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Abstract Background The Tibetan sheep is one of the three major primitive sheep breeds in China, representing a unique and high-quality genetic resource in the Qinghai-Tibet Plateau and neighboring high-altitude regions, exhibiting exceptional adaptability to high-altitude climatic environments. However, research on the genetic relationships among different populations of Tibetan sheep at the whole-genome level remains insufficient. This study aims to explore the population structure and historical dynamics among 11 Tibetan sheep populations, accurately assess the genetic diversity within the populations, and providing a theoretical basis for the development of targeted genetic breeding strategies for Tibetan sheep. Results In this study, a total of 10,884,454 high-quality SNPs were obtained. All Tibetan sheep populations exhibited varying degrees of linkage disequilibrium, with similar decay rates; among them, the WT population showed the fastest decay, while the TS population exhibited the slowest decay rate. Analyses using Tajima’s D and π indicated that the genetic diversity levels of the Tibetan sheep populations are generally low. Fst results revealed that most populations exhibited moderate to low levels of genetic differentiation. The effective population size among Tibetan sheep populations showed an increasing trend over time. The evolutionary relationships among Tibetan sheep populations reflect the correlation between their geographical locations and genomic genetic distances, while also indirectly confirming the impact of historical activities such as early human migration, admixture, fusion, and expansion on the population sizes and distributions of Tibetan sheep. Conclusions The results indicate that the genetic diversity levels and genetic differentiation among Tibetan sheep populations are relatively low. In this study, we identified the genetic characteristics of Tibetan sheep populations, which exhibit low levels of diversity, genetic differentiation, and a strong population structure. A deeper genomic exploration of the population structure and diversity status of Tibetan sheep populations will provide theoretical support for subsequent genetic breeding and diversity conservation efforts.

Abstract Guide Black-Fur sheep (GD) is a breed of Tibetan sheep (Ovis aries) that lives in the Qinghai–Tibetan plateau region at an altitude of over 4,000 m. However, a lack of genomic information has made it difficult to understand the high-altitude adaptation of these sheep. We sequenced and assembled the GD reference genome using PacBio, Hi-C, and Illumina sequencing technologies. The final assembled genome size was 2.73 Gb, with a contig N50 of 20.30 Mb and a scaffold N50 of 107.63 Mb. The genome is predicted to contain 20,759 protein-coding genes, of which 98.42 have functional annotations. Repeat elements account for approximately 52.2% of the genomic landscape. The completeness of the GD genome assembly is highlighted by a BUSCO score of 93.1%. This high-quality genome assembly provides a critical resource for future molecular breeding and genetic improvement of Tibetan sheep.

Abstract Background The Alpine Merino is a new breed of fine-wool sheep adapted to the cold and arid climate of the plateau in the world. It has been popularized in Northwest China due to its superior adaptability as well as excellent production performance. Those traits related to body weight, wool yield, and wool fiber characteristics, which are economically essential traits in Alpine Merino sheep, are controlled by QTL (Quantitative Trait Loci). Therefore, the identification of QTL and genetic markers for these key economic traits is a critical step in establishing a MAS (Marker-Assisted Selection) breeding program. Results In this study, we constructed the high-density genetic linkage map of Alpine Merino sheep by sequencing 110 F1 generation individuals using WGR (Whole Genome Resequencing) technology. 14,942 SNPs (Single Nucleotide Polymorphism) were identified and genotyped. The map spanned 2,697.86 cM, with an average genetic marker interval of 1.44 cM. A total of 1,871 high-quality SNP markers were distributed across 27 linkage groups, with an average of 69 markers per LG (Linkage Group). Among them, the smallest genetic distance is 19.62 cM for LG2, while the largest is 237.19 cM for LG19. The average genetic distance between markers in LGs ranged from 0.24 cM (LG2) to 3.57 cM (LG17). The marker density in the LGs ranged from LG14 (39 markers) to LG1 (150 markers). Conclusions The first genetic map of Alpine Merino sheep we constructed included 14,942 SNPs, while 46 QTLs associated with body weight, wool yield and wool fiber traits were identified, laying the foundation for genetic studies and molecular marker-assisted breeding. Notably, there were QTL intervals for overlapping traits on LG4 and LG8, providing potential opportunities for multi-trait co-breeding and further theoretical support for selection and breeding of ultra-fine and meaty Alpine Merino sheep.

