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The Chinese Qingyu pig is a typical domestic fatty pig breed and an invaluable indigenous genetic resource in China. Compared with the Landrace pig, the Qingyu pig has unique meat characteristics, including muscle development, intramuscular fat, and other meat quality traits. At present, few studies have explored epigenetic differences due to DNA methylation between the Qingyu pig and the Landrace pig. In this study, 30 Qingyu pigs and 31 Landrace pigs were subjected to reduced representation bisulfite sequencing (RRBS). Genome-wide differential DNA methylation analysis was conducted. Six genomic regions, including regions on Sus scrofa chromosome (SSC) 1: 266.09–274.23 Mb, SSC5: 0.88–10.68 Mb, SSC8: 41.23–48.51 Mb, SSC12: 45.43–54.38 Mb, SSC13: 202.15–207.95 Mb, and SSC14: 126.43–139.85 Mb, were regarded as key regions that may be associated with phenotypic differences between the Qingyu pig and the Landrace pig. Furthermore, according to further analysis, five differentially methylated genes (ADCY1, FUBP3, GRIN2B, KIT, and PIK3R6) were identified as key candidate genes that might be associated with meat characteristics. Our findings provide new insights into the differences in DNA methylation between the Qingyu pig and the Landrace pig. These results enrich the epigenetic research of the Chinese Qingyu pig.

Animal fat deposition has a significant impact on meat flavor and texture. However, the molecular mechanisms of fat deposition are not well understood. LncPLAAT3-AS is a naturally occurring transcript that is abundant in porcine adipose tissue. Here, we focus on the regulatory role of lncPLAAT3-AS in promoting preadipocyte proliferation and adipocyte differentiation. By overexpressing or repressing lncPLAAT3 expression, we found that lncPLAAT3-AS promoted the transcription of its host gene PLAAT3, a regulator of adipocyte differentiation. In addition, we predicted the region of lncPLAAT3-AS that binds to miR-503-5p and showed by dual luciferase assay that lncPLAAT3-AS acts as a sponge to absorb miR-503-5p. Interestingly, miR-503-5p also targets and represses PLAAT3 expression and helps regulate porcine preadipocyte proliferation and differentiation. Taken together, these results show that lncPLAAT3-AS upregulates PLAAT3 expression by absorbing miR-503-5p, suggesting a potential regulatory mechanism based on competing endogenous RNAs. Finally, we explored lncPLAAT3-AS and PLAAT3 expression in adipose tissue and found that both molecules were expressed at significantly higher levels in fatty pig breeds compared to lean pig breeds. In summary, we identified the mechanism by which lncPLAAT3-AS regulates porcine preadipocyte proliferation and differentiation, contributing to our understanding of the molecular mechanisms of lipid deposition in pigs.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeepin situHi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omics data revealed the tissue-dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic rewiring of long-range promoter-enhancer contacts, which likely contribute to the dramatic phenotypic differences between the parental pig breeds. This study also provides definitive evidence of structured homolog pairing in the pig genome which could facilitate regulatory interactions between homologous chromosomes. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, and consequently phenotypic changes in mammals.

A comprehensive transcriptomic survey of pigs can provide a mechanistic understanding of tissue specialization processes underlying economically valuable traits and accelerate their use as a biomedical model. Here we characterize four transcript types (lncRNAs, TUCPs, miRNAs, and circRNAs) and protein-coding genes in 31 adult pig tissues and two cell lines. We uncover the transcriptomic variability among 47 skeletal muscles, and six adipose depots linked to their different origins, metabolism, cell composition, physical activity, and mitochondrial pathways. We perform comparative analysis of the transcriptomes of seven tissues from pigs and nine other vertebrates to reveal that evolutionary divergence in transcription potentially contributes to lineage-specific biology. Long-range promoter–enhancer interaction analysis in subcutaneous adipose tissues across species suggests evolutionarily stable transcription patterns likely attributable to redundant enhancers buffering gene expression patterns against perturbations, thereby conferring robustness during speciation. This study can facilitate adoption of the pig as a biomedical model for human biology and disease and uncovers the molecular bases of valuable traits., A comprehensive transcriptomic survey of the pig could enable mechanistic understanding of tissue specialization and accelerate its use as a biomedical model. Here the authors characterize four distinct transcript types in 31 adult pig tissues to dissect their distinct structural and transcriptional features and uncover transcriptomic variability related to tissue physiology.

