Two-stage genome-wide association study for the identification of causal variants underlying hoof disorders in cattle

Feet and legs disorders influence dairy cattle breeding by their effect on animal welfare, economic losses due to lower production and fertility, costs of treatment, and problems with herd management. In our study, we estimated heritabilities and performed a 2-step GWAS for 3 traits describing hoof health: hoof health status defined by a veterinarian (HSV), hoof health status defined by a claw trimmer (HSC), and the total number of hoof disorders (NHD), scored in 1,998 Fleckvieh and 979 Braunvieh cows. The individuals were genotyped with a high-density (HD) panel consisting of 76,934 SNP. For significant genomic regions, the SNP information was enhanced by SNP imputed from the whole-genome sequence of Fleckvieh and Braunvieh bulls from the 1000 Bulls Genome project. The heritabilities were estimated to be 0.035 for HSV, 0.249 for HSC, and 0.279 for NHD. Based on the first-stage GWAS with SNP from the HD panel, 7 significant genomic regions on 6 chromosomes were defined: (1) 120 SNP spanning 15,522 bp on BTA1, including the TOPBP1 gene; (2) 4,139 SNP spanning 1,426,046 bp on BTA7, including the RIOK2 and RGMB genes; (3) 167 SNP spanning 167,352 bp on BTA13, including the C13H20orf194 gene; (4) 2 regions on BTA14, one harboring 1,071 SNP spanning 380,024 bp, including RRM2B and NCALD, and the other comprising 632 SNP spanning 385,111 bp, including STK3; (5) 328 SNP on BTA15, spanning 235,567 bp between FAM168A and PLEKHB1; and (6) 1,549 SNP on BTA22, spanning 596,101 bp in the neighborhood of PTPRG. Then, we conducted a second-stage GWAS based on SNP from whole-genome sequences within the significant regions obtained in the first stage of the analysis. For HSV, the highest additive effect was estimated for 23 SNP located within a region on BTA15, close to FAM168A, corresponding to a predicted gene sequence. For HSC, the highest additive effect was attributed to 44 SNP located within a region of BTA22 corresponding to 4 predicted gene sequences, with rs135082893 within a sequence encoding a microRNA. Another potential causal mutation for HSC was rs134142607 on BTA13, within the exon of C13H20orf194. For NHD, 33 SNP with the highest estimated effect were located on BTA7 within a region of a predicted gene positioned between RIOK2 and RGMB. On BTA14, all significant SNP were located in introns of STK3, which is responsible for the "abnormal gait" phenotype in mice.