Abstract Background Gangba sheep as a famous breed of Tibetan sheep, its wool color is mainly white and black. Gangba wool is economically important as a high-quality raw material for Tibetan blankets and Tibetan serge. However, relatively few studies have been conducted on the wool color of Tibetan sheep. Results To fill this research gap, this study conducted an in-depth analysis of two populations of Gangba sheep (black and white wool color) using whole genome resequencing to identify genetic variation associated with wool color. Utilizing PCA, Genetic Admixture, and N-J Tree analyses, the present study revealed a consistent genetic relationship and structure between black and white wool colored Gangba sheep populations, which is consistent with their breed history. Analysis of selection signatures using multiple methods (FST, π ratio, Tajima's D), 370 candidate genes were screened in the black wool group (GBB vs GBW); among them, MC1R, MLPH, SPIRE2, RAB17, SMARCA4, IRF4, CAV1, USP7, TP53, MYO6, MITF, MC2R, TET2, NF1, JAK1, GABRR1 genes are mainly associated with melanin synthesis, melanin delivery, and distribution. The enrichment results of the candidate genes identified 35 GO entries and 19 KEGG pathways associated with the formation of the black phenotype. 311 candidate genes were screened in the white wool group (GBW vs GBB); among them, REST, POU2F1, ADCY10, CCNB1, EP300, BRD4, GLI3, and SDHA genes were mainly associated with interfering with the differentiation of neural crest cells into melanocytes, affecting the proliferation of melanocytes, and inhibiting melanin synthesis. 31 GO entries and 22 KEGG pathways were associated with the formation of the white phenotype. Conclusions This study provides important information for understanding the genetic mechanism of wool color in Gangba, and provides genetic knowledge for improving and optimizing the wool color of Tibetan sheep. Genetic improvement and selective breeding to produce wool of specific colors can meet the demand for a diversity of wool products in the Tibetan wool textile market.

Abstract Background Chinese indigenous sheep are valuable resources with unique features and characteristics. They are distributed across regions with different climates in mainland China; however, few reports have analyzed the environmental adaptability of sheep based on their genome. We examined the variants and signatures of selection involved in adaptation to extreme humidity, altitude, and temperature conditions in 173 sheep genomes from 41 phenotypically and geographically representative Chinese indigenous sheep breeds to characterize the genetic basis underlying environmental adaptation in these populations. Results Based on the analysis of population structure, we inferred that Chinese indigenous sheep are divided into four groups: Kazakh (KAZ), Mongolian (MON), Tibetan (TIB), and Yunnan (YUN). We also detected a set of candidate genes that are relevant to adaptation to extreme environmental conditions, such as drought-prone regions (TBXT, TG, and HOXA1), high-altitude regions (DYSF, EPAS1, JAZF1, PDGFD, and NF1) and warm-temperature regions (TSHR, ABCD4, and TEX11). Among all these candidate genes, eight ABCD4, CNTN4, DOCK10, LOC105608545, LOC121816479, SEM3A, SVIL, and TSHR overlap between extreme environmental conditions. The TSHR gene shows a strong signature for positive selection in the warm-temperature group and harbors a single nucleotide polymorphism (SNP) missense mutation located between positions 90,600,001 and 90,650,001 on chromosome 7, which leads to a change in the protein structure of TSHR and influences its stability. Conclusions Analysis of the signatures of selection uncovered genes that are likely related to environmental adaptation and a SNP missense mutation in the TSHR gene that affects the protein structure and stability. It also provides information on the evolution of the phylogeographic structure of Chinese indigenous sheep populations. These results provide important genetic resources for future breeding studies and new perspectives on how animals can adapt to climate change.

Tibetan sheep have developed unique adaptations for survival in the Qinghai–Tibet Plateau environment. However, the functional genes and molecular mechanisms that regulate hypoxia adaptation have not been fully characterized. In this study, based on the whole-genome resequencing data for Tibetan sheep at different altitudes, the population differentiation index (FST) and nucleotide diversity ratio (θπ ratio) were evaluated in populations of 20 Oula sheep (3501 m altitude, OL), 20 Zashijia sheep (4369 m altitude, ZSJ), and 20 Awang sheep (4643 m altitude, AW) to reveal candidate loci related to high-altitude hypoxia. We screened 728 and 524 candidate genes in the AW vs. OL and ZSJ vs. OL groups, respectively, of which 134 genes were jointly screened. Candidate genes were mainly enriched in the Ras, melanoma, melanogenesis, VEGF, and PPAR signaling pathways. HIF1AN, PDGFA, PDGFD, ANXA2, SOCS2, NOXA1, WNT7B, MMP14, GNG2, ATF6, PGAM2, PPP3R1, GSTCD, and PPARA may play important roles in the high-altitude adaptation of Tibetan sheep. In conclusion, this study provides valuable insights into the genes and molecular mechanisms that underlie high-altitude hypoxia adaptation in Tibetan sheep.

