Markers For Sex Identification Research Articles

Identification of two effective sex-specific DNA markers in silver arowana (Osteoglossum bicirrhosum)

The silver arowana (Osteoglossum bicirrhosum), often referred to as a living fossil, presents significant challenges in sex identification based on morphological characteristics, with its sex determination mechanism remaining poorly understood. This issue poses considerable obstacles to its captive breeding efforts. Given the species' substantial value for both consumption and ornamental purposes, it is a critical candidate for aquaculture. Through genome sequencing, read mapping, and alignment analysis of five male and five female silver arowanas, along with one mixed pool of males, we successfully identified two sex-specific SNPs and confirmed a ZW model of sex determination in this ancient species. These two SNPs demonstrated remarkable accuracy, achieving 100 % success in genetic sex identification, as validated by Sanger sequencing of PCR products from 135 individuals. Both sex-specific SNPs are located approximately 26 kb upstream of the foxl2 gene, indicating a potentially pivotal role for the foxl2 gene in the sex-determination process of the silver arowana. Consequently, we have developed precise molecular markers for sex identification in the silver arowana, significantly enhancing reproductive efficiency and promoting further industrial development. This achievement lays a foundational basis for investigating the sex-determination mechanism of the silver arowana and other osteoglossid fish, thereby advancing aquaculture practices within this species.

Innovative approach for high-throughput exploiting sex-specific markers in Japanese parrotfish Oplegnathus fasciatus.

The use of sex-specific molecular markers has become a prominent method in enhancing fish production and economic value, as well as providing a foundation for understanding the complex molecular mechanisms involved in fish sex determination. Over the past decades, research on male and female sex identification has predominantly employed molecular biology methodologies such as restriction fragment length polymorphism, random amplification of polymorphic DNA, simple sequence repeat, and amplified fragment length polymorphism. The emergence of high-throughput sequencing technologies, particularly Illumina, has led to the utilization of single nucleotide polymorphism and insertion/deletion variants as significant molecular markers for investigating sex identification in fish. The advancement of sex-controlled breeding encounters numerous challenges, including the inefficiency of current methods, intricate experimental protocols, high costs of development, elevated rates of false positives, marker instability, and cumbersome field-testing procedures. Nevertheless, the emergence and swift progress of PacBio high-throughput sequencing technology, characterized by its long-read output capabilities, offers novel opportunities to overcome these obstacles. Utilizing male/female assembled genome information in conjunction with short-read sequencing data survey and long-read PacBio sequencing data, a catalog of large-segment (>100bp) insertion/deletion genetic variants was generated through a genome-wide variant site-scanning approach with bidirectional comparisons. The sequence tagging sites were ranked based on the long-read depth of the insertion/deletion site, with markers exhibiting lower long-read depth being considered more effective for large-segment deletion variants. Subsequently, a catalog of bulk primers and simulated PCR for the male/female variant loci was developed, incorporating primer design for the target region and electronic PCR (e-PCR) technology. The Japanese parrotfish (Oplegnathus fasciatus), belonging to the Oplegnathidae family within the Centrarchiformes order, holds significant economic value as a rocky reef fish indigenous to East Asia. The criteria for rapid identification of male and female differences in Japanese parrotfish were established through agarose gel electrophoresis, which revealed 2 amplified bands for males and 1 amplified band for females. A high-throughput identification catalog of sex-specific markers was then constructed using this method, resulting in the identification of 3,639 (2,786 INS/853 DEL, ♀ as reference) and 3,672 (2,876 INS/833 DEL, ♂ as reference) markers in conjunction with 1,021 and 894 high-quality genetic sex identification markers, respectively. Sixteen differential loci were randomly chosen from the catalog for validation, with 11 of them meeting the criteria for male/female distinctions. The implementation of cost-effective and efficient technological processes would facilitate the rapid advancement of genetic breeding through expediting the high-throughput development of sex genetic markers for various species. Our study utilized assembled genome information from male and female individuals obtained from PacBio, in addition to data from short-read sequencing data survey and long-read PacBio sequencing data. We extensively employed genome-wide variant site scanning and identification, high-throughput primer design of target regions, and e-PCR batch amplification, along with statistical analysis and ranking of the long-read depth of the variant sites. Through this integrated approach, we successfully compiled a catalog of large insertion/deletion sites (>100bp) in both male and female Japanese parrotfish.

