Structural variations (SVs) are a major contributor to genetic diversity and phenotypic variations, but their prevalence and functions in domestic animals are largely unexplored. Here we generated high-quality genome assemblies for 15 individuals from genetically diverse sheep breeds using Pacific Biosciences (PacBio) high-fidelity sequencing, discovering 130.3 Mb nonreference sequences, from which 588 genes were annotated. A total of 149,158 biallelic insertions/deletions, 6531 divergent alleles, and 14,707 multiallelic variations with precise breakpoints were discovered. The SV spectrum is characterized by an excess of derived insertions compared to deletions (94,422 vs. 33,571), suggesting recent active LINE expansions in sheep. Nearly half of the SVs display low to moderate linkage disequilibrium with surrounding single-nucleotide polymorphisms (SNPs) and most SVs cannot be tagged by SNP probes from the widely used ovine 50K SNP chip. We identified 865 population-stratified SVs including 122 SVs possibly derived in the domestication process among 690 individuals from sheep breeds worldwide. A novel 168-bp insertion in the 5′ untranslated region (5′ UTR) ofHOXB13is found at high frequency in long-tailed sheep. Further genome-wide association study and gene expression analyses suggest that this mutation is causative for the long-tail trait. In summary, we have developed a panel of high-quality de novo assemblies and present a catalog of structural variations in sheep. Our data capture abundant candidate functional variations that were previously unexplored and provide a fundamental resource for understanding trait biology in sheep.

Rumen microbiota are essential for maintaining digestive and metabolic functions, producing methane as a byproduct. Dairy heifers produce large amounts of methane based on fermentation of digested organic matter, with adverse consequences for feed efficiency and the environment. It is therefore important to understand the influence of host age on the relationship between microbiota and methane production. This study explored the age effect on the relationship between microbial communities and enteric methane production in dairy cows and heifers using high-throughput sequencing. Methane production and volatile fatty acid concentrations were age-related. Heifers (9–10 months) had lower methane production but higher methane production per dry matter intake (DMI). The acetate:propionate ratio decreased significantly with increasing age. Age-related microbiota changes in the rumen were reflected by a significant shift in bacterial taxa, but relatively stable archaeal taxa. Prevotella, Ruminococcus, Flavonifractor, Succinivibrio, and Methanobrevibacter were affected by age. This study revealed different associations between predominant bacterial phylotypes and Methanobrevibacter with increasing age. Prevotella was strongly correlated with Methanobrevibacter in heifers; howerver, in older cows (96–120 months) this association was replaced by a correlation between Succinivibrio and Methanobrevibacter. This shift may account for the age-related difference in rumen fermentation and methane production per DMI.

Fat tail in sheep presents a valuable energy reserve that has historically facilitated adaptation to harsh environments. However, in modern intensive and semi-intensive sheep industry systems, breeds with leaner tails are more desirable. In the present study, RNA sequencing (RNA-Seq) was applied to determine the transcriptome profiles of tail fat tissues in two Chinese sheep breeds, fat-rumped Altay sheep and thin-tailed Xinjiang fine wool (XFW) sheep, with extreme fat tail phenotype difference. Then the differentially expressed genes (DEGs) and their sequence variations were further analyzed. In total, 21,527 genes were detected, among which 3,965 displayed significant expression variations in tail fat tissues of the two sheep breeds (P< 0.05), including 707 upregulated and 3,258 downregulated genes. Gene Ontology (GO) analysis disclosed that 198 DEGs were related to fat metabolism. In Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the majority of DEGs were significantly enriched in “adipocytokine signaling,” “PPAR signaling,” and “metabolic pathways” (P< 0.05); moreover, some genes were involved in multiple pathways. Among the 198 DEGs, 22 genes were markedly up- or downregulated in tail fat tissue of Altay sheep, indicating that these genes might be closely related to the fat tail trait of this breed. A total of 41,724 and 42,193 SNPs were detected in the transcriptomic data of tail fat tissues obtained from Altay and XFW sheep, respectively. The distribution of seven SNPs in the coding regions of the 22 candidate genes was further investigated in populations of three sheep breeds with distinct tail phenotypes. In particular, the g.18167532T/C (Oar_v3.1) mutation of the ATP-binding cassette transporter A1 (ABCA1) gene and g.57036072G/T (Oar_v3.1) mutation of the solute carrier family 27 member 2 (SLC27A2) gene showed significantly different distributions and were closely associated with tail phenotype (P< 0.05). The present study provides transcriptomic evidence explaining the differences in fat- and thin-tailed sheep breeds and reveals numerous DEGs and SNPs associated with tail phenotype. Our data provide a valuable theoretical basis for selection of lean-tailed sheep breeds.

