Analysis of genetic evolutionary differences among four Tibetan pig populations in China.

Tibetan pigs are a locally bred domestic pig breed originating from the Tibetan Plateau in China. They can be categorized into four distinct groups based on their geographical locations: Sichuan Tibetan pigs, Tibetan pigs from Tibet, Yunnan Tibetan pigs, and Gansu Tibetan pigs. This study aimed to explore population diversity, genetic structure and selection signals among Tibetan pigs in four Chinese national nature reserves. The results show that there is different observed heterozygosity among Tibetan pig populations (0.1957-0.1978). Ratio of runs of homozygosity (Froh) calculation of four Tibetan pig populations by runs of homozygosity (ROH) revealed the presence of inbreeding within the population (0.0336-0.0378). Analysis of the genetic structure demonstrated distinct population stratification among the four Tibetan pig populations, with each showing relatively independent evolutionary directions. Furthermore, Five methods (FST, Piratio, ROD, Tajima's D, XP-CLR) were used to artificially select evolutionary trajectories. The results mainly involved processes such as DNA repair, immune regulation, muscle fat deposition and adaptation to hypoxia. In conclusion, this study enhances our understanding of the genetic characteristics of Tibetan pig populations and provides a theoretical reference for the conservation of resources across different populations of Tibetan pigs.

Whole-genome SNP allele frequency differences between Tibetan and Large white pigs reveal genes associated with skeletal muscle growth.

BACKGROUND: The skeletal muscle growth rate and body size of Tibetan pigs (TIB) are lower than Large white pigs (LW). However, the underlying genetic basis attributing to these differences remains uncertain. To address this knowledge gap, the present study employed whole-genome sequencing of TIB (slow growth) and LW (fast growth) individuals, and integrated with existing NCBI sequencing datasets of TIB and LW individuals, enabling the identification of a comprehensive set of genetic variations for each breed. The specific and predominant SNPs in the TIB and LW populations were detected by using a cutoff value of 0.50 for SNP allele frequency and absolute allele frequency differences (△AF) between the TIB and LW populations. RESULTS: A total of 21,767,938 SNPs were retrieved from 44 TIB and 29 LW genomes. The analysis detected 2,893,106 (13.29%) and 813,310 (3.74%) specific and predominant SNPs in the TIB and LW populations, and annotated to 24,560 genes. Further GO analysis revealed 291 genes involved in biological processes related to striated and/or skeletal muscle differentiation, proliferation, hypertrophy, regulation of striated muscle cell differentiation and proliferation, and myoblast differentiation and fusion. These 291 genes included crucial regulators of muscle cell determination, proliferation, differentiation, and hypertrophy, such as members of the Myogenic regulatory factors (MRF) (MYOD, MYF5, MYOG, MYF6) and Myocyte enhancer factor 2 (MEF2) (MEF2A, MEF2C, MEF2D) families, as well as muscle growth inhibitors (MSTN, ACVR1, and SMAD1); KEGG pathway analysis revealed 106 and 20 genes were found in muscle growth related positive and negative regulatory signaling pathways. Notably, genes critical for protein synthesis, such as MTOR, IGF1, IGF1R, IRS1, INSR, and RPS6KA6, were implicated in these pathways. CONCLUSION: This study employed an effective methodology to rigorously identify the potential genes associated with skeletal muscle development. A substantial number of SNPs and genes that potentially play roles in the divergence observed in skeletal muscle growth between the TIB and LW breeds were identified. These findings offer valuable insights into the genetic underpinnings of skeletal muscle development and present opportunities for enhancing meat production through pig breeding.

Whole-genome sequencing and assessment of a novel protein- and gossypol-degrading Bacillus subtilis strain isolated from intestinal digesta of Tibetan Pigs.

