Integrated analysis strategy of genome-wide functional gene mining reveals DKK2 gene underlying meat quality in Shaziling synthesized pigs.

BACKGROUND: Shaziling pig is a well-known indigenous breed in China who has superior meat quality traits. However, the genetic mechanism and genomic evidence underlying meat quality characteristics of Shaziling pigs are still unclear. To explore and investigate the germplasm characteristics of Shaziling pigs, we totally analyzed 67 individual's whole genome sequencing data for the first time (20 Shaziling pigs [S], 20 Dabasha pigs [DBS], 11 Yorkshire pigs [Y], 10 Berkshire pigs [BKX], 5 Basha pigs [BS] and 1 Warthog). RESULTS: A total of 2,538,577 SNPs with high quality were detected and 9 candidate genes which was specifically selected in S and shared in S to DBS were precisely mined and screened using an integrated analysis strategy of identity-by-descent (IBD) and selective sweep. Of them, dickkopf WNT signaling pathway inhibitor 2 (DKK2), the antagonist of Wnt signaling pathway, was the most promising candidate gene which was not only identified an association of palmitic acid and palmitoleic acid quantitative trait locus in PigQTLdb, but also specifically selected in S compared to other 48 Chinese local pigs of 12 populations and 39 foreign pigs of 4 populations. Subsequently, a mutation at 12,726-bp of DKK2 intron 1 (g.114874954 A > C) was identified associated with intramuscular fat content using method of PCR-RFLP in 21 different pig populations. We observed DKK2 specifically expressed in adipose tissues. Overexpression of DKK2 decreased the content of triglyceride, fatty acid synthase and expression of relevant genes of adipogenic and Wnt signaling pathway, while interference of DKK2 got contrary effect during adipogenesis differentiation of porcine preadipocytes and 3T3-L1 cells. CONCLUSIONS: Our findings provide an analysis strategy for mining functional genes of important economic traits and provide fundamental data and molecular evidence for improving pig meat quality traits and molecular breeding.

Combined Transcriptomics and Metabolomics Identify Regulatory Mechanisms of Porcine Vertebral Chondrocyte Development In Vitro.

Porcine body length is closely related to meat production, growth, and reproductive performance, thus playing a key role in the profitability of the pork industry. Cartilage development is critical to longitudinal elongation of individual vertebrae. This study isolated primary porcine vertebral chondrocytes (PVCs) to clarify the complex mechanisms of elongation. We used transcriptome and target energy metabolome technologies to confirm crucial genes and metabolites in primary PVCs at different differentiation stages (0, 4, 8, and 12 days). Pairwise comparisons of the four stages identified 4566 differentially expressed genes (DEGs). Time-series gene cluster and functional analyses of these DEGs revealed four clusters related to metabolic processes, cartilage development, vascular development, and cell cycle regulation. We constructed a transcriptional regulatory network determining chondrocyte maturation. The network indicated that significantly enriched transcription factor (TF) families, including zf-C2H2, homeobox, TF_bZIP, and RHD, are important in cell cycle and differentiation processes. Further, dynamic network biomarker (DNB) analysis revealed that day 4 was the tipping point for chondrocyte development, consistent with morphological and metabolic changes. We found 24 DNB DEGs, including the TFs NFATC2 and SP7. Targeted energy metabolome analysis showed that most metabolites were elevated throughout chondrocyte development; notably, 16 differentially regulated metabolites (DRMs) were increased at three time points after cell differentiation. In conclusion, integrated metabolome and transcriptome analyses highlighted the importance of amino acid biosynthesis in chondrocyte development, with coordinated regulation of DEGs and DRMs promoting PVC differentiation via glucose oxidation. These findings reveal the regulatory mechanisms underlying PVC development and provide an important theoretical reference for improving pork production.

Specific gastrointestinal microbiota profiles in Chinese Tan sheep are associated with lauric acid content in muscle.

