Comprehensive functional genome annotation is crucial to elucidate the molecular mechanisms of agronomic traits in livestock, yet systematic functional annotation of the sheep genome is lacking. Here, we generated 92 transcriptomic and epigenomic data sets from nine major tissues, along with whole‐genome data from 2357 individuals across 29 breeds worldwide, and 4006 phenotypic data related to tail fat weight. We constructed the first multi‐tissue epigenome atlas in terms of functional elements, chromatin states, and their functions and explored the utility of the functional elements in interpreting phenotypic variation during sheep domestication and improvement. Particularly, we identified a total of 753,723 nonredundant functional elements, with over 60% being novel. We found tissue‐specific promoters and enhancers related to sensory abilities and immune response that were highly enriched in genomic regions influenced by domestication, while longissimus dorsi tissue‐specific active enhancers and tail fat tissue‐specific active promoters were highly enriched in genomic regions influenced by breeding and improvement. Notably, a variant, Chr13:51760995A>C, located in an enhancer region, was identified as a causal variant for tail fat deposition based on multi‐layered data sets. Overall, this research provides foundational resources and a successful case for future investigations of complex traits in sheep through the integration of multi‐omics data sets.

Feed efficiency makes up a large part of sheep production, which also has a crucial impact on the economic benefits of producers. This study explores the polymorphism of Adenylyl cyclase 8 gene associated with feed efficiency and detects the expression characteristics of ADCY8 in ten tissues of Hu sheep. The polymorphism of ADCY8 was recognized by using PCR amplification and Sanger sequencing, KASPar technology was used for genotyping subsequently, and the relationship between SNP and RFI is also studied. The results indicated that an intronic mutation g.24799148 C > T (rs 423395741) was identified in ADCY8, and association analysis showed that the SNP g.24799148 C > T (rs 423395741) was significantly associated with RFI at 100–120, 100–140, 100–160, and 100–180 days (p < 0.05). The quantitative real-time PCR (qRT-PCR) result showed that ADCY8 was expressed in ten tissues, and the expression of ADCY8 gene in rumen tissue was significantly higher than in the other tested tissues. Therefore, these results indicated that the ADCY8 mutation locus may be used as a candidate molecular marker for evaluating the feed efficiency of Hu sheep.

Scrotal circumference is an important reproductive index of breeding rams, which has a high genetic correlation with ejaculation volume and semen quality. In this study, the scrotal circumference of 1353 male Hu sheep at different stages of development was measured and descriptive statistical analysis was performed. The results showed that the coefficient of variation of scrotal circumference at each stage was greater than 10%, and its heritability were moderately to high, ranging from 0.318 to 0.719. We used PCR amplification and Sanger sequencing to scan the polymorphisms of the IGFALS gene, and performed association analysis with the circumference of the scrotum at different stages. We identified a synonymous mutation g.918 G > C in exon 1 of the IGFALS gene, and this mutation was significantly associated with scrotal circumference at 100, 120, 140, 160 and 180 days (p < 0.05). Therefore, IGFALS gene polymorphism can be used as a molecular marker affecting scrotal circumference of Hu sheep, which can provide a reference for future molecular marker-assisted selection of scrotal circumference in sheep.

Hybridization effectively enhances breeding efficiency and significantly boosts sheep productivity. However, the epigenetic mechanisms underlying the superior production performance of crossbreds remain largely elusive. In this study, Hu sheep were crossbred with Suffolk rams used as the paternal line. We integrated RNA-seq, ATAC-seq, and CUT&Tag (H3K4me3, H3K4me1, H3K27ac, and H3K27me3) techniques to characterize epigenetic regulatory differences in the longissimus dorsi muscle between Hu sheep (HU) and crossbred progeny (SH). Phenotypic and transcriptomic analyses revealed that SH crossbred sheep exhibited superior growth performance (p < 0.05), and the upregulated genes in the Apelin signaling pathway were significantly correlated with eye muscle area (p < 0.05). Utilizing a Hidden Markov Model, we annotated 15 distinct chromatin states in both HU and SH sheep, systematically characterizing the dynamic epigenomic landscapes across the two breeds. In contrast to SH sheep, the genome of HU sheep exhibited enrichment of repressive chromatin modifications typified by H3K27me3. Strong active enhancers (EnhA) were significantly enriched within upregulated genes in SH. A total of 1862 SH-specific and 691 HU-specific EnhA elements were characterized in this study. Motif analysis revealed that SH-specific EnhA were enriched for myogenic MEF2 family motifs (p < 0.05), which promote muscle and vascular development. By integrating multi-omics data, we constructed a putative regulatory network potentially modulated by SH-specific enhancers, identifying CMKLR1, PPARGC1A, and TLE3 as the core hub genes. Collectively, this study provides a robust data resource, identifying candidate genes and regulatory elements associated with crossbreeding-related muscle phenotypes.