Photoperiod is one of the important factors leading to seasonal reproduction of sheep. However, the molecular mechanisms underlying the photoperiod regulation of seasonal reproduction remain poorly understood. In this study, we compared the expression profiles of mRNAs, lncRNAs, and circRNAs in uterine tissues from Sunite sheep during three different photoperiods, namely, the short photoperiod (SP), short transfer to long photoperiod (SLP), and long photoperiod (LP). The results showed that 298, 403, and 378 differentially expressed (DE) mRNAs, 171, 491, and 499 DE lncRNAs, and 124, 270, and 400 DE circRNAs were identified between SP and LP, between SP and SLP, and between LP and SLP, respectively. Furthermore, functional enrichment analysis showed that the differentially expressed RNAs were mainly involved in the GnRH signaling pathway, thyroid hormone synthesis, and thyroid hormone signaling pathway. In addition, co-expression networks of lncRNA–mRNA were constructed based on the correlation analysis between the differentially expressed RNAs. Our study provides new insights into the expression changes of RNAs in different photoperiods, which might contribute to understanding the molecular mechanisms of seasonal reproduction in sheep.

Circular RNA (circRNA), as an emerging class of noncoding RNA, has been found to play key roles in many biological processes. However, its expression profile in the hypothalamus, a powerful organ initiating the reproductive process, has not yet been explored. Therefore, we used RNA sequencing to explore the expression of circRNAs in the hypothalamus of sheep with the FecB ++ genotype. We totally identified 41,863 circRNAs from sheep hypothalamus, in which 333 (162 were upregulated, while 171 were downregulated) were differentially expressed in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF), moreover, 340 circRNAs (163 were upregulated, while 177 were downregulated) were differentially expressed in polytocous sheep in the luteal phase versus monotocous sheep in the luteal sheep (PL vs. ML). We also identified several key circRNAs including oar_circ_0018794, oar_circ_0008291, oar_circ_0015119, oar_circ_0012801, oar_circ_0010234, and oar_circ_0013788 through functional enrichment analysis and oar_circ_0012110 through a competing endogenous RNA network, most of which may participate in reproduction by influencing gonadotropin-releasing hormone (GnRH) activities or affecting key gene expression, indirectly or directly. Our study explored the overall expression profile of circRNAs in sheep hypothalamus, which potentially provides an alternative insight into the mechanism of sheep prolificacy without the effects of FecB mutation.

The diverse functions of long noncoding RNAs (lncRNAs), which execute their functions mainly through modulating the activities of their target genes, have been have been widely studied for many years (including a number of studies involving lncRNAs in the ovary and uterus). Herein, for the first time, we detect lncRNAs in sheep hypothalami with FecB++ through RNA Sequencing (RNA-Seq) and identify a number of known and novel lncRNAs, with 622 and 809 found to be differentially expressed in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML), respectively. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed based on the predicted target genes. The most highly enriched GO terms (at the molecular function level) included carbonyl reductase (NADPH), 15-hydroxyprostaglandin dehydrogenase (NADP+), and prostaglandin-E2 9-reductase activity in PF vs. MF, and phosphatidylinositol-3,5-bisphosphate binding in PL vs. ML was associated with sheep fecundity. Interestingly, the phenomena of valine, leucine, and isoleucine degradation in PL vs. ML, and valine, leucine, and isoleucine biosynthesis in PF vs. MF, were present. In addition, the interactome of lncRNA and its targets showed that MSTRG.26777 and its cis-targets ENSOARG00000013744, ENSOARG00000013700, and ENSOARG00000013777, and MSTRG.105228 and its target WNT7A may participate in the sheep reproductive process at the hypothalamus level. Significantly, MSTRG.95128 and its cis-target Forkhead box L1 (FOXG1) were shown to be upregulated in PF vs. MF but downregulated in PL vs. ML. All of these results may be attributed to discoveries of new candidate genes and pathways related to sheep reproduction, and they may provide new views for understanding sheep reproduction without the effects of the FecB mutation.