The biological mechanisms underlying meat quality remain unclear. Currently, many studies report that the gastrointestinal microbiota is essential for animal growth and performance. However, it is uncertain which bacterial species are specifically associated with the meat quality traits. In this study, 16S rDNA and metagenomic sequencing were performed to explore the composition and function of microbes in various gastrointestinal segments of Tan sheep and Dorper sheep, as well as the relationship between microbiota and meat quality (specifically, the fatty acid content of the muscle). In the ruminal, duodenal, and colonic microbiome, several bacteria were uniquely identified in respective breeds, including Agrobacterium tumefaciens, Bacteroidales bacterium CF, and several members of the family Oscillospiraceae. The annotation of GO, KEGG, and CAZYme revealed that these different bacterial species were linked to the metabolism of glucose, lipids, and amino acids. Additionally, our findings suggested that 16 microbial species may be essential to the content of fatty acids in the muscle, especially C12:0 (lauric acid). 4 bacterial species, including Achromobacter xylosoxidans, Mageeibacillus indolicus, and Mycobacterium dioxanotrophicus, were positively correlated with C12:0, while 13 bacteria, including Methanobrevibacter millerae, Bacteroidales bacterium CF, and Bacteroides coprosuis were negatively correlated with C12:0. In a word, this study provides a basic data for better understanding the interaction between ruminant gastrointestinal microorganisms and the meat quality traits of hosts.

Integrated Analysis of Transcriptome Expression Profiles Reveals miRNA-326-NKX3.2-Regulated Porcine Chondrocyte Differentiation.

The porcine body length trait is an essential factor affecting meat production and reproductive performance. It is evident that the development/lengthening of individual vertebrae is one of the main reasons for increases in body length; however, the underlying molecular mechanism remains unclear. In this study, RNA-seq analysis was used to profile the transcriptome (lncRNA, mRNA, and miRNA) of the thoracic intervertebral cartilage (TIC) at two time points (1 and 4 months) during vertebral column development in Yorkshire (Y) and Wuzhishan pigs (W). There were four groups: 1- (Y1) and 4-month-old (Y4) Yorkshire pigs and 1- (W1) and 4-month-old (W4) Wuzhishan pigs. In total, 161, 275, 86, and 126 differentially expressed (DE) lncRNAs, 1478, 2643, 404, and 750 DE genes (DEGs), and 74,51, 34, and 23 DE miRNAs (DE miRNAs) were identified in the Y4 vs. Y1, W4 vs. W1, Y4 vs. W4, and Y1 vs. W1 comparisons, respectively. Functional analysis of these DE transcripts (DETs) demonstrated that they had participated in various biological processes, such as cellular component organization or biogenesis, the developmental process, the metabolic process, bone development, and cartilage development. The crucial bone development-related candidate genes NK3 Homeobox 2 (NKX3.2), Wnt ligand secretion mediator (WLS), gremlin 1 (GREM1), fibroblast growth factor receptor 3 (FGFR3), hematopoietically expressed homeobox (HHEX), (collagen type XI alpha 1 chain (COL11A1), and Wnt Family Member 16 (WNT16)) were further identified by functional analysis. Moreover, lncRNA, miRNA, and gene interaction networks were constructed; a total of 55 lncRNAs, 6 miRNAs, and 7 genes formed lncRNA-gene, miRNA-gene, and lncRNA-miRNA-gene pairs, respectively. The aim was to demonstrate that coding and non-coding genes may co-regulate porcine spine development through interaction networks. NKX3.2 was identified as being specifically expressed in cartilage tissues, and it delayed chondrocyte differentiation. miRNA-326 regulated chondrocyte differentiation by targeting NKX3.2. The present study provides the first non-coding RNA and gene expression profiles in the porcine TIC, constructs the lncRNA-miRNA-gene interaction networks, and confirms the function of NKX3.2 in vertebral column development. These findings contribute to the understanding of the potential molecular mechanisms regulating pig vertebral column development. They expand our knowledge about the differences in body length between different pig species and provide a foundation for future studies.

Protein expression profiles in Meishan and Duroc sows during mid-gestation reveal differences affecting uterine capacity, endometrial receptivity, and the maternal-fetal Interface.