Numerous genes govern male reproduction, modulating testicular development and spermatogenesis. Our study leveraged RNA-Seq to explore candidate genes and pivotal pathways influencing fecundity in an F1 hybrid of Southdown × Hu sheep testes across four developmental milestones: M0 (0 months old, newborn), M3 (3 months old, sexually immature), M6 (6 months old, sexually mature), and Y1 (1 years old, adult). Histological examination using hematoxylins and eosin staining revealed that the cross-sectional area of the spermatid tubules and the number of supportive cells increased in the other groups, as compared to the M0 group. The cross-sectional area of the vasculature and the number of supporting cells were found to be significantly increased in all other groups in comparison to the M0 group. We conducted GO and KEGG analyses of the differentially expressed genes (DEGs) in the three comparison groups and identified key pathways, including cAMP, MAPK, ECM–receptor interactions, PI3K-Akt, and FOXO signaling, which are closely related to testicular development and spermatogenesis. Notably, alternative splicing (AS) events were markedly elevated in M6 and Y1 stages. Key genes like GATA4, GATA6, SMAD4, SOX9, YAP1, ITGB1 and MAPK1 emerged as significantly enriched in these pathways, potentially orchestrating the transition from immature to mature testes in sheep. These findings offer valuable insights into male reproductive potential and can inform strategies for optimizing animal breeding.

This study analyzed the flavor compounds in the meat of four indigenous breeds of Chinese sheep through the use of gas chromatography–ion mobility spectrometry (GC-IMS). GC-IMS provided information on the characteristics and strength of 71 volatile flavor compounds (monomers and dimers), with aldehydes, alcohols and ketones being the most abundant in all types of sheep meat. The compounds with higher intensity peaks in the sheep meat were aldehydes (n-nonanal, octanal, heptanal, 3-methylbutanal, and hexanal), alcohols (1-octen-3-ol, hexanol, and pentanol), ketones (3-hydroxy-2-butanon, 2-butanone, and 2-propanone), esters (methyl benzoate), and thiazole (trimethylthiazole). The volatile flavor components in the meat of the different breeds of sheep obtained via GC-IMS were further differentiated using principal component analysis. In addition, orthogonal partial least squares discriminant analysis (OPLS-DA) and variable importance on projection (VIP) were used to determine the characteristic flavor compounds in the meats of different breeds of sheep, and 21 differentially volatile components were screened out based on having a VIP above 1. These results indicate that GC-IMS combined with multivariate analysis is a convenient and powerful method for characterizing and discriminating sheep meat.

The liver plays a crucial role in metabolism, synthesis, biotransformation, secretion, and excretion. Hepatocytes are the main cells of the liver and can be used as a cell model to study liver function. The classic method of collagenase perfusion to isolate hepatocytes is a two-step technique that is time-consuming, labor-intensive, and has high technical requirements. Therefore, in this study, we compared different methods for isolating and culturing primary hepatocytes. We found that the 0.25% trypsin and 0.1 mg/mL type IV collagenase mixture at a 1:1 ratio showed the most efficient cell digestion, and William’s Medium E complete medium showed the best growth and proliferation. The isolated cells showed the typical irregular polygonal morphology of hepatocytes. Periodic acid–Schiff staining and immunofluorescence confirmed that the isolated cells were positive for glycogen and hepatocyte-specific markers cytokeratin 18, AFP, and albumin. On subculturing, stable cell lines were obtained. Therefore, we optimized the isolation and in vitro culture method to obtain highly pure (>95%) sheep primary hepatocytes from newborn sheep liver tissue.

Abstract Background Hu sheep and Tibetan sheep in China are characterized by fat tails and thin tails, respectively. Several transcriptomes have been conducted in different sheep breeds to identify the differentially expressed genes (DEGs) underlying this trait. However, these studies identified different DEGs in different sheep breeds. Results Hence, RNA sequencing was performed on Hu sheep and Tibetan sheep. We obtained a total of 45.57 and 43.82 million sequencing reads, respectively. Two libraries mapped reads from 36.93 and 38.55 million reads after alignment to the reference sequences. 2108 DEGs were identified, including 1247 downregulated and 861 upregulated DEGs. GO and KEGG analyses of all DEGs demonstrated that pathways were enriched in the regulation of lipolysis in adipocytes and terms related to the chemokine signalling pathway, lysosomes, and glycosaminoglycan degradation. Eight genes were selected for validation by RT–qPCR. In addition, the transfection of BMP2 overexpression into preadipocytes resulted in increased PPAR-γ expression and expression. BMP2 potentially induces adipogenesis through LOX in preadipocytes. The number of lipid drops in BMP2 overexpression detected by oil red O staining was also greater than that in the negative control. Conclusion In summary, these results showed that significant genes (BMP2, HOXA11, PPP1CC and LPIN1) are involved in the regulation of adipogenesis metabolism and suggested novel insights into metabolic molecules in sheep fat tails.

