A Robust Assay for Molecular Sexing of Emperor Penguins Using Degraded DNA and the Potential Applicability to Other Spheniscids
4 affiliations
- 1Moss Landing Marine Laboratories San José State University Moss Landing California USA
- 2School of Biological Sciences University of Canterbury Christchurch New Zealand
- 3New Zealand Institute for Earth Science Wellington New Zealand
- 4Gateway Antarctica, School of Earth and Environment University of Canterbury Christchurch New Zealand
Abstract
Molecular sexing is a widely used approach for identifying the sex of monomorphic birds, but existing primers often target relatively large products that are difficult to amplify from highly degraded DNA. In this study, we developed a new primer set (voEP_F/voEP_R/P2) based on allele‐specific polymerase chain reaction (ASPCR) to facilitate the sexing of emperor penguins ( Aptenodytes forsteri ) using non‐invasively collected guano. Our assay targets short sequences within a conserved exon of the chromodomain helicase DNA binding 1 ( CHD1 ) gene and can therefore be used with a broad range of sample types. We compared the performance of our new primer set in both plucked feathers and guano to the commonly used intron‐based sexing primers (2550F/2718R and P8/P2) and evaluated its sensitivity through a cross‐factorial fragmentation and dilution experiment of synthetic CHD1 templates. We then independently validated the accuracy of our assay in a small subset of feather samples using whole‐genome sequencing (WGS) data. Although P8/P2 was highly effective compared to 2550F/2718R for sex determination in emperor penguins, it was less reliable when used with guano samples. In contrast, the voEP primers consistently yielded higher amplification success and showed greater analytical sensitivity across both feathers and guano. Sequence alignments of CHD1 from multiple penguin species revealed conservation of the primer‐binding sites, suggesting that the newly designed primers can potentially be used with other Spheniscids. Our results demonstrate that the voEP assay is a robust and reliable alternative method for sex identification in penguins and may provide a solution for sexing with lower‐quality genetic samples.
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BibTeX
@article{ooi2026robust,
title = {{A Robust Assay for Molecular Sexing of Emperor Penguins Using Degraded DNA and the Potential Applicability to Other Spheniscids}},
author = {Vicky Ooi and Sarah R. Smith and Skye Davis and David R. Thompson and Matt H. Pinkerton and Sarah P. Flanagan and Michelle LaRue and Birgitte I. McDonald},
journal = {Ecology and Evolution},
year = {2026},
month = nov,
doi = {10.1002/ece3.74654},
url = {https://onlinelibrary.wiley.com/doi/10.1002/ece3.74654},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A Robust Assay for Molecular Sexing of Emperor Penguins Using Degraded DNA and the Potential Applicability to Other Spheniscids
AU - Vicky Ooi
AU - Sarah R. Smith
AU - Skye Davis
AU - David R. Thompson
AU - Matt H. Pinkerton
AU - Sarah P. Flanagan
AU - Michelle LaRue
AU - Birgitte I. McDonald
PY - 2026
DA - 2026/11/05
JO - Ecology and Evolution
DO - 10.1002/ece3.74654
UR - https://onlinelibrary.wiley.com/doi/10.1002/ece3.74654
AB - Molecular sexing is a widely used approach for identifying the sex of monomorphic birds, but existing primers often target relatively large products that are difficult to amplify from highly degraded DNA. In this study, we developed a new primer set (voEP_F/voEP_R/P2) based on allele‐specific polymerase chain reaction (ASPCR) to facilitate the sexing of emperor penguins ( Aptenodytes forsteri ) using non‐invasively collected guano. Our assay targets short sequences within a conserved exon of the chromodomain helicase DNA binding 1 ( CHD1 ) gene and can therefore be used with a broad range of sample types. We compared the performance of our new primer set in both plucked feathers and guano to the commonly used intron‐based sexing primers (2550F/2718R and P8/P2) and evaluated its sensitivity through a cross‐factorial fragmentation and dilution experiment of synthetic CHD1 templates. We then independently validated the accuracy of our assay in a small subset of feather samples using whole‐genome sequencing (WGS) data. Although P8/P2 was highly effective compared to 2550F/2718R for sex determination in emperor penguins, it was less reliable when used with guano samples. In contrast, the voEP primers consistently yielded higher amplification success and showed greater analytical sensitivity across both feathers and guano. Sequence alignments of CHD1 from multiple penguin species revealed conservation of the primer‐binding sites, suggesting that the newly designed primers can potentially be used with other Spheniscids. Our results demonstrate that the voEP assay is a robust and reliable alternative method for sex identification in penguins and may provide a solution for sexing with lower‐quality genetic samples.
SN - 2045-7758
SN - 2045-7758
ER -