Origin of purple asparagus cultivar 'Pacific Purple' based on the sequence of sex determination gene.

Garden asparagus is one of the most important crops worldwide. Since this crop is dioecious and male plants generally have higher yields compared to female plants, several DNA markers for sex identification have been developed for acceleration of asparagus breeding. Among these markers, Asp1-T7sp and MSSTS710 were found to be effective in sex determination for many asparagus cultivars. However, we previously found that these markers were not completely suitable for sex identification in the purple asparagus cultivar 'Pacific Purple'. There are two types of male individuals in this cultivar: One type is PP-m, which is identified the sex type by Asp1-T7sp and MSSTS710 markers, while the other type is PP-m* whose sex type is not identified by these markers. Since the sex identification markers are located on the non-recombining Y region, it was expected that the sequence around this region might be different between PP-m and PP-m*. In this study, the sequence of one of the sex-determining genes, MSE1/AoMYB35/AspTDF1, was analyzed, and a comparative analysis was conducted among PP-m and PP-m* of 'Pacific Purple', A. officinalis and related species A. maritimus. The results revealed that PP-m and PP-m* has the similar sequence of MSE1/AoMYB35/AspTDF1 gene from A. officinalis and A. maritimus, respectively. 'Pacific Purple' is a cultivar developed through polycross hybrid from Italian landrace 'Violetto d'Albenga' (VA), suggesting that VA originated from an interspecific crossing between A. officinalis and A. maritimus and that the pollen parent used in 'Pacific Purple' breeding contained two types of male individuals with different MSE1/AoMYB35/AspTDF1 sequence. As a result, PP-m and PP-m* of 'Pacific Purple' harbors the similar sequences of the MSE1/AoMYB35/AspTDF1 gene from A. officinalis and A. maritimus, respectively.

The Development of Genetic Sex Identification Markers and Evidence of a Male Heterogametic Sex Determination System in Red Shiner

AbstractThe Red Shiner Cyprinella lutrensis is of increasing management interest as an invasive species that negatively impacts many native fishes throughout North America. Trojan sex chromosome (TSC)‐carrying individuals could theoretically control invasive fish populations by skewing the sex ratio to 100% male. The efficacy of TSC‐based control programs requires an understanding of a population's sex determination system, yet such information is lacking for Red Shiner. We used single‐digest restriction site‐associated DNA sequencing to discover sex‐linked single‐nucleotide polymorphisms (SNPs), and we conducted a series of breeding experiments to uncover the sex determination system. All candidate sex‐linked SNPs that fit our selection criteria exhibited a pattern of male heterogamety. We developed two sex‐identification (sex‐ID) marker assays, XY_248 and XY_170, which showed phenotype–genotype concordance scores of 77.00% and 84.35%, respectively. These sex‐ID markers exhibited relatively high phenotype–genotype concordance in females (XY_248 = 96.30%; XY_170 = 98.61%), which allowed for selective breeding of phenotypically feminized genetic males. We observed a 3:1 male : female sex ratio in spawns from feminized males crossed with wild‐type males, indicative of a male heterogametic sex determination system (i.e., XY male/XX female). The discovery of a male heterogametic sex determination system, in combination with our two markers, increases the likelihood of developing an effective TSC eradication strategy for invasive Red Shiner populations.

Rare but Not Gone: A Relict Population of the Black Sea Ship Sturgeon Acipenser nudiventris Persists in the Rioni River, Georgia

Historically, the ship sturgeon (Acipenser nudiventris) occurred in the Aral, Caspian, Azov, and Black Sea basins. However, its numbers decreased dramatically during the 20th century. It is now considered extirpated from the Aral, Azov, and Black Seas, and has almost disappeared in the Caspian Sea. A. nudiventris is listed as Critically Endangered on the IUCN Red List and, in Georgia, the species has been undetected for the last three decades. We collected 22 sightings, including nine genetic samples taken from fin clips of ship sturgeon from the Rioni River in Georgia during 2020–2022. For the genetic samples, the mitochondrial DNA control region was used for species identification. Because cases of sturgeon inter-species hybridization have been reported in the Rioni River, we used species-specific diagnostic markers and ship sturgeon-specific microsatellite markers for detecting hybridization with other sturgeon species. In addition, we used a sex-specific marker for sex identification. Based on the maternal identification, all nine individuals are identified as ship sturgeon, representing one haplotype, and the haplotype is different from all other A. nudiventris haplotypes available in GenBank. Based on genetic analysis, the specimens did not show signs of hybridization with other locally occurring species. We conclude that ship sturgeon still live in the Rioni River, and are a remnant of an older, preexisting Black Sea ship sturgeon population.

