Developmental validation of a novel multiple genotyping assay with 23 pigeon STR loci

Pigeons (<i>Columba livia</i>) occupy an important place in pigeon racing due to their extraordinary flying and nesting abilities. In order to maintain fairness in these races through accurate individual identification of pigeons using forensic techniques, we developed a novel multiple genotyping system containing 23 pigeon short tandem repeat (STR) polymorphisms and a chromo-helicase DNA binding gene (CHD). This system underwent rigorous validation in accordance with the guidelines set by the Scientific Working Group on DNA Analysis Methods, including species specificity, sensitivity, repeatability, reproducibility, size precision, and a population study of 219 unrelated individual pigeons. The results demonstrated that the novel pigeon STR system was specific, reproducible, accurate and consistent. Complete profiles were obtained with 62.5 pg of pigeon DNA input. Furthermore, the combined discrimination power of the 23 STR loci reached 1-3.666 4 × 10^-19, while the combined exclusion power was 1-6.596 8 × 10^-5. These findings underscore the high polymorphism and informativeness of pigeon STR genetic markers, making them ideal for pigeon individual identification and parentage testing.