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With the intensive development of the sheep industry and increasing global temperatures, heat stress in sheep has become an increasingly severe and important issue in recent years. The level of N6-methyladenosine (m6A) RNA methylation changes in response to stress plays important roles in stress responses. However, the role of m6A in the heat stress response of sheep remains unclear. To explore this issue, we measured heat stress protein (HSP) expression, liver function indexes, m6A on RNA, m6A-related enzyme expression, and tissue damage in sheep that had been subjected to heat stress. At the transcriptome level, our results showed significant increases in m6A on RNA and increased mRNA levels of HSPs (HSP70, HSP90, and HSP110) and m6A-related enzymes [METTL3 (methyltransferase-like 3), METTL14 (methyltransferase-like 14), WTAP (wilms tumor 1-associated protein), FTO (fat mass and obesity-associated protein), ALKBH5 (alkB homologue 5), YTHDF1-3 (YTH domain family proteins), and YTHDC1-2 (YTH domain-containing proteins)] following heat stress. At the protein level, the expression of METTL3, YTHDF1-2, and YTHDC2 showed no significant differences following heat stress. However, in contrast to its mRNA level after heat stress, the protein expression of YTHDF3 was reduced, while the expression of HSPs (HSP70, HSP90, and HSP110), METTL14, WTAP, FTO, ALKBH5, YTHDF3, and YTHDC1 increased in line with their measured mRNA levels. Histological experiments revealed that heat stress caused varying degrees of damage to sheep liver tissue. Moreover, immunohistochemical staining indicated that the m6A-related enzymes were expressed in sheep hepatocytes, and differences in expression patterns were observed between the control and heat stress groups. In summary, differences in the level of m6A and the expression of m6A-related enzymes in the liver of sheep were observed after heat stress. This indicates that m6A is involved in the regulation of heat stress in sheep. Our findings provide a new avenue for studying the responses to heat stress in sheep.

Long-term natural and artificial selection leads to change in certain regions of the genome, resulting in selection signatures that can reveal genes associated with selected traits, such as horns (i.e., polled/horned), high-quality wool traits, and high-altitude hypoxia adaptability. These are complex traits determined by multiple genes, regulatory pathways, and environmental factors. A list of genes with considerable effects on horn and adaptability traits has not been found, although multiple quantitative trait loci (QTL) have been identified. Selection signatures could be identified using genetic differentiation (FST), polymorphism levels θπ, and Tajima’s D. This study aimed to identify selection signatures in fine-wool sheep and to investigate the genes annotated in these regions, as well as the biological pathways involved in horn and adaptability traits. For this purpose, the whole-genome sequence of 120 individuals from four breeds, which come from different elevations and habitats in China, was used to analyze selection signatures for horn and adaptability traits. Annotation of the consensus regions of FST and θπ ratios revealed a list of identified genes associated with polled/horned and high-altitude hypoxia adaptability traits, such as RXPF2, EERFC4, MSH6, PP1R12A, THBS1, ATP1B2, RYR2, and PLA2G2E. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified genes related primarily to mismatch repair, metabolism, vascular smooth muscle contraction, and cardiac muscle contraction. This is the first study to demonstrate that selection signatures play an important role in the polled/horned and high-altitude hypoxia adaptability traits of fine-wool sheep breeds that have undergone high-intensity selection and adapted to different ecological environments in China. Changes observed in the genome of fine-wool sheep may have acted on genomic regions that affect performance traits and provide a reference for genome design and breeding.