Objective: We investigated the temporal expression profiles of long noncoding RNA (lncRNA) and mRNA in the peripheral blood of pigs during development and identified the lncRNAs that are related to the blood-based immune system.Methods: Peripheral blood samples were obtained from the pigs at 0, 7, 28, and 180 days and 2 years of age. RNA sequencing was performed to survey the lncRNA and mRNA transcriptomes in the samples. Short time-series expression miner (STEM) was used to show temporal expression patterns in the mRNAs and lncRNAs. Gene ontology and Kyoto encyclopedia of genes and genomes analyses were performed to assess the genes’ biological relevance. To predict the functions of the identified lncRNAs, we extracted mRNAs that were nearby loci and highly correlated with the lncRNAs.Results: In total of 5,946 lncRNA and 12,354 mRNA transcripts were identified among the samples. STEM showed that most lncRNAs and mRNAs had similar temporal expression patterns during development, indicating the expressional correlation and functional relatedness between them. The five stages were divided into two classes: the suckling period and the late developmental stage. Most genes were expressed at low level during the suckling period, but at higher level during the late stages. Expression of several T-cell-related genes increased continuously during the suckling period, indicating that these genes are crucial for establishing the adaptive immune system in piglets at this stage. Notably, lncRNA TCONS-00086451may promote blood-based immune system development by upregulating nuclear factor of activated T-cells cytoplasmic 2 expression.Conclusion: This study provides a catalog of porcine peripheral blood-related lncRNAs and mRNAs and reveals the characteristics and temporal expression profiles of these lncRNAs and mRNAs during peripheral blood development from the newborn to adult stages in pigs.

Feed occupies a significant proportion in the production cost of pigs, and the feed efficiency (FE) in pigs is of utmost economic importance. Hence, the objective of this study is to identify single-nucleotide polymorphisms (SNPs) and candidate genes associated with FE-related traits, including feed conversion ratio (FCR) and residual feed intake (RFI). A genome-wide association study was conducted for FCR and RFI in 169 Yorkshire pigs using whole-genome sequencing data. A total of 23 and 33 suggestive significant SNPs ( P < 1 × 10−6) were detected for FCR and RFI, respectively. However, none of SNPs achieved the genome-wide significance threshold ( P < 5 × 10−8). Importantly, three common SNPs (SSC7:7987268, SSC13:42350250, and SSC13:42551718) were associated with both FCR and RFI. Additionally, the NEDD9 gene related to FCR and RFI traits was overlapped. This study detected novel SNPs on SSC7 and SSC13 common for FCR and RFI. These results provide new insights into the genetic mechanisms and candidate genes of FE-related traits in pigs.

Feed efficiency (FE) is a typical social affected trait. However, the mechanisms involved are not fully elucidated. According to the rank of residual feed intake (RFI)’s social genetic effect (SGE), ten high and low pigs were selected, named LRI and HRI groups. A sampling of ileal chyme after slaughter. Liquid chromatography-mass spectrometry (LC-MS) and 16S rRNA were conducted to investigate the relationship between the gut microbiome or metabolites and the SGE of RFI. The results showed significant differences between HRI and LRI groups. Compared with the HRI group, Escherichia, Eubacterium, and Gemmiger were enriched in the LRI group (P Escherichia and Fusobacterium. Therefore, Escherichia and Fusobacterium may influence the SGE of RFI through amino acid metabolism, thereby affecting feed efficiency.

Liver development is a complex process that is regulated by a series of signaling pathways. Three-dimensional (3D) chromatin architecture plays an important role in transcriptional regulation; nonetheless, its dynamics and role in the rapid transition of core liver functions during development and obesity-induced metabolic stress remain largely unexplored. To investigate the dynamic chromatin architecture during liver development and under metabolic stress, we generated high-resolution maps of chromatin architecture for porcine livers across six major developmental stages (from embryonic day 38 to the adult stage) and under a high-fat diet-induced obesity. The characteristically loose chromatin architecture supports a highly plastic genome organization during early liver development, which fundamentally contributes to the rapid functional transitions in the liver after birth. We reveal the multi-scale reorganization of chromatin architecture and its influence on transcriptional regulation of critical signaling processes during liver development, and show its close association with transition in hepatic functions (i.e., from hematopoiesis in the fetus to metabolism and immunity after birth). The limited changes in chromatin structure help explain the observed metabolic adaptation to excessive energy intake in pigs. These results provide a global overview of chromatin architecture dynamics associated with the transition of physiological liver functions between prenatal development and postnatal maturation, and a foundational resource that allows for future in-depth functional characterization.

