Haplotype-resolved 3D chromatin architecture of the hybrid pig.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-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-omic 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 differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.

Genetic parameter estimation for reproductive traits in QingYu pigs and comparison of carcass and meat quality traits to Berkshire×QingYu crossbred pigs.

OBJECTIVE: The QingYu pig is well known for its excellent meat quality attributes in Sichuan province, China. In order to improve its production efficiency, the determination of genetic factors contributing to quantifiable economic traits of livestock is important. Moreover, the cross-breeding of QingYu pigs with western breeds possessing strong growth attributes is an efficient way to improve the performance of this breed. METHODS: Here, the genetic parameters of several important reproductive traits of QingYu pigs were estimated, include total number born (TNB), number born alive, litter birth weight, individual birth weight, number of piglets weaned, litter weaning weight, and individual weaning weight. The data was analyzed using the ASReml 3.0 software (NSW Inc., Sydney, Australia). Furthermore, the effects of crossing Berkshire with QingYu (BQ) pigs on carcass and meat quality traits, as well as the effects of slaughter weight on carcass and meat quality of BQ were characterized. RESULTS: QingYu pigs exhibited superior reproductive traits. The TNB available to QingYu pigs was more than 8 per parity. The observed repeatability of the reproductive traits of the QingYu pigs was between 0.10 and 0.23. The significantly correlated genetic and phenotypic of reproduction traits were consistent. Interestingly, the BQ pigs exhibited improved carcass quality, with a significant increase in loin muscle area, lean percentage and reduction in sebum percentage. As a result, BQ had higher L45min, lower cooking scores, and lower drip loss. In addition, the loin muscle area, body length, and sebum percentage were significantly higher in 90 and 100 kg animals. Cooking loss showed a significant increase at 80 kg, and marbling increased significantly from 90 kg. CONCLUSION: The results of this study suggest that QingYu pigs exhibit excellent reproductive properties and heritability of these traits. Crossing with Berkshire is an efficient strategy to improve the carcass and meat quality of QingYu pigs for commercial operations. Furthermore, it appears as though the optimal slaughter weight of BQ pigs is at approximately 90 kg.

The Pro-angiogenesis Of Exosomes Derived From Umbilical Cord Blood Of Intrauterine Growth Restriction Pigs Was Repressed Associated With MiRNAs.

Dysfunctional umbilical cord blood (UCB) is a key factor for the development of intrauterine growth restriction (IUGR) in utero. Poor degrees of angiogenesis were observed during IUGR development. Here, it was demonstrated that NV-EXO (normal piglet's Umbilical Veins derived exosomes) promoted angiogenesis within the subdued pro-angiogenesis context of IV-EXO (IUGR piglet's Umbilical Veins derived exosomes). Investigation of the miRNA transcriptome of umbilical cord vein and artery exosomes between IUGR and normal littermates showed significant differences between umbilical veins from normal (NV) and IUGR (IV) piglets. Similar patterns were observed in normal (NA) and IUGR (IA) umbilical arteries as well. Moreover, the miRNAs expession level was more stable in NV. Further analysis revealed that miRNAs related to angiogenesis exhibited aberrant expression in IUGR pigs. The miRNA expression patterns between IUGR and normal piglets showed great difference. Expression of miR-150 in the tissues and UCB exosomes of IUGR pigs was significantly decreased. Up-regulation of miR-150 was able to increase proliferation, migration and tube formation of Human umbilical vein endothelial cells (HUVECs), suggesting a pro-angiogenic role. Furthermore, the data demonstrated that UCB derived miRNAs participate in fetal epigenetic regulation during pregnancy, suggesting a novel possible explanation for abnormal embryologic vascular development and several congenital cardiovascular diseases.

The regulation of skeletal muscle fiber-type composition by betaine is associated with NFATc1/MyoD.

Increasing evidence indicates that muscular dysfunction or alterations in skeletal muscle fiber-type composition not only are involved in muscle metabolism and function but also can limit functional capacity. Therefore, understanding the mechanisms regulating key events during skeletal myogenesis is necessary. Betaine is a naturally occurring component of commonly eaten foods. Here, we showed that 10 mM betaine supplementation in vitro significantly repressed myoblast proliferation and enhanced myoblast differentiation. This effect can be mediated by regulation of miR-29b-3p. Further analysis showed that betaine supplementation in vitro regulated skeletal muscle fiber-type composition through the induction of NFATc1 and the negative regulation of MyoD expression. Furthermore, mice fed with 10 mM betaine in water for 133 days showed no impairment in overall health. Consistently, betaine supplementation increased muscle mass, promoted muscle formation, and modulated the ratio of fiber types in skeletal muscle in vivo. These findings shed light on the diverse biological functions of betaine and indicate that betaine supplementation may lead to new therapies for diseases such as muscular dystrophy or other diseases related to muscle dysfunction. KEY MESSAGES: Betaine supplementation inhibits proliferation and promotes differentiation of C2C12 myoblasts. Betaine supplementation regulates fast to slow muscle fiber-type conversion and is associated with NFATc1/MyoD. Betaine supplementation enhances skeletal myogenesis in vivo. Betaine supplementation does not impair health of mice.