The Landscape of Genome-Wide and Gender-Specific Microsatellites in Indo-Pacific Humpback Dolphin and Potential Applications in Cetacean Resource Investigation

Microsatellites are one of the important genome characterizations that can be a valuable resource for variety identification, genetic diversity, phylogenetic analysis, as well as comparative and conservation genomics research. Here, we developed comprehensive microsatellites through genome-wide mining for the threatened cetacean Indo-Pacific humpback dolphin (Sousa chinensis). We found 87,757 microsatellites with 2–6 bp nucleotide motifs, showing that about 32.5 microsatellites per megabase comprises microsatellites sequences. Approximately 97.8% of the markers developed in this study were consistent with the published identified markers. About 75.3% microsatellites were with dinucleotide motifs, followed by tetranucleotide motifs (17.4%), sharing the same composition pattern as other cetaceans. The microsatellites were not evenly distributed in the S. chinensis genome, mainly in non-coding regions, with only about 0.5% of the markers located in coding regions. The microsatellite-containing genes were mainly functionally enriched in the methylation process, probably demonstrating the potential impacts of microsatellites on biological functions. Polymorphic microsatellites were developed between different genders of S. chinensis, which was expected to lay the foundation for genetic diversity investigation in cetaceans. The specific markers for a male Indo-Pacific humpback dolphin will provide comprehensive and representative male candidate markers for sex identification, providing a potential biomolecular tool for further analysis of population structure and social behavior of wild populations, population trend evaluation, and species conservation management.

Characterisation and cross-amplification of sex-specific genetic markers in Australasian Egerniinae lizards and their implications for understanding the evolution of sex determination and social complexity

Sex is a pervasive factor that underpins functional phenotypic variation across a range of traits. Although sex can usually be distinguished morphologically, in some species this is not possible. The development of genetic markers for sex identification is, thus, key if we are to incorporate sex into an understanding of ecological or evolutionary process. Here we develop genetic markers for the identification of sex within an iconic Australian lizard group, the Egernia group, which is notable for its complex social behaviour. We used restriction-site associated DNA sequencing to characterise sex-specific genetic sequences for a key member of the group, Liopholis whitii, and designed primers for four of these putative sex-specific sequences. These primers amplified across some, but not all, species of the group. Our results provided several important insights. They suggest conservatism of a XX/XY sex determination system within the group as well as sex-specific genomic regions that appear independent of the conserved genomic regions identified in other skink species. More broadly, the development of sex markers for the Egernia group opens up a range of potential research questions related to the role that sex plays in the mediation of social behaviour and, through this, the emergence and stability of social life.

Development and characterization of microsatellite markers in the small Indian mongoose (Urva auropunctata).

The small Indian mongoose (Urva auropunctata) is one of the world's worst invasive alien species and eradication programs are ongoing worldwide. The development of individual and sex identification markers will improve their management. We searched for novel mongoose microsatellite markers using genome-wide screening and identified 115,265 tetra-nucleotide repeat loci. Of 96 loci tested, 17 were genotyped in 28 mongooses from the Okinawa population. The genetic diversity analysis showed that the average expected and observed heterozygosity and number of alleles were 0.55, 0.56, and 2.94, respectively. Of 17 loci, one deviated from Hardy-Weinberg equilibrium and six loci pairs were likely linked to each other. However, we succeed in identifying all individuals using all of the microsatellite loci. The novel sex identification markers worked successfully in a test using sex known samples. Our novel microsatellite and sex identification markers should be useful in studies of individual identification and population genetics of the mongoose.