BACKGROUND: With the rapid development of animal husbandry, the demand for protein feed resources is increasing. Cottonseed meal (CSM) and soybean meal (SBM) are rich sources of protein. However, their application is limited due to the existence of anti-nutrients, which can be harmful to the digestion and absorption. A strain of Bacillus subtilis (Mafic-Y7) was isolated from digesta of intestines of Tibetan pigs. The strain showed high protease activity, which helps in degrading proteinic anti-nutritional factors in grain meal and in vitro degradation of free gossypol. In order to better understand this isolated strain, whole genome of Mafic-Y7 strain was sequenced and analyzed. Different effects on various grain meals were identified. RESULT: The GC-depth Poisson distributions showed no bias suggesting high-quality genome assembly of Mafic-Y7. The whole genome sequencing showed that one chromosome with 4,248,845 base pairs(bp)and the genes total length with 3,736,524 bp was predicted in Mafic-Y7. Additionally, Mafic-Y7 possessed 4,254 protein-coding genes, and several protease genes were annotated by aligning them with databases. There are 55 protease genes, one phytase gene and one laccase gene were annotated in the gene sequence of Mafic-Y7. The average nucleotide identity between Mafic-Y7 and the GCA-000009045.1 homologous genome was 0.9938, suggesting a close genetic relationship between them at the species level. Compared with the closest four whole genomes, Mafic-Y7 was annotated the most abundant of protease genes (55 genes). The fermentation supernatant of Mafic-Y7 could increase the content of small peptides, water-soluble proteins, and acid-soluble proteins in vitro by 411%, 281% and 317% in SBM and 420%, 257% and 338% in CSM. After fermentation in grain meal by Mafic-Y7, the degradation rate of anti-nutritional factors in SBM, such as trypsin inhibitor, glycinin, and ß-conglycinin was greater than 70%, and lectin was greater than 30%. The degradation rates of anti-nutritional factors in CSM, such as gossypol and phytic acid, were 82% and 26%, respectively.

Prevalence and Characteristics of Salmonella from Tibetan Pigs in Tibet, China.

This study aimed to understand the epidemiological characteristics of Salmonella in Tibetan pigs. We isolated, identified, and examined via antimicrobial susceptibility testing on Salmonella from Tibetan pigs breeder farms and slaughterhouses in Tibet, China. A genetic evolutionary tree was constructed on the basis of whole genome sequencing (WGS). A total of 81 Salmonella isolates were isolated from 987 samples. The main serovars were Salmonella Typhimurium and Salmonella London in Tibetan pigs. The isolated Salmonella Typhimurium isolates subjected to antimicrobial susceptibility testing showed varying degrees of resistance to ß-lactams, aminoglycosides, fluoroquinolones, sulfonamides, tetracyclines, and amphenicols. WGS analysis was performed on 20 Salmonella Typhimurium isolates in Tibet (n = 10), Jiangsu (n = 10), and 205 genome sequences downloaded from the Enterobase database to reveal their epidemiological and genetic characteristics. They were divided into two clusters based on core genome single-nucleotide polymorphisms: Cluster A with 112 isolates from Tibet and other regions in China and Cluster B with 113 isolates from Jiangsu and other regions. The isolates in Cluster A were further divided into two subclusters: A-1 with 40 isolates including Tibet and A-2 with 72 isolates from other regions. Virulence factors analysis revealed that all isolates from Tibet carried adeG, but this observation was not as common in Salmonella isolates from Jiangsu and other regions of China. Antibiotic resistance genes (ARGs) analysis showed that all isolates from Tibet carried blaTEM-55 and rmtB, which were absent in Salmonella isolates from Jiangsu and other regions of China. Genetic characteristic analysis and biofilm determination indicated that the biofilm formation capabilities of the isolates from Tibet were stronger than those of the isolates from Jiangsu and other regions of China. Our research revealed the epidemic patterns and genomic characteristics of Salmonella in Tibetan pigs and provided theoretical guidance for the prevention and control of local salmonellosis.

Genetic Adaptations of the Tibetan Pig to High-Altitude Hypoxia on the Qinghai-Tibet Plateau.

The Tibetan Plateau's distinctive high-altitude environment, marked by extreme cold and reduced oxygen levels, presents considerable survival challenges for both humans and mammals. Natural selection has led to the accumulation of adaptive mutations in Tibetan pigs, enabling them to develop distinctive adaptive phenotypes. Here, we aim to uncover the genetic mechanisms underlying the adaptation of Tibetan pigs to high-altitude hypoxia. Therefore, we conducted a systematic analysis of 140 whole-genome sequencing (WGS) data points from different representing pig populations. Our analysis identified a total of 27,614,561 mutations, including 22,386,319 single-nucleotide variants (SNVs) and 5,228,242 insertions/deletions (INDELs, size < 50 bp). A total of 11% (2,678,569) of the SNVs were newly identified in our project, significantly expanding the dataset of genetic variants in Tibetan pigs. Compared to other pig breeds, Tibetan pigs are uniquely adapted to high-altitude environments, exhibiting the highest genetic diversity and the lowest inbreeding coefficient. Employing the composite of multiple signals (CMS) method, we scanned the genome-wide Darwinian positive selection signals and identified 32,499 Tibetan pig positively selected SNVs (TBPSSs) and 129 selected genes (TBPSGs), including 213 newly discovered genes. Notably, we identified eight genes (PHACTR1, SFI1, EPM2A, SLC30A7, NKAIN2, TNNI3K, and PLIN2) with strong nature selection signals. They are likely to improve cardiorespiratory function and fat metabolism to help Tibetan pigs become adapted to the high-altitude environment. These findings provide new insights into the genetic mechanisms of high-altitude adaptation and the adaptive phenotypes of Tibetan pigs.