BACKGROUND: Embryonic mortality is a major concern in the commercial swine industry and primarily occurs early in gestation, but also during mid-gestation (~ days 50-70). Previous reports demonstrated that the embryonic loss rate was significant lower in Meishan than in commercial breeds (including Duroc). Most studies have focused on embryonic mortality in early gestation, but little is known about embryonic loss during mid-gestation. RESULTS: In this study, protein expression patterns in endometrial tissue from Meishan and Duroc sows were examined during mid-gestation. A total of 2170 proteins were identified in both breeds. After statistical analysis, 70 and 114 differentially expressed proteins (DEPs) were identified in Meishan and Duroc sows, respectively. Between Meishan and Duroc sows, 114 DEPs were detected at day 49, and 98 DEPs were detected at day 72. Functional enrichment analysis revealed differences in protein expression patterns in the two breeds. Around half of DEPs were more highly expressed in Duroc at day 49 (DUD49), relative to DUD72 and Meishan at day 49 (MSD49). Many DEPs appear to be involved in metabolic process such as arginine metabolism. Our results suggest that the differences in expression affect uterine capacity, endometrial matrix remodeling, and maternal-embryo cross-talk, and may be major factors influencing the differences in embryonic loss between Meishan and Duroc sows during mid-gestation. CONCLUSIONS: Our data showed differential protein expression pattern in endometrium between Meishan and Duroc sows and provides insight into the development process of endometrium. These findings could help us further uncover the molecular mechanism involved in prolificacy.

Study on the Characteristics of Coarse Feeding Tolerance of Ding'an Pigs: Phenotypic and Candidate Genes Identification.

Ding'an (DA) pig, a prominent local breed in Hainan Province, exhibits notable advantages in coarse feeding tolerance and high-quality meat. To explore the potential genetic mechanism of coarse feeding tolerance in DA pigs, 60-day-old full sibling pairs of DA and DLY (Duroc-Landrace-Yorkshire) pigs were subjected to fed normal (5%) and high (10%) crude fiber diets for 56 days, respectively. The findings showed that increasing the crude fiber level had no impact on the apparent digestibility of crude fiber, intramuscular fat, and marbling scores in DA pigs, whereas these factors were significantly reduced in DLY pigs (p < 0.05). Through differential expression analysis and Weighted Gene Co-expression Network Analysis (WGCNA) of the colonic mucosal transcriptome data, 65 and 482 candidate genes with coarse feeding tolerance in DA pigs were identified, respectively. Joint analysis screened four key candidate genes, including LDHB, MLC1, LSG1, and ESM1, potentially serving as key regulated genes for coarse feeding tolerance. Functional analysis revealed that the most significant pathway enriched in differential genes associated with coarse feeding tolerance in Ding'an pigs was the signaling receptor binding. The results hold substantial significance for advancing our understanding of the genetic mechanisms governing coarse feeding tolerance in Ding'an pigs.

Differential Regulation of Male-Hormones-Related Enhancers Revealed by Chromatin Accessibility and Transcriptional Profiles in Pig Liver.

Surgical castration can effectively avoid boar taint and improve pork quality by removing the synthesis of androstenone in the testis, thereby reducing its deposition in adipose tissue. The expression of genes involved in testis-derived hormone metabolism was altered following surgical castration, but the upstream regulatory factors and underlying mechanism remain unclear. In this study, we systematically profiled chromatin accessibility and transcriptional dynamics in liver tissue of castrated and intact full-sibling Yorkshire pigs. First, we identified 897 differentially expressed genes and 6864 differential accessible regions (DARs) using RNA- and ATAC-seq. By integrating the RNA- and ATAC-seq results, 227 genes were identified, and a significant positive correlation was revealed between differential gene expression and the ATAC-seq signal. We constructed a transcription factor regulatory network after motif analysis of DARs and identified a candidate transcription factor (TF) SP1 that targeted the HSD3B1 gene, which was responsible for the metabolism of androstenone. Subsequently, we annotated DARs by incorporating H3K27ac ChIP-seq data, marking 2234 typical enhancers and 245 super enhancers involved in the regulation of all testis-derived hormones. Among these, four typical enhancers associated with HSD3B1 were identified. Furthermore, an in-depth investigation was conducted on the androstenone-related enhancers, and an androstenone-related mutation was identified in a newfound candidatetypical enhancer (andEN) with dual-luciferase assays. These findings provide further insights into how enhancers function as links between phenotypic and non-coding area variations. The discovery of upstream TF and enhancers of HSD3B1 contributes to understanding the regulatory networks of androstenone metabolism and provides an important foundation for improving pork quality.

