Data from: Genome-wide association study for tobiano spotting coat color in Korean Jeju × Thoroughbred horse population

Korean Jeju horse features a small to medium frame size and stature having varieties of coat colors including grey, milky, spotted and bay1. Crossbreeding with Thoroughbred has been a long tradition in Korea to have intermediate frame size horse suitable for racing, leisure activities and meat production2. Tobiano white-spotting pattern is preferred by many horse breeders and owners which is inherited as an autosomal dominant trait1,3. Polymorphisms in proto-oncogene receptor tyrosine kinase (KIT) gene were strongly associated with tobiano and sabino coat color pattern in American and European horse breeds3,5. In addition, ECA3 inversion locus near KIT gene also significantly associated with tobiano spotting pattern in horses4,5. Here, we report SNPs and harbored genes associated with tobiano coat color in a crossbred horse population through genome-wide SNP association analysis.

In this study, coat color patterns of 142 crossbred horses (Jeju indigenous horse × Thoroughbred) were verified according to previously described methods3,5 and coded as “0” or “1” (Figure S1). Genomic DNA was extracted from blood samples using DNeasy 96 blood DNA extraction Kit (QIAGEN, USA). Genotyping was performed using Equine SNP 50K BeadChip (Illumina, San Diego, USA) at AGBD, NIAS, Korea. A total of 45,204 SNPs passed the set quality control criteria as minor allele frequency (MAF<0.01), missing genotype (GENO>0.10) and Hardy-Weinberg equilibrium (HWE<0.0001) in 142 individuals. Finally, a set of 16,223 independent SNPs were generated through –indep –pairwise 25 5 0.25 pruning6.

Genome wide association study revealed 18 Bonferroni adjusted SNPs located on chromosome 3 (ECA3) significantly associated with tobiano coat color in the studied population (Figure 1, Table S1). These SNPs broadly plotted on 40 Mbp region (from 39 to 79 Mbp) of ECA3 which might be due to high LD among the significant SNPs. The most significant association was found between ECA3 inversion locus (77657979 bp) and tobiano pattern (P< 5.56×10-17) while the second highest association was detected with SNP rs68555258 (48853535 bp; P< 4.04×10-12, Table S1). Apart from this, genotypes of ECA3 inversion locus by pyrosequencing matched perfectly in our study (Table S2). All tobiano horses (n = 37) were heterozygous for inversion locus (+/To) while solid-colored horses (n = 103) were homozygous (+/+). ECA3 inversion is located 70 kbp downstream from KIT gene which possessed causal variants for tobiano pattern in horse5,6. The Biomart and Variant Effect Predictor tools in Ensembl Genome Browser (http://www.ensembl.org/index.html) identified several significant SNPs harboring genes of which GPRIN3, ARHGAP24, SCFD2, RASSL11B and WDFY3 are noticeable (Table S3). Based on our genome wide association and inversion locus genotyping results, it is suggested that ECA3 inversion locus variant is either causative or completely linked with causative variants for tobiano coat color in this crossbred horse population. Moreover, a set of putative mutations in other genes might be in high LD with causative variants that could be explored for functional annotations associated with tobiano coat color pattern in horse population.

韩国济州马（Jeju horse）体型偏小至中等，毛色多样，涵盖灰色、乳白色、花斑色以及枣色¹。在韩国，与纯血马（Thoroughbred）杂交培育出体型中等的杂交马，用于赛马、休闲活动及肉用生产，已是悠久传统²。托比亚诺白斑毛色表型（tobiano white-spotting pattern）受众多马繁育者与马主青睐，该性状以常染色体显性遗传（autosomal dominant trait）方式遗传¹,³。此前研究证实，原癌基因受体酪氨酸激酶（KIT）基因的多态性与欧美马品种中的托比亚诺白斑及萨比诺（sabino）毛色表型显著相关³,⁵。此外，位于KIT基因附近的ECA3倒位位点同样与马的托比亚诺白斑表型存在显著关联⁴,⁵。本研究通过全基因组单核苷酸多态性（SNP）关联分析，报告了杂交马群体中与托比亚诺毛色表型相关的SNP及关联基因。

本研究参照已发表方法³,⁵对142匹杂交马（济州本土马×纯血马）的毛色表型进行鉴定，并编码为"0"或"1"（补充图S1）。采用DNeasy 96血液DNA提取试剂盒（QIAGEN，美国）从血液样本中提取基因组DNA。本研究在韩国国家农业科学研究院（NIAS）的农业生物技术实验室（AGBD），使用马用SNP 50K基因分型芯片（Illumina，美国圣地亚哥）完成基因分型。在142个个体中，共45204个SNP通过了预设质量控制标准：次要等位基因频率（minor allele frequency, MAF<0.01）、基因型缺失率（GENO>0.10）以及哈迪-温伯格平衡（Hardy-Weinberg equilibrium, HWE<0.0001）。最终通过–indep –pairwise 25 5 0.25 修剪步骤⁶，得到16223个独立SNP。

全基因组关联分析显示，在研究群体中，位于3号染色体（ECA3）上的18个经邦费罗尼校正的SNP与托比亚诺毛色表型显著相关（图1、补充表S1）。这些SNP广泛分布于ECA3的40 Mb区间（39~79 Mb），这可能源于显著关联SNP间存在高度连锁不平衡（linkage disequilibrium, LD）。其中最显著的关联出现在ECA3倒位位点（77657979 bp）与托比亚诺表型之间（P<5.56×10^-17），其次为SNP rs68555258（48853535 bp；P<4.04×10^-12，补充表S1）。此外，本研究通过焦磷酸测序得到的ECA3倒位位点基因型结果与分型结果完全一致（补充表S2）。所有托比亚诺毛色马（n=37）均为倒位位点的杂合子（+/To），而纯色马（n=103）则为纯合子（+/+）。ECA3倒位位于KIT基因下游70 kb处，该基因此前被证实存在导致马托比亚诺表型的致病变异⁵,⁶。通过Ensembl基因组浏览器（Ensembl Genome Browser，http://www.ensembl.org/index.html）中的Biomart和变异效应预测器工具，我们鉴定出多个携带显著关联SNP的基因，其中GPRIN3、ARHGAP24、SCFD2、RASSL11B及WDFY3较为突出（补充表S3）。基于本研究的全基因组关联分析及倒位位点基因分型结果，我们推测：在该杂交马群体中，ECA3倒位位点变异要么为托比亚诺毛色表型的致病变异，要么与致病变异完全连锁。此外，其他基因中的一批潜在突变可能与致病变异存在高度连锁不平衡，可进一步探究其与马群体中托比亚诺毛色表型相关的功能注释。