Developmental validation of VeriFiler™ Plus PCR Amplification Kit: A 6-dye multiplex assay designed for casework samples

The VeriFiler™ Plus PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 23 autosomal markers (D3S1358, vWA, D16S539, CSF1PO, D6S1043, D8S1179, D21S11, D18S51, D5S818, D2S441, D19S433, FGA, D10S1248, D22S1045, D1S1656, D13S317, D7S820, Penta E, Penta D, TH01, D12S391, D2S1338, and TPOX), a quality indicator system, and two sex-identification markers. Combined, the markers satisfy the requirements of the Chinese National autosomal DNA database as well as expanded CODIS (Combined DNA Index System). The VeriFiler Plus kit was developed with an improved Master Mix which incorporates the brighter TED™ dye, and accommodates a higher sample loading volume thus allowing for increased sensitivity and enabling maximum information recovery from challenging casework samples including touch, degraded, and inhibited samples. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

Simple Nested Allele-Specific approach with penultimate mismatch for precise species and sex identification of tiger and leopard.

Accurate species and sex identification of non-invasive and forensic samples of the tiger and leopard is still confusing when using the allele-specific methods. We designed allele-specific methods with penultimate nucleotide mismatch in a nested manner for the exact identification and double-checking of forensic samples. The mismatch design is a novel concept in species and sex identification, making the allele-specific targeting precise. We developed three sets of markers, a 365bp outer and a 98bp inner marker for nested tiger species identification assay, 136bp leopard specific marker, and carnivore sex identification markers. We validated the method with tissue/blood forensic samples of various felids and herbivorous available in our lab and on known fecal samples from Vandalur Zoo. We also collected 37 scat samples at diverse stages of deterioration from the Mudumalai Tiger Reserve, Tamil Nadu, India. The 365bp targeted markers resulted in 70.2% (n = 22; 22/37) amplification success, while the 98bp FAM-labelled marker amplified 89% (n=33; 33/37) scat samples independently. The 136bp leopard markers answered four scat samples (11%) unrequited by the tiger specific markers. We evaluated species and the sex identification with these markers in another 190 non-invasive samples provided by the Mudumalai Tiger Reserve authorities. Among which 56.3% (n= 107) of samples were recognized as tiger (64 male and 43 female) and 38.9% (n= 74) as leopard (41 male and 33 female). The method supersedes any other previous methods in this regard by its high accuracy and simplicity.

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.

Applicability of two male specific DNA markers for sex identification of various Asparagus species

Applicability of two male specific DNA markers for sex identification of various <i>Asparagus</i> species

Effectiveness and potential application of sex-identification DNA markers in tunas

Sex-identification DNA markers are useful tools for sexing organisms that lack externally visible sexual dimorphism, and thus, they provide biological information for ecological and evolutionary studies. Tunas of the genus Thunnus (Scombridae), which comprises 8 species, lack sexual dimorphism of external morphology or coloration. In this study, we applied recently developed genotypic sex-identification markers for Pacific bluefin tuna to other tuna species to evaluate their effectiveness in sex identification. A sex-identification marker named ‘primer pair II’ demonstrated relatively high effectiveness in all tuna species, except southern bluefin tuna. Primer pair II was further tested in 209 albacore individuals collected during the scientific observer program onboard Japanese commercial long-line vessels, and it demonstrated robust performance for genotypic sex identification. The sex ratio of this albacore sample (1:1.4) significantly deviated from the expected 1:1 with the dominance of males, and the mean body size of males was higher than that of females. As all cross-species amplifications of the male-specific markers, except those for the southern bluefin tuna, were male-heterozygous polymorphisms, it is likely that a male-heterozygous sex-associated region exists in the Thunnus genome. The evolution of sex-determination systems in tunas was analyzed by ancestral state reconstruction, which showed that a common ancestor, before the evolution of the genus, possessed the male-heterozygous sex-associated genome region.

Autosomal sdY Pseudogenes Explain Discordances Between Phenotypic Sex and DNA Marker for Sex Identification in Atlantic Salmon

Despite the key role that sex-determination plays in evolutionary processes, it is still poorly understood in many species. In salmonids, which are among the best studied fishes, the master sex-determining gene sexually dimorphic on the Y-chromosome (sdY) has been identified. However, sdY displays unexplained discordance to the phenotypic sex, with a variable frequency of phenotypic females being reported as genetic males. Multiple sex determining loci in Atlantic salmon have also been reported, possibly as a result of recent transposition events in this species. We hypothesized the existence of an autosomal copy of sdY, causing apparent discordance between phenotypic and genetic sex, that is transmitted in accordance with autosomal inheritance. To test this, we developed a qPCR methodology to detect the total number of sdY copies present in the genome. Based on the observed phenotype/genotype frequencies and linkage analysis among 2,025 offspring from 64 pedigree-controlled families of accurately phenotyped Atlantic salmon, we identified both males and females carrying one or two autosomal copies of sdY in addition to the Y-specific copy present in males. Patterns across families were highly consistent with autosomal inheritance. These autosomal sdY copies appear to have lost the ability to function as a sex determining gene and were only occasionally assigned to the actual sex chromosome in any of the affected families.