Background The Chinese Chenghua pig (CHP) is a typical Chinese domestic fatty pig breed with superior meat quality characteristics, while the Yorkshire pig (YP) has the characteristics of fast growth and a high rate of lean meat. Long term natural selection and artificial selection resulted in great phenotypic differences between the two breeds, including growth, development, production performance, meat quality, and coat color. However, genome-wide DNA methylation differences between CHP and YP remain unclear. Results DNA methylation data were generated for muscle tissues of CHP and YP using reduced representation bisulfite sequencing (RRBS). In this study, a total of 2,416,211 CpG sites were identified. Besides, the genome-wide DNA methylation analysis revealed 722 differentially methylated regions (DMRs) and 466 differentially methylated genes (DMGs) in pairwise CHP vs. YP comparison. Six key genomic regions (Sus scrofa chromosome (SSC)1:253.47–274.23 Mb, SSC6:148.71–169.49 Mb, SSC7:0.25–9.86 Mb, SSC12:43.06–61.49 Mb, SSC14:126.43–140.95 Mb, and SSC18:49.17–54.54 Mb) containing multiple DMRs were identified, and differences of methylation patterns in these regions may be related to phenotypic differences between CHP and YP. Based on the functional analysis of DMGs, 8 DMGs (ADCY1, AGBL4, EXOC2, FUBP3, PAPPA2, PIK3R1, MGMT and MYH8) were considered as important candidate genes associated with muscle development and meat quality traits in pigs. Conclusions This study explored the difference in meat quality between CHP and YP from the epigenetic point of view, which has important reference significance for the local pork industry and pork food processing.

Gene expression profiles of blood can reflect the physiopathologic status of the immune system. The dynamic microRNA (miRNA) expression profiles of peripheral blood from pigs at different developmental stages, and how differential expression of miRNAs might relate to immune system development, are unknown. In this study, peripheral blood samples taken at five developmental stages were used to construct 15 miRNA libraries (three biological replicates/stage): 0 days (newborn), 30 days (weaning), 60 days (weaned), and 180 and 360 days (puberty). We identified 295 known mature miRNAs. Hierarchical clustering of the miRNA expression profile showed significant differences between individuals at the neonatal and postnatal stages. Functional enrichment analysis revealed that miRNAs differentially expressed between pairwise comparisons of the developmental stages were over-represented in immune-related pathways such as toll-like receptor signaling. The time-course of expression of the over-representated miRNAs exhibited a pattern of steady decline over time, for both the complete miRNA compendium and immune-related miRNAs. We identified six marker miRNAs that were highly negatively correlated with chronologic age and enriched for genes involved in immune-related pathways. This study of a peripheral blood miRNA transcriptome offers insight into immune system development in swine and provides a resource for pig genome annotation.

Efforts have been made to characterize the high-altitude adaption in Tibetan pigs and identified vast of genes or genomic regions undergone natural selection. Nonetheless, information concerning gene expression and DNA methylation changes response to low-altitude acclimation in Tibetan pigs is long overdue. To explore the exceptional mechanisms of gene expression and DNA methylation that are induced by low altitude environments in Tibetan pigs, we performed a comparative transcriptomic and DNA methylation analysis of skeletal muscle between indigenous Tibetan pigs that reside in high altitude regions (~4000m) and their counterparts that migrated to the geographically neighboring low-altitude regions (~500m) for nearly ten generations. Many genes that related to hypoxia response (EGLN3 and FLT1) and energy metabolism (TFB2M) were differentially expressed, but without significant DNA methylation changes. We also found genes embedded in differentially methylated regions were mainly involved in 'Starch and sucrose metabolism', 'glucuronosyltransferase activity' processes. Specifically, our results showed increased SIN3A mRNA expression, with hypomethylation status of its promoter, in longissimus dorsi muscle of low-altitude Tibetan pig. Another gene, CACNG6, showed decreasing expression level with an elevated methylation in its intron 3. These results indicated DNA-methylation-mediated expression alterations in low-altitude acclimation. We envision that this study will serve as a valuable resource for mammal acclimation research and agricultural food industry.