SNPs associated with important traits of fine-wool sheep that were previously obtained through genome-wide association analysis screening were verified and analyzed. A total of 20 SNPs related to birth weight, bundle strength, cleaning rate, and fiber diameter were screened using whole-genome resequencing, and the SNPshot assay was used to detect and analyze polymorphisms. This study found that, among the 20 SNPs associated with important traits in Alpine Merino sheep, 8 were monomorphic and 12 were polymorphic, of which 6 showed moderate polymorphisms and 6 showed low polymorphisms. The heterozygosity of the 12 polymorphic loci ranged from 0.10 to 0.49, the effective number of alleles ranged from 1.11 to 1.98, and the polymorphic information content ranged from 0.09 to 0.37. The chi-square test showed that only RHPN2:g.42678119T>G and RALYL:g.90030866A>G were in Hardy–Weinberg disequilibrium (p < 0.05); the other loci were in equilibrium (p > 0.05). These SNPs associated with important traits in Alpine Merino sheep provide a theoretical basis for genomic selection and molecular design breeding.

The SLIT3 gene encodes a secreted protein, and the ZNF280B gene is a member of the transcription factor family. Both genes have multiple biological functions. This study was conducted to investigate the association between SLIT3 and ZNF280B gene polymorphisms and wool fiber diameter and to determine potential molecular marker sites for breeding sheep with fine wool. We used Kompetitive Allele-Specific PCR to type the single nucleotide polymorphism (SNP) loci in the SLIT3 and ZNF280B genes within 1081 Alpine Merino sheep and associated these SNPs with wool fiber diameter. The results revealed one SNP in SLIT3 and ZNF280B, which were each related to sheep fiber diameter. The wool fiber diameters of sheep with the CC genotype in SLIT3 g.478807C>G and AA genotype in ZNF280B g.677G>A were the smallest and differed significantly from the diameters of other genotypes (p < 0.05). These results suggest potential molecular marker sites for fine-wool sheep breeding.

Wool color is controlled by a variety of genes. Although the gene regulation of some wool colors has been studied in relative depth, there may still be unknown genetic variants and control genes for some colors or different breeds of wool that need to be identified and recognized by whole genome resequencing. Therefore, we used whole genome resequencing data to compare and analyze sheep populations of different breeds by population differentiation index and nucleotide diversity ratios (Fst and θπ ratio) as well as extended haplotype purity between populations (XP-EHH) to reveal selection signals related to wool coloration in sheep. Screening in the non-white wool color group (G1 vs. G2) yielded 365 candidate genes, among which PDE4B, GMDS, GATA1, RCOR1, MAPK4, SLC36A1, and PPP3CA were associated with the formation of non-white wool; an enrichment analysis of the candidate genes yielded 21 significant GO terms and 49 significant KEGG pathways (p < 0.05), among which 17 GO terms and 21 KEGG pathways were associated with the formation of non-white wool. Screening in the white wool color group (G2 vs. G1) yielded 214 candidate genes, including ABCD4, VSX2, ITCH, NNT, POLA1, IGF1R, HOXA10, and DAO, which were associated with the formation of white wool; an enrichment analysis of the candidate genes revealed 9 significant GO-enriched pathways and 19 significant KEGG pathways (p < 0.05), including 5 GO terms and 12 KEGG pathways associated with the formation of white wool. In addition to furthering our understanding of wool color genetics, this research is important for breeding purposes.

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics ChromiumTM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (Apale and Adark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, Apale and Adark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs.

There is a genetic difference between Hu sheep (short/fat-tailed sheep) and Tibetan sheep (short/thin-tailed sheep) in tail type, because of fat metabolism. Previous studies have mainly focused directly on sheep tail fat, which is not the main organ of fat metabolism. The function of miRNAs in sheep liver fat metabolism has not been thoroughly elucidated. In this study, miRNA-Seq was used to identify miRNAs in the liver tissue of three Hu sheep (short/fat-tailed sheep) and three Tibetan sheep (short/thin-tailed sheep) to characterize the differences in fat metabolism of sheep. In our study, Hu sheep was in a control group, we identified 11 differentially expressed miRNAs (DE miRNAs), including six up-regulated miRNAs and five down-regulated miRNAs. Miranda and RNAhybrid were used to predict the target genes of DE miRNAs, obtaining 3,404 target genes. A total of 115 and 67 GO terms as well as 54 and 5 KEGG pathways were significantly (padj < 0.05) enriched for predicted 3,109 target genes of up-regulated and 295 target genes of down-regulated miRNAs, respectively. oar-miR-432 was one of the most up-regulated miRNAs between Hu sheep and Tibetan sheep. And SIRT1 is one of the potential target genes of oar-miR-432. Furthermore, functional validation using the dual-luciferase reporter assay indicated that the up-regulated miRNA; oar-miR-432 potentially targeted sirtuin 1 (SIRT1) expression. Then, the oar-miR-432 mimic transfected into preadipocytes resulted in inhibited expression of SIRT1. This is the first time reported that the expression of SIRT1 gene was regulated by oar-miR-432 in fat metabolism of sheep liver. These results could provide a meaningful theoretical basis for studying the fat metabolism of sheep.

