Molecular sexing by targeting W-chromosome specific sequences in chicken

Present investigation was carried out to evaluate and develop single-plex and multiplex PCR based sex differentiation methods in chicken by targeting W-chromosome specific repetitive sequences like XhoI and EcoRI families and assess their accuracy and reliability, so to address various issues related to poultry breeding, conservation and evolutionary studies since most of the conventional methods of sexing are not foolproof. Simple PCR with two primers’ pairs viz. W3/W5 and USP1/USP3 was used to amplify specific sequences viz. XhoI and EcoRI repeats, respectively in 10 genomic DNA samples, five of each sex. It was followed by multiplex PCR to exclude possibility of amplification failure employing primers’ pairs USP1/USP3 and CPE15F/CPE15R (targeting sequences located in both Z and W chromosomes). Simple PCR with W5/W3 and USP1/USP3 primers revealed only one band of 415bp and 370bp, respectively, which were female–specific as no band was seen in any of the male samples with either of the primers pair. Multiplex PCR revealed two amplicons of 370bp and 250bp in heterogametic (ZW) females, whereas males, being homogametic (ZZ), demonstrated only one band of 250bp because the targeted EcoRI sequence was absent in males. This study assessed the multiplex PCR method, employing one set of primers targeting W-chromosome and other primers pair targeting Z-chromosome, suitable for accurate and reliable sex differentiation in chicken. The results of this investigation can be used in early and accurate sex differentiation in chicken.