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Simple Summary: We performed a genome-wide association study of 100 sheep genomes to investigate the hereditary basis of the four-horn phenotype (FHP) in sheep. The results revealed that a total of 3 InDels (e.g., CHR2: g.133,742,709delA and g.133,743,215insC) and 14 SNPs (e.g., CHR2: g.133,727,513C > T and CHR16: g.40,351,378G > A) in CHR2 and CHR16 were significantly associated with the four-horn trait. Furthermore, HOXD1 and ADAMTS12 annotated from both CHR sequences were the key candidate gene loci contributing to FHP in sheep. This study provided a novel understanding of the Mendelian genetic basis for four horns in sheep. Horns are an important breeding trait for sheep. However, no widely recognized viewpoint on the regulatory genes and mechanisms of horns is available, and the genetic basis of the four-horn phenotype (FHP) is unclear. This work conducted a genome-wide association study with 100 sheep genomes from multiple breeds to investigate the genetic basis of the FHP. The results revealed three significant associations (corrected as p < 1.64 × 10−8) of the InDels (CHR2: g.133,742,709delA, g.133,743,215insC, and g.133,743,940delT) for FHP in the intergenic sequence (IGS) between the MTX2 and the LOC105609047 of CHR2. Moreover, 14 significant associations (corrected as p < 1.42 × 10−9) of SNPs with the FHP phenotype were identified in CHR2 and CHR16, including five (e.g., CHR16: g.40,351,378G > A and g.40,352,577G > A) located in the intron of the ADAMTS12 gene, eight (e.g., CHR2: g.133,727,513C > T and g.133,732,145T > G) in the IGS between MTX2 and LOC105609047, and only one (CHR2: g.133,930,761A > G) in the IGS between HOXD1 and MTX2. Obvious divergence was also observed in genotype patterns between the FHP and others (two horns and hornless) in the HOXD1 and ADAMTS12 gene regions. An extremely significant linkage also occurred between Loci I and Loci II within 100 individuals (LD = −156.02186, p < 0.00001). In summary, our study indicated that the genomic sequences from CHR2 and CHR16 contributed to the FHP in sheep, specifically the key candidate genes HOXD1 and ADAMTS12. These results improved our understanding of the Mendelian genetic basis of the FHP in sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: In our research, candidate genes related to sheep-milk production were revealed by a genome-resequencing analysis and a genome-signal-selection analysis, and a RT-qPCR experiment was performed to prove the expression levels of these candidate genes, the results showed that the FCGR3A gene's expression level had a significant negative relationship with sheep-milk production. Natural selection and domestication have shaped modern sheep populations into a vast range of phenotypically diverse breeds. Among these breeds, dairy sheep have a smaller population than meat sheep and wool sheep, and less research is performed on them, but the lactation mechanism in dairy sheep is critically important for improving animal-production methods. In this study, whole-genome sequences were generated from 10 sheep breeds, including 57 high-milk-yield sheep and 44 low-milk-yield sheep, to investigate the genetic signatures of milk production in dairy sheep, and 59,864,820 valid SNPs (Single Nucleotide Polymorphisms) were kept after quality control to perform population-genetic-structure analyses, gene-detection analyses, and gene-function-validation analyses. For the population-genetic-structure analyses, we carried out PCA (Principal Component Analysis), as well as neighbor-joining tree and structure analyses to classify different sheep populations. The sheep used in our study were well distributed in ten groups, with the high-milk-yield-group populations close to each other and the low-milk-yield-group populations showing similar classifications. To perform an exact signal-selection analysis, we used three different methods to find SNPs to perform gene-annotation analyses within the 995 common regions derived from the fixation index (FST), nucleotide diversity (Ɵπ), and heterozygosity rate (ZHp) results. In total, we found 553 genes that were located in these regions. These genes mainly participate in the protein-binding pathway and the nucleoplasm-interaction pathway, as revealed by the GO- and KEGG-function-enrichment analyses. After the gene selection and function analyses, we found that FCGR3A, CTSK, CTSS, ARNT, GHR, SLC29A4, ROR1, and TNRC18 were potentially related to sheep-milk-production traits. We chose the strongly selected genes, FCGR3A, CTSK, CTSS, and ARNT during the signal-selection analysis to perform a RT-qPCR (Reale time Quantitative Polymerase Chain Reaction) experiment to validate their expression-level relationship with milk production, and the results showed that FCGR3A has a significant