This experiment was conducted to study the effects of dietary energy level on the growth performance and meat quality of weaned Alpine Merino lambs. The study ran for a total of 104 days (20-day pretrial, 84-day trial). From three groups of test lambs, we randomly selected ten lambs per group to determine slaughter performance, meat quality characteristics, and organ indexes. The slaughter performances of the lambs improved as the dietary energy level increased. The live weight before the slaughter of the lambs was significantly higher in the high group than in the low and medium groups. The carcass weight was significantly higher in the high group than in the low group. Dietary energy level had little effect on the organ weight of lambs. Meat quality differed among the three dietary energy levels. The muscle yellowness and redness scores decreased significantly as the energy levels increased. The C18:0, C21:0, C20:1, C18:2n6c, and C20:2 contents in the muscle were significantly higher in the high group than in the medium and low groups. The C18:3n6 content in the muscle was significantly higher in the low group than in the medium group. The C20:5n3 content in the longissimus dorsi was significantly higher in the high group than in the medium and low groups. The monounsaturated and unsaturated fatty acid contents in the muscle were significantly higher in the high group than in the low group. A dietary energy level of 10.5 MJ/kg is suitable for fattening weaned male Alpine Merino lambs.

Wool fineness affects the quality of wool, and some studies have identified about forty candidate genes that affect sheep wool fineness, but these genes often reveal only a certain proportion of the variation in wool thickness. We further explore additional genes associated with the fineness of sheep wool. Whole-genome resequencing of eight sheep breeds was performed to reveal selection signals associated with wool fineness, including four coarse wool and four fine/semi-fine wool sheep breeds. Multiple methods to reveal selection signals (Fst and θπ Ratio and XP-EHH) were applied for sheep wool fineness traits. In total, 269 and 319 genes were annotated in the fine wool (F vs. C) group and the coarse wool (C vs. F) group, such as LGR4, PIK3CA, and SEMA3C and NFIB, OPHN1, and THADA. In F vs. C, 269 genes were enriched in 15 significant GO Terms (p < 0.05) and 38 significant KEGG Pathways (p < 0.05), such as protein localization to plasma membrane (GO: 0072659) and Inositol phosphate metabolism (oas 00562). In C vs. F, 319 genes were enriched in 21 GO Terms (p < 0.05) and 16 KEGG Pathways (p < 0.05), such as negative regulation of focal adhesion assembly (GO: 0051895) and Axon guidance (oas 04360). Our study has uncovered genomic information pertaining to significant traits in sheep and has identified valuable candidate genes. This will pave the way for subsequent investigations into related traits.

Crossbreeding improves and enhances meat quality and is widely used in sheep production; however, the molecular mechanisms underlying the meat quality of various crossbred sheep remain unknown. In this study, male Southdown, Suffolk and Hu sheep were crossbred with female Hu sheep, and the transcriptomes and metabolomes of the longissimus dorsi muscle of the F1 generation were sequenced to explore how different sire breeds affect meat quality. The results showed that 631 differentially expressed genes and 119 significantly altered metabolites contributed to muscle development characteristics and meat quality-related diversity (P < 0.05). These genes and metabolites were significantly enriched in lipid metabolism pathways, including arachidonic acid metabolism and PPAR signaling. Several candidate genes were associated with muscle growth, such as MYLK3, MYL10, FIGN, MYH8, MYOM3, LMCD1, and FLRT1. Among these, MYH8 and MYL10 participated in regulating muscle growth and development and were correlated with meat quality-related fatty acid levels (|r| > 0.5 and p < 0.05). We selected mRNA from four of these genes to verify the accuracy of the sequencing data via qRT-PCR. Our findings provide further insight into the key genes and metabolites involved in muscle growth and meat quality in hybrid sheep populations.