Simple Summary Cashmere goats are a unique husbandry resource in China. These goats are well known for producing the highest cashmere yield and best fiber quality in the world. Although cashmere is highly valued and also known as “fiber gem” and “soft gold”, few studies have examined the genetic basis of cashmere traits in cashmere goats. Here, we identified selection signals by comparing Fst and XP-EHH (the cross population extend haplotype homozygosity test) of a non-cashmere breed (Huanghuai goat) with those of two cashmere breeds (Inner Mongolia and Liaoning cashmere goats). Two genes (WNT10A and CSN3) were potentially associated with cashmere traits. This information may be valuable for studying the genetic uniqueness of cashmere goats and elucidating the mechanisms underlying cashmere traits in cashmere goats. Abstract Inner Mongolia and Liaoning cashmere goats in China are well-known for their cashmere quality and yield. Thus, they are great models for identifying genomic regions associated with cashmere traits. Herein, 53 Inner Mongolia cashmere goats, Liaoning cashmere goats and Huanghuai goats were genotyped, and 53,347 single-nucleotide polymorphisms (SNPs) were produced using the Illumina Caprine 50K SNP chip. Additionally, we identified some positively selected SNPs by analyzing Fst and XP-EHH. The top 5% of SNPs had selection signatures. After gene annotation, 222 and 173 candidate genes were identified in Inner Mongolia and Liaoning cashmere goats, respectively. Several genes were related to hair follicle development, such as TRPS1, WDR74, LRRC14, SPTLC3, IGF1R, PADI2, FOXP1, WNT10A and CSN3. Gene enrichment analysis of these cashmere trait-associated genes related 67 enriched signaling pathways that mainly participate in hair follicle development and stem cell pluripotency regulation. Furthermore, we identified 20 overlapping genes that were selected in both cashmere goat breeds. Among these overlapping genes, WNT10A and CSN3, which are associated with hair follicle development, are potentially involved in cashmere production. These findings may improve molecular breeding of cashmere goats in the future.

Tibetan goat is an ancient breed, which inhabits the adverse conditions of the plateaus in China. To investigate the role of selection in shaping its genomes, we genotyped Tibetan goats (Nagqu Prefecture, above 4500 m) and three lowland populations (Xinjiang goats, Taihang goats and Huanghuai goats). The result of PCA, neighbor-joining (N-J) tree and model-based clustering showed that the genetic structure between the Tibetan goat and the three lowland populations has significant difference. As demonstrated by the di statistic, we found that some genes were related to the high-altitude adaptation of Tibetan goats. Functional analysis revealed that these genes were enriched in the VEGF (vascular endothelial growth factor) signaling pathway and melanoma, suggesting that nine genes (FGF2, EGFR, AKT1, PTEN, MITF, ENPEP, SIRT6, KDR, and CDC42) might have important roles in the high-altitude adaptation of Nagqu Tibetan goats. We also found that the LEPR gene was under the strongest selection (di value = 16.70), and it could induce upregulation of the hypoxic ventilatory response. In addition, five genes (LEPR, LDB1, EGFR, NOX4 and FGF2) with high di values were analyzed using q-PCR. Among them, we found that LEPR, LDB1 and FGF2 exhibited higher expression in the lungs of the Tibetan goats, LEPR, EGFR and LDB1 exhibited higher expression in the hearts of the Huanghuai goat. Our results suggest that LEPR, LDB1, EGFR and FGF2 genes may be related to the high-altitude adaptation of the goats. These findings improve our understanding of the selection of the high-altitude adaptability of the Nagqu Tibetan goats and provide new theoretical knowledge for the conservation and utilization of germplasm resources.

To date, molecular genetics and population studies in Tibetan sheep (Ovis aries) have been limited, and little is known about the phylogenetic evolution and phylogeography of Tibetan sheep populations. The aim of the present research was to explore phylogeography and phylogenetic evolution of Tibetan sheep populations, on the basis of mitochondrial DNA (mtDNA) gene MT-CYB (1140 base pairs). Our dataset consisted of 641 MT-CYB sequences from the same amount of animals belonging to 15 populations of Tibetan sheep living in the Qinghai&ndash, Tibetan Plateau, China. Haplotype and nucleotide diversities were 0.748 &plusmn, 0.010 and 0.003 &plusmn, 0.001, respectively. The analysis of phylogeography revealed the presence of two formerly described haplogroups in 15 populations of Tibetan sheep, however only one haplogroup was present in Awang sheep. Moreover, 641 Tibetan sheep were distributed into a minimum of two clusters by clustering analysis. The 15 Tibetan sheep populations and 19 reference populations of 878 individuals were separated into six main groups based on their substitutions per site, from which we constructed a phylogenetic tree. Minor differences in branching order of various taxa between trees acquired from either gene were observed. This study provides insights on the origins and phylogenetic evolution of populations residing in the Qinghai&ndash, Tibetan Plateau, which will aid information of future conservation programs aimed at conserving this valuable genetic resource.