negative relationship with sheep-milk production, while other three genes did not show any positive or negative relations. In this study, it was discovered and proven that the candidate gene FCGR3A potentially contributes to the milk production of dairy sheep and a basis was laid for the further study of the genetic mechanism underlying the strong milk-production traits of sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: A unique structure of ruminants, the horn trait is not only closely related to natural and sexual selection but is also an important trait for polled sheep breeding. RXFP2 may be a crucial gene in regulating sheep horn. However, the underlying genetic mechanisms of sheep horn development remain largely unknown. In this study, we investigated the gene expression profile of the horn buds in sheep fetuses using RNA-seq technology. We identified 68 differentially expressed genes in the horn buds of 105-day-old Altay sheep fetuses, including RXFP2, FOXL2, and TNN. Further, we found that the Wnt signaling pathway may be responsible for horn development. Our study provides new possible marker genes for horn development, which may promote our understanding of the genetic mechanisms of sheep horn formation. Horns, also known as headgear, are a unique structure of ruminants. As ruminants are globally distributed, the study of horn formation is critical not only for increasing our understanding of natural and sexual selection but also for the breeding of polled sheep breeds to facilitate modern sheep farming. Despite this, a significant number of the underlying genetic pathways in sheep horn remain unclear. In this study, to clarify the gene expression profile of horn buds and investigate the key genes in horn bud formation, RNA-sequencing (RNA-seq) technology was utilized to investigate differential gene expression in the horn buds and adjacent forehead skin of Altay sheep fetuses. There were only 68 differentially expressed genes (DEGs) identified, consisting of 58 up-regulated genes and 10 down-regulated genes. RXFP2 was differentially up-regulated in the horn buds and had the highest significance (p-value = 7.42 × 10−14). In addition, 32 DEGs were horn-related genes identified in previous studies, such as RXFP2, FOXL2, SFRP4, SFRP2, KRT1, KRT10, WNT7B, and WNT3. Further, Gene Ontology (GO) analysis showed that the DEGs were mainly enriched with regard to growth, development, and cell differentiation. Pathway analysis revealed that the Wnt signaling pathway may be responsible for horn development. Further, through combining the protein–protein interaction networks of the DEGs, it was found that the top five hub genes, namely, ACAN, SFRP2, SFRP4, WNT3, and WNT7B, were also associated with horn development. Our results suggest that only a few key genes, including RXFP2, are involved in bud formation. This study not only validates the expression of candidate genes identified at the transcriptome level in previous studies but also provides new possible marker genes for horn development, which may promote our understanding of the genetic mechanisms of horn formation. [ABSTRACT FROM AUTHOR]

Tibetan sheep are one of the three major coarse sheep breeds in China, and they possess a long history of formation. However, few studies have been conducted on the identification of Tibetan sheep breeds at the molecular level. In this study, a total of 448 individuals from 24 Tibetan sheep populations in the 5 regions of Tibet, Qinghai, Gansu, Yunnan, and Sichuan were analyzed using the Affymetrix Ovine SNP 600K high-density chip to construct specific single-nucleoside polymorphism (SNP) genetic marker sets of Tibetan sheep breeds. Firstly, the genetic structure analysis showed that Yunnan–Tibetan sheep (NL, Ninglang; JC, Jianchuan), Zuogong (ZG), Heizang (HZ), Gongga (GG,) and Tao sheep (TS) can be clearly distinguished from other Tibetan sheep populations. Next, based on the population differentiation index FST, the PCA and NJ tree results showed that only 60 specific SNPs can successfully separate Tibetan sheep in the Yunnan region from Tibetan sheep in other regions, and the distinguishing effect on Yunnan–Tibetan sheep reached 100%. Using the same method, we found that 4 Tibetan sheep breeds, including Zuogong (ZG, 20 SNPs), Heizang (HZ, 60 SNPs), Gongga (GG, 60 SNPs), and Tao sheep (TS, 30 SNPs), can also be distinguished from other Tibetan sheep populations with only a few SNP loci (20–60), and the distinguishing effect reached 100%. Overall, we successfully obtained a Yunnan region-specific (60 SNPs) genetic marker set and 4 breed-specific SNP genetic marker sets (20–60 SNPs) for the first time for the identification of Tibetan sheep breeds at the molecular level. These made up for the lack of genetic marker sets for the identification of Tibetan sheep breeds, and provided a genomic basis for the scientific classification and accurate identification of livestock and poultry genetic resources on the Qinghai–Tibet Plateau. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigated the family structure, parent–child pair and inbreeding coefficient of a Tan sheep breeding farm in Ningxia, China. The results can be used as the theoretical basis for selecting excellent breeding stock and making an apposite mating plan; furthermore, it can provide an important scientific basis for the conservation and reproduction of Tan sheep germplasm resources. Tan sheep is a special breed of locally protected sheep in China, one of the best quality meat sheep in the world. Due to the unclear pedigree of the rams on the Ningxia Tan sheep breeding farm, we investigated 74 rams in the field and explored a new method for family division. Genomic DNA was extracted from the blood of breeding rams. Using Plink software, GCTA tools and R language, we analyzed the genetic structure, kinship, and inbreeding coefficient of the breeding sheep, which revealed the genetic relationship between the individuals. The results showed that there was no obvious clustering phenomenon in the PCA, and the genetic background of the samples was similar. The G matrix and IBS distance matrix indicated that most individuals were far away from each other. Paternity testing identified 24 pairs of unknown parent–child pairs, and all the Tan sheep could be divided into 12 families, which provided a reference for sheep breeding. The average inbreeding coefficient based on the ROH of this population was 0.049, so there was a low degree of inbreeding and the rams in the field were able to maintain high genetic diversity. Overall, we explored a more accurate method through paternity and kinship analysis; it provides a scientific basis for pedigree construction, which has an important application value for Tan sheep breeding. [ABSTRACT FROM AUTHOR]

Summary: The multiple teats trait is common in many species of mammals and is considered related to lactation ability in swine. However, in Hu sheep, related gene research is still relatively limited. In this study, a genome‐wide association study was used to identify genetic markers and genes related to the number of teats in the Hu sheep population, a native Chinese sheep breed. A single marker method and several multi‐locus methods were utilized. A total of 61 SNPs were found to be related to the number of teats. Among these, 11 SNPs and one SNP were consistently detected by two and three multi‐locus models respectively. Four SNPs were concordantly identified between the single marker and multi‐locus methods. We also performed quantitative real‐time PCR testing of these identified candidate genes, identifying three genes with significantly different expression. Our study suggested that the LHFP, DPYSL2, and TDP‐43 genes may be related to the number of teats in sheep. The combination of single and multi‐locus GWAS detected additional SNPs not found with only one model. Our results provide new and important insights into the genetic mechanisms of the mammalian multiparous teat phenotype. These findings may be useful for future breeding and understanding the genetics of sheep and other livestock. [ABSTRACT FROM AUTHOR]

Coat colour is one of the most important economic traits of sheep and is mainly used for breed identification and characterization. This trait is determined by the biochemical function, availability and distribution of phaeomelanin and eumelanin pigments. In our study, we conducted a genome-wide association study to identify candidate genes and genetic variants associated with coat colour in 75 Chinese Tan sheep using the ovine 600K SNP BeadChip. Accordingly, we identified two significant SNPs (rs409651063 at 14.232 Mb and rs408511664 at 14.228 Mb) associated with coat colour in the MC1R gene on chromosome 14 with −log10(P) = 2.47E-14 and 1.00E-13, respectively. The consequence of rs409651063 was a missense variant (g.14231948 G>A) that caused an amino acid change (Asp105Asn); however, the second SNP (rs408511664) was a synonymous substitution and is an upstream variant (g.14228343G>A). Moreover, our PCR analysis revealed that the genotype of white sheep was exclusively homozygous (GG), whereas the genotypes of black-head sheep were mainly heterozygous (GA). Interestingly, allele-specific expression analysis (using the missense variant for the skin cDNA samples from black-head sheep) revealed that only the G allele was expressed in the skin covered with white hair, while both the G and A alleles were expressed in the skin covered with black hair. This finding indicated that the missense mutation that we identified is probably responsible for white coat colour in Tan sheep. Furthermore, qPCR analysis of MC1R mRNA level in the skin samples was significantly higher in black-head than white sheep and very significantly higher in GA than GG individuals. Taken together, these results help to elucidate the genetic mechanism underlying coat colour variation in Chinese indigenous sheep. [ABSTRACT FROM AUTHOR]

