Long divergent haplotypes introgressed from wild sheep are associated with distinct morphological and adaptive characteristics in domestic sheep.

The worldwide sheep population comprises more than 1000 breeds. Together, these exhibit a considerable morphological diversity, which has not been extensively investigated at the molecular level. Here, we analyze whole-genome sequencing individuals of 1,098 domestic sheep from 154 breeds, and 69 wild sheep from seven Ovis species. On average, we detected 6.8%, 1.0% and 0.2% introgressed sequence in domestic sheep originating from Iranian mouflon, urial and argali, respectively, with rare introgressions from other wild species. Interestingly, several introgressed haplotypes contributed to the morphological differentiations across sheep breeds, such as a RXFP2 haplotype from Iranian mouflon conferring the spiral horn trait, a MSRB3 haplotype from argali strongly associated with ear morphology, and a VPS13B haplotype probably originating from urial and mouflon possibly associated with facial traits. Our results reveal that introgression events from wild Ovis species contributed to the high rate of morphological differentiation in sheep breeds, but also to individual variation within breeds. We propose that long divergent haplotypes are a ubiquitous source of phenotypic variation that allows adaptation to a variable environment, and that these remain intact in the receiving population probably due to reduced recombination.

Genomic analyses of wild argali, domestic sheep, and their hybrids provide insights into chromosome evolution, phenotypic variation, and germplasm innovation.

Understanding the genetic mechanisms of phenotypic variation in hybrids between domestic animals and their wild relatives may aid germplasm innovation. Here, we report the high-quality genome assemblies of a male Pamir argali (O ammon polii, 2n = 56), a female Tibetan sheep (O aries, 2n = 54), and a male hybrid of Pamir argali and domestic sheep, and the high-throughput sequencing of 425 ovine animals, including the hybrids of argali and domestic sheep. We detected genomic synteny between Chromosome 2 of sheep and two acrocentric chromosomes of argali. We revealed consistent satellite repeats around the chromosome breakpoints, which could have resulted in chromosome fusion. We observed many more hybrids with karyotype 2n = 54 than with 2n = 55, which could be explained by the selfish centromeres, the possible decreased rate of normal/balanced sperm, and the increased incidence of early pregnancy loss in the aneuploid ewes or rams. We identified genes and variants associated with important morphological and production traits (e.g., body weight, cannon circumference, hip height, and tail length) that show significant variations. We revealed a strong selective signature at the mutation (c.334C > A, p.G112W) in TBXT and confirmed its association with tail length among sheep populations of wide geographic and genetic origins. We produced an intercross population of 110 F2 offspring with varied number of vertebrae and validated the causal mutation by whole-genome association analysis. We verified its function using CRISPR-Cas9 genome editing. Our results provide insights into chromosomal speciation and phenotypic evolution and a foundation of genetic variants for the breeding of sheep and other animals.

Molecular identification and phylogenetic analysis of gastrointestinal nematodes in different populations of Kazakh sheep.

Gastrointestinal nematode (GIN) infection in sheep has been recognized globally as a major problem challenging animal health and production. The objective of this study is to use a molecular diagnosis of the prevalence for gastrointestinal nematode (GIN) dominant species of Kazakh sheep and its hybrid (Kazakh × Texel). The internal transcribed spacer 2 (ITS-2) sequences of ribosomal DNA (rDNA) were used as the target sequence. In the study, three dominant species of nematodes, namely Haemonchus contortus, Trichostrongylus spp., and Teladorsagia (Ostertagia) circumcincta from the Kazakh sheep and the F1 and F2 generations of Texel × Kazakh sheep hybrids were subjected to molecular identification and phylogenetic analysis. The fecal and single larva genomic DNA were extracted and amplified by PCR using specific primers to determine the infection rate of the three nematode species. In addition, the PCR products were sequenced and analyzed using bioinformatics methods to construct a phylogenetic tree. The results showed that all the three species had their ITS-2 specific amplified. According to the sequence homology analysis of PCR products, the results showed a high homology (above 98.5% homology) with H. contortus, Trichostrongylus spp., T. circumcincta ITS-2 sequences in GenBank. Phylogenetic analysis showed that the ITS-2 sequences of the three species were on the same branch as the ITS-2 sequences of the same species in NCBI. And on different branches from those of the ITS-2 sequences of different families, genera and species. Sequences carried out on three species from different samples showed a close relationship and little genetic difference in phylogenetic tree. The infection rates based on fecal DNA were 35.59, 25.55, and 11.24% for H. contortus, Trichostrongylus spp., and T. circumcincta, respectively. While the infection rates based on larva DNA, were 24.07, 18.89, and 13.26% for H. contortus, Trichostrongylus spp., and T. circumcincta, respectively. The seasonal prevalence of the three dominant species in spring was significantly higher than that in autumn and winter. And there was no significant difference between Kazakh, F1 and F2 sheep considering the infection rate of the studied three species of nematodes. This study provides valuable molecular approaches for epidemiological surveillance and for assisting in the control of Nematodirus infection in sheep.

