Genome-Wide Detection of Copy Number Variants in Chinese Indigenous Horse Breeds and Verification of CNV-Overlapped Genes Related to Heat Adaptation of the Jinjiang Horse

Abstract

In the present study, genome-wide CNVs were detected in a total of 301 samples from 10 Chinese indigenous horse breeds using the Illumina Equine SNP70 Bead Array, and the candidate genes related to adaptability to high temperature and humidity in Jinjiang horses were identified and validated. We determined a total of 577 CNVs ranging in size from 1.06 Kb to 2023.07 Kb on the 31 pairs of autosomes. By aggregating the overlapping CNVs for each breed, a total of 495 CNVRs were detected in the 10 Chinese horse breeds. As many as 211 breed-specific CNVRs were determined, of which 64 were found in the Jinjiang horse population. By removing repetitive CNV regions between breeds, a total of 239 CNVRs were identified in the Chinese indigenous horse breeds including 102 losses, 133 gains and 4 of both events (losses and gains in the same region), in which 131 CNVRs were novel and only detected in the present study compared with previous studies. The total detected CNVR length was 41.74 Mb, accounting for 1.83% of the total length of equine autosomal chromosomes. The coverage of CNVRs on each chromosome varied from 0.47% to 15.68%, with the highest coverage on ECA 12, but the highest number of CNVRs was detected on ECA1 and ECA24. A total of 229 genes overlapping with CNVRs were detected in the Jinjiang horse population, which is an indigenous horse breed unique to the southeastern coast of China exhibiting adaptability to high temperature and humidity. The functional annotation of these genes showed significant relation to cellular heat acclimation and immunity. The expression levels of the candidate genes were validated by heat shock treatment of various durations on fibroblasts of horses. The results show that the expression levels of HSPA1A were significantly increased among the different heat shock durations. The expression level of NFKBIA and SOCS4 declined from the beginning of heat shock to 2 h after heat shock and then showed a gradual increase until it reached the highest value at 6 h and 10 h of heat shock, respectively. Breed-specific CNVRs of Chinese indigenous horse breeds were revealed in the present study, and the results facilitate mapping CNVs on the whole genome and also provide valuable insights into the molecular mechanisms of adaptation to high temperature and humidity in the Jinjiang horse.

Copy Number Variation (CNV): A New Genomic Insight in Horses

“…In addition, although several CNVRs were observed in all the chromosomes ( Figure 2 ), the coverage of CNVRs in each chromosome varied from 1.9% in ECA14 and ECA16 to 19.2% in ECA12. These results in terms of variability agree with most of the previous studies carried out on horses [ 9 , 12 , 13 , 14 , 16 , 19 , 20 , 21 , 22 , 23 , 24 ]. However, several of them reported that ECA12 is particularly enriched in CNVRs, including a cluster of genes associated with the development of olfactory receptors (ORs) [ 13 , 18 , 19 ].…”

“…Although the number of deletions and duplications detected differed among chromosomes ( Table S1 ), an interesting finding was the fact that the number of duplications (12,987) exceeded the number of deletions (6915) in most of them, with the sole exception of ECA1, ECA4, and ECA29, in which the opposite pattern was observed. These results are similar to other studies [ 9 , 12 , 13 , 14 , 16 , 24 ], which reported more gains than losses in several horse populations. Although CNVs can be a source of wide variability at the same locus, duplications are more likely to occur in large CNVs than deletions, since they are more tolerated by the genome since no loss of genetic material occurs [ 40 , 41 ].…”

“…Among these, 109 (31 duplications, 19 deletions, and 59 mixed) were present in at least 5% of the PRE population analyzed. These results are higher than those obtained in several studies reported to date in other equine breeds, which range from 0.6 to 3.7% [ 13 , 14 , 20 , 21 , 22 , 23 , 24 ], but lower than those obtained by Schurink, et al [ 9 ] and Metzger, et al [ 19 ] using PennCNV software. In addition, although several CNVRs were observed in all the chromosomes ( Figure 2 ), the coverage of CNVRs in each chromosome varied from 1.9% in ECA14 and ECA16 to 19.2% in ECA12.…”

“…Differences in CNVRs have been successfully employed as a method of differentiation of horse breeds [ 12 ], since they allow us to find variants and regions which may be associated with its historical evolution, adaptability to a specific environment, or an improvement in the phenotype in traits of interest, such as the grey coat [ 50 ]. For this reason, we compared CNVRs identified in this study with those previously reported in eleven different reports [ 9 , 12 , 13 , 14 , 16 , 19 , 20 , 21 , 23 , 24 ] by generating a consensus list including all the different CNVRs reported previously ( n = 8292; Table S3 ). Our analysis showed that 61% (614) of the CNVRs which we detected in PRE overlapped with some of these, whereas near 40% were described for the first time in this study.…”

“…The first report was published by Doan, et al [ 13 ], which suggested that CNVs are common in the horse genome and may modulate the biological processes underlying different traits. Thereafter, several studies have reported the association of CNVs with diseases [ 9 , 14 , 15 , 16 ], chromosomal abnormalities [ 17 , 18 ], and phenotypic traits [ 19 , 20 , 21 , 22 ], as well as reported CNV regions overlapping with several genes associated with the reproductive system [ 23 ] or adaptability to high temperature and humidity [ 24 ]. However, the largest study assessing the CNV landscape in several European horse breeds was recently published by Sole, et al [ 12 ] where they identified CNV regions overlapping with QTLs previously associated with changes in fertility, coat color, conformation, and temperament.…”