Background Species living at high altitude are subject to strong selective pressures due to inhospitable environments (e.g., hypoxia, low temperature, high solar radiation, and lack of biological production), making these species valuable models for comparative analyses of local adaptation. Studies that have examined high-altitude adaptation have identified a vast array of rapidly evolving genes that characterize the dramatic phenotypic changes in high-altitude animals. However, how high-altitude environment shapes gene expression programs remains largely unknown. Findings We generated a total of 910 Gb of high-quality RNA-seq data for 180 samples derived from 6 tissues of 5 agriculturally important high-altitude vertebrates (Tibetan chicken, Tibetan pig, Tibetan sheep, Tibetan goat, and yak) and their cross-fertile relatives living in geographically neighboring low-altitude regions. Of these, ∼75% reads could be aligned to their respective reference genomes, and on average ∼60% of annotated protein coding genes in each organism showed FPKM expression values greater than 0.5. We observed a general concordance in topological relationships between the nucleotide alignments and gene expression–based trees. Tissue and species accounted for markedly more variance than altitude based on either the expression or the alternative splicing patterns. Cross-species clustering analyses showed a tissue-dominated pattern of gene expression and a species-dominated pattern for alternative splicing. We also identified numerous differentially expressed genes that could potentially be involved in phenotypic divergence shaped by high-altitude adaptation. Conclusions These data serve as a valuable resource for examining the convergence and divergence of gene expression changes between species as they adapt or acclimatize to high-altitude environments.

Increasing evidence suggests that micro (mi)RNAs play important roles in various biological process. To evaluate the roles of miRNA in the porcine liver, we investigated the dynamic profiles of microRNAomes using liver tissue from pigs during the embryonic period (embryonic day 90), weaning stage (postnatal day 30), and adult stage (7 years old). A total of 186 unique miRNAs were differentially expressed during liver development. We also identified that 17, 13, and 6 miRNAs were specifically abundant at embryonic day 90, postnatal day 30, and at 7 years, respectively. Besides regulating basic cellular roles in development, miRNAs expressed at the three developmental stages also participated in regulating “embryonic liver development,” “early hepatic growth and generating a functioning liver,” and “energy metabolic processes,” respectively. Our study indicates that miRNAs are extensively involved in liver development, and provides a valuable resource for the further elucidation of miRNA regulatory roles during liver development.

Uncovering genetic variation through resequencing is limited by the fact that only sequences with similarity to the reference genome are examined. Reference genomes are often incomplete and cannot represent the full range of genetic diversity as a result of geographical divergence and independent demographic events. To more comprehensively characterize genetic variation of pigs (Sus scrofa), we generated de novo assemblies of nine geographically and phenotypically representative pigs from Eurasia. By comparing them to the reference pig assembly, we uncovered a substantial number of novel SNPs and structural variants, as well as 137.02-Mb sequences harboring 1737 protein-coding genes that were absent in the reference assembly, revealing variants left by selection. Our results illustrate the power of whole-genome de novo sequencing relative to resequencing and provide valuable genetic resources that enable effective use of pigs in both agricultural production and biomedical research.

BackgroundCrossbreeding is effective for improving performance in poultry and livestock, which is mainly attributed to heterosis. For pork production, a classic three-way crossbreeding system of Duroc × (Landrace × Yorkshire) (DLY) is widely used to produce terminal crossbred pigs with stable and prominent performance. Nonetheless, studies on the transmission of genetic information and gene expression pattern of DLY have been limited.FindingsWe analyzed population-scale SNPs based on 30 individuals from these three purebreds and identified 529.93 K SNPs of breed-of-origin of alleles. We also applied whole-genome sequencing of ten individuals from a DLY pig family as well as transcriptome of four representative tissues (adipose, skeletal muscle, heart, and liver) for six DLY individuals. Based on above, we identified a large number of high-confidence ASE genes, among which four ASE genes (KMO,PLIN4,POPDC3andUGT1A6) were found to be shared over all DLY individuals.ConclusionWe suggest DLY is a more effective strategy of three-way crossbreeding among these three purebreds from genetic aspect. We suppose the numerous breed-of-origin of alleles have close association with improved performance of crossbred individuals. ASE may also play important roles on DLY three-way crossbreeding system. Our findings are valuable for understanding the transmission of genetic information and the gene expression in DLY three-way crossbreeding and may be used to guide breeding and production of pigs in the future.