Hare's affairs: Lessons learnt from a noninvasive genetic monitoring for tracking mountain hare individuals.

Systematic monitoring of individuals and their abundance over time has become an important tool to provide information for conservation. For genetic monitoring studies, noninvasive sampling has emerged as a valuable approach, particularly so for elusive or rare animals. Here, we present the 5‐year results of an ongoing noninvasive genetic monitoring of mountain hares (Lepus timidus) in a protected area in the Swiss Alps. We used nuclear microsatellites and a sex marker to identify individuals and assign species to noninvasively collected feces samples. Through including a marker for sex identification, we were able to assess sex ratio changes and sex‐specific demographic parameters over time. Male abundance in the area showed high fluctuations and apparent survival for males was lower than for females. Generally, males and females showed only little temporary migration into and out of the study area. Additionally, using genotyped tissue samples from mountain hares, European hares (Lepus europaeus) and their hybrids, we were able to provide evidence for the first occurrence of a European hare in the study area at an elevation of 2,300 m a.s.l. in spring 2016. For future monitoring studies, we suggest to include complementary analysis methods to reliably infer species identities of the individuals analyzed and, thus, not only monitor mountain hare individual abundance, but also assess the potential threats given through competitive exclusion by and hybridization with the European hare.

Molecular Sex Identification in the Hardy Rubber Tree (Eucommia ulmoides Oliver) via ddRAD Markers.

Eucommia ulmoides, also known as the industrially and medicinally important hardy rubber tree, is the sole species of Eucommiaceae. Nevertheless, its dioecious property hinders sex recognition by traditional morphological observation at very early developmental stages, thus inhibiting breeding and economic cropping. In this study, double-digest restriction site-associated DNA sequencing (ddRAD-seq) was applied to screen sex-linked molecular markers for sex identification and investigation of the sex determination system in 20 male and female E. ulmoides individual plants, respectively. In consequence, five candidate male-specific loci but no female-specific loci were predicated among the 183,752 male and 147,122 female catalogue loci by bioinformatics analysis. Subsequent PCR (polymerase chain reaction) amplification and Sanger sequencing examinations were performed on another 24 individuals, 12 for each sex, from a separate population. One ideal sex-linked locus, MSL4, was identified among the five putative male-specific loci that were found using ddRAD data. MSL4 is 479 bp in length and highly conserved in all the male individuals, suggesting its feature of being stable and repeatable. Our results also indicated that the sex of E. ulmoides is likely determined genetically. In short, this study provides a consistent and reproducible ddRAD marker (MSL4) that is able to discriminate male from female seedlings in E. ulmoides, which will be valuable for rapid breeding practice and better commercial production of this economically important tree.

Vitellogenin in the European cave salamander, Proteus anguinus: Its characterization and dynamics in a captive female as a basis for non-destructive sex identification.

Vitellogenin (Vtg) is a precursor protein of egg yolk proteins in oviparous and ovoviviparous vertebrates. Except in a case of exposure to estrogenic endocrine disruptors, Vtg is a female-specific protein and could be used as a molecular marker for sex identification. This would be especially useful in the case of the endangered European cave salamander Proteus anguinus in which sexes are indistinguishable according to external morphology, which hinders the establishment of a successful captive breeding program. Here we describe the identification, partial characterization, and purification of Vtg from P. anguinus. Vtg was identified in the plasma of a vitellogenic proteus female with visible oocytes. The identification of this protein was accomplished by mass spectrometry analysis. Two-dimensional gel electrophoresis revealed proteus Vtg as a mix of 190 kDa isoforms with isoelectric points in the pH range 5.3-6.0. Vtg was purified from proteus blood by gel filtration followed by anion-exchange chromatography. Using specific staining of SDS-PAGE gels, the Vtg was found to be phosphorylated and lipidated. Unlike the case in some other aquatic vertebrates, in P. anguinus, Vtg was not present in detectable amounts in cutaneous mucus. Degradation of oocytes in the captive vitellogenic female was accompanied by simultaneous decrease of Vtg concentration. Over a period of 10 months, the concentration of Vtg dropped from maximal to sub-detectable. Our results show that Vtg is a promising molecular marker for sex identification and ovary maturation in P. anguinus, which could contribute to the development of a viable program for captive reproduction of this unique species.