Four-horned sheep are an ideal animal model for illuminating the genetic basis of horn development. The objective of this study was to locate the genetic region responsible for the four-horned phenotype and to verify a previously reported polled locus in three Chinese breeds. A genome-wide association study ( GWAS) was performed using 34 two-horned and 32 four-horned sheep from three Chinese indigenous breeds: Altay, Mongolian and Sishui Fur sheep. The top two significant single nucleotide polymorphisms ( SNPs) associated with the four-horned phenotype were both located in a region spanning positions 132.6 to 132.7 Mb on sheep chromosome 2. Similar locations for the four-horned trait were previously identified in Jacob, Navajo-Churro, Damara and Sishui Fur sheep, suggesting a common genetic component underlying the four-horned phenotype. The two identified SNPs were both downstream of the metaxin 2 ( MTX2) gene and the HOXD gene cluster. For the top SNP- OAR2:g.132619300G>A-the strong associations of the AA and AG genotypes with the four-horned phenotype and the GG genotype with the two-horned phenotype indicated the dominant inheritance of the four-horned trait. No significant SNPs for the polled phenotype were identified in the GWAS analysis, and a PCR analysis for the detection of the 1.8-kb insertion associated with polled sheep in other breeds failed to verify the association with polledness in the three Chinese breeds. This study supports the hypothesis that two different loci are responsible for horn existence and number. This study contributes to the understanding of the molecular regulation of horn development and enriches the knowledge of qualitative traits in domestic animals. [ABSTRACT FROM AUTHOR]

The dairy sheep industry is an important but lacking part of the small ruminant industry. For a sheep breeding program, in addition to wool and meat use, sheep milk can also be processed into high-end dairy products such as cheese and milk powder and bring high economic interests for businesses home and abroad. With increasing interest in sheep milk, the content of which is becoming increasingly clearer, people have found that the nutritional value of sheep milk is higher than that of goat milk and cow milk, with abundant fat yield, protein percentage, and mineral contents, which provide a good opportunity for the development of the sheep milk industry. This review will introduce some dairy sheep breeds with the highest milk production worldwide and compare sheep milk nutrition contents with other ruminants' milk. Moreover, genes influencing lactation or mammary gland growth like CSN2, SLC2A2, SCD, and SOCS2, which have been revealed in recent studies to significantly affect milk production and milk composition traits will be discussed. For the SLC2A2 gene, working as an important solute carrier to transport small molecular nutrition from blood to milk and SOCS2 gene mutation as an indicator of mastitis, in addition, other genes have been detected that correlate with milk traits, which will be introduced in the review. Some personal opinions into future sheep milk development will be given in the final part of the text. Although the research of sheep milk genetic factors has achieved some progress in recent years, there is still a long way to go. [ABSTRACT FROM AUTHOR]

Homozygosity of long sequence genotypes are a result of parents transmitting identical haplotypes, which can be used to estimate their auto-zygosity. Therefore, we used high-density SNP Chip data to characterize the auto-zygosity of each breed according to the occurrence and distribution of runs of homozygosity (ROH). Subsequently, we identified the genomic regions with high runs of homozygosity frequencies within individuals of each breed. We selected 96 sheep samples from five local Chinese sheep breeds belonging to different geographical locations. We identified 3046 ROHs within the study breed individuals, among which the longer segments (>1–5 Mb) were dominant. On average, ROH segments covered about 12% of the genomes; the coverage rate of OAR20 was the lowest and that of OAR2 was the highest. The distribution analysis of runs of homozygosity showed that the detected ROH mainly distributed between >26 and 28 Mb. The Hetian and Hu sheep showed the lowest ROH distribution. The estimation of homozygosity level reflects the history of modern and ancient inbreeding, which may affect the genomes of Chinese indigenous sheep breeds and indicate that some animals have experienced recent self-pollination events (Yabuyi, Karakul and Wadi). In these sheep breeds, the genomic regions were assumed to be under selection signatures frequently in line with long ROH. These regions included candidate genes associated with disease resistance traits (5S_rRNA), the innate and adaptive immune response (HERC2 and CYFIP1), digestion and metabolism (CENPJ), growth (SPP1), body size and developments (GJB2 and GJA3). This study highlighted new insights into the ROH patterns and provides a basis for future breeding and conservation strategies of Chinese sheep breeds. [ABSTRACT FROM AUTHOR]