[Regulatory mechanism for lncRNAs in skeletal muscle development and progress on its research in domestic animals].

Skeletal muscle is an essential tissue to maintain the normal functions of an organism. It is also closely associated with important economic performance, such as carcass weight, of domestic animals. In recent years, studies using high-throughput sequencing techniques have identified numerous long non-coding RNAs (lncRNAs) with myogenic functions involved in regulation of gene expression at multiple levels, including epigenetic, transcriptional and post-transcriptional regulation. These lncRNAs target myogenic factors, which participate in all processes of skeletal muscle development, including proliferation, migration and differentiation of skeletal muscle stem cells, proliferation, differentiation and fusion of myocytes, muscle hypertrophy and conversion of muscle fiber types. In this review, we summarize the functional roles of lncRNAs in regulation of myogenesis in humans and mice, describe the methods for the analysis of lncRNA function, discuss the progress of lncRNA research in domestic animals, and highlight the current problems and challenges in lncRNA research on livestock production. We hope to provide a useful reference for research on lncRNA in domestic animals, thereby further identifying the molecular regulatory mechanisms in skeletal muscle growth and development.

Betaine Supplementation Enhances Lipid Metabolism and Improves Insulin Resistance in Mice Fed a High-Fat Diet.

Obesity is a major driver of metabolic diseases such as nonalcoholic fatty liver disease, certain cancers, and insulin resistance. However, there are no effective drugs to treat obesity. Betaine is a nontoxic, chemically stable and naturally occurring molecule. This study shows that dietary betaine supplementation significantly inhibits the white fat production in a high-fat diet (HFD)-induced obese mice. This might be due to betaine preventing the formation of new white fat (WAT), and guiding the original WAT to burn through stimulated mitochondrial biogenesis and promoting browning of WAT. Furthermore, dietary betaine supplementation decreases intramyocellular lipid accumulation in HFD-induced obese mice. Further analysis shows that betaine supplementation reduced intramyocellular lipid accumulation might be associated with increasing polyunsaturated fatty acids (PUFA), fatty acid oxidation, and the inhibition of fatty acid synthesis in muscle. Notably, by performing insulin-tolerance tests (ITTs) and glucose-tolerance tests (GTTs), dietary betaine supplementation could be observed for improvement of obesity and non-obesity induced insulin resistance. Together, these findings could suggest that inhibiting WAT production, intramyocellular lipid accumulation and inflammation, betaine supplementation limits HFD-induced obesity and improves insulin resistance.

Analysis of mitochondrial DNA sequence and copy number variation across five high-altitude species and their low-altitude relatives.

High-altitude inhospitable environments impose a formidable life challenge for the local animals. Training and exposure to high-altitude environments produce both distinct physiological and phenotypic characteristics. The mitochondrion, an organelle crucial for the energy production, plays an important role in hypoxia adaptation. In this study, we investigated the mitochondrial DNA (mtDNA) polymorphism and copy number variation between the population pairs from distinct altitudes across the multi-species. Higher mitochondrial DNA control region's genetic diversity is conspicuous in high-altitude animals versus low-altitude relatives. We also found an accordant decrease of mtDNA copy number in most of the tissues from high-altitude animals. Compared to mammals, chickens have significantly distinct mitogenomic characteristics, and more significant changes in the skeletal muscle mtDNA copy number between high- and low-altitude individuals. Our study catches a snapshot of the biological similarities and differences in the mitochondrial high-altitude acclimation across the species.

Comparative transcriptomics of 5 high-altitude vertebrates and their low-altitude relatives.

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.

Dynamic gene expression profiles during postnatal development of porcine subcutaneous adipose.