Recent evidence suggests that testosterone deficiency can dramatically decrease the quality of sperm. MicroRNAs (miRNAs) are conserved mediators of post-transcriptional gene regulation in eukaryotes. However, the systemic regulation and function of miRNAs in sperm quality decline induced by testosterone deficiency has not been investigated. Here, we found that the sperm apoptosis was significantly enhanced and the sperm motility was dramatically decreased in hemicastrated pigs. We then used small RNA sequencing to detect miRNA profiles of sperm from pigs with prepubertal hemicastration (HC) and compared them with control libraries. We identified 16 differentially expressed (DE) miRNAs between the sperm of prepubertal HC and control (CT) pigs. Functional enrichment analysis indicated that the target genes of these DE miRNAs were mainly enriched in apoptosis-related pathways including the p53, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) pathways. Furthermore, gain- and loss-of-function analyses demonstrated potential anti-apoptotic effects of the DE miRNAs miR-26a-5p and let-7g-5p on sperm cells. The luciferase reporter assay confirmed that PTEN and PMAIP1 are targets of miR-26a-5p and let-7g-5p, respectively. Spearman’s correlation analysis revealed significantly positive correlations between the sperm and its corresponding seminal plasma exosomes regarding the miRNA expression levels. In conclusion, testosterone deficiency-induced changes in the miRNA components of seminal plasma exosomes secreted by the genital tract may partially elucidate sperm miRNAome alterations, which are further responsible for the decline of sperm motility.

Recent evidence suggests that testosterone deficiency can dramatically decrease the quality of sperm. MicroRNAs (miRNAs) are conserved mediators of post-transcriptional gene regulation in eukaryotes. However, the systemic regulation and function of miRNAs in sperm quality decline induced by testosterone deficiency has not been investigated. Here, we found that the sperm apoptosis was significantly enhanced and the sperm motility was dramatically decreased in hemicastrated pigs. We then used small RNA sequencing to detect miRNA profiles of sperm from pigs with prepubertal hemicastration (HC) and compared them with control libraries. We identified 16 differentially expressed (DE) miRNAs between the sperm of prepubertal HC and control (CT) pigs. Functional enrichment analysis indicated that the target genes of these DE miRNAs were mainly enriched in apoptosis-related pathways including the p53, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) pathways. Furthermore, gain- and loss-of-function analyses demonstrated potential anti-apoptotic effects of the DE miRNAs miR-26a-5p and let-7g-5p on sperm cells. The luciferase reporter assay confirmed that PTEN and PMAIP1 are targets of miR-26a-5p and let-7g-5p, respectively. Spearman’s correlation analysis revealed significantly positive correlations between the sperm and its corresponding seminal plasma exosomes regarding the miRNA expression levels. In conclusion, testosterone deficiency-induced changes in the miRNA components of seminal plasma exosomes secreted by the genital tract may partially elucidate sperm miRNAome alterations, which are further responsible for the decline of sperm motility.

The recently developed high-throughput chromatin conformation capture (Hi-C) technology enables us to explore the spatial architecture of genomes, which is increasingly considered an important regulator of gene expression. To investigate the changes in three-dimensional (3D) chromatin structure and its mediated gene expression during adipogenesis and myogenesis, we comprehensively mapped 3D chromatin organization for four cell types (3T3-L1 pre-adipocytes, 3T3-L1-D adipocytes, C2C12 myoblasts, and C2C12-D myotubes). We demonstrate that the dynamic spatial genome architecture affected gene expression during cell differentiation. A considerable proportion (~22%) of the mouse genome underwent compartment A/B rearrangement during adipogenic and myogenic differentiation, and most (~80%) upregulated marker genes exhibited an active chromatin state with B to A switch or stable A compartment. More than half (65.4%-73.2%) of the topologically associating domains (TADs) are dynamic. The newly formed TAD and intensified local interactions in the Fabp gene cluster indicated more precise structural regulation of the expression of pro-differentiation genes during adipogenesis. About half (32.39%-59.04%) of the differential chromatin interactions (DCIs) during differentiation are promoter interactions, although these DCIs only account for a small proportion of genome-wide interactions (~9.67% in adipogenesis and ~4.24% in myogenesis). These differential promoter interactions were enriched with promoter-enhancer interactions (PEIs), which were mediated by typical adipogenic and myogenic transcription factors. Differential promoter interactions also included more differentially expressed genes than nonpromoter interactions. Our results provide a global view of dynamic chromatin interactions during adipogenesis and myogenesis and are a resource for studying long-range chromatin interactions mediating the expression of pro-differentiation genes.