Study on the difference of gut microbiota in DLY and Diqing Tibetan pigs induce by high fiber diet.

In order to investigate the regularity of fecal microorganisms changes in Landrace × Large White × Duroc (DLY) and Diqing Tibetan pigs (TP) induced by dietary fiber, and further explore the buffering effect of different intestinal flora structures on dietary stress. DLY (n = 15) and TP (n = 15) were divided into two treatments. Then, diet with 20% neutral detergent fiber (NDF) was supplemented for 9 days. Our results showed that the feed conversion efficiency of TP was significantly higher (p < 0.05) than that of DLY. The fecal microorganisms shared by the two groups gradually increased with the feeding cycle. In addition, the dispersion of Shannon, Simpson, ACE and Chao of TP decreased. Also, we found that the fecal microorganisms of TP (R2 = 0.2089, p < 0.01) and DLY (R2 = 0.3982, p < 0.01) showed significant differences in different feeding cycles. With the prolongation of feeding cycle, the similarity of fecal microbial composition between DLY and TP increased. Our study strongly suggests that the complex environment and diet structure have shaped the unique gut microbiota of TP, which plays a vital role in the buffering effect of high-fiber diets.

Cryptosporidium infection induced the dropping of SCFAS and dysbiosis in intestinal microbiome of Tibetan pigs.

The infection of Cryptosporidium in pigs causes digestive system ailments, diarrhea and weight loss serving as an economic burden, especially in newborn animals. The bacterial fermentation products of short-chain fatty acids have important roles in immune function, microbiota regulation, osmotic balance and metabolism. However, till now little knowledge is available about the effect of Cryptosporidium infection on microbiota and SCFAs in plateau pigs. Hence, we performed this study to explore the response of microbiota and SCFAs in the natural infection of Cryptosporidium in Tibetan pigs. Cryptosporidium positive (infected, G) and negative samples (healthy, J) in our previous study were used for high throughputsequencing and Gas Chromatography-Mass Spectrometer analysis. Over 81 000 and 74 000 filtered sequences were detected in healthy and infected Tibetan pigs, respectively. Lower sample richness and evenness were observed in Cryptosporidium infection, as alpha diversity analysis found that chao1 (p < 0.05), faith_pd (p < 0.05), and observed_features in group G were significantly lower than pigs in group J. A total of 4 and 27 significant different phyla and genera were found between group G and J. The changed genera were Psychrobacter, Desemzia, Succiniclasticum, Treponema, Campylobacter, Atopobium, Olsenella, Pediococcus, Peptococcus, Sharpea, Desulfovibrio, Acinetobacter, Rhodococcus, Anaerostipes, Turicibacter, Lactobacillus, RFN20, Phascolarctobacterium, Roseburia, Megasphaera, Streptococcus, Blautia, Lachnospira, rc4_4, Gemmiger, Dorea, Oribacterium and Prevotella, which affected the microbiota functions with 360 abundance changed enzymes, and pathways in L1, L2 and L3 levels of KEGG. The concentration of acetic acid (p < 0.01), butyric acid (p < 0.05) and caproic acid (p < 0.01) were lower in group G. In conclusion, the present study herein uncovered that the host responses to Cryptosporidium infection in Tibetan pigs with 27 of significantly changed genera decreased SCFAs in pigs, which may provide insights in further developing novel therapy against this protozoan.

Divergence of Liver Lipidomes in Tibetan and Yorkshire Pigs Living at Different Altitudes.