A better understanding of the control of lipogenesis is of critical importance for both human and animal physiology. This requires a better knowledge of the changes of gene expression during the process of adipose tissue development. Thus, the objective of the current study was to determine the effects of development on subcutaneous adipose tissue gene expression in growing and adult pigs. Here, we present a comprehensive investigation of mRNA transcriptomes in porcine subcutaneous adipose tissue across four developmental stages using digital gene expression profiling. We identified 3,274 differential expressed genes associated with oxidative stress, immune processes, apoptosis, energy metabolism, insulin stimulus, cell cycle, angiogenesis and translation. A set of universally abundant genes (ATP8, COX2, COX3, ND1, ND2, SCD and TUBA1B) was found across all four developmental stages. This set of genes may play important roles in lipogenesis and development. We also identified development-related gene expression patterns that are linked to the different adipose phenotypes. We showed that genes enriched in significantly up-regulated profiles were associated with phosphorylation and angiogenesis. In contrast, genes enriched in significantly down-regulated profiles were related to cell cycle and cytoskeleton organization, suggesting an important role for these biological processes in adipose growth and development. These results provide a resource for studying adipose development and promote the pig as a model organism for researching the development of human obesity, as well as being used in the pig industry.

Deciphering the microRNA transcriptome of skeletal muscle during porcine development.

MicroRNAs (miRNAs) play critical roles in many important biological processes, such as growth and development in mammals. Various studies of porcine muscle development have mainly focused on identifying miRNAs that are important for fetal and adult muscle development; however, little is known about the role of miRNAs in middle-aged muscle development. Here, we present a comprehensive investigation of miRNA transcriptomes across five porcine muscle development stages, including one prenatal and four postnatal stages. We identified 404 known porcine miRNAs, 118 novel miRNAs, and 101 miRNAs that are conserved in other mammals. A set of universally abundant miRNAs was found across the distinct muscle development stages. This set of miRNAs may play important housekeeping roles that are involved in myogenesis. A short time-series expression miner analysis indicated significant variations in miRNA expression across distinct muscle development stages. We also found enhanced differentiation- and morphogenesis-related miRNA levels in the embryonic stage; conversely, apoptosis-related miRNA levels increased relatively later in muscle development. These results provide integral insight into miRNA function throughout pig muscle development stages. Our findings will promote further development of the pig as a model organism for human age-related muscle disease research.

Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs.

BACKGROUND: Age-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach. RESULTS: We observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age. CONCLUSIONS: This study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research.

Whole-genome sequencing of Berkshire (European native pig) provides insights into its origin and domestication.

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.

Intrinsic features in microRNA transcriptomes link porcine visceral rather than subcutaneous adipose tissues to metabolic risk.

MicroRNAs (miRNAs) are non-coding small RNA ∼22 nucleotides in length that can regulate the expression of a wide range of coding genes at the post-transcriptional level. Visceral adipose tissues (VATs) and subcutaneous adipose tissues (SATs), the two main fat compartments in mammals, are anatomically, physiologically, metabolically, and clinically distinct. Various studies of adipose tissues have focused mainly on DNA methylation, and mRNA and protein expression, nonetheless little research sheds directly light on the miRNA transcriptome differences between these two distinct adipose tissue types. Here, we present a comprehensive investigation of miRNA transcriptomes across six variant porcine adipose tissues by small RNA-sequencing. We identified 219 known porcine miRNAs, 97 novel miRNA*s, and 124 miRNAs that are conserved to other mammals. A set of universally abundant miRNAs (i.e., miR-148a-3p, miR-143-3p, miR-27b-3p, miR-let-7a-1-5p, and miR-let-7f-5p) across the distinct adipose tissues was found. This set of miRNAs may play important housekeeping roles that are involved in adipogenesis. Clustering analysis indicated significant variations in miRNA expression between the VATs and SATs, and highlighted the role of the greater omentum in responding to potential metabolic risk because of the observed enrichment in this tissue of the immune- and inflammation-related miRNAs, such as the members of miR-17-92 cluster and miR-181 family. Differential expression of the miRNAs between the VATs and SATs, and miRNA target prediction analysis revealed that the VATs-specific enriched miRNAs were associated mainly with immune and inflammation responses. In summary, the differences of miRNA expression between the VATs and SATs revealed some of their intrinsic differences and indicated that the VATs might be closely associated with increased risk of metabolic disorders.

Mitochondrial DNA evidence indicates the local origin of domestic pigs in the upstream region of the Yangtze River.