MiRNAs regulate the expression of target genes in diverse cellular processes and hence play important roles in different physiological processes, yet little is known about the stomach microRNAome (miRNAome) of the Tibetan pig. The objective of this experiment was to investigate differentially expressed stomach miRNAs participating in digestion. Firstly, we isolated total RNA by Trizol reagent from three Tibetan and three Yorkshire purebred pigs stomach samples at 90-day-old. Secondly, a comprehensive analysis of Tibetan and Yorkshire pig stomach miRNAomes was performed by small RNA sequencing in the Illumina HiSeq 2000 system. Finally, SYBR Green Real-time RT-PCR was performed to validate the differentially expressed miRNAs. We identified 318 unique miRNAs, 260 were co-expressed in both libraries, 17 and 31 miRNAs were specifically expressed in Tibetan and Yorkshire pigs respectively. Fifty six differentially expressed miRNAs were identified by the identifying differentially expressed genes 6 (IDEG6). Kyoto encyclopedia of genes and genomes analysis revealed that some of the differentially expressed miRNAs were associated with protein and fat digestion. Two differentially expressed miRNAs (miR-214-3p and ssc-un39) participating in the digestion of lipid were identified. Additionally, qRT-PCR results suggested that a higher expression of miR-214-3p in the Tibetan pig stomach could lead to relatively lower expression of calcium-dependent phospholipase A2, which is an enzyme important for the digestion of glycerol phospholipid. This study has delineated the different stomach miRNAs expression patterns of Tibetan and Yorkshire pigs, which would help explain the regulatory mechanisms of miRNAs in digestion of Tibetan pigs, and contribute to utilize a the unique digestion merits of Tibetan pig in future porcine hybridization breeding.

IL-15 cDNA of Tibetan pig was firstly cloned from its activated lymphocytes, and then was sub-cloned into VR1020 to construct recombinant VRTIL-15 plasmid to study the in vitro and in vivo biological effects on animal. The VRTIL-15 was entrapped with chitosan modified with PEG-PEI (CS-PEG-PEI) to transfect HEK293 cells for the preliminary study of its expression in eukaryotic cells. The total RNA of HEK293 cell was isolated in 48h, and the successful expression of IL-15 was detected by RT-PCR and the supernatant of HEK293 cells was found to stimulate significant proliferation of lymphoblasts of pig. Subsequently, VRTIL-15 packed with CS-PEG-PEI was utilized to intramuscularly inoculate Kunming female mice at the age of 21 days. Their bloods were collected before and after inoculation on 1, 2, 3, 4 and 5 weeks to detect the changes of innate and adaptive immunity of animals. The results were found that Th and Tc, specific antibody to FMD, IgG, IgG1, IgG2a content markedly increased in the blood of treated mice compared with the control group (P

Growth performance and meat quality are important traits for the pig industry and consumers. Adipose tissue is the main site at which fat storage and fatty acid synthesis occur. Therefore, we combined high-throughput transcriptomic sequencing in adipose and muscle tissues with the quantification of corresponding phenotypic features using seven Chinese indigenous pig breeds and one Western commercial breed (Yorkshire). We obtained data on 101 phenotypic traits, from which principal component analysis distinguished two groups: one associated with the Chinese breeds and one with Yorkshire. The numbers of differentially expressed genes between all Chinese breeds and Yorkshire were shown to be 673 and 1056 in adipose and muscle tissues, respectively. Functional enrichment analysis revealed that these genes are associated with biological functions and canonical pathways related to oxidoreductase activity, immune response, and metabolic process. Weighted gene coexpression network analysis found more coexpression modules significantly correlated with the measured phenotypic traits in adipose than in muscle, indicating that adipose regulates meat and carcass quality. Using the combination of differential expression, QTL information, gene significance, and module hub genes, we identified a large number of candidate genes potentially related to economically important traits in pig, which should help us improve meat production and quality.

