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.

我们开发了一种可靠的非侵入式PCR方法，用于鉴定Gentoo企鹅（Pygoscelis papua））基于染色体盒解旋酶DNA结合（CHD）基因的扩增。设计了两组嵌套引物（F1 / R1，F2 / R2）来扩增CHD1W和CHD1Z基因的内含子。粪便样品在含有PCR缓冲液的优化裂解试剂中裂解。将粪便裂解物用作模板DNA以进行PCR扩增。巢式PCR后，进行琼脂糖凝胶电泳，扩增子表现出性别特异性条带模式（雌性为211 bp和315 bp，雄性为211 bp）。在14个测试样品中（7个雄性和7个雌性），我们的特异性引物没有扩增失败，基因型鉴定的结果与基于排卵行为和配对组合（基于Gentoo企鹅的一夫一妻制）的性别记录相匹配。南极侏儒（Pygoscelis antarctica）和企鹅国王（Aptenodytes patagonicus）。总之，我们使用从粪便样品中提取的DNA成功地对Gentoo企鹅进行了性别鉴定，并进行了性别特异性CHD基因的巢式PCR。