Rapid Identification of the SNP Mutation in the ABCD4 Gene and Its Association with Multi-Vertebrae Phenotypes in Ujimqin Sheep Using TaqMan-MGB Technology

Abstract

Ujimqin sheep, known for its distinctive multi-vertebrae phenotypes (T13L7, T14L6, and T14L7) and economic value, has garnered significant attention. However, conventional phenotypic detection methods suffer from low efficiency and high costs. In this study, based on a key SNP locus (ABCD4 gene, Chr7:89393414, C > T) identified through a genome-wide association study (GWAS), a TaqMan-MGB (minor groove binder) genotyping system was developed. the objective was to establish a high-throughput and efficient molecular marker-assisted selection (MAS) tool. Specific primers and dual fluorescent probes were designed to optimize the reaction system. Standard plasmids were adopted to validate genotyping accuracy. A total of 152 Ujimqin sheep were subjected to TaqMan-MGB genotyping, digital radiography (DR) imaging, and Sanger sequencing. the results showed complete concordance between TaqMan-MGB and Sanger sequencing, with an overall agreement rate of 83.6% with DR imaging. For individuals with T/T genotypes (127/139), the detection accuracy reached 91.4%. This method demonstrated high specificity, simplicity, and cost-efficiency, significantly reducing the time and financial burden associated with traditional imaging-based approaches. the findings indicate that the TaqMan-MGB technique can accurately identify the T/T genotype at the SNP site and its strong association with the multi-vertebrae phenotypes, offering an effective and reliable tool for molecular breeding of Ujimqin sheep.

Keywords: ABCD4 gene; TaqMan-MGB detection; Ujimqin sheep; molecular marker; multi-vertebrae; single nucleotide polymorphism.

Figure 1

Results of DR imaging detection method for detecting spinal phenotype in Ujimqin sheep: (A) T13L6 type Ujimqin sheep; (B) T13L7 type multi-vertebra Ujimqin sheep; (C) T14L6 type multi-vertebra Ujimqin sheep; (D) T14L7 type multi-vertebra Ujimqin sheep; Note: Red dots indicate thoracic vertebrae; green dots indicate lumbar vertebrae.

Figure 2

SNP Genotyping of Standard Plasmids: (A) Sanger sequencing results of C/C genotype plasmids; (B) Sanger sequencing results of T/T genotype plasmids; (C) scatter plot confirms clear separation of three genotypes by fluorescence signals, validating assay specificity. The scatter plot of standard plasmids (blue square: SNP (89393414, C > T) locus genotype is “T/T” homozygous; green triangle: SNP (89393414, C > T) locus genotype is “C/T” heterozygous; yellow dots: SNP (89393414, C > T) locus genotype is “C/C” homozygous; red circles: the standard sample); Scatter plot confirms clear separation of three genotypes by fluorescence signals, validating assay specificity. (D) Dissolution curve of C/C genotype plasmid; (E) dissolution curves of C/C and T/T genotype mixed plasmids; (F) dissolution curve of T/T genotype plasmid.

Figure 3

Genetic testing and DR imaging detection results of T13L6 Type Ujimqin Sheep: (A) Taqman-MGB genotyping results with specific responses to FAM fluorescence signals; (B) Sanger sequencing results of amplification products with specific responses to FAM fluorescence signals; (C) DR imaging detection results of Ujimqin sheep with normal vertebral traits; (D) Taqman-MGB genotyping results that simultaneously respond to HEX and FAM fluorescence signals; (E) Sanger sequencing results of amplification products that simultaneously respond to both HEX and FAM fluorescence signals; (F) DR imaging detection results that simultaneously respond to HEX and FAM fluorescence signals. Note: Red dots indicate thoracic vertebrae; green dots indicate lumbar vertebrae.

Figure 4

This genetic testing and DR imaging results of T13L7, T14L6, and T14L7 types: (A,B) Genetic testing results showing HEX fluorescence-specific detection. (A) TaqMan-MGB genotyping. (B) Sanger sequencing of amplification products. (C–E) DR imaging detection results of Ujimqin sheep with multi-vertebrae traits. (C) T13L7(genotype T/T), (D) T14L6(genotype T/T), (E) T14L7(genotype T/T). Note: Red dots indicate thoracic vertebrae; green dots indicate lumbar vertebrae.