The adhesive heterogeneity of different compartments of oral mucosal rete ridges.

Rete ridges play a critical role in maintaining epidermal structure and mechanical properties. Notably, rete ridges can be divided into three compartments: the base, slope and tip. The present study aims to explore whether these three compartments have distinct adhesive functions. We collected 28 normal masticatory mucosae to prepare paraffin-embedded sections. Immunohistochemistry and immunofluorescent staining were used to analyse the expression pattern of integrin α6 and ß4 in different compartments of the rete ridges. To observe whether the different compartments had distinct adhesive forces, dermal-epidermal junction separation experiments were performed by peeling the oral epithelium from the lamina propria after treatment with cold saline for 72 h. The results showed that integrin α6 and ß4 prefer the basal layer keratinocytes closely adjacent to the base compartment of the rete ridges. The oral mucosal epithelium separated from the underlying lamina propria at the tip of rete ridges when they were peeled after the cold saline treatment. In conclusion, the adhesive force of the basal layer keratinocytes at the base of the rete ridges is stronger than at the tip.

Whole-Genome Sequencing of Native Sheep Provides Insights into Rapid Adaptations to Extreme Environments.

Global climate change has a significant effect on extreme environments and a profound influence on species survival. However, little is known of the genome-wide pattern of livestock adaptations to extreme environments over a short time frame following domestication. Sheep (Ovis aries) have become well adapted to a diverse range of agroecological zones, including certain extreme environments (e.g., plateaus and deserts), during their post-domestication (approximately 8-9 kya) migration and differentiation. Here, we generated whole-genome sequences from 77 native sheep, with an average effective sequencing depth of ∼5× for 75 samples and ∼42× for 2 samples. Comparative genomic analyses among sheep in contrasting environments, that is, plateau (>4,000 m above sea level) versus lowland (<100 m), high-altitude region (>1500 m) versus low-altitude region (<1300 m), desert (<10 mm average annual precipitation) versus highly humid region (>600 mm), and arid zone (<400 mm) versus humid zone (>400 mm), detected a novel set of candidate genes as well as pathways and GO categories that are putatively associated with hypoxia responses at high altitudes and water reabsorption in arid environments. In addition, candidate genes and GO terms functionally related to energy metabolism and body size variations were identified. This study offers novel insights into rapid genomic adaptations to extreme environments in sheep and other animals, and provides a valuable resource for future research on livestock breeding in response to climate change.

Removal of a large number of foreign bodies in the maxillofacial region with navigation system.

The removal of a large number of foreign bodies in the maxillofacial region can be complicated and challenging. In this case report, a specific strategy for removal of multiple foreign bodies in the maxillofacial region is presented. A girl diagnosed with 24 foreign bodies in her left lower face and neck underwent the surgery using a computer-assisted navigation system. A specific strategy was used in this case, which included from one sub-region to the next and from the easiest to the most difficult. This strategy is recommended for the removal of a large number of foreign bodies in the maxillofacial region with a navigation system.

Effects of vertebral number variations on carcass traits and genotyping of Vertnin candidate gene in Kazakh sheep.