Colonic Microbiota Improves Fiber Digestion Ability and Enhances Absorption of Short-Chain Fatty Acids in Local Pigs of Hainan.

Compared to commercial breeds, Chinese local pig breeds have a greater ability to digest dietary fiber, which may be due to differences in intestinal microbiota. In this study, we fed Ding'an and DLY pigs high and low levels of dietary fiber, respectively, to investigate factors contributing to high dietary fiber adaption in Ding'an pigs. Twelve Ding'an pigs and DLY pigs were randomly divided into a 2 (diet) × 2 (breed) factorial experiment (n = 3). Compared with commercial pigs, Ding'an pigs have a stronger ability to digest dietary fiber. Prevotella was more prevalent in Ding'an pigs than in DLY pigs, which may be an important reason for the stronger ability of fiber degradation in Ding'an pigs. When the effects of feed and breed factors are considered, differences in abundance of 31 species and 14 species, respectively, may result in a greater ability of fiber degradation in Ding'an pigs. Among them, Prevotella. sp. CAG:520 may be a newly discovered bacterium related to fiber degradation, which positively correlated with many fiber-degrading bacteria (r > 0.7). We also found that the concentration of plant metabolites with anti-inflammatory and antioxidant effects was higher in the colonic chyme of Ding'an pigs after increasing the fiber content, which resulted in the downregulated expression of inflammatory factors in colonic mucosa. Spearman's correlation coefficient revealed a strong correlation between microbiota and the apparent digestibility of dietary fiber (r > 0.7). The mRNA expressions of SLC16A1, PYY, and GCG were significantly increased in the colonic mucosa of Ding'an pigs fed on high-fiber diets, which indicates that Ding'an pigs have an enhanced absorption of SCFAs. Our results suggested that an appropriate increase in dietary fiber content can reduce the inflammatory response and improve feed efficiency in Ding'an pigs, and differences in the intestinal microbial composition may be an important reason for the difference in the fiber degradation capacity between the two breeds of pigs.

Novel Haplotype in the HHEX Gene Promoter Associated with Body Length in Pigs.

The screening of important candidate genes and the identification of genetic markers are important for molecular selection in the pig industry. The hematopoietically expressed homeobox (HHEX) gene plays an important role in embryonic development and organogenesis; however, the genetic variation and expression pattern of the porcine HHEX gene remains to be clarified. In this study, semiquantitative RT-PCR and immunohistochemistry results showed the specific expression of the HHEX gene in porcine cartilage tissues. A novel haplotype consisting of two SNPs rs80901185 (T > C) and rs80934526 (A > G) was detected in the promoter region of the HHEX gene. The expression of the HHEX gene was significantly higher in Yorkshire pigs (TA haplotype) than in Wuzhishan pigs (CG haplotype), and a population analysis showed that this haplotype was significantly associated with body length. An analysis subsequently revealed that the -586 to -1 bp region of the HHEX gene promoter showed the highest activity. Furthermore, we found that the activity of the TA haplotype was significantly higher than that of the CG haplotype by changing the potential binding of transcription factors YY1 and HDAC2. In summary, we conclude that the porcine HHEX gene may contribute to the breeding of pigs for body length traits.

The Novel-miR-659/SPP1 Interaction Regulates Fat Deposition in Castrated Male Pigs.

