Sex identification based on the CHD gene from Gentoo penguin (Pygoscelis papua ) fecal DNA samples

We developed a reliable noninvasive PCR method for sex identification of the Gentoo penguin (Pygoscelis papua) based on the amplification of the chromobox-helicase DNA-binding (CHD) gene. Two sets of nested primers (F1/R1, F2/R2) were designed to amplify the introns of the CHD1W and CHD1Z genes. The fecal samples were lysed in an optimized lysis reagent containing PCR buffer. The fecal lysate was employed as the template DNA to perform PCR amplification. Following nested PCR, agarose gel electrophoresis was performed, and the amplicons exhibited a sex-specific band pattern (211 bp and 315 bp in females, 211 bp in males). There was no amplification failure of our specific primers in 14 tested samples (7 males and 7 females), and the results of genotypic identification matched the records of sex based on oviposition behavior and pairing combination (based on the monogamy of the Gentoo penguin). The preliminary results showed that these nested primers could also be used for sex identification of Chinstrap penguins (Pygoscelis antarctica) and King penguins (Aptenodytes patagonicus). In conclusion, we successfully sexed Gentoo penguins using DNA extracted from fecal samples and performed nested PCR of sex-specific CHD genes.