The Tibetan pig is a characteristic breed of the Qinghai-Tibet Plateau with distinct physiological and meat quality attributes. The liver lipid profile can offer an important perspective to explore the uniqueness of Tibetan pigs. A quantitative comparison of liver lipidomes revealed significant differences in the lipid profiles between Tibetan and Yorkshire pigs raised at different altitudes. The abundance of lipids in the livers of pigs raised at a high altitude was higher than that of pigs raised at a lower altitude, whereas the abundance of lipids in the livers of Yorkshire pigs was higher than that of Tibetan pigs raised at the same altitude. Of the 1101 lipids identified, 323 and 193 differentially abundant lipids (DALs) were identified in the pairwise comparisons of Tibetan and Yorkshire pigs raised at different altitudes, respectively. The DALs of Tibetan pigs consisted mainly of 161 triglycerides, along with several acylcarnitines, represented by carnitine C2:0, and significant changes in the abundance of some phospholipids. The DALs of Yorkshire pigs were more complex, with significant increases in the abundance of triglycerides, cholesteryl esters, and free fatty acids, and decreases in the abundance of some phospholipids. This research provides strong theoretical and data support for the high-quality development of the highland livestock industry.

Complete genome sequencing and investigation on the fiber-degrading potential of Bacillus amyloliquefaciens strain TL106 from the tibetan pig.

BACKGROUND: Cellulolytic microorganisms are considered a key player in the degradation of feed fiber. These microorganisms can be isolated from various resources, such as animal gut, plant surfaces, soil and oceans. A new strain of Bacillus amyloliquefaciens, TL106, was isolated from faeces of a healthy Tibetan pigs. This strain can produce cellulase and shows strong antimicrobial activity in mice. Thus, in this study, to better understand the strain of B. amyloliquefaciens TL106 on degradation of cellulose, the genome of the strain TL106 was completely sequenced and analyzed. In addition, we also explored the cellulose degradation ability of strain TL106 in vitro. RESULTS: TL106 was completely sequenced with the third generation high-throughput DNA sequencing. In vitro analysis with enzymatic hydrolysis identified the activity of cellulose degradation. TL106 consisted of one circular chromosome with 3,980,960 bp and one plasmid with 16,916 bp, the genome total length was 3.99 Mb and total of 4,130 genes were predicted. Several genes of cellulases and hemicellulase were blasted in Genbank, including ß-glucosidase, endoglucanase, ß-glucanase and xylanase genes. Additionally, the activities of amylase (20.25 U/mL), cellulase (20.86 U/mL), xylanase (39.71 U/mL) and ß-glucanase (36.13 U/mL) in the fermentation supernatant of strain TL106 were higher. In the study of degradation characteristics, we found that strain TL106 had a better degradation effect on crude fiber, neutral detergent fiber, acid detergent fiber, starch, arabinoxylan and ß-glucan of wheat and highland barley . CONCLUSIONS: The genome of B. amyloliquefaciens TL106 contained several genes of cellulases and hemicellulases, can produce carbohydrate-active enzymes, amylase, cellulase, xylanase and ß-glucanase. The supernatant of fermented had activities of strain TL106. It could degrade the fiber fraction and non-starch polysaccharides (arabinoxylans and ß-glucan) of wheat and highland barley. The present study demonstrated that the degradation activity of TL106 to crude fiber which can potentially be applied as a feed additive to potentiate the digestion of plant feed by monogastric animals.

Molecular detection and phylogenetic analysis of porcine circovirus type 3 in Tibetan pigs on the Qinghai-Tibet Plateau of China.

