale stocking program could occur, we believe that future work on the TYC strategy for Brook Trout is warranted.

Identification of the molecular sex‐differentiation period in the siberian sturgeon

Sexual development prior to gonadal sex differentiation is regulated by various molecular mechanisms. In fish, a "molecular sex-differentiation period" has been identified in species for which sex can be ascertained prior to gonadal sex differentiation. The present study was designed to identify such a period in a species for which no genetic sex markers or monosex populations are available. Siberian sturgeons undergo a slow sex-differentiation process over several months, so gonad morphology and gene expression was tracked in fish from ages 3-27 months to identify the sex-differentiation period. The genes amh, sox9, and dmrt1 were selected as male gonad markers; cyp19a1a and foxl2a as female gonad markers; and cyp17a1 and ar as markers of steroid synthesis and steroid receptivity. Sex differentiation occurred at 8 months, and was preceded by a molecular sex-differentiation period at 3-4 months, at which time all of the genes except ar showed clear expression peaks. amh and sox9 expression seemed to be involved in male sexual development whereas dmrt1, a gene involved in testis development in metazoans, unexpectedly showed a pattern similar to those of the genes known to be involved in female gonadal sex differentiation (cyp19a1 and foxl2a). In conclusion, the timing of and gene candidates involved with molecular sex differentiation in the Siberian sturgeon were identified.

Gonadal sex differentiation and effects of dietary methyltestosterone treatment in sablefish (Anoplopoma fimbria).

Methods for sex control are needed to establish monosex aquaculture of sablefish (Anoplopoma fimbria). Here we conducted the first characterization of sex differentiation by histology and hormonal sex reversal experiment in sablefish. Ovarian differentiation was first discernible at ~80 mm fork length (FL) and characterized by development of lamellar structures and onset of meiosis. Testes exhibited a dual-lobe appearance over much of their length and remained non-meiotic until males were ≥520 mm FL (2 years post-fertilization). Juveniles with undifferentiated gonads were provided diets containing 0 (control), 5 or 50 mg 17α-methyltestosterone (MT)/kg for 2 months. Following treatment, controls possessed either ovaries or non-meiotic testes, whereas MT-treated fish exhibited meiotic testes (60% of the fish), intersex gonads (~30%), or gonads that appeared sterile (~10%). A genetic sex marker revealed that all intersex fish were genetic females, although other females appeared to be completely sex reversed (i.e., neomales). One year after treatment, MT-treated fish possessed non-meiotic testes similar to control males or intersex gonads with reduced ovarian features, presumably due to atresia following MT withdrawal. Milt collected from neomales and genetic males 3 years post-treatment permitted sperm motility analyses; however, neomale sperm were virtually immotile. These results demonstrated that sablefish are differentiated gonochorists and that MT treatment from 76 to 196 mm FL induced permanent masculinization of a portion of the genetic females, but acquisition of sperm motility was impaired. Earlier administration of MT may be necessary to sex reverse a higher proportion of genetic females and reduce negative effects on fertility.

Male adventitious roots of Rumex thyrsiflorus Fingerh. as a source of genetically stable micropropagated plantlets

Rumex thyrsiflorus Fingerh. is one of the few dioecious plant species that have sex chromosomes. The chromosome constitution of females is 2n = 12A + XX and 2n = 12A + XY1Y2 of males. It is a medicinally important plant species and has also been the object of studies on the structure and function of sex chromosomes and sex ratio. An efficient plant regeneration protocol was developed from karyologically stable male roots that had been derived from a long-term liquid culture. The root segments were grown on MS medium supplemented with the following plant growth regulators: 2.4-D, NAA, kinetin, BAP and TDZ. The highest frequency (81.73 %) of adventitious shoot formation (16.27 shoots/explant) was obtained on MS + 0.5 mg/l TDZ. Regenerated shoots were successfully rooted on ½ MS + 2 % sucrose + 0.5 mg/l IBA and acclimated to in vivo conditions. Histological analysis revealed indirect (via callus) adventitious shoot formation. The cells of the morphogenetic callus were surrounded by a fibrillar structure that was similar to the extracellular matrix. Molecular analysis based on genetic sex markers confirmed that all of the root explants were male. The genetic stability of the regenerated plantlets was confirmed using random amplified polymorphic DNA analysis. This is the first report concerning the micropropagation protocol for R. thyrsiflorus Fingerh. from male roots derived from a long-term liquid culture, which offers a unique opportunity to obtain true-to-type plants of the same sex.

Identification and validation of a new male sex-specific ISSR marker in pointed gourd (Trichosanthes dioica Roxb.).

The aim of the present study was to develop a genetic sex marker for the pointed gourd (Trichosanthes dioica Roxb.) to allow gender determination at any stage in the life cycle. Screening of genomic DNA with intersimple sequence repeat (ISSR) primers was used to discover sex-specific touch-down polymerase chain reaction (Td-PCR) amplification products. Using pooled DNA from male and female genotypes and 42 ISSR primers, a putative male specific marker (~550 bp) was identified. DNA marker specific to male is an indication of existence of nonepigenetic factors involved in gender development in pointed gourd. The ISSR technique has proved to be a reliable technique in gender determination of pointed gourd genotypes at the seedling phenophase. The sex marker developed here could also be used as a starting material towards sequence characterization of sex linked genes for better understanding the developmental as well as evolutionary pathways in sexual dimorphism.

A Transgenic Medaka Line with Visible Markers for Genotypic and Phenotypic Sex

Accurate genotyping of sex is required for correct interpretation in any in vivo assays with endocrine disrupting chemicals (EDCs). Visible markers for genotypic sex, if reliable, simplify assays because time-consuming PCR-based genotyping can be skipped. Here, we describe a line of Japanese medaka with a brain-expressed green fluorescent protein (GFP) transgene inserted near the sex-determining locus. When used with a white pigment cell marker, genotypic sex can be determined reliably as early as 3 days after fertilization (well before gonadal sex differentiation). No recombinants were found in more than 2000 progenies. We also introduced a strong ovarian GFP marker into the line with these genetic sex markers, so that phenotypic sex can also be determined reliably at 8 days after hatching. Well-known sex reversal protocols using exogenous steroid treatments of embryos were monitored by this transgenic line, demonstrating the line to be a useful tool for in vivo studies utilizing gonadal sex differentiation of the medaka, especially for screenings of potential estrogenic and androgenic EDCs.