Abstract Background The quality and yield of wool determine the economic value of the fine-wool sheep. Therefore, discovering markers or genes relevant to wool traits is the cornerstone for the breeding of fine-wool sheep. In this study, we used the Illumina HiSeq X Ten platform to re-sequence 460 sheep belonging to four different fine-wool sheep breeds, namely, Alpine Merino sheep (AMS), Chinese Merino sheep (CMS), Aohan fine-wool sheep (AHS) and Qinghai fine-wool sheep (QHS). Eight wool traits, including fiber diameter (FD), fiber diameter coefficient of variance (FDCV), fiber diameter standard deviation (FDSD), staple length (SL), greasy fleece weight (GFW), clean wool rate (CWR), staple strength (SS) and staple elongation (SE) were examined. A genome-wide association study (GWAS) was performed to detect the candidate genes for the eight wool traits. Results A total of 8.222 Tb of raw data was generated, with an average of approximately 8.59X sequencing depth. After quality control, 12,561,225 SNPs were available for analysis. And a total of 57 genome-wide significant SNPs and 30 candidate genes were detected for the desired wool traits. Among them, 7 SNPs and 6 genes are related to wool fineness indicators (FD, FDCV and FDSD), 10 SNPs and 7 genes are related to staple length, 13 SNPs and 7 genes are related to wool production indicators (GFW and CWR), 27 SNPs and 10 genes associated with staple elongation. Among these candidate genes, UBE2E3 and RHPN2 associated with fiber diameter, were found to play an important role in keratinocyte differentiation and cell proliferation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results, revealed that multitude significant pathways are related to keratin and cell proliferation and differentiation, such as positive regulation of canonical Wnt signaling pathway (GO:0090263). Conclusion This is the first GWAS on the wool traits by using re-sequencing data in Chinese fine-wool sheep. The newly detected significant SNPs in this study can be used in genome-selective breeding for the fine-wool sheep. And the new candidate genes would provide a good theoretical basis for the fine-wool sheep breeding.

Abstract Background Copy number variation (CNV) is an important source of genetic variation that has a significant influence on phenotypic diversity, economically important traits and the evolution of livestock species. In this study, the genome-wide CNV distribution characteristics of 32 fine-wool sheep from three breeds were analyzed using resequencing. Results A total of 1,747,604 CNVs were detected in this study, and 7228 CNV regions (CNVR) were obtained after merging overlapping CNVs; these regions accounted for 2.17% of the sheep reference genome. The average length of the CNVRs was 4307.17 bp. “Deletion” events took place more frequently than “duplication” or “both” events. The CNVRs obtained overlapped with previously reported sheep CNVRs to variable extents (4.39–55.46%). Functional enrichment analysis showed that the CNVR-harboring genes were mainly involved in sensory perception systems, nutrient metabolism processes, and growth and development processes. Furthermore, 1855 of the CNVRs were associated with 166 quantitative trait loci (QTL), including milk QTLs, carcass QTLs, and health-related QTLs, among others. In addition, the 32 fine-wool sheep were divided into horned and polled groups to analyze for the selective sweep of CNVRs, and it was found that the relaxin family peptide receptor 2 (RXFP2) gene was strongly influenced by selection. Conclusions In summary, we constructed a genomic CNV map for Chinese indigenous fine-wool sheep using resequencing, thereby providing a valuable genetic variation resource for sheep genome research, which will contribute to the study of complex traits in sheep.

Dongxiang tribute sheep have a history of use in food dishes such as “Dongxiang Handgrip,” which dates back hundreds of years and is a favorite halal food in northwestern China. However, little is known about the mutton quality characteristics of Dongxiang tribute sheep. Here, we measured the sensory characteristics, nutritional quality, and flavor substances to comprehensively evaluate the mutton quality characteristics of these sheep. The mutton qualities of Dongxiang tribute, Tibetan, Ujumqin, and Hu sheep were comprehensively evaluated by membership function. Subsequently, the volatile components in mutton samples from 30 Dongxiang tribute sheep were detected via gas chromatography and ion mobility spectrometry (GC-IMS), and their fingerprints were established. The result of meat quality revealed that the shear force, the contents of protein, essential amino acid (EAA), non-essential amino acid (NEAA), and n-6/n-3 ratio of Dongxiang tribute mutton were better than the other three breeds. Membership functions were calculated for 10 physical and chemical indexes of mutton quality, and the comprehensive membership function values of the four breeds in order of highest to lowest mutton quality were Tibetan sheep (0.76) > Dongxiang tribute sheep (0.49) > Hu sheep (0.46) > Ujumqin sheep (0.33). Thirty volatile compounds were identified via GC-IMS: seven alcohols, eight aldehydes, five ketones, two esters, two phenols, one ether, one furan, one acid, two hydrocarbons, and one pyrazine. Ketones, aldehydes, and alcohols were the main volatile compounds forming the flavor of Dongxiang tribute sheep mutton. The reliability of the results was validated by PCA (principal component analysis) and similarity analyses. Our results provide reference value for consumers of mutton in China.