BACKGROUND: Porcine circovirus type 3 (PCV3) has been confirmed to infect pigs, posing a health risk and making pigs more susceptible to other pathogens. After the first report of PCV3 infection in the United States, its prevalence was determined in pigs suffering from clinical digestive or respiratory diseases in several other regions, including the Sichuan and Gansu provinces of China. In this study, we describe the frequency of PCV3 detection in Tibetan pigs inhabiting three different provinces surrounding the Qinghai-Tibet Plateau of China. METHODS: A total of 316 samples from diarrheic animals and 182 samples from healthy animals were collected in a randomized manner. Conventional PCR was applied for PCV3 DNA detection. The conserved regions of the PCV3 gene were analyzed with MEGA 7.1 software to design specific primers to sequence entire Cap genes in PCV3 strains, and the sequences were then used to confirm the subtypes of PCV3 in the positive samples. Prediction of the amino acid sequences by nucleotide sequence translation was also performed to compare the point mutations in the entire Cap protein. Twenty PCV3 whole-genomic sequences were used for genome phylogenetic analyses of PCV3 and sequence alignments with 22 other reference strains. RESULTS: We found that the prevalence of the virus was significantly higher in samples from pigs with diarrhoea than that in samples from healthy pigs. Phylogenetic analysis of Cap proteins demonstrated that the 20 PCV3 strains formed three clades, including PCV3a (8/20, 40.00%), PCV3b (5/20, 25%) and PCV3c (7/20, 35.00%). The complete genome sequence revealed that these strains formed one branch in the phylogenetic tree. Sequence analysis showed that the Cap proteins of the 20 different viral strains shared between 95.84 and 99.18% nucleotide identity. Cap protein sequence analyses showed that the positivity rate of PCV3a was highest in the samples from pigs with diarrhoea. In comparison, PCV3c was the most elevated subtype in the healthy samples. There was no mutation at a specific site in the amino acid sequences of the entire Cap protein from different PCV3 subtype strains from heathy samples. There was a mutation at site 113 in PCV3a, site 129 in PCV3b, and site 116 in PCV3c. CONCLUSION: Our present data provide evidence that PCV3 is prevalent in Tibetan pigs at high altitudes in China, and the higher prevalence rates of the PCV3a and PCV3b subtypes in samples from pigs with diarrhoea further indicate that the genotypes should not be neglected during surveys of the pathogenicity of PCV3. Phylogenetic and genetic diversity analyses suggested that the continuous evolution, adaptation and mechanisms of pathogenicity of PCV3 in Tibetan pigs living in this special environment should be further studied.

Primary characterization of the immune responses in Tibetan pigs infected with Chinese Tibet isolate of Trichinella spiralis.

BACKGROUND: Trichinellosis, caused by Trichinella spiralis, is a serious foodborne parasitic zoonosis. Tibetan pig is an infrequent, endemic plateau pig species, mainly distributed in Tibet Plateau, China. Because of the free-range system, Tibetan pigs are at risk of infection with Trichinella. The present study aimed to primarily profile the characteristics of T. spiralis infection in Tibetan pigs, including IgG levels, larvae burdens, and cytokines. RESULTS: The immune responses to Chinese Tibet T. spiralis isolate infection in Tibetan pigs with different doses were investigated in a tracking duration of 49 days. The muscle larvae per gram (lpg) were evaluated at 105 days post-infection (dpi). The results showed that the mean larval number of T. spiralis in Tibetan pigs increased with infective dose, with average lpg values of 3.5, 50.4 and 115.6 for Tibetan pigs infected with 200, 2,000, and 20,000 muscle larvae (ML) of T. spiralis. The anti-Trichinella IgG increased with inoculum dose and dpi, and peaked at 49 dpi. The kinetics of cytokines in the sera was detected by microarray, including interferon-γ (IFN-γ), interleukin (IL)-1ß, IL-8, IL-12, IL-4, IL-6, IL-10, Granulocyte-macrophage Colony Stimulating Factor (GM-CSF), tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß1. The Th1/Th2 mixed cytokines were detectable in all samples. Interleukin-12 demonstrated the highest concentration compared to other cytokines and peaked at 42 dpi. Almost all cytokines were maintained at a high level at 42 dpi. Additionally, we also report a Trichinella seropositive rate of 43.9 % (18 out of 41) from field samples of Tibetan pigs. CONCLUSIONS: The present study showed an increased Th1/Th2 mixed cytokines in Tibetan pigs elicited by T. spiralis. The high seroprevalence of Trichinella infection in field samples of Tibetan pigs further raises serious concern for the prevention and control of trichinellosis in this host for public health safety.

Evaluating genetic diversity and identifying priority conservation for seven Tibetan pig populations in China based on the mtDNA D-loop.

OBJECTIVE: Tibetan pigs, an excellent species unique to China, face serious threats, which in turn affects the development and utilization of the outstanding advantages of plateau hypoxia adaptability and reduces their genetic diversity. Therefore, a discussion of measures to conserve this genetic resource is necessary. The method, based on genetic diversity, genetic divergence and total genetic contribution rate of population, reflects the priority conservation order and varies depending on the three different purposes of conservation. METHODS: We analyzed mitochondrial DNA control region (D-loop) variation in 1,201 individuals from nine Tibetan pig populations across five provinces and downloaded 564 mtDNA D-loop sequences from three indigenous pig breeds in Qinghai, Sichuan, and Yunnan Provinces distributed near the Tibetan pigs. RESULTS: We analyzed three different aspects: Changdu Tibetan pigs have the highest genetic diversity, and from the perspective of genetic diversity, the priority conservation is Changdu Tibetan pigs. Hezuo Tibetan pigs have the highest genetic contribution, so the priority conservation is Hezuo Tibetan pigs in the genetic contribution aspect. Rkaze Tibetan pigs were severely affected by indigenous pig breeds, so if considering from the perspective of introgression, the priority conservation is Rkaze Tibetan pigs. CONCLUSION: This study evaluated genetic diversity and comprehensively assessed conservation priority from three different aspects in nine Tibetan pig populations.