Castration is usually used to remove boar taint in commercial pork production, but the adipose accumulation was increased excessively, which affected the meat quality of pigs. Based on our previous study, secreted phosphoprotein 1 (SPP1) was significantly differentially expressed between castrated and intact male pigs. However, the role of SPP1 in regulating adipose growth and fat storage caused by castration is unknown. In this study, SPP1 was identified to inhibit adipogenesis by the expression of adipogenic markers PPARγ and FABP4 as well as Oil red staining assay during differentiation of porcine bone marrow mesenchymal stem cells (pBMSCs). Subsequently, testosterone was used to treat pBMSCs to simulate the androgen status of intact pigs. Compared with the control groups without testosterone, the SPP1 expression in the testosterone group was markedly increased in the late stage of pBMSCs differentiation. Furthermore, novel-miR-659 was predicted by TargetScan and miRDB to target SPP1 and verified through a dual-luciferase reporter assay. Oil Red O staining assay indicated that novel-miR-659 overexpression significantly promoted adipogenesis, whereas novel-miR-659 inhibition suppressed adipogenesis. The expressions of adipogenic markers PPARγ and FABP4 showed the same tendency. Taken together, our study found that the targeted interaction between novel-miR-659 and SPP1 is involved in regulation of fat deposition in castrated male pigs.

miR-F4-C12 Functions on the Regulation of Adipose Accumulation by Targeting PIK3R1 in Castrated Male Pigs.

MicroRNAs (miRNAs) constitute small regulatory molecules for a wide array of biological activities (18~24 nucleotides in length), including adipogenesis and adipose deposition. Their effect is, however, incompletely defined in inducing fat accumulation in castrated male pigs. Based on our study, four nine-times miRNAs were selected to examine their functions in adipose formation activities. In 3T3-L1 cells and backfat tissues of castrated and intact male pigs, miR-F4-C12 was identified as a factor in adipose development utilizing quantitative real-time PCR (qRT-PCR). Further, miR-F4-C12 was identified to promote fat development, suggesting that miR-F4-C12 was involved in adipogenesis. Moreover, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was proposed by the TargetScan, miRDB and starBase as a target of miR-F4-C12 and verified through a two-luciferase reporter assay. The over-expression of miR-F4-C12 dramatically decreases the PIK3R1 protein level in 3T3-L1 cells. The mRNA and protein levels of PIK3R1 in castrated pigs are reduced relative to intact pigs, providing further evidence that PIK3R1 is involved in regulating adipose accumulation. These results suggest that miR-F4-C12 involves adipose development and may regulate subcutaneous adipose tissue accumulation by targeting PIK3R1 in castrated male pigs.

Integrated Analysis of miRNA-mRNA Network Reveals Different Regulatory Patterns in the Endometrium of Meishan and Duroc Sows during Mid-Late Gestation.

Embryo loss is a major factor affecting profitability in the pig industry. Embryonic mortality occurs during peri-implantation and mid-late gestation in pigs. Previous investigations have shown that the embryo loss rate in Meishan pigs is significantly lower than in commercial breeds. Most studies have focused on embryonic mortality during early gestation, but little is known about losses during mid-late gestation. In this study, we performed a transcriptome analysis of endometrial tissue in mid-late gestation sows (gestation days 49 and 72) sampled from two breeds (Meishan (MS) and Duroc (DU)) that have different embryo loss rates. We identified 411, 1113, 697, and 327 differentially expressed genes, and 14, 36, 57, and 43 differentially expressed miRNAs in four comparisons (DU49 vs. DU72, DU49 vs. MS49, DU72 vs. MS72, and MS49 vs. MS72), respectively. Subsequently; seven differentially expressed mRNAs and miRNAs were validated using qPCR. Functional analysis suggested the differentially expressed genes and miRNAs target genes mainly involved in regulation of hormone levels, blood vessel development, developmental process involved in reproduction, embryonic placenta development, and the immune system. A network analysis of potential miRNA-gene interactions revealed that differentially expressed miRNAs in Meishan pigs are involved in the response to estradiol and oxygen levels, and affect angiogenesis and blood vessel development. The binding site on ssc-miR-503 for epidermal growth factor (EGF) and the binding site on ssc-miR-671-5p for estrogen receptor α (ESR1) were identified using a dual luciferase assay. The results of this study will enable further exploration of miRNA-mRNA interactions important in pig pregnancy and will help to uncover molecular mechanisms affecting embryonic mortality in pigs during mid-late gestation.

