Molecular sexing assays in 114 mammalian species: In silico sequence reanalysis and a unified graphical visualization of diagnostic tests

Molecular‐based methods for identifying sex in mammals have a wide range of applications, from embryo manipulation to ecological studies. Various sex‐specific or homologous genes can be used for this purpose, PCR amplification being a common method. Over the years, the number of reported tests and the range of tested species have increased greatly. The aim of the present analysis was to retrieve PCR‐based sexing assays for a range of mammalian species, gathering the gene sequences from either the articles or online databases, and visualize the molecular design in a uniform manner. For nucleotide alignment and diagnostic test visualization, the following genomic databases and tools were used: NCBI, Ensembl Nucleotide BLAST, ClustalW2, and NEBcutter V2.0. In the 45 gathered articles, 59 different diagnostic tests based on eight different PCR‐based methods were developed for 114 mammalian species. Most commonly used genes for the analysis were ZFX, ZFY, AMELX, and AMELY. The tests were most commonly based on sex‐specific insertions and deletions (SSIndels) and sex‐specific sequence polymorphisms (SSSP). This review provides an overview of PCR‐based sexing methods developed for mammals. This information will facilitate more efficient development of novel molecular sexing assays and reuse of previously developed tests. Development of many novel and improvement of previously developed tests is also expected with the rapid increase in the quantity and quality of available genetic information.     

Fast and reliable sexing of prosimian and human DNA

Molecular sexing of mammals is normally done by PCR amplification of Y chromosomal fragments, or coamplification of homologous fragments from both sex chromosomes. Existing primers are often unreliable for distantly related species due to mutations in primer regions. Currently there are no published primers for the sexing of prosimian DNA. We show that an existing method (using the zinc finger protein) based on a size difference between the X and Y homologs does not work in prosimians. Multiple alignments of distantly related mammalian species from Genbank and genome databases enabled us to identify conserved regions in the amelogenin gene. Using these conserved regions, we can target species that have no sequence information. We designed a single, conserved primer pair that is useful for fast and reliable molecular sexing of prosimian primates. A single PCR yields two fragments in males and only one in females, which are easily separated with the use of agarose gels. Amplification of separable fragments was successful in seven species of lemurs, as well as humans.     

PCR-based sexing in conservation biology: Wrong answers from an accurate methodology?

Molecular tests of sex based on the polymerase chain reaction (PCR) are now commonplace in conservation biology, routinely guiding management decisions. While molecular approaches to sexing can be highly reliable, current practices may leave an undesirable level of uncertainty in the sexes identified, because researchers focus on determining the sex-specific nature of a test, largely ignoring the accuracy of the test to correctly sex individuals. This latter step requires considerably more known-sex individuals. We argue that, due to the well-known technical problems associated with PCR amplification, the demonstrated potential for sexing errors and few known-sex individuals being available from threatened species, conservationists should place greater emphasis on verifying the sexes identified with PCR tests. We propose that all individuals of the sex indistinguishable from an amplification failure (e.g., females in mammals XX, males in birds ZZ) should be verified with a second independent sex test. Such a consensus approach to molecular sexing would reduce errors that could arise due to technical failure and PCR anomalies, but may also reduce field and laboratory bookkeeping errors.     

Multiple molecular approaches yield no evidence for sex‐determining genes in lake sturgeon (Acipenser fulvescens)

Common DNA‐based sexing assays have been widely used for the conservation and management of mammals and birds. However, many fishes do not have genetic sex determination and in those that do, the plasticity of the genes involved means that species‐specific assays are normally required. Such DNA‐sexing markers would be especially valuable in lake sturgeon (Acipenser fulvescens) because of their sexual monomorphism, delayed sexual maturity, and conservation status. We tried to identify genetic differences between male and female lake sturgeon using several different molecular genetic methods, including randomly amplified polymorphic DNA, representational difference analyses, subtractive hybridization, and a candidate gene approach. Ultimately, a number of genes were identified but none was sex‐specific. Although the ultimate mechanism of sex determination is yet unknown, it is possible that sex determination is environmental in lake sturgeon, especially since recent studies have also failed to identify sex determination genes in other sturgeon species.     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Noninvasive molecular sexing: An evaluation and validation of the SRY‐ and amelogenin‐based method in three new lemur species

Many lemur species are arboreal, elusive, and/or nocturnal and are consequently difficult to approach, observe and catch. In addition, most of them are endangered. For these reasons, non‐invasive sampling is especially useful in primates including lemurs. A key issue in conservation and ecological studies is to identify the sex of the sampled individuals to investigate sex‐biased dispersal, parentage, social organization and population sex ratio. Several molecular tests of sex are available in apes and monkeys, but only a handful of them work in the lemuriform clade. Among these tests, the coamplification of the SRY gene with the amelogenin X gene using strepsirhine‐specific X primers seems particularly promising, but the reliability and validity of this sexing test have not been properly assessed yet. In this study, we (i) show that this molecular sexing test works on three additional lemur species (Microcebus tavaratra, Propithecus coronatus and P. verreauxi) from two previously untested genera and one previously untested family, suggesting that these markers are likely to be universal among lemurs and other strepsirrhines; (ii) provide the first evidence that this PCR‐based sexing test works on degraded DNA obtained from noninvasive samples; (iii) validate the approach using a large number of known‐sex individuals and a multiple‐tubes approach, and show that mismatches between the field sex and the final molecular consensus sex occur in less than 10% of all the samples and that most of these mismatches were likely linked to incorrect sex determinations in the field rather than genotyping errors. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