OBJECTIVE: The vertebral number is associated with body length and carcass traits, which represents an economically important trait in farm animals. The variation of vertebral number has been observed in a few mammalian species. However, the variation of vertebral number and quantitative trait loci in sheep breeds have not been well addressed. METHODS: In our investigation, the information including gender, age, carcass weight, carcass length and the number of thoracic and lumbar vertebrae from 624 China Kazakh sheep was collected. The effect of vertebral number variation on carcass weight and carcass length was estimated by general linear model. Further, the polymorphic sites of Vertnin (VRTN) gene were identified by sequencing, and the association of the genotype and vertebral number variation was analyzed by the one-way analysis of variance model. RESULTS: The variation of thoracolumbar vertebrae number in Kazakh sheep (18 to 20) was smaller than that in Texel sheep (17 to 21). The individuals with 19 thoracolumbar vertebrae (T13L6) were dominant in Kazakh sheep (79.2%). The association study showed that the numbers of thoracolumbar vertebrae were positively correlated with the carcass length and carcass weight, statistically significant with carcass length. To investigate the association of thoracolumbar vertebrae number with VRTN gene, we genotyped the VRTN gene. A total of 9 polymorphic sites were detected and only a single nucleotide polymorphism (SNP) (rs426367238) was suggested to associate with thoracic vertebral number statistically. CONCLUSION: The variation of thoracolumbar vertebrae number positively associated with the carcass length and carcass weight, especially with the carcass length. VRTN gene polymorphism of the SNP (rs426367238) with significant effect on thoracic vertebral number could be as a candidate marker to further evaluate its role in influence of thoracolumbar vertebral number.

BIEC2-808543 SNP in the LCORL Gene is Associated with Body Conformation in the Yili Horse.

Recently, a SNP (BIEC2-808543) was demonstrated to be associated with equine body size in horses. In this study, we genotyped BIEC2-808543 SNPs in 314 Yili horses in order to evaluate the association between genotype and body composition traits, such as body weight, withers height, chest circumference, and cannon circumference. Results indicate significant associations between polymorphisms of this SNP and body conformation in Yili horse populations. Based on these results, we hypothesize that BIEC2-808543 is strongly related to body conformation of Yili horses and has the potential to be used for marker-assisted selection.

A 76-base pair duplication within the enhancer region of the HMX1 gene causes sheep microtia.

Sheep congenital microtia is characterized by underdeveloped ears and provides an ideal basis for studying human microtia. This study identified the causal mutation and regulatory mechanisms underlying this disorder. Whole-genome association analysis was conducted using 23 ear tissue samples from sheep with microtia and 28 samples from normal-eared sheep. A significant correlation was found between microtia and a 76-base pair duplication in the enhancer region of the HMX1 gene. Further analysis of offspring phenotypes confirmed an autosomal dominant inheritance pattern. Genotypic analysis showed that individuals that are homozygous for this duplication were earless, heterozygous individuals exhibited shortened ears, and wild-type individuals had normal ears. Moreover, luciferase assays confirmed that this duplication increased HMX1 gene expression, and duplication knock-in mice also exhibited shorter and narrower external ears compared to wild-type mice. Transcriptomic analysis further demonstrated that this duplication enhanced HMX1 gene expression in animal models. This study characterized the causal regulatory mutation underlying sheep microtia.

Expression of 2A peptide mediated tri-fluorescent protein genes were regulated by epigenetics in transgenic sheep.

A number of gene therapy applications and basic research would benefit from vectors expressing multiple genes. In this study, we constructed 2A peptide based tricistronic lentiviral vector and generated transgenic lambs by injecting lentivirus carrying the tricistronic vector into perivitelline space of zygotes. Of 7 lambs born, 2 lambs (#6 and #7) carried the transgene. However, no fluorescent proteins were identified in transgenic sheep. To investigate why the transgene was silenced in transgenic sheep, we analyzed the methylation status of transgene. The methylation level of CMV promoter was 76.25% in #6, and 64.7% in #7. In the coding region of three fluorescent protein genes, methylation levels were extremely high, with the average level of 98.3% in #6 and 98.4% in #7 respectively. Furthermore, the ratio of GFP(+) cells were increased significantly when the fibroblasts derived from the transgenic sheep were treated with 5-azaC and/ or TSA. Our results showed that 2A peptide based tricistronic construct was subjected to hypermethylation in transgenic sheep. Moreover, the silencing could be relieved by treating with methytransferase inhibitor and/or deacetylase inhibitor.

