Development and Validation of a Novel Forensic STR Multiplex Assay for Domestic Pigeon (Columba livia domestica)

The domestic pigeon (Columba livia domestica), recognized as one of the earliest species to be domesticated by humans, has sustained a profound and enduring association with human society. These birds fulfill significant social and economic roles, particularly in the context of pigeon racing competitions. To ensure fairness and authenticity in such events, as well as to address concerns related to meat fraud and adulteration, we have developed a six-color fluorescent multiplex PCR amplification system incorporating 24 short tandem repeat (STR) loci and the chromobox helicase DNA-binding gene (CHD) specific to domestic pigeons. The multiplex assay underwent comprehensive validation in accordance with the guidelines established by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Committee on Wildlife Forensic Science Guidelines, encompassing evaluations of PCR conditions, precision, species specificity, sensitivity, stability, repeatability, and reproducibility. Furthermore, population genetic analyses and evaluations using simulated forensic case samples were conducted. The assay's applicability to closely related species was also evaluated. Our results show that the developed pigeon STR genotyping system exhibits high levels of accuracy, specificity, repeatability, stability, and robustness. Complete genotyping profiles were obtained with only 125 pg of domestic pigeon genomic DNA. Analysis of 248 unrelated domestic pigeons obtained from a local market in Shanghai, China, yielded the cumulative probability of exclusion (CPE) and the combined power of discrimination (CPD) values of 0.999999659383865638452 and 0.99999999999999999999918448912103896, respectively. Additionally, the majority of primers effectively amplified loci in congeneric species, indicating the assay's broad applicability. These results suggest that the multiplex system is highly polymorphic and well-suited for individual identification and kinship analysis in domestic pigeons, with potential applicability extending to related species.