The aim of this study was to examine the correlation between the platelet-derived growth factor-D (PDGF-D) gene and sheep tail type character and explore the potential underlying mechanism. A total of 533 sheep were included in this study. Polymorphic sites were examined by Pool-seq, and individual genotype identification and correlation analysis between tail type data were conducted using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) method. JASPART website was used to predict transcription factor binding sites in the promoter region with and without PDGF-D gene mutation. The effect of PDGF-D on adipogenic differentiation of sheep preadipocytes was investigated. Two single nucleotide polymorphism sites were identified: g.4122606 C &gt, G site was significantly correlated with tail length, and g.3852134 C &gt, T site was significantly correlated with tail width. g.3852134 C &gt, T was located in the promoter region. Six transcription factor binding sites were eliminated after promoter mutation, and three new transcription factor binding sites appeared. Expression levels of peroxisome proliferator-activated receptor gamma (PPAR&gamma, ) and lipoproteinlipase (LPL) were significantly up-regulated upon PDGF-D overexpression. Oil red O staining showed increased small and large oil drops in the PDGF-D overexpression group. Together these results indicate the PDGF-D gene is an important gene controlling sheep tail shape and regulating sheep tail fat deposition to a certain degree.

MicroRNA (miRNA) is a sort of endogenous ~20-25 nt non-coding RNAs, and it can regulate a variety of biological events. We found the miR-378 may involve in regulating the muscle development of sheep during our previous research. However, the molecular mechanism of miR-378 regulating myoblast proliferation is still unclear. In this research, we predicted that BMP2 (Bone morphogenetic protein 2) was the target gene of miR-378 and the BMP-Smad signal pathway that BMP2 participated in playing an important role in the muscle development. Therefore, we tried to determine whether miR-378 influence myoblast proliferation of sheep through the BMP-Smad signal pathway. The results indicated that inhibit BMP-Smad signal pathway by interfering Smad4 to promote proliferation of sheep myoblasts; promote BMP-Smad signal pathway by interfering Smad7 to inhibit proliferation of sheep myoblasts; over-expression miR-378 promotes BMP-Smad signal pathway and myoblast proliferation in sheep; interfering miR-378 inhibits BMP-Smad signal pathway and myoblast proliferation in sheep. However, when both of which functioned at the myoblast, miR-378 could not fully depend on BMP-Smad signal pathway to regulate myoblast proliferation. In sum, both miR-378 and BMP-Smad can influence the proliferation of myoblast, but miR-378 does not target the 3' UTR of sheep BMP2.

Simple Summary Body weight traits are economically important in the sheep industry, and it is critical to explore their underlying genetic architecture. Hence, four body weight traits, including birth, weaning, yearling, and adult weights were examined. Through a genome-wide association study on Chinese fine-wool sheep, several candidate single-nucleotide polymorphisms (SNPs) and genes were found potentially associated with the traits of interest. The results of this study may facilitate the potential use of the genes involved in growth and production traits for the genetic improvement of productivity in sheep. Abstract Body weight is an important economic trait for sheep and it is vital for their successful production and breeding. Therefore, identifying the genomic regions and biological pathways that contribute to understanding variability in body weight traits is significant for selection purposes. In this study, the genome-wide associations of birth, weaning, yearling, and adult weights of 460 fine-wool sheep were determined using resequencing technology. The results showed that 113 single nucleotide polymorphisms (SNPs) reached the genome-wide significance levels for the four body weight traits and 30 genes were annotated effectively, including AADACL3, VGF, NPC1, and SERPINA12. The genes annotated by these SNPs significantly enriched 78 gene ontology terms and 25 signaling pathways, and were found to mainly participate in skeletal muscle development and lipid metabolism. These genes can be used as candidate genes for body weight in sheep, and provide useful information for the production and genomic selection of Chinese fine-wool sheep.

