Haplotype Variation of <scp> <i>TBXT</i> </scp> Enhancer Contributes to Tail Length Diversity of Sheep Through Modulation of Gene Expression

The T-bound notochord enhancer (TNE) is a conserved cis-regulatory element that governs the expression of T-box transcription factor T (TBXT) to regulate tail bud patterning and notochord formation in vertebrates. However, the role of ovine TNE (oTNE) variations in tail divergence remains unclear. Here, we cloned and analyzed oTNE sequences from 575 sheep representing five groups with diverse tail patterns. The distinct haplotypes, which consisted of 19 mutations exhibiting strong linkage disequilibrium, were constructed. Notably, a unique oTNE haplotype (H001) was predominantly distributed in tailless fat-rumped sheep, while H002 and H003 collectively accounted for the majority of haplotypes in long-tailed breeds. To further validate this association, we measured caudal vertebral number by X-ray imaging and tail length manually in a segregating F2 population (n = 79) derived from the cross of Texel ram (long-tailed) with Kazakh ewe (fat-rumped tailless), revealing that the oTNE haplotypes were associated with caudal vertebrae number and tail length. Functional analysis by luciferase reporter assays in UCH-1 cells revealed significant differences in activity among the haplotypes compared to the blank control. oTNE enhancer contains a T-box binding motif enabling TBXT-mediated self-regulation. Under the regulatory influence of TBXT on oTNE, we further found that H001 exhibited significantly higher enhancer activity than the other haplotypes. Thus, haplotypes within oTNE alter its transcriptional regulation of TBXT, providing a mechanistic explanation for tail morphology variation in sheep. This study not only provides evidence to further understand the cis-regulatory mechanisms underlying tail length in sheep but also lays a foundation for short-tail sheep breeding.