Phenotypic Stability of Sex and Expression of Sex Identification Markers in the Adult Yesso Scallop Mizuhopecten yessoensis throughout the Reproductive Cycle

Simple SummaryBivalve sex is thought to fluctuate depending on environmental conditions. So far, there has been no investigation on the phenotypic stability of sex in the commercially important Yesso scallop Mizuhopecten yessoensis. The present study revealed that the sex of the Yesso scallop is stable after initial sex differentiation and that this species maintains a sex-stable maturation system throughout its life. In addition, gonad differentiation for each sex was precisely characterized by using molecular markers throughout the maturational cycle.The objective of the present study was to analyze the phenotypic stability of sex after sex differentiation in the Yesso scallop, which is a gonochoristic species that has been described as protandrous. So far, no study has investigated in detail the sexual fate of the scallop after completion of sex differentiation, although bivalve species often show annual sex change. In the present study, we performed a tracking experiment to analyze the phenotypic stability of sex in scallops between one and two years of age. We also conducted molecular marker analyses to describe sex differentiation and gonad development. The results of the tracking experiment revealed that all scallops maintained their initial sex phenotype, as identified in the last reproductive period. Using molecular analyses, we characterized my-dmrt2 and my-foxl2 as sex identification markers for the testis and ovary, respectively. We conclude by proposing that the Yesso scallop is a sex-stable bivalve after its initial sex differentiation and that it maintains a sex-stable maturation system throughout its life. The sex-specific molecular markers identified in this study are useful tools to assess the reproductive status of the Yesso scallop.

Seminal Plasma Exosomes: Promising Biomarkers for Identification of Male and Pseudo-Males in Cynoglossus semilaevis.

In mammals, small RNAs enclosed in exosomes have been identified as appropriate signatures for disease diagnosis. However, there is limited information on exosomes derived from seminal plasma, and few studies have reported analyzed the composition of exosomes and enclosed small RNAs in fish. The half-smooth tongue sole (Cynoglossus semilaevis) is an economically important fish for aquaculture, and it exhibits sexual dimorphism: the female gender show higher growth rates and larger body sizes than males. Standard karyotype analysis and artificial gynogenesis tests have revealed that this species uses heterogametic sex determination (ZW/ZZ), and so-called sex-reversed pseudo-males exist. In this study, we successfully identified exosomes in the seminal plasma of C. semilaevis; to the best of our knowledge, this is the first report of exosomes in fish seminal plasma. Analysis of the nucleotide composition showed that miRNAs were dominant in the exosomes, and the miRNAs were sequenced and compared to identify signature miRNAs as sexual biomarkers. Moreover, target genes of the signature miRNAs were predicted by sequence matching and annotation. Finally, four miRNAs (dre-miR-141-3P, dre-miR-10d-5p, ssa-miR-27b-3p, and ssa-miR-23a-3p) with significant differential expression in the males and pseudo-males were selected from the signature candidate miRNAs as markers for sex identification, and their expression profiles were verified using real-time quantitative PCR. Our findings could provide an effective detection method for sex differentiation in fish.

Widely Applicable PCR Markers for Sex Identification in Birds

To aid in avian sex determination if birds are not sexually dimorphic and/or they are sexually immature, several molecular assays involving the polymerase chain reaction (PCR) have been developed. To test in a variety of domestic and wild avian species applicability of five sexing assays: previously described four assays based on nucleotide sequence differences between the Z and W copy of the chicken chromodomain-helicase-DNA-binding protein gene (CHD1Z and CHD1W), and a new sexing marker using the ubiquitin associated protein 2 (UBAP2) gene sequence. At least one molecular sexing marker was successful in 84 out of 88 examined species across 13 avian orders. These assays may be useful in breeding management of domestic and wild birds as well as in studies of avian ecology, population genetics, embryology and transgenesis.