We report the sequencing at 131× coverage, de novo assembly and analyses of the genome of a female Tibetan wild boar. We also resequenced the whole genomes of 30 Tibetan wild boars from six major distributed locations and 18 geographically related pigs in China. We characterized genetic diversity, population structure and patterns of evolution. We searched for genomic regions under selection, which includes genes that are involved in hypoxia, olfaction, energy metabolism and drug response. Comparing the genome of Tibetan wild boar with those of neighboring Chinese domestic pigs further showed the impact of thousands of years of artificial selection and different signatures of selection in wild boar and domestic pig. We also report genetic adaptations in Tibetan wild boar that are associated with high altitudes and characterize the genetic basis of increased salivation in domestic pig.

Determination of an optimal set/number of internal control microRNA (miRNA) genes is a critical, but often undervalued, detail of quantitative gene expression analysis. No validated internal genes for miRNA quantitative PCR (q-PCR) in pig milk were available. We compared the expression stability of six porcine internal control miRNA genes in pig milk from different lactation periods (1 h, 3 days, 7 days, 14 days, 21 days, and 28 days postpartum), using an EvaGreen q-PCR approach. We found that using the three most stable internal control genes to calculate the normalization factor is sufficient for producing reliable q-PCR expression data. We also found that miRNAs are superior to ribosomal RNA (rRNA) and snRNA, which are commonly used as internal controls for normalizing miRNA q-PCR data. In terms of economic and experimental feasibility, we recommend the use of the three most stable internal control miRNA genes (miR-17, -107 and -103) for calculating the normalization factors for pig milk samples from different lactation periods. These results can be applied to future studies aimed at measuring miRNA abundance in porcine milk.

Determination of an optimal number/set of endogenous control (EC) microRNA (miRNA) genes is a critical but often an underappreciated aspect of quantitative gene expression analysis. In this study, the expression stabilities of 13 selected porcine EC miRNA genes were compared in all 47 tissue-specific normal tissues, 10 types of adipose tissue, and four types of muscle tissue using an EvaGreen quantitative PCR approach. Seven, 12, and 11 genes exhibited credible stability in the three groups, respectively. Our analysis clearly showed that three optimal EC genes are adequate for an accurate normalization, which correlated well with the theoretical optimal number (r ≥ 0.841). In terms of economical and experimental feasibility, we recommend the use of the three most stable EC miRNA genes for calculating the normalization factor, that is, ssc-miR-17, -103 and -107 for all 47 different tissues. We also suggest that two sets of EC miRNA genes are appropriate for 10 types of adipose tissue (ssc-miR-17, -107 and -24) and four types of muscle tissue (ssc-miR-17, -23a and -103), respectively. We envision that these results will serve as a valuable reference for other studies aimed at measuring tissue-specific miRNA abundance in porcine samples.

To normalize a set of quantitative real-time PCR (q-PCR) data, it is essential to determine an optimal number/set of housekeeping genes, as the abundance of housekeeping genes can vary across tissues or cells during different developmental stages, or even under certain environmental conditions. In this study, of the 20 commonly used endogenous control genes, 13, 18 and 17 genes exhibited credible stability in 56 different tissues, 10 types of adipose tissue and five types of muscle tissue, respectively. Our analysis clearly showed that three optimal housekeeping genes are adequate for an accurate normalization, which correlated well with the theoretical optimal number (r ≥ 0.94). In terms of economical and experimental feasibility, we recommend the use of the three most stable housekeeping genes for calculating the normalization factor. Based on our results, the three most stable housekeeping genes in all analysed samples (TOP2B, HSPCB and YWHAZ) are recommended for accurate normalization of q-PCR data. We also suggest that two different sets of housekeeping genes are appropriate for 10 types of adipose tissue (the HSPCB, ALDOA and GAPDH genes) and five types of muscle tissue (the TOP2B, HSPCB and YWHAZ genes), respectively. Our report will serve as a valuable reference for other studies aimed at measuring tissue-specific mRNA abundance in porcine samples.