Morphogenesis of rete ridges in human oral mucosa: a pioneering morphological and immunohistochemical study.

OBJECTIVE: One of the major impediments in tissue-engineered oral mucosa (TEOM) is the lack of rete ridge (RR) structures that can weaken the connection between the epidermis and dermis. This study aimed to investigate the native morphology of RRs as well as the expression of extracellular signal-regulated kinase 1/2 (ERK1/2), Ki67, and keratin-19, which are related to cell mechanotransduction, proliferation, and stemness in the oral epidermis, respectively. METHODS: RR characteristics, including type, density, length, and width, were analyzed in the masticatory mucosa (Mm) and lining mucosa (Lm) sites of 52 specimens. The expression of ERK1/2, Ki67, and keratin-19 was assessed by immunohistochemistry. ERK1/2 activation by masticatory stimuli was confirmed in vitro by loading pressure onto cultured keratinocytes isolated from the specimens. RESULTS: Three types of RR were found. The RRs in the Mm and Lm differed. The length and percentage of ERK1/2-positive (%ERK1/2+) basal layer cells had a negative correlation (p = 0.004), whereas the length and %Ki67+ basal layer cells had a positive correlation (p = 0.013). The %ERK1/2+ basal layer cells and %keratin-19+ basal layer cells had a negative relationship (p = 0.011). ERK1/2 activation in the oral epithelium was induced by pressure and propagated in cultured keratinocytes. CONCLUSION: RRs are longer in the Mm, which may result from the topical basal cell proliferation and migration induced by masticatory pressure via ERK1/2 activation. Our findings preliminarily interpret RR histomorphology as influenced by oral masticatory pressure. Results may benefit future studies on RR development and reconstruction in TEOM models to enhance the epidermis-dermis connection.

Genome-Wide Association Analysis of Muscle pH in Texel Sheep × Altay Sheep F2 Resource Population.

pH was one of the important meat quality traits, which was an important factor affecting the storage/shelf life and quality of meat in meat production. In order to find a way to extend the storage/shelf life, the pH values (pH45min, pH24h, pH48h and pH72h) of the longissimus dorsi muscles in F2 individuals of 462 Texel sheep × Altay sheep were determined, genotyping was performed using Illumina Ovine SNP 600 K BeadChip and whole genome resequencing technology, a genome-wide association analysis (GWAS) was used to screen the candidate genes and molecular markers for pH values related to the quality traits of mutton, and the effects of population stratification were detected by Q-Q plots. The results showed that the pH population stratification analysis did not find significant systemic bias, and there was no obvious population stratification effect. The results of the association analysis showed that 28 SNPs significantly associated with pH reached the level of genomic significance. The candidate gene associated with pH45min was identified as the CCDC92 gene by gene annotation and a search of the literature. Candidate genes related to pH24h were KDM4C, TGFB2 and GOT2 genes. The candidate genes related to pH48h were MMP12 and MMP13 genes. The candidate genes related to pH72h were HILPDA and FAT1 genes. Further bioinformatics analyses showed 24 gene ontology terms and five signaling pathways that were significantly enriched (p ≤ 0.05). Many terms and pathways were related to cellular components, processes of protein modification, the activity of protein dimerization and hydrolase activity. These identified SNPs and genes could provide useful information about meat and the storage/shelf life of meat, thereby extending the storage/shelf life and quality of meat.

The critical role of myostatin in differentiation of sheep myoblasts.

Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

Correction: An epidemiological study of gastrointestinal nematode and Eimeria coccidia infections in different populations of Kazakh sheep.

[This corrects the article DOI: 10.1371/journal.pone.0251307.].

Regulatory mechanism of oral mucosal rete peg formation.