Background: Fat tail is a unique trait in sheep acquired during domestication. Several genomic analyses have been conducted in sheep breeds from limited geographic origins to identify the genetic factors underlying this trait. Nevertheless, these studies obtained different candidates. The results of these regional studies were easily biased by the breed structures. Results: To minimize the bias and distinguish the true candidates, we used an extended data set of 968 sheep representing 18 fat-tailed breeds and 14 thin-tailed breeds from around the world, and integrated two statistical tests to detect selection signatures, including Genetic Fixation Index (FST) and difference of derived allele frequency (ΔDAF). The results showed that platelet derived growth factor D (PDGFD) exhibited the highest genetic differentiation between fat- and thin-tailed sheep breeds. Analysis of sequence variation identified that a 6.8-kb region within the first intron of PDGFD is likely the target of positive selection and contains regulatory mutation(s) in fat-tailed sheep. Histological and gene expression analyses demonstrated that PDGFD expression is associated with maturation and hemostasis of adipocytes. Further retrospective analysis of public transcriptomic datasets revealed that PDGFD expression is down-regulated during adipogenesis in both human and mouse, and is higher in fat tissues of obese individuals than that in lean individuals. Conclusions: These results reveal that PDGFD is the predominant factor for the fat tail phenotype in sheep by contributing to adiopogenesis and maintaining the hemostasis of mature adipocytes. This study provides insights into the selection of fat-tailed sheep and has important application to animal breeding, as well as obesity-related human diseases. [ABSTRACT FROM AUTHOR]

Through long term natural and artificial selection, domestic sheep (Ovis aries) have become adapted to a diverse range of agro-ecological environments and display multiple phenotypic traits. Characterization of diversity and selection signature is essential for genetic improvement, understanding of environmental adaptation, as well as utilization and conservation of sheep genetic resources. Here, we aimed to assess genomic diversity, population structure, and genomic selection among five Chinese native sheep breeds using 600K high density SNP genotypes. A total of 96 animals of the five breeds were selected from different geographical locations with extremely dry or humid conditions. We found a high proportion of informative SNPs, ranging from 93.3% in Yabuyi to 95.5% in Wadi, Hu, and Hetian sheep. The average pairwise population differentiation (FST) between the breeds was 0.048%, ranging from 0.022% to 0.054%, indicating their low to moderate differentiation. PCA, ADMIXTURE, and phylogenetic tree analyses revealed a clustering pattern of the five Chinese sheep breeds according to their geographical distribution, tail type, coat color, body size, and breeding history. The genomic regions under putative selection identified by FST and XP-EHH approaches frequently overlapped across the breeds, and spanned genes associated with adaptation to extremely dry or humid environments, innate and adaptive immune responses, and growth, wool, milk, and reproduction traits. The present study offers novel insight into genomic adaptation to dry and humid climates in sheep among other domestic animals and provides a valuable resource for further investigation. Moreover, it contributes useful information to sustainable utilization and conservation of sheep genetic resources. [ABSTRACT FROM AUTHOR]