The giant panda (Ailuropoda melanoleuca) is one of the world's most endangered mammals, and it has evolved several unusual biological and behavioral traits. During puberty, pregnancy, lactation, and involution, the mammary gland undergoes profound morphological and functional changes. A large number of microRNAs (miRNAs) have been identified to be involved in mammary gland development and lactation. In this study, we identified 202 conserved mature miRNAs, corresponding to 147 pre-miRNAs, in giant panda peripheral blood using a small RNA-sequencing approach. In addition, 27 miRNA families and 29 miRNA clusters were identified. We analyzed the arm selection preference of pre-miRNAs and found that: 1) most giant panda pre-miRNAs generated one-strand miRNAs, and the 5p-arm only miRNAs have a higher expression level than 3p-arm only miRNAs; 2) there were more 5p-arm dominant miRNAs than 3p-arm dominant miRNAs; and 3) 5p-arm dominant miRNAs have a larger fold change within miRNA pairs than 3p-arm dominant miRNAs. Expression of 12 lactation-related miRNAs was detected across late pregnancy and early lactation stages by qPCR, and seven miRNAs were identified as clustered in one significant model. Most of these clustered miRNAs exhibited inhibitory roles in proliferation and differentiation of mammary epithelial cells. Functional analysis highlighted important roles of the seven as signed miRNAs in mammary development and metabolic changes, including blood vessel morphogenesis, macromolecule biosynthesis, cell cycle regulation, and protein transport.

MicroRNAs (miRNAs) play an important role in the modulation of various metabolic processes in the liver, yet little is known about the liver microRNAome (miRNAome) of the Tibetan pig. Here we used the Yorkshire pig as a control to analyze the Tibetan pig-specific liver miRNAome, and for preliminary investigation of differentially expressed miRNAs participating in metabolism. A comprehensive analysis of Tibetan and Yorkshire pig liver miRNAomes by small RNA sequencing identified 362 unique miRNAs. Among these, 304 were co-expressed in both libraries, and 10 and 48 miRNAs were specifically expressed. Differential expression analysis of miRNAs, miRNA target prediction and KEGG analysis revealed that differentially expressed miRNAs were associated mainly with the metabolism of glucose, lipid and protein. Six differentially expressed miRNAs (miR-34a, miR-326, miR-1, miR-335, miR-185 and miR-378) participating in the metabolism of glucose and lipid were identified. Additionally, qPCR results revealed that a lower expression of miR-34a in Tibetan pig liver may promote gluconeogenesis by increasing the expression of Sirtuin type 1 (Sirt1); a lower expression of miR-1 in Tibetan pig liver may promote the synthesis and accumulation of lipid by increasing the expression of Liver X receptor α (LXRα); and a lower expression of miR-185 in Tibetan pig liver may promote the uptake of cholesterol from blood and secretion of bile by increasing the expression of the scavenger receptor class B type I (SR-BI). Our results provide new information and understanding of porcine miRNA profiles, which may help explain the regulatory mechanisms of miRNAs in the metabolic functions of Tibetan pig liver, and provide new biomarkers to assist in the development of Tibetan pig breeding characteristics.

Domesticated organisms have experienced strong selective pressures directed at genes or genomic regions controlling traits of biological, agricultural or medical importance. The genome of native and domesticated pigs provide a unique opportunity for tracing the history of domestication and identifying signatures of artificial selection. Here we used whole-genome sequencing to explore the genetic relationships among the European native pig Berkshire and breeds that are distributed worldwide, and to identify genomic footprints left by selection during the domestication of Berkshire. Numerous nonsynonymous SNPs-containing genes fall into olfactory-related categories, which are part of a rapidly evolving superfamily in the mammalian genome. Phylogenetic analyses revealed a deep phylogenetic split between European and Asian pigs rather than between domestic and wild pigs. Admixture analysis exhibited higher portion of Chinese genetic material for the Berkshire pigs, which is consistent with the historical record regarding its origin. Selective sweep analyses revealed strong signatures of selection affecting genomic regions that harbor genes underlying economic traits such as disease resistance, pork yield, fertility, tameness and body length. These discoveries confirmed the history of origin of Berkshire pig by genome-wide analysis and illustrate how domestication has shaped the patterns of genetic variation.