A Rapid, Polymerase Chain Reaction-Based Procedure for Identifying Mutant Restricted Ovulator Chickens

Abstract

Females of the restricted ovulator (RO) strain of White Leghorn chickens fail to lay eggs upon photostimulation and exhibit endogenous hyperlipidemia and atherosclerotic lesions. A mutation in the gene specifying the oocyte vitellogenesis receptor (OVR), a 95-kDa membrane protein that normally mediates the massive uptake of yolk precursors from the serum, is responsible for this abnormal phenotype. Because a single nucleotide substitution (G → C) is responsible for the defective OVR, a PCR-based procedure, described herein, was developed in order to provide a rapid and accurate method for identifying chickens possessing the mutant allele. Polymerase chain reaction-amplified fragments of apparently identical size (~ 400 bp) were obtained from genomic DNA using primer pairs specific for either the wild-type or mutant genes. Through cloning and sequencing of the PCR-amplified products, the fragment sizes were determined to be 413 bp each, which included an intron sequence. Polymerase chain reaction-amplified genomic DNA from wild-type (ovr+/ovr+) males, heterozygous carrier (ovr+/ovr−) males, and wild-type (−/ovr+) females all yielded a 413 bp fragment when a primer pair specific for the wild-type gene was used. Because female chickens are heterogametic (ZW), no PCR product was observed in the case of the mutant (−/ovr−) females. When the primer pair specific for the mutant gene was employed, PCR-amplification of genomic DNA from both heterozygous carrier (ovr+/ovr−) males and mutant (−/ovr−) females, but not wild-type (ovr+/ovr+) males or (−/ovr+) females, also yielded a 413-bp fragment. Employment of the present rapid and accurate procedure would be expected to obviate the need for conventional progeny testing while reducing the time required to identify RO carrier males and mutant females from approximately 1 yr to several days.