Combining QTL Mapping, GWAS and RNA Sequencing to Resolve a QTL Affecting Bone Strength to the Functional Gene

Bone fractures are a major welfare problem in laying hens. High incidences of skeletal damage are being reported over a range of husbandry systems. Our previous work has shown that genetic factors underlie the variation in the susceptibility of individual birds to osteoporosis and bone fracture (Bishop et al., 2000), and a quantitative trait locus (QTL) of large effect, located on Chromosome 1, was characterised in a F2 population of hens divergently selected for bone strength (Dunn et al., 2007).

In this study, we refined the QTL in the F2 by adding SNP markers. We then tested the SNPs from the refined region on the 2006 generation of the White Leghorn line from which the F2 was derived after 4 generations of divergent selection.  Subsequently, birds from the 2011 generation were genotyped for the QTL haplotype and birds with alternative QTL haplotypes were selected for qPCR of candidate genes and RNA sequencing. 

For the SNPs with the most consistent effects, the tibial breaking strength for the alternative haplotypes in the 2006 population was 200.4 vs 218.1 Newtons (p<0.002). This SNP marker is also significantly associated (P=0.019) with cortical bone density (mmAl equiv/mm³). The values for each haplotype were 0.044 vs 0.050.

The RNA sequencing showed strong differential expression for a single candidate gene in the QTL region with clear allelic imbalance suggesting a cis-acting effect within the candidate gene.  

BISHOP, S. C., et al 2000. British Poultry Science, 41,33-40.

DUNN, I. C. et al, 2007. Animal Genetics, 38, 45-49.