Dosage compensation is a mechanism first proposed by Susumu Ohno, whereby X inactivation balances X gene output between males (XY) and females (XX), while X upregulation balances X genes with autosomal gene output. These mechanisms have been actively studied in Drosophila and mice, but research regarding them lags behind in domestic species. It is unclear how the X chromosome is regulated in the sheep male germline. To address this, using single-cell RNA sequencing, we analyzed testes in three important developmental stages of sheep. We observed that the total RNA per cell from X and autosomes peaked in SSCs and spermatogonia and was then reduced in early spermatocytes. Furthermore, we counted the detected reads per gene in each cell type for X and autosomes. In cells experiencing dose compensation, close proximity to MSL (male-specific lethal), which is regulated the active X chromosome and was observed. Our results suggest that there is no dose compensation in the pre-meiotic germ cells of sheep testes and, in addition, MSL1 and MSL2 are expressed in early germ cells and involved in regulating mammalian X-chromosome inactivation and activation.

The short-tailed phenotype has long been considered one of the best traits for population genetic improvement in sheep breeding. In short-tailed sheep, not only is tail fat eliminated but also the pubic area is exposed due to the lack of a tail covering, giving them an advantage in reproduction. Recent studies have shown that two linked mutations in sheep TBXT at nucleotides 333 and 334 are associated with the short-tailed phenotype. In the population of short-tailed sheep, several heterozygous mutants of this gene are found. In our research, we used high-resolution melting (HRM) to identify homozygous and heterozygous genotypes in a flock of short-tailed sheep and compared the results with those of Sanger sequencing, which were identical. This demonstrates that our established HRM method, a rapid and inexpensive genotyping method, can be used to identify homozygous and heterozygous individuals in short-tailed sheep flocks.

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 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.