Sexing free-ranging brown bears Ursus arctos using hairs found in the field

As an aid to the management of the Pyrenean population of the brown bear Ursus arctos , a sexing method based on the amplification of a Y chromosome specific sequence has been developed, and tested using hairs found in the field as a source of DNA. This method involves a two-step polymerase chain reaction (PCR) which allows the detection of a very small amount of DNA, probably a single SRY gene molecule. The sex can reliably be identified using about 50pg of DNA extract as template. It is possible that this approach could, with adjustments, be used to identify the sex of other species of eutherian mammals.     

Fluorescence in situ hybridization: a new method for determining primary sex ratio in ants

The haplodiploid sex determining system in Hymenoptera, whereby males develop from haploid eggs and females from diploid eggs, allows females to control the primary sex ratio (the proportion of each sex at oviposition) in response to ecological and/or genetic conditions. Surprisingly, primary sex ratio adjustment by queens in eusocial Hymenoptera has been poorly studied, because of difficulties in sexing the eggs laid. Here, we show that fluorescence in situ hybridization (FISH) can be used to accurately determine the sex (haploid or diploid) of eggs, and hence the primary sex ratio, in ants. We first isolated the homologue coding sequences of the abdominal-A gene from 10 species of 8 subfamilies of Formicidae. Our data show that the nucleotide sequence of this gene is highly conserved among the different subfamilies. Second, we used a sequence of 4.5 kbp from this gene as a DNA probe for primary sex ratio determination by FISH. Our results show that this DNA probe hybridizes successfully with its complementary DNA sequence in all ant species tested, and allows reliable determination of the sex of eggs. Our proposed method should greatly facilitate empirical tests of primary sex ratio in ants.     

Rapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvae

Two different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female‐specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W‐specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2‐EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2‐EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co‐amplified the CpW2‐EcoRI sequence to identify the W chromosome and the Z‐linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth.     

Using genotyping data to assign markers to their chromosome type and to infer the sex of individuals: a Bayesian model‐based classifier

The recent democratization of next‐generation‐sequencing‐based approaches towards nonmodel species has made it cost‐effective to produce large genotyping data sets for a wider range of species. However, when no detailed genome assembly is available, poor knowledge about the organization of the markers within the genome might hamper the optimal use of this abundant information. At the most basic level of genomic organization, the type of chromosome (autosomes, sex chromosomes, mitochondria or chloroplast in plants) may remain unknown for most markers which might be limiting or even misleading in some applications, particularly in population genetics. Conversely, the characterization of sex‐linked markers allows molecular sexing of the individuals. In this study, we propose a Bayesian model‐based classifier named detsex, to assign markers to their chromosome type and/or to perform sexing of individuals based on genotyping data. The performance of detsex is further evaluated by a comprehensive simulation study and by the analysis of real data sets from various origins (microsatellite and SNP data derived from genotyping assay designs and NGS experiments). Irrespective of the origin of the markers or the size of the data set, detsex was proved efficient (i) to identify the sex‐linked markers, (ii) to perform molecular sexing of the individuals and (iii) to perform basic quality check of the genotyping data sets. The underlying structure of the model also allows to consider each of these potential applications either separately or jointly.     

Evolutionary stability of sex chromosomes in snakes

Amniote vertebrates possess various mechanisms of sex determination, but their variability is not equally distributed. The large evolutionary stability of sex chromosomes in viviparous mammals and birds was believed to be connected with their endothermy. However, some ectotherm lineages seem to be comparably conserved in sex determination, but previously there was a lack of molecular evidence to confirm this. Here, we document a stability of sex chromosomes in advanced snakes based on the testing of Z-specificity of genes using quantitative PCR (qPCR) across 37 snake species (our qPCR technique is suitable for molecular sexing in potentially all advanced snakes). We discovered that at least part of sex chromosomes is homologous across all families of caenophidian snakes (Acrochordidae, Xenodermatidae, Pareatidae, Viperidae, Homalopsidae, Colubridae, Elapidae and Lamprophiidae). The emergence of differentiated sex chromosomes can be dated back to about 60 Ma and preceded the extensive diversification of advanced snakes, the group with more than 3000 species. The Z-specific genes of caenophidian snakes are (pseudo)autosomal in the members of the snake families Pythonidae, Xenopeltidae, Boidae, Erycidae and Sanziniidae, as well as in outgroups with differentiated sex chromosomes such as monitor lizards, iguanas and chameleons. Along with iguanas, advanced snakes are therefore another example of ectothermic amniotes with a long-term stability of sex chromosomes comparable with endotherms.     

