Jinyong Wang

Genomic analyses identify distinct patterns of selection in domesticated pigs and Tibetan wild boars

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.

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.

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.

Expression profiling analysis for genes related to meat quality and carcass traits during postnatal development of backfat in two pig breeds

The competitive equilibrium of fatty acid biosynthesis and oxidation in vivo determines porcine subcutaneous fat thickness (SFT) and intramuscular fat (IMF) content. Obese and lean-type pig breeds show obvious differences in adipose deposition; however, the molecular mechanism underlying this phenotypic variation remains unclear. We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with meat quality and carcass traits in backfat at five growth stages (1–5 months) of Landrace (a leaner, Western breed) and Taihu pigs (a fatty, indigenous, Chinese breed). Variance analysis (ANOVA) revealed that differences in the expression of 25 genes in Landrace pigs were significant (FDR adjusted permutation, P<0.05) among 5 growth stages. Gene class test (GCT) indicated that a gene-group was very significant between 2 pig breeds across 5 growth stages (PErmineJ<0.01), which consisted of 23 genes encoding enzymes and regulatory proteins associated with lipid and steroid metabolism. These findings suggest that the distinct differences in fat deposition ability between Landrace and Taihu pigs may closely correlate with the expression changes of these genes. Clustering analysis revealed a very high level of significance (FDR adjusted, P<0.01) for 2 gene expression patterns in Landrace pigs and a high level of significance (FDR adjusted, P<0.05) for 2 gene expression patterns in Taihu pigs. Also, expression patterns of genes were more diversified in Taihu pigs than those in Landrace pigs, which suggests that the regulatory mechanism of micro-effect polygenes in adipocytes may be more complex in Taihu pigs than in Landrace pigs. Based on a dynamic Bayesian network (DBN) model, gene regulatory networks (GRNs) were reconstructed from time-series data for each pig breed. These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of adipose metabolism between the two pig breeds; from these results, some potential key genes could be identified. Quantitative, real-time RT-PCR (QRT-PCR) was used to verify the microarray data for five modulated genes, and a good correlation between the two measures of expression was observed for both 2 pig breeds at different growth stages (R=0.874±0.071). These results highlight some possible candidate genes for porcine fat characteristics and provide some data on which to base further study of the molecular basis of adipose metabolism.

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.

MicroRNA transcriptomes relate intermuscular adipose tissue to metabolic risk.

Intermuscular adipose tissue is located between the muscle fiber bundles in skeletal muscles, and has similar metabolic features to visceral adipose tissue, which has been found to be related to a number of obesity-related diseases. Although various miRNAs are known to play crucial roles in adipose deposition and adipogenesis, the microRNA transcriptome of intermuscular adipose tissue has not, until now, been studied. Here, we sequenced the miRNA transcriptomes of porcine intermuscular adipose tissue by small RNA-sequencing and compared it to a representative subcutaneous adipose tissue. We found that the inflammation- and diabetes-related miRNAs were significantly enriched in the intermuscular rather than in the subcutaneous adipose tissue. A functional enrichment analysis of the genes predicted to be targeted by the enriched miRNAs also indicated that intermuscular adipose tissue was associated mainly with immune and inflammation responses. Our results suggest that the intermuscular adipose tissue should be recognized as a potential metabolic risk factor of obesity.

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.

Distinct Expression Patterns of Genes Associated with Muscle Growth and Adipose Deposition in Tibetan Pigs: A Possible Adaptive Mechanism for High Altitude Conditions

Zhu, Li, Mingzhou Li, Xuewei Li, Surong Shuai, Haifeng Liu, Jinyong Wang, Anan Jiang, Yiren Gu, Kai Zhang, Xiaokun Teng, and Zhongrong Jiang. Distinct expression patterns of genes associated with muscle growth and adipose deposition in Tibetan pigs: a possible adaptive mechanism for high altitude conditions. High Alt. Med. Biol. 10:45-55, 2009.-To investigate the genetic mechanisms underlying high altitude adaptations in Tibetan pigs, changes in the expression of 140 genes associated with muscle growth and adipose deposition in the longissimus dorsi muscle were studied at various growth stages in Tibetan, Landrace, and Meishan pigs using microarray analysis. Analysis of variance (ANOVA) revealed significant differences in the expression of 13 genes (p < 0.05) and highly significant differences in the expression of 15 genes (p < 0.01) among the three pig breeds at 2 months. Differences in the expression of 7 genes were significant (p < 0.05) and differences in the expression of 10 genes were very significant (p < 0.01) in Tibetan pigs from 2 to 8 months. Tibetan pigs had significantly lower body weight than Landrace and Meishan pigs at 2 months and a larger myofiber cross-sectional area (CSA). Cluster analysis showed two significant (p < 0.01) gene expression patterns in Tibetan pigs, in addition to strong downregulation or upregulation of genes between 2 and 8 months. These results indicate that, in Tibetan pigs aged 2 to 8 months, the growth intensity of skeletal muscle is higher than that of intramuscular fat (IMF). The genes that exhibited downregulation were mainly those controlling adipose deposition, whereas the genes that were upregulated were primarily involved in adipose metabolism and skeletal muscle growth. These results are consistent with the unique genetic characteristics of Tibetan pigs, which have likely adapted to the unusual ecological conditions in high altitude areas.

Comprehensive variation discovery and recovery of missing sequence in the pig genome using multiple de novo assemblies

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.

microRNA profiling in three main stages during porcine spermatogenesis

AbstractBackgroundSpermatogenesis is an intricate biological event wherein an undifferentiated spermatogonium develops into mature sperms. MicroRNAs are a type of single strand small non-coding RNA molecule and are implicated in the regulation of many crucial pathways during cell proliferation, apoptosis, and differentiation.MethodHere, we present a comprehensive comparison of miRNA expression profiling in three main stages during porcine spermatogenesis using high-throughput sequencing.ResultsWe built three small RNA libraries for the testis, the epididymis and the ejaculated sperm from a Landrace boar, and in total obtained 3821 precursor hairpins encoding for 4761 mature miRNAs, of which 23 are miRNA*. Notably, 940 precursor miRNAs produced both the 5’- and 3’- strands as sister pairs, indicating the distinctive expression patterns of germ cell miRNAs. Additionally, 418 out of 710 co-expressed miRNAs were identified as being differentially expressed between libraries (P < 0.001). Apart from the sexual specific X chromosome, many miRNAs were found to be located on chromosome 12, which may play potential roles in spermatogenesis according to the result of synteny analysis with human and mouse. The Gene Ontology and KEGG pathway analysis revealed that the target genes of co-expressed miRNAs were highly involved in the cell cycle process, metal ion binding, modification of plasma membrane, and the p53 signal pathway.