Tail adipose as one of the important functional tissues can enhance hazardous environments tolerance for sheep. The objective of this study was to gain insight into the underlying development mechanisms of this trait. A quantitative analysis of protein abundance in ovine tail/rump adipose tissue was performed between Chinese local fat- (Kazakh, Hu and Lanzhou) and thin-tailed (Alpine Merino, Tibetan) sheep in the present study by using lable-free approach. Results showed that 3400 proteins were identified in the five breeds, and 804 were differentially expressed proteins, including 638 up regulated proteins and 83 down regulated proteins in the tail adipose tissues between fat- and thin-tailed sheep, and 8 clusters were distinguished for all the DEPs’ expression patterns. The differentially expressed proteins are mainly associated with metabolism pathways and peroxisome proliferator activated receptor signaling pathway. Furthermore, the proteomics results were validated by quantitative real-time PCR and Western Blot. Our research has also suggested that the up-regulated proteins ACSL1, HSD17β4, FABP4 in the tail adipose tissue might contribute to tail fat deposition by facilitating the proliferation of adipocytes and fat accumulation in tail/rump of sheep. Particularly, FABP4 highly expressed in the fat-tail will play an important role for tail fat deposition. Our study might provide a novel view to understanding fat accumulation in special parts of the body in sheep and other animals.

The aim of this study was to investigate the genetic mechanisms underlying wool production by characterizing the skin protein profile and determining the proteomic changes that occur as a consequence of development in wool-producing sheep using a label-free proteomics approach. Samples were collected at four stages during gestation (87, 96, 102, and 138 days), and every two consecutive stages were statistically compared (87 versus 96, 96 versus 102, and 102 versus 138 days). We identified 227 specific proteins in the sheep proteome that were present in all four stages, and 123 differentially abundant proteins (DAPs). We also observed that the microstructure of the secondary follicles changed significantly during the development of the fetal skin hair follicle. The screened DAPs were strictly related to metabolic and skin development pathways, and were associated with pathways such as the glycolysis/gluconeogenesis. These analyses indicated that the wool production of fine wool sheep is regulated via a variety of pathways. These findings provide an important resource that can be used in future studies of the genetic mechanisms underlying wool traits in fine wool sheep, and the identified DAPs should be further investigated as candidate markers for predicting wool traits in sheep. Significance Wool quality (fiber diameter, length, etc.) is an important economic trait of fine wool sheep that is determined by secondary follicle differentiation and re-differentiation. Secondary follicles of fine wool sheep developed from a bud (87 days), and underwent differentiation (96 days) and rapid growth (102 days) until maturity (138 days) during gestation. Comparative analysis based on differential proteomics of these four periods could provide a better understanding of the wool growth mechanism of fine wool sheep and offer novel strategies for improving fine wool quality by breeding.