The fat tail is a unique characteristic of sheep that represents energy reserves and is a complex adaptative mechanism of fat-tailed sheep to environmental stress. MicroRNA plays a significant role as regulators at the posttranscriptional level, but no studies have explained the molecular mechanisms of miRNA which regulate fat deposition in sheep tails. In this study, mRNA and miRNA analysis examined tail fat tissue from three Hu fat-tailed and three Tibetan thin-tailed sheep. After aligning to the reference sequences, 2,108 differentially expressed genes and 105 differential expression miRNAs were identified, including 1,247 up- and 861 downregulated genes and 43 up- and 62 downregulated miRNAs. Among these differentially expressed miRNAs, oar-miR-432 was one of the most downregulated miRNAs between Hu sheep and Tibetan sheep, and 712 genes were predicted to be targeted by oar-miR-432, 80 of which overlapped with DEGs. The Gene Ontology analysis on these genes showed that BMP2, LEP, GRK5, BMP7, and RORC were enriched in fat cell differentiation terms. The genes for BMP2 targeted by oar-miR-432 were examined using dual-luciferase assay. The oar-miR-432 mimic transfected into preadipocytes resulted in increased expression of BMP2. The marker gene PPAR-γ of fat differentiation had a lower expression than the negative control on days 0, 2, and 4 after induced differentiation. The decrease in the number of lipids in the oar-miR-432 mimic group detected by oil red O stain was also less than that in the negative control. This is the first study to reveal the fat mechanisms by which oar-miR-432 inhibits fat differentiation and promotes the expression of BMP2 in sheep tails.

Tibetan sheep have lived on the Qinghai-Tibet Plateau for a long time, and after long-term natural selection, they have shown stable genetic adaptability to high-altitude environments. However, little is known about the molecular mechanisms of the long non-coding (lnc)RNAs involved in the adaptation of Tibetan sheep to hypoxia. Here, we collected lung tissues from high-altitude Tibetan sheep and low-altitude Hu sheep for RNA sequencing to study the regulatory mechanisms of the lncRNAs and mRNAs in the adaptation of Tibetan sheep to hypoxia. We identified 254 differentially expressed lncRNAs and 1,502 differentially expressed mRNAs. We found 20 pairs of cis-regulatory relationships between 15 differentially expressed lncRNAs and 14 protein-coding genes and two pairs of trans-regulatory relationships between two differentially expressed lncRNAs and two protein-coding genes. These differentially expressed mRNAs and lncRNA target genes were mainly enriched in pathways related to lipid metabolism and immune function. Interaction network analysis showed that 17 differentially expressed lncRNAs and 15 differentially expressed mRNAs had an interactive relationship. Additionally, we used six differentially expressed lncRNAs and mRNAs to verify the accuracy of the sequencing data via qRT-PCR. Our results provide a comprehensive overview of the expression patterns of the lncRNAs and mRNAs involved in the adaptation of Tibetan sheep to hypoxia, laying a foundation for further analysis of the adaptations of plateau animals.

MicroRNA (miRNA) is a kind of noncoding RNA whose function involved in various biological processes in neuronal maturation and adipocyte cells, such as differentiation, proliferation, development, apoptosis, and metabolism. Herein, miRNA-Seq was used to identify miRNAs in the tail fat tissue of Hu sheep (short-fat-tailed) and Tibetan sheep (short-thin-tailed). In this study, 155 differentially expression miRNAs (DE miRNAs) were identified, including 78 up-regulated and 77 down-regulated. Among these DE miRNAs, 17 miRNAs were reported and related with lipid metabolism. MiRanda and RNAhybrid software were used to predict the target genes of DE miRNAs, obtaining the number of targeting relationships is 38553. Target genes were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 742 terms and 302 single pathways are enriched, including lipid metabolic process, response to lipid, cellular lipid catabolic process, lipid catabolic process, cellular lipid metabolic process, inositol lipid-mediated signaling, calcium channel activity, PI3K-Akt signaling pathway, MAPK signaling pathway, ECM-receptor interaction, AMPK signaling pathway, Wnt signaling pathway and TGF-beta signaling pathway. Notably, miR-379-5p was associated with tail fat deposition of sheep. Dual-Luciferase reporter assays showed miR-379-5p and HOXC9 had targeted relationship. The result of RT-qPCR showed that the expression trend of miR-379-5p and HOXC9 was opposite. miR-379-5p was down-regulated and highly expressed in tail adipose tissue of Tibetan sheep. HOXC9 was highly expressed in adipose tissue of Hu sheep. These results could provide a meaningful theoretical basis for studying the molecular mechanisms of sheep tail adipogenesis.

