Whole-Genome Resequencing of Ujimqin Sheep Identifies Genes Associated with Vertebral Number

Abstract

The number of vertebrae is a crucial economic trait that can significantly impact the carcass length and meat production in animals. However, our understanding of the quantitative trait loci (QTLs) and candidate genes associated with the vertebral number in sheep (Ovis aries) remains limited. To identify these candidate genes and QTLs, we collected 73 Ujimqin sheep with increased numbers of vertebrae (T13L7, T14L6, and T14L7) and 23 sheep with normal numbers of vertebrae (T13L6). Through high-throughput genome resequencing, we obtained a total of 24,130,801 effective single-nucleotide polymorphisms (SNPs). By conducting a selective-sweep analysis, we discovered that the most significantly selective region was located on chromosome 7. Within this region, we identified several genes, including VRTN, SYNDIG1L, LTBP2, and ABCD4, known to regulate the spinal development and morphology. Further, a genome-wide association study (GWAS) performed on sheep with increased and normal vertebral numbers confirmed that ABCD4 is a candidate gene for determining the number of vertebrae in sheep. Additionally, the most significant SNP on chromosome 7 was identified as a candidate QTL. Moreover, we detected two missense mutations in the ABCD4 gene; one of these mutations (Chr7: 89393414, C > T) at position 22 leads to the conversion of arginine (Arg) to glutamine (Gln), which is expected to negatively affect the protein's function. Notably, a transcriptome expression profile in mouse embryonic development revealed that ABCD4 is highly expressed during the critical period of vertebral formation (4.5-7.5 days). Our study highlights ABCD4 as a potential major gene influencing the number of vertebrae in Ujimqin sheep, with promising prospects for future genome-assisted breeding improvements in sheep.

Keywords: ABCD4 gene; Ujimqin sheep; vertebral number; whole-genome resequencing.

Figure 1

Four types of vertebral numbers in Ujimqin sheep. (A–D) show the DR images of the thoracolumbar vertebrae in Ujimqin Sheep with T13L6, T13L7, T14L6, and T14L7 traits. The red marks indicate the thoracic vertebra, and the green marks represent the lumbar vertebra. (E) Statistical bar chart of vertebral number in adult sheep in 2020 and lambs born in 2021. The X-axis represents the phenotype of the sheep, and the Y-axis represents the number of sheep.

Figure 2

Selection analyses identified a candidate region. (A) Pi value in 50 kb sliding windows with 5 kb steps in T13L6 (blue line) and multi-vertebral sheep (red line) from 88.5 to 91.0 Mb on chromosome 7. The shaded region indicates 89.25–90.0 Mb screened by Pi, and the red dotted box region represents 89.0–89.55 Mb screened by Fst. The shadows of the other colors represent genes in this region. (B) Schematic diagram of ABCD4 exon structure and two missense mutations in ABCD4 exon. X-axis represents the physical distance on chromosome 7. (C) Fst distributions plotted for sheep chromosome 7.

Figure 3

(A) Genome-wide association study for vertebral number in Ujimqin sheep. Manhattan plots of GWAS results. The X-axis shows SNPs across chromosomes from SSC1 to SSC26, and the Y-axis represents the −log10(p) value. The top line indicates the genome-wide significant thresholds (p < 1 × 10⁻⁶). (B) Manhattan plots for SSC7 of GWAS results. The top line indicates the genome-wide significant thresholds (p < 10⁻⁶). (C) Q–Q plot of GWAS analysis. (D) Heat map of linkage disequilibrium between significant SNPs on SSC7. The X-axis and Y-axis represent SNPs.

Figure 4

Statistics of mutation types in ABCD4 gene (Chr7: 89379307–89394764). (A) Pie plot of SNP types in ABCD4 gene (Chr7: 89379307–89394764). (B) Bar plot of base types in ABCD4 gene (Chr7: 89379307–89394764). The X-axis represents the mutation number, and the Y-axis represents the mutation type. (C) The three-dimensional protein structure of ABCD4 (sheep) is predicted using Alpha-Fold, and the location of the protein structure is affected by missense mutations in the ABCD4 gene.

Figure 5

Sanger sequencing of the three candidate SNPs in the sheep ABCD4 gene (sequencing of complementary chain). The shadows in figures (A–C) indicate the locations of candidate mutations and base types in sheep with different vertebral numbers.

Figure 6

The expression of ABCD4 and related gene enrichment analysis. (A) Gene Ontology (GO) analysis of the top 100 genes co-expressed with ABCD4 obtained using GEPIA2. Green indicates the top 10 GO terms of the molecular function, yellow indicates the top 10 GO terms of the cellular component, and blue indicates the top 10 GO terms of the biological process. The X-axis represents the number of genes, and the Y-axis represents the GO term. (B) The expression of the ABCD4 gene in mice during the embryonic development period (from 2 cells to E11.5). The X-axis represents the developmental stage, and the Y-axis represents TPM (transcripts per kilobase million). (C) Co-expression network of 40 genes co-expressed with ABCD4 was obtained using the STRING tool.