Simple Summary LncRNAs may play important role in many biological processes. The aims of this research were to identify potential lncRNAs active in skeletal muscle of the Texel and Ujumqin sheep and investigate their functions. Overall, 2002 lncRNA transcripts were found, some of which may be related to muscle development. The findings obtained here should promote understanding of the regulatory functions of lncRNAs in ovine muscle development and potentially also in other mammals. Abstract Long noncoding RNAs (lncRNAs) are increasingly being recognized as key regulators in many cellular processes. However, few reports of them in livestock have been published. Here, we describe the identification and characterization of lncRNAs in ovine skeletal muscle. Eight libraries were constructed from the gastrocnemius muscle of fetal (days 85 and 120), newborn and adult Texel and Ujumqin sheep. The 2002 identified transcripts shared some characteristics, such as their number of exons, length and distribution. We also identified some coding genes near these lncRNA transcripts, which are particularly associated with transcriptional regulation- and development-related processes, suggesting that the lncRNAs are associated with muscle development. In addition, in pairwise comparisons between the libraries of the same stage in different breeds, a total of 967 transcripts were differentially expressed but just 15 differentially expressed lncRNAs were common to all stages. Among them, we found that TCONS_00013201 exhibited higher expression in Ujumqin samples, while TCONS_00006187 and TCONS_00083104 were higher in Texel samples. Moreover, TCONS_00044801, TCONS_00008482 and TCONS_00102859 were almost completely absent from Ujumqin samples. Our results suggest that differences in the expression of these lncRNAs may be associated with the muscular differences observed between Texel and Ujumqin sheep breeds.

Male reproductive capacity is essential for animal breeding and reproduction. In males, the testes produce sperm and secrete androgen, processes which require precise regulation by multiple proteins. The composition of proteins in the ram testes has not yet been studied systematically, thus, the application of proteomics to explore differential protein regulation during ram testes development is of great significance. In the present study, ram testes were studied at five different developmental phases to assess postnatal differences in protein regulation. Two dimensional electrophoresis (2-DE) was used to separate ram testes proteins at each developmental phase, yielding 45 different proteins, 37 of which were identified by Matrix Assisted Laser Desorption Ionization-Time of Flight-Time of Flight-Mass Spectrometry (MALDI-TOF/TOF-MS). Gene Ontology (GO) was used to specifically annotate the biological process, cellular composition, and molecular function of each identified protein. Most of the identified proteins were involved in structural formation, development, reproduction, and apoptosis of the testicular spermatogenic tissue and spermatozoa. Quantitative real time PCR (qRT-PCR), western blot and immunohistochemical methods were used to verify the proteins, and the results were consistent with that of 2-DE. The proteins that were different in abundance that were identified in this study can be used as biomarkers in future studies of ram reproduction.

Double sex and mab-3 related transcription factor 1 (Dmrt1), an evolutionarily conserved gene, is a sex-related gene expressed in male gonads, that is involved in the regulation of sex differentiation, testicular development and reproductive function maintenance. Until now, functional studies on the Dmrt1 gene in sheep (Ovis aries) have been lacking. In this study, testis, heart, liver, spleen, lung, kidney and longissimus dorsi muscle tissues were collected from Small-Tail Han sheep at 0, 2, 5, 12 and 24months after birth (mab). Dmrt1 expression and cellular localization were detected in various testicular tissues by quantitative real time PCR (qRT-PCR), western blot and immunohistochemistry methods. The morphological structures of testicular tissues at different developmental stages were observed by hematoxylineosin (HE) staining. The Dmrt1 mRNA expression levels in 12 and 24 mab sheep were significantly higher than those in 0 and 2 mab sheep (P0.05), and Dmrt1 protein expression showed a similar trend. The qRT-PCR results in various tissues at 12 mab showed that Dmrt1 mRNA was predominantly expressed in testes. Immunohistochemical staining in testes at different developmental stages showed that Dmrt1 protein immunoreactive responses were mainly localized in Sertoli cells and gonocytes at 0, 2 and 5 mab, while they were localized in spermatocytes, sperm cells and some spermatogonia and Sertoli cells at 12 and 24 mab. We speculate that the Dmrt1 gene plays a vital role in postnatal sheep spermatogenesis, perhaps by regulating the maturation and functional maintenance of Sertoli cells, the proliferation and differentiation of gonocytes in prepubertal sheep testes, and the mitosis and meiosis of germ cells in adult sheep, but the specific mechanisms underlying these phenomena must be further studied and verified.