A simple and cost-effective denaturation-reannealing multiplex PCR (drmPCR) method for discriminating late-feathering genotypes in male Korean indigenous breed chickens.

Feather-based autosexing visually distinguishes the sex of day-old chicks to optimize commercial poultry production. Crossbreeding early-feathering (EF) males (Zk+/Zk+) with late-feathering (LF) females (ZK/W) yields LF male and EF female progeny for rapid identification. However, maintaining this system requires accurately distinguishing homozygous (ZK/ZK) form heterozygous (ZK/Zk+) LF males in parental lines. Currently, the reliance on specialized equipment limits the routine use of quantitative genotyping for this screening in large breeding populations. To overcome this limitation, this study evaluated a cost-effective denaturation-reannealing multiplex PCR (drmPCR) method for LF male genotyping in Korean Native Chicken (KNC) and Korean Ogye (KO) population. The developed assay integrates a denaturation-reannealing step with following multiplex amplification of the LF-specific K-junction and a PRLR control. This pre-treatment significantly improves the dosage-dependent separation of band ratios, effectively classifying LF KNC and KO males into either homozygous or heterozygous, although small portions of samples were not clearly classified using this method. However, drmPCR demonstrated higher accuracy than standard quantitative PCR (qPCR) when compared with droplet digital (ddPCR)-defined LF genotype patterns. Thus, drmPCR offers a rapid, cost-effective first-line screening tool for LF genotyping in poultry breeding without the need of specialized equipment or skills, highly suitable for large-scale screening of LF male genotypes in poultry industry.