No Variation in the Binding Sequence of W-Chromosome-Specific Primer P0: Reliable Universal Sex Determination of Genetically Distant Birds

We previously developed a W-chromosome-specific primer (P0) for universal avian sexing, in conjunction with the P2 and P8 primers. In our prior study, 18 bird species across 12 orders were tested to assess the applicability of this approach. However, the reliability of the test needs further confirmation with a larger sample size due to potential sequence polymorphism in the PCR primer binding site. To validate the reliability of P0, we examined the conservation of nucleotide sequences in the Chromodomain-Helicase DNA-binding 1 (CHD1) gene on the W and Z chromosomes, focusing on the P0 binding site in Domestic Chickens (Gallus domesticus), Saunders’s Gulls (Larus saundersi ), and White Storks (Ciconia boy-ciana). We found no variation in the P0 binding site of 90 Domestic Chickens, 37 Saunders’s Gulls, and 39 White Storks. In addition, there was no interspecies poly-morphism among the three species except one nucleotide, indicating conservation of the sequence on both chromosomes targeted by P0.