Porcine epidemic diarrhea: an emerging disease in Tibetan pigs in Tibet, China.

Porcine epidemic diarrhea virus (PEDV) is a devastating cause of diarrhea in pigs worldwide, and has caused enormous economic losses in the pork industry. To date, however, it is still unclear whether Tibetan pigs from the Tibetan Plateau of China are exposed to PED. This study was to investigate the seroprevalence of PED in Tibetan pigs in Tibet, China. A total of 364 serum samples were collected from Tibetan pigs in Nyingchi in 2014 and 2015. Each sample was assayed for PED antibodies by commercial enzyme-linked immunosorbent assay (ELISA) kits. Our results showed that the seroprevalence of PED in Tibetan pigs was 39.56% with the further distribution of 26.37% and 52.75% in Nyingchi and Mainling (odds ratio was more commonly affected in Mainling than Nyingchi), respectively. Moreover, the seroprevalence of PED was 65.28%, 35.83%, and 28.57% in juveniles, sub adults, and adults, respectively. The prevalence was found as 40.63% in males and 38.37% in females. The present survey indicated that the infection of PED in Tibetan pigs was widely spread in Tibet, which should arouse public concern of the threat to the health of Tibetan pigs and the development of breeding industry in this unique region of the world.

Seroprevalence and risk factors associated with Pseudorabies virus infection in Tibetan pigs in Tibet.

BACKGROUND: Pseudorabies (PR) is an important emerging infectious disease that is characterized by fever, extreme itching and encephalomyelitis. However, it is still unclear whether Tibetan pigs are exposed to Pseudorabies virus (PRV) or not. The present study was conducted to investigate the seroprevalence of PRV infection in Tibetan pigs in Nyingchi area of Tibet through enzyme-linked immunosorbent assay (ELISA). A total of 368 serum samples from Tibetan pigs were collected during 2015. RESULTS: Results showed that 58 (15.76%) samples were found positive for PRV antibodies with further distribution of 18.23%, 13.42% and 6.25% from Nyingchi, Mainling and Gongbo'gyamda areas on the Tibetan plateau, respectively; along with 12.10%, 17.71% and 17.57% prevalence of PRV in juveniles, sub-adults and adults, respectively. The prevalence of PRV infection between male (14.61%) and female (16.84%) showed non-significant difference (P > 0.05). The risk factors of infection were found to be associated with feed type, age and altitude. CONCLUSIONS: The present study depicts a serious concern with a new emerging infectious disease in Tibetan pigs in Tibet, China.

Prevalence of hepatitis E virus genotype 4 of probable human origin in Tibetan pigs from the Qinghai-Tibetan Plateau, China.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. In 2018-2022, we investigated the presence of HEV RNA in 1233 stool samples collected in the Qinghai-Tibetan Plateau, including humans (16), Tibetan pigs (624), yaks (312), sheep (267), and dogs (14). HEV RNA was only detected in Tibetan pig faecal samples (18.27%, 114/624). To perform molecular characterization of HEV strains in Tibetan pigs, we obtained 21 complete HEV genome sequences between 2018 and 2022. Sequence comparisons showed that 21 HEV strains from Tibetan pigs shared the mean nucleotide identities with the reference HEV strains ranging between 82.9% and 94.9% and 89.3% and 92.1% similarities with human HEV strains. Phylogenetic analysis confirmed that all HEV strains were genotype 4, closely related to human HEV strains. Sequence recombinant analysis showed five potential recombinant strains identified in this study, of which SWU/D18/2018 (GenBank No. MK410044) was recombinant with human and swine HEV strains, located 6509-6878 nt from the recombination point. Based on the Bayesian evolutionary trees, we found that most HEV strains diverged later than human HEV (16 Tibetan pig HEV strains diverged later than 1979, and seven human HEV strains diverged earlier than 1979). Therefore, we speculated that the prevalence of HEV 4 in Tibetan pigs possibly originated from humans in the Qinghai-Tibetan Plateau.

Genetic characterization of Tibetan pigs adapted to high altitude under natural selection based on a large whole-genome dataset.

