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. 2021 Jun 28;11(7):1924.
doi: 10.3390/ani11071924.

Comparative Analysis for Genetic Characterization in Korean Native Jeju Horse

Wooseok Lee  1 , Seyoung Mun  1   2 , Song-Yi Choi  3 , Dong-Yep Oh  4 , Yong-Soo Park  5 , Kyudong Han  1   6
Affiliations

Comparative Analysis for Genetic Characterization in Korean Native Jeju Horse

Wooseok Lee et al. Animals (Basel). .

Abstract

The Jeju horse is a native Korean species that has been breeding on Jeju Island since the 13th century. Their shape has a distinct appearance from the representative species, Thoroughbred. Here, we performed a comparison of the Jeju horse and Thoroughbred horse for the identification of genome-wide structure variation by using the next-generation sequencing (NGS) technique. We generated an average of 95.59 Gb of the DNA sequence, resulting in an average of 33.74 X sequence coverage from five Jeju horses. In addition, reads obtained from WGRS data almost covered the horse reference genome (mapped reads 98.4%). Based on our results, we identified 1,244,064 single nucleotide polymorphisms (SNPs), 113,498 genomic insertions, and 114,751 deletions through bioinformatics analysis. Interestingly, the results of the WGRS comparison indicated that the eqCD1a6 gene contains signatures of positive natural selection in Jeju horses. The eqCD1a6 gene is known to be involved in immunity. The eqCD1a6 gene of Jeju horses commonly contained 296 variants (275 SNPs and 21 INDELs) that were compared with its counterpart of two Thoroughbred horses. In addition, we used LOAA, digital PCR, to confirm the possibility of developing a molecular marker for species identification using variant sites. As a result, it was possible to confirm the result of the molecular marker with high accuracy. Nevertheless, eqCD1a6 was shown to be functionally intact. Taken together, we have found significant genomic variation in these two different horse species.

Keywords: Equus ferus caballus; Jeju horse; SNP; Thoroughbred; WGRS; digital PCR; eqCD1a6.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The appearance of pure Jeju horses. This representative photo of a Jeju horse was taken at a grazing ranch on Jeju Island, Korea. As described in the text, the Jeju horse has a small height, a large head, and a thick, short morphology.
Figure 2
Figure 2
Venn diagram of genomic variants identified from genome comparison with the horse reference genome. In comparative analysis with the horse reference genome (Equus caballus, equCab3: January 2018), we screened the number of SNPs and INDELs to find unique variants shared with the five Jeju horses. The number of SNPs (a), small insertions (b), and deletions (c), which are common in the five Jeju horses compared with the horse reference genome, are annotated at each Venn diagram. The number of variants highlighted in the bold letter is common to Jeju horse. (a) For SNPs, common variants are 2,758,242 loci, which account for 22.54% of the total. (b) For small insertions, 233,819 loci, 33.63% of the total. (c) For small deletions, 240,738 loci, 31.5% of the total.
Figure 3
Figure 3
Second comparison of genomic variants between Jeju horse and Thoroughbred. Using the variants compared to the horse reference genes for the first time, secondly, we selected unique variants for the five Jeju horses by comparing and analyzing the genome data of one Thoroughbred obtained in this study. Through this process with another Thoroughbred genome, we were able to accurately distinguish the variant calling of Jeju horse. (a) Among the 2,758,242 SNPs identified from the first comparison, a total of 1,244,064 (45.1%) were unique in Jeju horse. (b) A total of 113,498 insertions (45.1%) and (c) 114,751 deletions (48.54%) were detected in Jeju horse.
Figure 4
Figure 4
Third comparison of SNP variants using open-access SNP data for horses. By comparing open access SNP data published at the Broad Institute (add URL), Ensembl (add URL), and NCBI (add URL), 408,601 out of 1,244,064 SNPs accounting for 1.66% of all the variants in the SNP database (SNPdb) were finally identified. The number of final Jeju horse-specific SNPs is highlighted in the bold letter.
Figure 5
Figure 5
Gene ontology (GO) enrichment analysis of genes with non-synonymous SNPs in Jeju horses. A total of 106 of 788 are significantly associated with cardiac development and blood circulation. Each term is represented by a circle node, where its size is proportional to the number of input genes falling into that term. Its color represents its GO cluster identity (i.e., nodes of the same color belong to the same cluster). The small nodes interacting with circle nodes denote the genes that show associations with the GO cluster.
Figure 6
Figure 6
Amino acid (AA) sequence comparison of eqCD1A6 gene. Using BioEdit, eqCD1A6 sequences were visually compared to detect sequence signatures that distinguished between the two species. (a) The location of Jeju horse-specific SNPs in the exon region of the eqCD1a6 gene. Most of the SNPs are located in exon 2 and exon 3. (b) AA sequences of the eqCD1A6 gene searched by NCBI blast were aligned. Dots indicate AA sequences identical in nine eqCD1A6 isoforms. Orange lines denote the 37 conservative AA substitution spots in Jeju-horses. The eqCD1A6 protein domains, including the signal peptide, α-1, α-2, α-3, transmembrane, and cytoplasmic tail, are boxed with annotations.
Figure 7
Figure 7
Sliding-window analysis of Jeju horse and Thoroughbred eqCD1a6 genes. Sliding-window analysis of dN/dS ratios was performed along the length of the eqCD1a6 gene, comparing Jeju horse and Thoroughbred gene sequences. dN/dS is plotted against base-pair coordinates in the coding sequence. dN/dS ratios of 1.0 indicate neutral evolution, while ratios of <1.0 are indicative of purifying selection.
Figure 8
Figure 8
A molecular marker was applied to Jeju horse and Thoroughbred samples in digital PCR assays. A schematic dot plot diagram showing the molecular marker result. The yellow cluster on the plot expresses the control droplets. The green cluster (FAM) and red cluster (SFC620) express the positive droplets for Jeju horse-specific and Thoroughbred-specific, respectively.
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