Rete pegs are finger-like structures that are formed during the development and wound healing process of the skin and oral mucosa, and they provide better mechanical resistance and nutritional supply between the epithelium and dermis. An increasing number of studies have shown that rete pegs have physiological functions, such as resisting bacterial invasion, body fluid loss, and other harmful changes, which indicate that rete pegs are important structures in natural skin and oral mucosa. Although a great deal of progress has been made in scaffold materials and construction methods for tissue-engineered skin and oral mucosa in recent years, construction of the oral mucosa with functional rete pegs remains a major challenge. In this review, we summarized current research on the progress on formation of rete pegs in human oral mucosa as well as its molecular basis and regulatory mechanism, which might provide new ideas for functional construction of tissue-engineered skin and oral mucosa.

Podosome formation in the murine palatal mucosae: Its proteolytic role in rete peg formation.

BACKGROUND: Basement membrane remodeling is an indispensable factor for oral mucosal rete peg formation, but how the basement membrane is remodeled remains unclear. Our previous study indicated that keratinocyte growth factor induces the assembly of podosomes, which are dynamic organelles critical for matrix remodeling in human immortalized oral epithelial cells. This study explores podosome formation and its role in basement membrane remodeling during murine oral mucosal rete peg formation. METHODS: Perinatal murine palatal tissue slices were obtained from embryonic day 17.5 (E 17.5) to postnatal day 10.5 (P 10.5) BALB/c mice. Rete peg formation was observed by hematoxylin and eosin (HE) staining. Proteolysis of the basement membrane was detected by immunofluorescence staining. The assembly of podosomes and their correlation with basement membrane proteolysis were investigated by laser scanning confocal microscopy. RESULTS: The shape of basal layer keratinocytes at the sites of emerging rete pegs changed from typically polygonal to spindle-shaped. Basement membrane proteolysis, indicated by decreased type IV collagen (Col IV) staining, was detected during rete peg formation. Classical markers for podosomes, including cortactin/Tks5, WASP, and matrix metalloproteinase foci, were easily observed at the spindle-shaped cells. Podosomes were visible in regions where there was a significant decrease in Col IV staining. CONCLUSIONS: These observations indicated that podosomes form at the front of the emerging rete peg and may play a pivotal role in basement membrane remodeling during rete peg formation.

In vitro and in vivo synergistic anti-tumor effect of LIN28 inhibitor and metformin in oral squamous cell carcinoma.

Cancer stem cell therapy is becoming a focal point for oral squamous cell carcinoma (OSCC). They can be regulated by tumor glucose metabolism, whereas the regulation is not fully investigated in OSCC. Herein, we studied the synergistic anti-tumor effect of a LIN28 inhibitor C1632 and hypoglycemic medication metformin in OSCC. In this study, OSCC cell lines SCC9 and CAL27 were treated with C1632 and metformin respectively or synergistically. First, western blotting was performed to detect the expression level of LIN28 and its downstream molecule HMGA2. Second, MTT assay was conducted to assess cell proliferation. Next, wound healing assay and transwell assay were applied to evaluate cell migration. Then, xenograft mouse experiment was done to explore anti-tumor effect in vivo. Finally, western blotting was used to investigate the pharmacological mechanisms of the synergistic effect oft he two medication. Results showed that LIN28 and HMGA2 expression decreased significantly in SCC9 and CAL27 cells under 240 µM C1632 treatment for 72 h. These effects were synergized under combined treatment for 24 h. Cell proliferation ability and migration ability of both cell lines decreased significantly under respective and combined treatment. In xenograft mouse experiment, tumor weights decreased by 48% under 40 mg/kg/3d C1632 treatment, 53% under 250 mg/kg/d metformin treatment and 91% under combined treatment for 18 days. Tumor volumes decreased by 32%, 57% and 47% under C1632, metformin and combined treatment respectively. These results indicated that C1632 and metformin exerts synergistic anti-tumor effects in OSCC cell lines SCC9 and CAL27, and also inhibits xenograft tumor growth in vivo.

An epidemiological study of gastrointestinal nematode and Eimeria coccidia infections in different populations of Kazakh sheep.