The Qinghai-Tibet Plateau is a valuable genetic resource pool, and the high-altitude adaptation of Tibetan pigs is a classic example of the adaptive evolution of domestic animals. Here, we report the presence of Darwinian positive selection signatures in Tibetan pigs (TBPs) using 348 genome-wide datasets (127 whole-genome sequence datasets (WGSs) and 221 whole-genome single-nucleotide polymorphism (SNP) chip datasets). We characterized a high-confidence list of genetic signatures related response to high-altitude adaptation in Tibetan pigs, including 4,598 candidate SNPs and 131 candidate genes. Functional annotation and enrichment analysis revealed that 131 candidate genes are related to multiple systems and organs in Tibetan pigs. Notably, eight of the top ten novel genes, RALB, NBEA, LIFR, CLEC17A, PRIM2, CDH7, GK5 and FAM83B, were highlighted and associated with improved adaptive heart functions in Tibetan pigs high-altitude adaptation. Moreover, genome-wide association analysis revealed that 29 SNPs were involved in 13 candidate genes associated with at least one adaptive trait. In particular, among the top ten candidate genes, CLEC17A is related to a reduction in hemoglobin (HGB) in Tibetan pigs. Overall, our study provides a robust SNP/gene list involving genetic adaptation for Tibetan pig high-altitude adaptation, and it will be a valuable resource for future Tibetan pig studies.

The Role of the MYL4 Gene in Porcine Muscle Development and Its Molecular Regulatory Mechanisms.

Muscle growth stands as a pivotal economic trait within pig production, governed by a complex interplay of multiple genes, each playing a role in its quantitative manifestation. Understanding the intricate regulatory mechanisms of porcine muscle development is crucial for enhancing both pork yield and quality. This study used the GSE99749 dataset downloaded from the GEO database, conducting a detailed analysis of the RNA-seq results from the longissimus dorsi muscle (LD) of Tibetan pigs (TP), Wujin pigs (WJ) and large white pigs (LW) at 60 days of gestation, representing diverse body sizes and growth rates. Comparative analyses between TPvsWJ and TPvsLW, along with differential gene expression (DEG) analysis, functional enrichment analysis, and protein-protein interaction (PPI) network analysis, revealed 1048 and 1157 significantly differentially expressed genes (p < 0.001) in TPvsWJ and TPvsLW, respectively. With stricter screening criteria, 37 DEGs were found to overlap between the 2 groups. PPI analysis identified MYL5, MYL4, and ACTC1 as the three core genes. This article focuses on exploring the MYL4 gene. Molecular-level experimental validation, through overexpression and interference of the MYL4 gene combined with EDU staining experiments, demonstrated that overexpression of MYL4 significantly promoted the proliferation of porcine skeletal muscle satellite cells (PSMSC), while interference with MYL4 inhibited their proliferation. Furthermore, by examining the effects of overexpressing and interfering with the MYL4 gene on the muscle hypertrophy marker Fst gene and the muscle degradation marker FOXO3 gene, the pivotal role of the MYL4 gene in promoting muscle growth and preventing muscle degradation was further confirmed. These findings offer a new perspective on the molecular mechanisms behind porcine muscle growth and development, furnishing valuable data and insights for muscle biology research.

Exploring the role of the CapG gene in hypoxia adaptation in Tibetan pigs.

Introduction: The CapG gene, which is an actin-binding protein, is prevalent in eukaryotic cells and is abundantly present in various pathways associated with plateau hypoxia adaptation. Tibetan pigs, which have inhabited high altitudes for extended periods, provide an excellent research population for investigating plateau hypoxia adaptation. Results: This study focused on Tibetan pigs and Yorkshire pigs residing in Nyingchi, Tibet. The blood physiological data of Tibetan pigs were found to be significantly higher than those of Yorkshire pigs, including RBC, HGB, HCT, MCH, and MCHC. The SNP analysis of the CapG gene identified six sites with mutations only present in Tibetan pigs. Notably, the transcription factors at sites C-489T, C-274T, and A-212G were found to be altered, and these sites are known to be associated with hypoxia adaptation and blood oxygen transportation. The mRNA expression of the CapG gene exhibited highly significant differences in several tissues, with the target proteins predominantly higher in the Yorkshire pig compared to the Tibetan pig. Specifically, a notable difference was observed in the lung tissues. Immunohistochemistry analysis revealed high expression levels of CapG proteins in the lung tissues of both Tibetan and Yorkshire pigs, primarily localized in the cytoplasm and cell membrane. Conclusion: The CapG gene plays a significant role in regulating hypoxia adaptation in Tibetan pigs. This study provides a theoretical basis for the conservation and utilization of Tibetan pig resources, the breeding of highland breeds, epidemic prevention and control, and holds great importance for the development of the highland livestock economy.