Previous studies have indicated two main domestic pig dispersal routes in East Asia: one is from the Mekong region, through the upstream region of the Yangtze River (URYZ) to the middle and upstream regions of the Yellow River, the other is from the middle and downstream regions of the Yangtze River to the downstream region of the Yellow River, and then to northeast China. The URYZ was regarded as a passageway of the former dispersal route; however, this assumption remains to be further investigated. We therefore analyzed the hypervariable segements of mitochondrial DNA from 513 individual pigs mainly from Sichuan and the Tibet highlands and 1,394 publicly available sequences from domestic pigs and wild boars across Asia. From the phylogenetic tree, most of the samples fell into a mixed group that was difficult to distinguish by breed or geography. The total network analysis showed that the URYZ pigs possessed a dominant position in haplogroup A and domestic pigs shared the same core haplotype with the local wild boars, suggesting that pigs in group A were most likely derived from the URYZ pool. In addition, a region-wise network analysis determined that URYZ contains 42 haplotypes of which 22 are unique indicating the high diversity in this region. In conclusion, our findings confirmed that pigs from the URYZ were domesticated in situ.

Identification of suitable endogenous control microRNA genes in normal pig tissues.

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.

Repertoire of porcine microRNAs in adult ovary and testis by deep sequencing.

BACKGROUND: MicroRNAs (miRNAs), a large family of short endogenous RNAs known to post-transcriptionally repress gene expression, participate in the regulation of almost every cellular process. Changes in miRNA expression are associated with many pathologies. Ovarian folliculogenesis and testicular spermatogenesis are complex and coordinated biological processes, in which tightly regulated expression and interaction of a multitude of genes could be regulated by these miRNAs. Identification and preliminary characterization of gonad-specific miRNAs would be a prerequisite for a thorough understanding of the role that miRNA-mediated posttranscriptional gene regulation plays in mammalian reproduction. METHOD: Here, we present the identification of a repertoire of porcine miRNAs in adult ovary and testis using deep sequencing technology. A bioinformatics pipeline was developed to distinguish authentic mature miRNA sequences from other classes of small RNAs represented in the sequencing data. RESULTS: Using this approach, we detected 582 precursor hairpins (pre-miRNAs) encoding for 732 mature miRNAs, of which 673 are unique. Statistically, 224 unique miRNAs (out of 673, 33.28%) were identified which had significant differential expression (DE) between ovary and testis libraries (P < 0.001). Most of DE miRNAs located on the X chromosome (X-linked miRNAs) (24 out of 34, 70.59%) significantly up-regulated in ovary versus testis (P < 0.001). Predictably, X-linked miRNAs are expressed in a testis-preferential or testis-specific pattern. To explore the potential for co-expression among genomic location clusters of X-linked miRNAs, we surveyed the relationship between the distance separating miRNA loci and the coordinate expression patterns of 32 high confidence X-linked miRNAs in seven normal pig tissues using the real-time quantitative PCR (q-PCR) approach. Our results show that proximal pairs of miRNAs are generally co-expressed implying that miRNAs within 50 kb of genomic bases are typically derived from a common transcript. CONCLUSIONS: The present study characterizes the miRNA transcriptome of adult porcine gonads, with an emphasis on the co-expression patterns of X-linked miRNAs. Our report should facilitate studies of the organ-specific reproductive roles of miRNAs.

MicroRNAome of porcine pre- and postnatal development.

The domestic pig is of enormous agricultural significance and valuable models for many human diseases. Information concerning the pig microRNAome (miRNAome) has been long overdue and elucidation of this information will permit an atlas of microRNA (miRNA) regulation functions and networks to be constructed. Here we performed a comprehensive search for porcine miRNAs on ten small RNA sequencing libraries prepared from a mixture of tissues obtained during the entire pig lifetime, from the fetal period through adulthood. The sequencing results were analyzed using mammalian miRNAs, the precursor hairpins (pre-miRNAs) and the first release of the high-coverage porcine genome assembly (Sscrofa9, April 2009) and the available expressed sequence tag (EST) sequences. Our results extend the repertoire of pig miRNAome to 867 pre-miRNAs (623 with genomic coordinates) encoding for 1,004 miRNAs, of which 777 are unique. We preformed real-time quantitative PCR (q-PCR) experiments for selected 30 miRNAs in 47 tissue-specific samples and found agreement between the sequencing and q-PCR data. This broad survey provides detailed information about multiple variants of mature sequences, precursors, chromosomal organization, development-specific expression, and conservation patterns. Our data mining produced a broad view of the pig miRNAome, consisting of miRNAs and isomiRs and a wealth of information of pig miRNA characteristics. These results are prelude to the advancement in pig biology as well the use of pigs as model organism for human biological and biomedical studies.