[Display omitted] • We find two functional mutations driving the finer and denser wool production. • One C/A missense variant of KRT74 drive finer wool production in Chinese fine wool sheep. • The C-KRT74 can activate the KRT74 protein and affect wool fineness. • One T/C SNP in the regulatory region upstream of EDAR drive denser wool production in fine wool sheep. • The C-to-T mutation can upregulate EDAR mRNA expression and potentially affect wool density. Fine-wool sheep are the most common breed used by the wool industry worldwide. Fine-wool sheep have over a three-fold higher follicle density and a 50% smaller fiber diameter than coarse-wool sheep. This study aims to clarify the underlying genetic basis for the denser and finer wool phenotype in fine-wool breeds. Whole-genome sequences of 140 samples, Ovine HD630K SNP array data of 385 samples, including fine, semi-fine, and coarse wool sheep, as well as skin transcriptomes of nine samples were integrated for genomic selection signature analysis. Two loci at keratin 74 (KRT74) and ectodysplasin receptor (EDAR) were revealed. Fine-scale analysis in 250 fine/semi-fine and 198 coarse wool sheep narrowed this association to one C/A missense variant of KRT74 (OAR3:133,486,008, P = 1.02E-67) and one T/C SNP in the regulatory region upstream of EDAR (OAR3:61,927,840, P = 2.50E-43). Cellular over-expression and ovine skin section staining assays confirmed that C-KRT74 activated the KRT74 protein and specifically enlarged cell size at the Huxley's layer of the inner root sheath (P < 0.01). This structure enhancement shapes the growing hair shaft into the finer wool than the wild type. Luciferase assays validated that the C-to-T mutation upregulated EDAR mRNA expression via a newly created SOX2 binding site and potentially led to the formation of more hair placodes. Two functional mutations driving finer and denser wool production were characterized and offered new targets for genetic breeding during wool sheep selection. This study not only provides a theoretical basis for future selection of fine wool sheep breeds but also contributes to improving the value of wool commodities. [ABSTRACT FROM AUTHOR]

Adipose-derived stem cells (ADSCs) that are enriched in adipose tissue with multilineage differentiation potential have become an important tool in therapeutic research and tissue engineering. Certain breeds of sheep exhibit a unique fat tail trait such that tail tissue accounts for approximately 10 % of body weight and can provide an excellent source of ADSCs. Here, we describe isolation of primary ADSCs from ovine embryonic fat tail tissues that displayed high self-renewal capacity, multilineage differentiation and excellent adipogenic ability. Through transcriptome analysis covering ADSCs differentiating into adipocytes, 37 transcription factors were involved in early transcriptional events that initiate a regulatory cascade of adipogenesis; the entire adipogenic activity consists of a reduction in proliferation ability and upregulation of genes related to lipid generation and energy metabolism, as well as several genes associated with myogenesis. Furthermore, Comparative transcriptome analysis across species (sheep, human, and mouse) revealed enhanced basal metabolic ability in differentiating ovine ADSCs, which may relate to the excellent adipogenic capability of these cells. We also identified a small evolutionarily conserved gene set, consisting of 21 and 22 genes exhibiting increased and decreased expression, respectively. Almost half (20) of these genes have not previously been reported to regulate adipogenesis in mammals. In this study, we identified important regulators that trigger ovine adipocyte differentiation, main biological pathways involved in adipogenesis as well as the evolutionarily conserved genes governing adipogenic process across species. Our study provides a novel excellent biomaterial and novel genes regulating adipogenesis for cellular transplantation therapy and investigations of fat metabolism. Transcriptomic characteristics of differentiating human, mouse, and sheep adipose-derived stem cells toward adipocyte, including the crucial genes encoding transcription factors with increased expression or decreased expression at each stage of adipogenesis of ovine ADSCs (inner circle), the specifically expressed genes by sheep (top arc) and human cells (bottom arc), as well as the evolutionary conservative genes set regulating adipogenesis in vitro across species. [Display omitted] • The clear physical distinction of fat tail, the large volume of fat tissue, and readily available donor animals makes fat tail sheep excellent low-cost sources of adipose-derived stem cells (ADSCs) for research and clinical applications. • We depicted the standard procedures to isolate ADSCs from ovine embryonic tail tissue and perform multi-lineages differentiation, and the isolated ADSCs exhibit excellent adipogenic ability. • Several important genes encoding transcription factors were involved in early transcriptional events that initiate a regulatory cascade of adipogenesis. • The enhanced basal metabolic ability in differentiating ovine ADSCs and one small evolutionarily conserved gene set were discovered by comparative transcriptome analysis across species. • In summary, this study provides a novel excellent biomaterial and novel genes regulating adipogenesis for cellular transplantation therapy and investigations of fat metabolism. [ABSTRACT FROM AUTHOR]