Reptile DNA Sexing: Easier Than You Ever Thought

Post provided by Lukáš Kratochvíl and Michail Rovatsos

The sand lizard (Lacerta agilis).

The sand lizard (Lacerta agilis).

Many researchers, breeders and hobbyists need to know sex of their animals. Sometimes it’s easy – in sexually dimorphic species you only have to look. In other species or juveniles it’s often not so straightforward though. And it’s often impossible – but sometimes essential – in embryos or in tissue samples. Determining sex from DNA is the most practical option, or sometimes even the only possibility, in these cases.

Molecular sexing is routinely used in mammals and birds, but until now it has only been available for a handful of reptile species. Many people didn’t believe that this situation would improve considerably any time soon. But why?

Difficulties with Reptile Sex Determination

In contrast to mammals and birds, reptiles were thought to have evolutionary unstable systems of sex determination, and because of this scientists wouldn’t be able to develop a widely applicable and effective method for studying it. It is true that there is a wide range of ways that sex can be determined in reptiles: from well-differentiated sex chromosomes to temperature-dependent sex determination.

In lineages with differentiated sex chromosomes, males and females differ in number of copies of genes linked to sex chromosomes (illustrated in red color). These differences can be quantified in DNA samples and used for molecular sexing.

In lineages with differentiated sex chromosomes, males and females differ in number of copies of genes linked to sex chromosomes (in red). These differences can be quantified in DNA samples and used for molecular sexing.

In species with temperature-dependent sex determination, the sex of an individual is set by incubation temperature and there’s no difference in the genotypes of males and females. So, for these species it’s impossible to determine sex from DNA. Many lineages of reptiles have genotypic sex determination based on the presence of either XX/XY or ZZ/ZW sex chromosomes though. Individuals with XX or ZW sex chromosomes are females, XY or ZZ individuals are males. For these species, molecular sexing is an option.

Previous strategies for molecular sexing in reptiles were based on determination of sex-specific differences in repetitive sequences in genomes. These are notoriously dynamic and demonstrate significant inter- and intraspecies variation. So such methods aren’t able to ensure accurate sex determination of reptiles from closely related species, let alone unrelated populations! In the August 2017 issue of Methods in Ecology and Evolution, we argue that the situation in reptiles is not as hopeless as it may seem and that it’s quite realistic to assume that we can in fact determine sex molecularly in around 4000 species of lizards and snakes (i.e. in nearly half of all living squamate reptiles).

Lacertid lizards, advanced snakes and iguanas cover around 4000 living species, so molecular sexing is now potentially available for around 40% of squamate reptiles.

Lacertid lizards, advanced snakes and iguanas cover around 4000 living species, so molecular sexing is now potentially available for around 40% of squamate reptiles.

How is it possible? We stress that sex determination is in fact not so unstable in reptiles, in comparison to mammals and birds. Sex chromosomes seem to be evolutionary conserved in several lineages of lizards and snakes and, because of this, protein coding genes located on sex chromosomes can be used as reliable markers for sex identification.

Making Molecular Sexing Widely Applicable

Well-differentiated sex chromosomes differ in gene content. The differentiated unpaired Y and W chromosomes used to be degenerated and lack many genes which are still present on their X and Z counterparts. This means that, in lineages with differentiated sex chromosomes, females used to have a different number of copies of genes linked to sex chromosomes than males (twice as many copies of X-linked genes in XX/XY lineages and only half of copies of Z-linked genes in ZZ/ZW lineages). And just the measurement of differences in number of copies of X- or Z-linked genes in DNA sample by quantitative Polymerase Chain Reaction (qPCR) can be used for molecular sexing.

In order to apply this method, we have to know at least some X- or Z-linked genes in a given lineage. This isn’t always easy as sex chromosomes evolved among reptiles several times, Y and W chromosomes must be adequately degenerated, and it works only within a lineage where species keeps the same sex chromosomes. As for any other method of molecular sexing, testing in several animals with known sex is recommended for any previously unstudied species.

Where does qPCR-based Molecular Sexing Method Work?

We designed multiple primers for sex-linked genes (molecular sexing markers), which were tested in over 90 species of reptiles. The qPCR-based molecular sexing method was proven to work accurately in lacertid lizards (ZZ/ZW), advanced snakes (ZZ/ZW) and most iguanas (XX/XY). These lineages have largely conserved sex chromosomes and include around 4000 species, for which molecular sexing is widely-applicable .

The Florida softshell turtle, Apalone ferox.

The Florida softshell turtle (Apalone ferox).

Recently, the same technique was developed for softshell turtles. As many of these turtles are endangered and sexing of juveniles is extremely difficult for several years after hatching, this method could be particularly useful for conservation and breeding projects. We expect our molecular sexing method will be available for more reptile lineages soon. In fact, we’re currently working on multiple reptilian lineages, to reveal their sex chromosomes and design primers of sex-linked genes, suitable for the qPCR-based molecular sexing method.

To find out more about qPCR-based molecular sexing, read our Methods in Ecology and Evolution article ‘Molecular sexing applicable in 4000 species of lizards and snakes? From dream to real possibility’.

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