Targeted next-generation sequencing for identifying genes related to horse temperament

It is a fundamental challenge to discover the association of genetic traits with phenotypic traits. In this study, we aimed to identify possible genetic traits related to horse temperament. Based on previous findings, we selected 71 candidate genes related to temperamental trait and examined them in the human and horse reference genomes (hg38 and equCab2, respectively). We found 16 orthologous genes and, by comparing with the human reference genome, 17 homologous genes in the horse reference genome. We designed probes specific for the 33 horse genes. Using the probes, we built sequencing libraries of the genomic DNA samples from eight aggressive and eight docile horses, and sequenced the constructed libraries using the Illumina Hiseq2500 platform. Through the analysis of the targeted exome sequences, we identified single nucleotide polymorphisms (SNPs) in the genes. SNPs could be served as genetic markers to evaluate aggressive or docile levels of horses. To examine whether any genetic variants are associated with horse temperament, we performed genome-wide association study (GWAS) using the SNP data. GWAS analysis identified ten variants (p-value <0.05) which could be related to horse temperament. We validated the variants using Sanger sequencing. The most significant variants were found in MAOA (c.1164+41T>C) and AR (c.1047+27G>T) genes with 8.09 × 10⁻⁴p-value. We suggest that the variants might be used to assess horse temperament and to determine superior horses for riding or racing.