Ssc-miR-101-3p inhibits hypoxia-induced apoptosis and inflammatory response in alveolar type-II epithelial cells of Tibetan pigs via targeting FOXO3.

Tibetan pigs are a unique swine strain adapted to the hypoxic environment of the plateau regions in China. The unique mechanisms underlying the adaption by Tibetan pigs, however, are still elusive. Only few studies have investigated hypoxia-associated molecular regulation in the lung tissues of animals living in the plateau region of China. Our previous study reported that ssc-miR-101-3p expression significantly differed in the lung tissues of Tibetan pigs at different altitudes, suggesting that ssc-miR-101-3p plays an important role in the adaptation of Tibetan pigs to high altitude. To understand the underlying molecular mechanism, in this study, the target genes of ssc-miR-101-3p and their functions were analyzed via various methods including qRT-PCR and GO and KEGG pathway enrichment analyses. The action of ssc-miR-101-3p was investigated by culturing alveolar type-II epithelial cells (ATII) of Tibetan pigs under hypoxic conditions and transfecting ATII cells with vectors overexpressing or inhibiting ssc-miR-101-3p. Overexpression of ssc-miR-101-3p significantly increased the proliferation of ATII cells and decreased the expression of inflammatory and apoptotic factors. The target genes of ssc-miR-101-3p were significantly enriched in FOXO and PI3K-AKT signaling pathways required to mitigate lung injury. Further, FOXO3 was identified as a direct target of ssc-miR-101-3p. Interestingly, ssc-miR-101-3p overexpression reversed the damaging effect of FOXO3 in the ATII cells. In conclusion, ssc-miR-101-3p targeting FOXO3 could inhibit hypoxia-induced apoptosis and inflammatory response in ATII cells of Tibetan pigs. These results provided new insights into the molecular mechanisms elucidating the response of lung tissues of Tibetan pigs to hypoxic stress.

Investigation of SNPs in differentially expressed genes and proteins reveals the genetic basis of skeletal muscle growth differences between Tibetan and Large White pigs.

Objective: Skeletal muscle growth is an important economic trait for meat production, with notable differences between Tibetan pigs (TIBPs, a slow-growing breed) and Large White pigs (LWPs, a fast-growing breed). However, the genetic underpinnings of this disparity remain unclear. Methods: In the current study, we integrated differentially expressed genes (DEGs) and proteins (DEPs) from 60-day-old embryonic muscle tissue, along with whole-genome single nucleotide polymorphisms (SNPs) displaying absolute allele frequency differences (ΔAF) of 0.5 or more between the TIBP and LWP breeds, to unravel the genetic factors influencing skeletal muscle growth. Results: Our analysis revealed 3499 DEGs and 628 DEPs with SNPs having a ΔAF equal to or greater than 0.5. Further functional analysis identified 145 DEGs and 23 DEPs involved in biological processes related to skeletal muscle development, and 22 DEGs and 3 DEPs implicated in the mTOR signaling pathway, which is known for positively regulating protein synthesis. Among these genes, several DEGs and DEPs, enriched with TIPB-specific SNPs in regulatory or/and coding regions, showed marked ΔAF between the TIBP and LWP breeds, including MYF5, MYOF, ASB2, PDE9A, SDC1, PDGFRA, MYOM2, ACVR1, ZIC3, COL11A1, TGFBR1, EDNRA, TGFB2, PDE4D, PGAM2, GRK2, SCN4B, CACNA1S, MYL4, IGF1, and FOXO1. Additionally, genes such as CAPN3, MYOM2, and PGAM2, identified as both DEPs and DEGs related to skeletal muscle development, contained multiple TIBP-specific and LWP-predominant SNPs in regulatory and/or coding regions, underscoring significant ΔAF differences between the two breeds. Conclusion: s: This comprehensive investigation of SNPs in DEGs and DEPs identified a significant number of SNPs and genes related to skeletal muscle development during the prenatal stage. These findings not only shed light on potential causal genes for muscle divergence between the TIBP and LWP breeds but also offer valuable insights for pig breeding strategies aimed at enhancing meat production.