Detection and assay of polymorphism in reproductive gene loci in a commercial broiler breeder population for use in association studies

A candidate gene approach has been adopted to determine if variance in reproductive traits in commercial broiler breeder stock can be associated with alleles of genes involved in reproduction. Alleles with favourable associations may be useful for marker assisted selection for improved reproductive performance.

Initial research focused on genes encoding for growth hormone receptor (GHR), gonadotrophin releasing hormone (GnRH) and its receptor (GnRHR), and neuropeptide Y (NPY), using published cDNA sequence information. Primer pairs designed for the genomic DNA sequence were used for Base Excision Sequence Scanning (BESS T-Scan™, Epicentre Technologies) analysis or bulk scanning with panels of restriction enzymes.

In the GHR gene, two linked polymorphic sites (A and B) were detected in intron 5. In a sample of 40 males from a pedigree broiler dam line, alleles T+ and T- at site A were estimated to have frequencies 0.67 and 0.33, whereas frequencies of alleles T+ and T- at site B were 0.33 and 0.67. A diagnostic test based on the presence or absence of Eco72I or NspI restriction enzyme sites was devised to distinguish the two alleles at site A in a PCR-generated DNA fragment from intron 5. This confirmed the genotyping carried out using the BESS T-Scan method.

A polymorphic region that contained a 4 base insertion was discovered at -746 bp in the NPY gene promoter. It was characterised by presence or absence of a DraI restriction site, the allele frequencies being 0.22 and 0.78, respectively. About 65% (4 kb) of the GnRH gene sequence has been scanned in a broiler dam line, but no polymorphism has been found so far.

The BESS T-Scan methodology facilitates the process of identifying single nucleotide polymorphisms in the candidate genes. The alleles revealed are being used to establish possible associations with reproductive traits in a broiler dam line.

Supported by MAFF (LS2002 ‘Candidate genes for reproductive efficiency in poultry’) and by a Royal Society/NATO Postdoctoral Fellowship for M. Romanov.