This is an epidemiological study on the gastrointestinal nematode (GIN) and Eimeria coccidia infections in Kazakh sheep and the F1 and F2 generations of Kazakh × Texel sheep crosses. A total of 7599 sheep fecal samples were collected from the Zhaosu County and Nilka County in Ili Kazakh Autonomous Prefecture in the four seasons-spring, summer, autumn, and winter of 2019. The parasite causing the infection was identified by the saturated saline floating method, and the infection intensity was calculated by the modified McMaster method. SPSS19.0 was used to evaluate the differences in the fecal egg count (FEC) of for GIN and the fecal oocyst count (FOC) value of for coccidia per sample. The results showed that there were nine types of sheep GIN infections and Eimeria coccidia in these two counties of Ililocations, with the dominant parasite species of Haemonchus contortus, Trichostrongylus spp., and Ostertagia spp as the predominant parasites in the sheep. Most of the GIN and coccidia infections in these two regions were mild and moderate. The mean log (FEC) of GIN infection in the Zhaosu area was significantly higher than that in the Nilka area, whereas the mean log (FOC) of coccidia infection in Zhaosu was significantly lower than that of Nilka. The mean log (FEC) of GIN infection in the four seasons was the highest in spring, followed by in summer, then in autumn, and the lowest in winter. The mean log (FOC) of coccidia infection was the highest in spring, followed by in autumn, and was the lowest in summer and winter. The mean log (FEC) of GIN infection and log (FOC) of coccidia infection of Kazakh sheep was significantly higher than the F1 generation, which was then significantly higher than the F2 generation of summer. A positive correlation was found between the EPG and OPG levels in the sheep. These results showed that the GIN and coccidia infection intensities of the F1 generation sheep of Kazakh ×Texel crosses were significantly lower than that of Kazakh sheep paving the way for marker-based resistance selection.

A Hu sheep genome with the first ovine Y chromosome reveal introgression history after sheep domestication.

The Y chromosome plays key roles in male fertility and reflects the evolutionary history of paternal lineages. Here, we present a de novo genome assembly of the Hu sheep with the first draft assembly of ovine Y chromosome (oMSY), using nanopore sequencing and Hi-C technologies. The oMSY that we generated spans 10.6 Mb from which 775 Y-SNPs were identified by applying a large panel of whole genome sequences from worldwide sheep and wild Iranian mouflons. Three major paternal lineages (HY1a, HY1b and HY2) were defined across domestic sheep, of which HY2 was newly detected. Surprisingly, HY2 forms a monophyletic clade with the Iranian mouflons and is highly divergent from both HY1a and HY1b. Demographic analysis of Y chromosomes, mitochondrial and nuclear genomes confirmed that HY2 and the maternal counterpart of lineage C represented a distinct wild mouflon population in Iran that diverge from the direct ancestor of domestic sheep, the wild mouflons in Southeastern Anatolia. Our results suggest that wild Iranian mouflons had introgressed into domestic sheep and thereby introduced this Iranian mouflon specific lineage carrying HY2 to both East Asian and Africa sheep populations.

An atlas of CNV maps in cattle, goat and sheep.

Copy number variation (CNV) is the most prevalent type of genetic structural variation that has been recognized as an important source of phenotypic variation in humans, animals and plants. However, the mechanisms underlying the evolution of CNVs and their function in natural or artificial selection remain unknown. Here, we generated CNV region (CNVR) datasets which were diverged or shared among cattle, goat, and sheep, including 886 individuals from 171 diverse populations. Using 9 environmental factors for genome-wide association study (GWAS), we identified a series of candidate CNVRs, including genes relating to immunity, tick resistance, multi-drug resistance, and muscle development. The number of CNVRs shared between species is significantly higher than expected (P<0.00001), and these CNVRs may be more persist than the single nucleotide polymorphisms (SNPs) shared between species. We also identified genomic regions under long-term balancing selection and uncovered the potential diversity of the selected CNVRs close to the important functional genes. This study provides the evidence that balancing selection might be more common in mammals than previously considered, and might play an important role in the daily activities of these ruminant species.