Choosing a DNA primer set for Bird Sexing

What is a primer set?

A primer set is a pair (or trio or more) of short DNA sequences used in a process called PCR (Polymerase Chain Reaction) to define the region of DNA to be amplified.

When sexing birds using PCR, the primers bind to specific parts of a bird’s sex chromosomes (known as Z and W chromosomes), and enable the PCR to specifically amplify the DNA from one primer to the other.

Currently, there is no single universal primer set that can determine the sex of all bird species. However, we offer four primer sets for bird sexing (of which two are commonly used), and between them you should be able to determine the sex of most bird species.

Bird sexing primer search tool

If you’re looking for a primer set for sexing your bird species, try our tool below, and search by species (common name, or scientific name) to see which primer set we would suggest. If your specific species is not included, you can also search by genus to give you a suggestion of which primer set to test.

The table shows which primers have previously been found to work when sexing different species of bird species. This is not a complete list, and there will be exceptions – please use it as a helpful guide, and ask us for help if you would like advice.

How else can I find out which primers to use?

If your bird species (or genus) is missing from our search tool, you could also:

• Consult other relevant literature to determine which primer set has been shown to work well with particular species in the past. For example, you could look for specific articles by searching in Google Scholar using the bird species name and a primer or primer set name (2550F, CHD1F, P0/P2/P8).
• Validate the assay with known male and female birds of that species to ensure a reliable result.
• Test all four primer sets on a species to achieve a consensus result that can then be used to identify the most appropriate primer sex for that species. In rare cases none of the primer sets we supply may be appropriate, and additional species-specific primer sets may be required.
• Test primers on a large enough group of samples of a species to be sure to detect both male and female birds. If the results indicate the presence of both male and female samples then the primers used are probably appropriate.
• Please note that in some cases additional primer sets beyond the four covered here may be needed. For example, anyone interested in sexing parrots should read our blog post on a new strategy for sexing different parrot species here, and see Table 1 of Kroczak et al., 2021 for information on which primers produce reliable results for which species.

Key bird sexing literature and resources

Here are some helpful publications to guide your choice of primer set, which we have included in our search tool above. They cover a wide variety of bird species that have been sexed using the primer sets CHD1F/CHD1R, 2550F/2718R, P0/P2/P8, and CHD1LF/CHD1LR, and most include detailed tables of results where you can search for your specific species.

🐦Fridolfsson & Ellegren (1999). A simple and universal method for molecular sexing of non-ratite birds. Journal of avian biology, 116-121. https://doi.org/10.2307/3677252 .

Fridolfsson & Ellegren (1999) developed the 2550F/2718R primer set and tested it on 50 bird species from 11 orders.

🐦Han et al. (2009). A simple and improved DNA test for avian sex determination. The Auk, 126(4), 779-783. https://doi.org/10.1525/auk.2009.08203

Han et al. (2009) developed the P0/P2/P8 primer set as an improvement of the P2/P8 primer set (which often produced bands of very similar sizes), and tested it on 18 bird species across 12 orders.

🐦Lee et al. (2010). A novel strategy for avian species and gender identification using the CHD gene. Molecular and cellular probes, 24(1), 27-31. https://doi.org/10.1016/j.mcp.2009.08.003

Lee et al. (2010) developed the CHD1F/CHD1R primer set and tested it on 58 species from 58 genera, 28 families and 12 orders. Subscription access but available on Academia.edu here.

🐦Çakmak et al. (2017). Comparison of three different primer sets for sexing birds. Journal of Veterinary Diagnostic Investigation, 29(1), 59-63. https://doi.org/10.1177/1040638716675197

Çakmak et al. (2017) tested 77 species from 14 orders with CHD1F/CHD1R, 2550F/2718R, and P2/P8 (part of the P0/P2/P8 set). A very useful supplementary table can be downloaded here.

🐦Liang et al. (2019). Sex identification of pigeons using polymerase chain reaction analysis with simple DNA extraction. Avian Biology Research, 12(2), 45-48. https://doi.org/10.1177/1758155919832141

Liang et al. (2019) developed the CHD1LF/CHD1LR primer set for sexing domestic pigeons. It can be found on Researchgate.org here.

🐦Kroczak et al. (2021). New Bird sexing strategy developed in the order Psittaciformes involves multiple markers to avoid sex misidentification: Debunked myth of the Universal DNA marker. Genes, 12(6), 878. https://doi.org/10.3390/genes12060878

Kroczak et al. (2021) tested a range of primers for sexing parrots, including 2550F/2718R (under the name CHD1iA). This article provides a very useful indication of which parrot species will work with 2550F/2718R, and those that will need other primers.

🐦Liu et al. (2024). Sex identification of birds in Taipei Zoo. Zoo Biology, 43(3), 268-275. https://doi.org/10.1002/zoo.21828

Liu et al. (2024) tested 166 bird species with CHD1F/CHD1R, 2550F/2718R, and P2/P8 (part of the P0/P2/P8 set). A very useful supplementary table of results can be downloaded here.

Want help?

If you have any questions about which primers can be used with which bird species, please get in touch and we will offer as much advice as we can.