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.

In this study, SweAMI FISH fluorescence in situ hybridization and immunofluorescence were used to analyze the spatiotemporal expression characteristics of the TBXT gene and Brachyury protein in 16-day-old Hulunbuir sheep embryos and 19-day-old Hu sheep embryos and to explore their regulatory effects on the development of sheep tails and related organs. The study showed the following: At 16 days of embryonic age, the TBXT gene was concentratedly expressed in the heart, somites, neural tube, and mesonephros of both sheep breeds; at 19 days of embryonic age, it was concentratedly expressed in the limb ectoderm and tail bud of Hulunbuir sheep, and in the midgut and tail bud of Hu sheep. At 16 days of embryonic age, the BRACHYURY protein was concentratedly expressed in the neural tube, somites, brain vesicles, and mesonephros of both sheep breeds; at 19 days of embryonic age, it was concentratedly expressed in the heart and hindgut of Hulunbuir sheep, and in the tail bud and hindgut of Hu sheep. In summary, this shows that there are differences in the temporal and spatial expressions of the TBXT gene and BRACHYURY protein between the two sheep breeds. There are also site-specific and time-specific differences in the regulation of the above genes and proteins during tail and related organ development between the two breeds, which confirms that the molecular regulation pathways of tail and related organ development are different between the two breeds. This study provides an experimental basis for screening molecular markers related to goat tail development and breed improvement.

This study designed three sgRNAs (sgRNA139, sgRNA128, and sgRNA109) targeting the prolactin gene receptor (PRLR) in fetal cattle, utilized Cas9 to cleave endogenous DNA, and screened stable cell lines for somatic cell nuclear transfer experiments to investigate the impact of different editing sites on embryonic development. The results showed that sgRNA139 had the highest cleavage efficiency (Fcut = 0.65, Indels = 42.19%), while sgRNA109 had the lowest (Fcut = 0.45, Indels = 35.31%). No significant differences were observed in cell growth status after electroporation (p > 0.05), and the transfection efficiency exceeded 90% after five days of culture. In the evaluation of key embryonic development indicators, sgRNA109 significantly reduced the cleavage rate and blastocyst rate (p < 0.01), whereas sgRNA139 showed no significant effect on the cleavage rate (p > 0.05), but its blastocyst rate was slightly lower than that of the control group (p > 0.05). This study demonstrates that highly specific sgRNAs and stable edited cell lines used as donor cells can significantly regulate the later stages of embryonic development. This study not only provides new experimental evidence for the functional study of the PRLR but also lays an important theoretical foundation for the innovation of molecular breeding technologies in dairy cattle.

This study aimed to develop an effective tail typing detection technology based on the TaqMan probe technology for genotyping different sheep tail types. A total of 122 Hulun Buir short-tailed sheep and 50 Hu sheep were enrolled in the study to compare their tail morphologies, lengths, and widths. Through the Sanger sequencing of loci 333 and 334 in the second exon of the T gene, distinct genotypes of various types of Hulun Buir short-tailed sheep and Hu sheep were identified. In addition, the TaqMan probe technology was employed to genotype the two SNP loci of the T gene in the two types of sheep. It was observed that the second exon of the T gene in Hulun Buir short-tailed sheep at loci 333 and 334 exhibited two genotypes, CT/CT and CT/GG, but this feature was not detected for the T gene in Hu sheep. The detection accuracy of the TaqMan probe technology for sheep tail types exceeded 70%, suggesting that it is an effective tail-typing detection technology. This study provides a solid economic foundation and theoretical ideas that will improve the breeding of short-tailed sheep.