Green peafowl (Pavo muticus), a representative of the Southeast Asia Phasianidae, is assessed as globally endangered with populations that are declining. It is highly sensitive to human disturbance and habitat change, two threats that are unlikely to be removed soon in present range countries, and that increase the need for long-term continuous effort for in situ conservation. Captive breeding for reintroduction is potentially an important measure for localized population restoration. But green peafowl can hybridize with blue peafowl (P. cristatus) and produce fertile offspring that can backcross with ancestral species. Hybrids backcrossed to green peafowl for a few generations are indistinguishable from pure green peafowl in appearance. Such hybrids must be excluded from captive breeding programs to avoid anthropogenic genetic contamination. In this study, we developed technology to identify hybrids using mitochondrial Cyt b and COI gene sequences, and 16 microsatellites. We studied maternal species assignment for excluding individuals of the blue peafowl maternal line and genetic clustering analysis to exclude individuals containing blue peafowl nuclear genes. Maternal species assignment was accomplished using K2P pairwise genetic distance computed using transition nucleotide substitution. Nuclear genetic clustering analysis was performed using STRUCTURE (assuming K = 2) and NewHybrids. The method was validated using reference pure green (n = 18) and blue (n = 21) peafowl and known hybrids (n = 12). Two criteria were set up to discriminate hybrids based on the probability that an individual is either pure green or pure blue peafowl at 95% confidence. This method was applied to identification of 59 peafowl of ambiguous genetic composition. Our study provides an example for establishing a purity test workflow for other species that can hybridize and has potential for use in reintroduction of genetically pure green peafowl.

绿孔雀（Pavo muticus）是东南亚Ph科的代表，被评估为全球濒临灭绝的种群，数量正在下降。它对人为干扰和栖息地变化高度敏感，这两种威胁在目前的覆盖范围内的国家中不太可能很快消除，并且增加了对原位保护进行长期持续努力的需要。圈养繁殖以重新引入可能是恢复本地种群的重要措施。但是绿孔雀可以与蓝孔雀（P. cristatus）杂交）并繁殖出可以与祖先物种回交的可育后代。与绿孔雀回交了几代的杂种在外观上与纯绿孔雀没有区别。此类杂种必须从圈养繁殖计划中排除，以避免人为遗传污染。在这项研究中，我们开发了利用线粒体Cyt b和COI基因序列以及16个微卫星鉴定杂种的技术。我们研究了排除蓝孔雀母系的个体的母体物种分配，并进行了遗传聚类分析以排除包含蓝孔雀核基因的个体。母体物种分配是通过使用过渡核苷酸取代计算的K2P成对遗传距离完成的。使用STRUCTURE（假设K = 2）和NewHybrids进行了核遗传聚类分析。使用参考纯绿色（n = 18）和蓝色（n = 21）孔雀以及已知的杂种（n = 12）验证了该方法。建立了两个标准，以基于95％置信度下一个人是纯绿色或蓝色孔雀的概率来区分杂种。该方法用于鉴定59只孔雀的遗传组成不明确。我们的研究为建立可杂交的其他物种的纯度测试工作流程提供了一个实例，并有潜力用于重新引入基因纯绿孔雀。该方法用于鉴定59只孔雀的遗传组成不明确。我们的研究为建立一个可以杂交的其他物种的纯度测试流程提供了一个示例，该物种可以杂交，并有潜力重新引入基因纯绿孔雀。该方法用于鉴定59只孔雀的遗传组成不明确。我们的研究为建立可杂交的其他物种的纯度测试工作流程提供了一个实例，并有潜力用于重新引入基因纯绿孔雀。