A critique of avian CHD‐based molecular sexing protocols illustrated by a Z‐chromosome polymorphism detected in auklets

The sexes of non‐ratite birds can be determined routinely by PCR amplification of the CHD‐Z and CHD‐W genes. CHD‐based molecular sexing of four species of auklets revealed the presence of a polymorphism in the Z chromosome. No deviation from a 1:1 sex ratio was observed among the chicks, though the analyses were of limited power. Polymorphism in the CHD‐Z gene has not been reported previously in any bird, but if undetected it could lead to the incorrect assignment of sex. We discuss the potential difficulties caused by a polymorphism such as that identified in auklets and the merits of alternative CHD‐based sexing protocols and primers.     

High‐resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples

High‐resolution melting (HRM) analysis is a very attractive and flexible advanced post‐PCR method with high sensitivity/specificity for simple, fast and cost‐effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real‐time PCR systems, along with improved saturating DNA‐binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex‐specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed‐tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real‐time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.     

Uncertainty in determination of sex from harvested bobcats

Considerable uncertainty often exists in estimates of demographic parameters based on data collected from harvested furbearer species. We used molecular genetic techniques to estimate rates of error in 2 methods of sex determination of harvested bobcats (Lynx rufus): manual examination of the carcass (field sex) and laboratory-based maximum canine root area (MRA sex). Error rates were high for both sexing techniques, and were associated with age and an age–sex interaction for the field and MRA sexing methods, respectively. These findings do not support the use of the field methods for identifying sex of harvested bobcats. The MRA method may be effective for determining sex of older bobcats but is limited by considerable overlap between sexes in juveniles and yearlings. If critical demographic parameters are estimated from harvest data, efforts should be made to identify and reduce rates of error before data are used to assess population status. © 2011 The Wildlife Society.     

Development of genetic sexing strains in Lepidoptera: from traditional to transgenic approaches

The sterile insect technique (SIT) is currently being used for the control of many agricultural pests, including some lepidopteran species. The SIT relies on the rearing and release of large numbers of genetically sterile insects into a wild population. The holokinetic chromosomes of Lepidoptera respond differently to radiation than do species where there is a localized centromere. This difference has enabled a variation of the SIT to be developed for Lepidoptera where a substerilizing dose of radiation is given to the insects before their release with the result that a certain level of sterility is inherited by the F1 offspring. The development of genetic sexing strains for fruit flies, enabling the release of males only, has resulted in enormous economic benefits in the mass rearing and has increased the efficiency of the field operations severalfold. This article outlines Mendelian approaches that are currently available to separate large numbers of males and females efficiently for different lepidopteran species and describes their difficulties and constraints. Successful transgenesis in several lepidopteran species opens up new possibilities to develop genetic sexing strains. The proposal to develop genetic sexing strains described in this article takes advantage of the fact that in Lepidoptera, the female is the heterogametic sex, with most species having aWZ sex chromosome pair, whereas the males are ZZ. This means that if a conditional lethal gene can be inserted into the W chromosome, then all females should die after the application of the restrictive condition. The assumptions made to accommodate this model are discussed, and the advantages to be gained for control programs are elucidated.     

A novel method of caenophidian snake sex identification using molecular markers based on two gametologous genes

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.     

Sex determination and differentiation in organisms other than higher plants

The understanding of sex determination in general, but in particular in mammals, has been a subject of scientific speculation for a long time. It has been shown that in many vertebrate and invertebrate species, the sex of an individual is determined by the individual's chromosomal constitution. Initial studies of classical genetic searching for sex-transforming mutations and the scrupulous analyses of modified phenotypes have shed light on the mechanism(s) of sex-determination. They paved the road to successful studies at molecular level. After a brief review on sex determination in chosen model species, the “Drosophila system” is presented to exemplify a possible general principle for sex determinism.     

Low rates of X‐Y recombination,not turnovers,account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup)

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X‐Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex‐linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X‐Y recombination in males. Phylogenetic analyses of sex‐linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X‐Y homomorphy and fine‐scale sequence similarity in these species do not stem from recent sex‐chromosome turnovers, but from occasional X‐Y recombination.     

Improving the reliability of molecular sexing of birds using a W-specific marker

Molecular techniques for identifying sex of birds utilize length differences between CHD-Z and CHD-W introns, but in some cases these methods can lead to sexing errors. Here we show that an additional W-specific primer can be used in conjunction with a pre-existing sexing primer pair to dramatically improve the reliability of molecular sexing methods. We illustrate the approach with American coots (Fulica americana), a species with CHD-Z polymorphism that could not be accurately sexed using traditional methods. We developed a reverse primer GWR2 designed to sit within the intron of the W chromosome and amplify a distinctively small DNA fragment that serves as a W-specific marker. Analysis of known-sex individuals indicates that this W-specific primer provides an efficient and reliable protocol to identify the sex of F. americana. The development of such sex-specific primers will likely increase the reliability of molecular sexing methods in other birds as well. Comparisons between CHD-Z alleles of coots and common moorhens (Gallinula chloropus) revealed that CHD-Z polymorphism evolved separately in these two closely related species. We discuss the implications of repeated evolution of CHD-Z polymorphisms among birds.