Transcriptomic Analysis of Coding Genes and Non-Coding RNAs Reveals Complex Regulatory Networks Underlying the Black Back and White Belly Coat Phenotype in Chinese Wuzhishan Pigs.

Coat color is one of the most important characteristics for distinguishing Chinese indigenous pig breeds. In Wuzhishan pigs, the animals have black on the back and white on the abdomen. However, the molecular genetic basis of this phenotype is unclear. In this study, we used high-throughput RNA sequencing to compare expression profiles of coding and non-coding RNAs from white and black skin samples obtained from individual Wuzhishan pigs. The expression profiling revealed that 194 lncRNAs (long non-coding RNAs), 189 mRNAs (messenger RNAs), and 162 miRNAs (microRNAs) had significantly different levels of expression (|log2 fold change| > 1, p-value < 0.05) in white and black skin. Compared to RNA levels in black skin, white skin had higher levels of expression of 185 lncRNAs, 181 mRNAs, and 23 miRNAs and lower levels of expression of 9 lncRNAs, 8 mRNAs, and 139 miRNAs. Functional analysis suggested that the differentially expressed transcripts are involved in biological processes such as melanin biosynthesis, pigmentation and tyrosine metabolism. Several key genes involved in melanogenesis, including MLANA, PMEL, TYR, TYRP1, DTC, TRPM1 and CAMK2A, had significantly different levels of expression in the two skin tissues. Potential lncRNA⁻miRNA⁻gene interactions were also examined. A total of 15 lncRNAs, 11 miRNAs and 7 genes formed 23 lncRNA⁻miRNA⁻gene pairs, suggesting that complex regulatory networks of coding and non-coding genes underlie the coat color trait in Wuzhishan pigs. Our study provides a foundation for understanding how lncRNA, miRNA and genes interact to regulate coat color in black-back/white-belly pigs. We also constructed lncRNA⁻miRNA⁻gene interaction networks to elucidate the complex molecular mechanisms underlying skin physiology and melanogenesis. The results extend our knowledge about the diversity of coat color among different domestic animals and provide a foundation for studying novel mechanisms that control coat color in Chinese indigenous pigs.

Runting and Stunting Syndrome Is Associated With Mitochondrial Dysfunction in Sex-Linked Dwarf Chicken.

Runting and stunting syndrome (RSS) in chicken are commonly known as "frozen chicken." The disease is characterized by lower body weight and slow growth and the incidence rate is widely 5%-20% in sex-linked dwarf (SLD) chickens. However, the etiology of RSS in chickens has plagued researchers for several decades. In this study, histopathology studies demonstrated that the hepatocytes of the RSS chickens contain many mitochondria with damaged and outer and inner membrane along with vacuolar hydropic degeneration. No mtDNA mutation was detected, but our microarray data showed that RSS chickens exhibited abnormal expression of genes, many of which are involved in oxidative phosphorylation (OXPHOS) and fatty acid metabolism. In particular, nuclear gene IGF2BP3 was upregulated in RSS chickens' liver cells. The abnormal expression of these genes is likely to impair the OXPHOS, resulting in reduced ATP synthesis in the hepatocytes of the RSS chickens, which may in turn leads to poor weight gain and retarded growth or stunting of chicks. Our findings suggest that mitochondria dysfunction rather than chronic inflammation is responsible for the reduced growth and RSS in SLD chickens. Mutations in GHR have been shown to compromise mitochondrial function in SLD chickens. Since the mitochondrial damage in the RSS chicken is more severe, we suggest that extra genes are likely to be affected to exacerbate the phenotype.