Crossbreeding significantly improves meat production performance in sheep; however, whether hybridization changes the meat quality characteristics of lambs is uncertain. We analyzed the effects of three different hybrid sires on muscle fiber characteristics (MFCs), fatty acid composition (FAC), and volatile flavor compounds (VFCs) in lambs under identical feeding conditions. Compared with those of purebred lambs, the muscle fiber diameter and cross-sectional areas of the crossbred lambs were significantly decreased (p < 0.05), and the collagen fiber content was significantly increased (p < 0.05). The numbers and area ratios of the fast and slow muscle fibers did not significantly differ between the purebred and crossbred lambs, but the expressions of four MyHC gene types differed significantly (p < 0.05). Twenty-three fatty acids were identified in both the purebred and crossbred lambs, of which thirteen were differentially expressed (p < 0.05). Saturated fatty acid (SFA) contents in the crossbred lambs were significantly increased (p < 0.05), whereas the monounsaturated fatty acid content was significantly decreased (p < 0.05). Polyunsaturated fatty acid/SFA and n-6/n-3 ratios were significantly lower in the crossbred lambs than in the purebred lambs (p < 0.05). Twenty-five VFCs were identified among the three hybrids, and aldehydes were the main VFCs. Eleven VFCs were differentially expressed in the crossbred lambs (p < 0.05). Hybrid sires affected the MFCs, FAC, and VFCs of the F1 lambs, thus providing a reference for high-quality mutton production.

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.

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.

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–Tibetan Plateau, China. Haplotype and nucleotide diversities were 0.748 ± 0.010 and 0.003 ± 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–Tibetan Plateau, which will aid information of future conservation programs aimed at conserving this valuable genetic resource.

Bone morphogenetic protein 2 (BMP2) is strongly selected in both fat-tailed and thin-tailed sheep and may be a candidate gene for sheep tail type selection. However, the mechanism of action of BMP2 in sheep tail fat deposition remains unclear. This study investigated genetic variation and haplotype combinations of the BMP2 gene in sheep with different tail types, aiming to reveal the molecular mechanism of BMP2 in sheep tail fat deposition. We detected a total of three single nucleotide polymorphisms (SNPs) (g.48401619 T > A, g.48401272 C > A, and g.48401136 C > T) among 533 sheep. The alleles and genotype frequencies of these SNPs were in Hardy–Weinberg equilibrium and showed significant correlations with tail length. Linkage disequilibrium existed between the g.48401272 C > A and g.48401136 C > T sites, where CACT was the predominant genotype. At the cellular level, the expression levels of peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) were upregulated after BMP2 overexpression; there were significantly higher levels of PPARγ than controls at 0 d and 1 d, and of LPL than controls at 1 d and 7 d. These results indicate that the BMP2 gene may participate in sheep tail fat deposition and could be used for molecular-marker-assisted selection of sheep tail type.

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.

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 > G site was significantly correlated with tail length, and g.3852134 C > T site was significantly correlated with tail width. g.3852134 C > 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 (PPARγ) 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.

With the intensified and large-scale development of sheep husbandry and global warming, sheep heat stress has become an increasingly important issue. However, little is known about the molecular mechanisms related to sheep responses to heat stress. In this study, transcriptomic analysis of liver tissues of sheep in the presence and absence of heat stress was conducted, with the goal of identifying genes and pathways related to regulation when under such stress. After a comparison with the sheep reference genome, 440,226,436 clean reads were obtained from eight libraries. A p-value ≤ 0.05 and fold change ≥ 2 were taken as thresholds for categorizing differentially expressed genes, of which 1137 were identified. The accuracy and reliability of the RNA-Seq results were confirmed by qRT-PCR. The identified differentially expressed genes were significantly associated with 419 GO terms and 51 KEGG pathways, which suggested their participation in biological processes such as response to stress, immunoreaction, and fat metabolism. This study’s results provide a comprehensive overview of sheep heat stress-induced transcriptional expression patterns, laying a foundation for further analysis of the molecular mechanisms of sheep heat stress.

Clinical mastitis is still an intractable problem for sheep breeding. The natural immunologic mechanisms of the mammary gland against infections are not yet understood. For a better understanding of the disease-associated proteins during clinical mastitis in meat sheep, we performed two-dimensional electrophoresis (2-DE)-based comparative proteomic analyses of mammary tissues, including from healthy mammary tissues (HMTs) and from mammary tissues with clinical mastitis (CMMTs). The 2-DE results showed that a total of 10 up-regulated and 16 down-regulated proteins were identified in CMMTs when compared to HMTs. Of these, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses revealed that most proteins were associated with immune responses or metabolisms. The results of qRT-PCR and Western blot for randomly selected four differentially expressed proteins (DEPs) including superoxide dismutase [Mn] (SOD2), annexin A2 (ANAX2), keratin 10 (KRT10) and endoplasmic reticulum resident protein 29 (ERP29) showed that their expression trends were consistent with 2-DE results except ANXA2 mRNA levels. This is an initial report describing the 2-DE-based proteomics study of the meat sheep mammary gland with clinical mastitis caused by natural infection, which provides additional insight into the immune and metabolic mechanisms during sheep mastitis.

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.