Integration Analysis of Hair Follicle Transcriptome and Proteome Reveals the Mechanisms Regulating Wool Fiber Diameter in Angora Rabbits.

Fiber diameter is an important characteristic that determines the quality and economic value of rabbit wool. This study aimed to investigate the genetic determinants of wool fiber diameter through an integration analysis using transcriptomic and proteomic datasets from hair follicles of coarse and fine wool from Angora rabbits. Using a 4D label-free technique, we identified 423 differentially expressed proteins (DEPs) in hair follicles of coarse and fine wool in Angora rabbits. Eighteen DEPs were examined using parallel reaction monitoring, which verified the reliability of our proteomic data. Functional enrichment analysis revealed that a set of biological processes and signaling pathways related to wool growth and hair diameter were strongly enriched by DEPs with fold changes greater than two, such as keratinocyte differentiation, skin development, epidermal and epithelial cell differentiation, epidermis and epithelium development, keratinization, and estrogen signaling pathway. Association analysis and protein-protein interaction network analysis further showed that the keratin (KRT) family members, including KRT77, KRT82, KRT72, KRT32, and KRT10, as well as CASP14 and CDSN, might be key factors contributing to differences in fiber diameter. Our results identified DEPs in hair follicles of coarse and fine wool and promoted understanding of the molecular mechanisms underlying wool fiber diameter variation among Angora rabbits.

Genome-wide scan for runs of homozygosity in Asian wild boars and Anqing six-end-white pigs.

Inbreeding and loss of genetic diversity are serious problems in local Chinese pig breeds. Runs of homozygosity (ROHs) are contiguous lengths of homozygous genotypes that can provide information on inbreeding levels, mating schemes, selection pressure, and genetic events. The Anqing six-end-white (AQ) pig, an important autochthonous pig breed bred in the Anhui province of China, plays a key role in the local pig industry. In recent decades, AQ pigs have become a closed breed with a small population size in conservation farms, raising the issue of inbreeding decline. In this study, we used 10× resequencing to detect the whole genome of 24 AQ pigs and six Asian wild boars (AWBs). We used the Plink software to analyze the homozygosity of the genome and the distribution of ROHs in the genome based on the resequencing data. We obtained 935.04 Gb of raw data from the resequencing results and more than 45 822 239 SNPs. Additionally, we identified 28 702 ROHs. Compared with AWB, AQ pigs had higher ROH numbers, longer ROH rates, and higher FROH values, which revealed that artificial selection influenced ROH distribution and genome inbreeding level. A total of 307 and 205 ROH islands were identified in the AQ pigs and AWBs respectively. The genes of ROH islands in AQ pigs were mainly enriched in immune biological processes. Our findings provide a useful reference for developing breeding programs to maintain genetic diversity and germplasm resources in AQ pigs.

Transcriptomic comparison of liver tissue between Anqing six-end-white pigs and Yorkshire pigs based on RNA sequencing.

Chinese indigenous pig and Western commercial pig breeds show different patterns of lipid metabolism, fat deposition, and fatty acid composition; for these reasons, they have become vitally important models of energy metabolism and obesity in humans. To compare the mechanisms underlying lipid metabolism between Yorkshire pigs (lean type) and Anqing six-end-white pigs (obese type), the liver transcriptomes of six castrated boars with a body weight of approximately 100 kg (three Yorkshire and three Anqing) were analyzed by RNA-seq. The total number of reads produced for each liver sample ranged from 47.05 to 62.6 million. Among 362 differentially expressed genes, 142 were up-regulated and 220 were down-regulated in Anqing six-end-white pigs. Based on these data, 79 GO terms were significantly enriched. The top 10 (the 10 with lowest corrected P-value) significantly enriched GO terms were identified, including lipid metabolic process and carboxylic acid metabolic process. Pathway analysis revealed three significantly enriched KEGG pathways including PPAR signaling pathway, steroid hormone biosynthesis, and retinol metabolism. Based on protein-protein interaction networks, multiple genes responsible for lipid metabolism were identified, such as PCK1, PPARA, and CYP7A1, and these were considered promising candidate genes that could affect porcine liver lipid metabolism and fat deposition. Our results provide abundant transcriptomic information that will be useful for animal breeding and biomedical research.

Transcriptional analysis of deoxynivalenol-induced apoptosis of sow ovarian granulosa cell.

Litter size is one of the most important economic traits in pig production. Recent studies identified that deoxynivalenol (DON), a widespread toxin in fodder, was associated with animal prolificacy. However, the underlying mechanisms have not yet been completely elucidated. Here, we used porcine ovary granulosa cells (pGCs) as a vector to establish DON concentration-time models and performed cell morphology and transcriptome analysis to identify and analyse the effects of DON on reproductive performance in swine. The results showed that DON can induce morphological changes and apoptosis of pGCs, while inhibiting cell proliferation. Moreover, these effects of DON on pGCs were dose-dependent. After treatment of pGCs with different concentrations of DON, the percentage of cells in S phase and G2/M phase increased. RNA-seq analyses revealed 5,937 differentially expressed genes, of which 1995 were down-regulated and 3,942 were up-regulated after DON treatment. KEGG enrichment analysis indicated important metabolic pathways such as IL-17 signalling pathway, eukaryotic ribosome synthesis pathway, RNA transport pathway and RNA degradation. Based on our results, we speculate that the effects of DON are related to the DNA damage process. Our study provides novel insights and a foundation to further understand the effect of DON on swine prolificacy.

Genomic analysis reveals selection signatures of the Wannan Black pig during domestication and breeding.

OBJECTIVE: The Wannan Black pig is a typical Chinese indigenous, disease-resistant pig breed with high fertility, and a crude-feed tolerance that has been bred by artificial selection in the south of Anhui province for a long time. However, genome variation, genetic relationships with other pig breeds, and domestication, remain poorly understood. Here, we focus on elucidating the genetic characteristics of the Wannan Black pig and identifying selection signatures during domestication and breeding. METHODS: We identified the whole-genome variation in the Wannan Black pig and performed population admixture analyses to determine genetic relationships with other domesticated pig breeds and wild boars. Then, we identified the selection signatures between the Wannan Black pig and Asian wild boars in 100-kb windows sliding in 10 kb steps by using two approaches: the fixation index (FST) and π ratios. RESULTS: Resequencing the Wannan Black pig genome yielded 501.52 G of raw data. After calling single-nucleotide variants (SNVs) and insertions/deletions (InDels), we identified 21,316,754 SNVs and 5,067,206 InDels (2,898,582 inserts and 2,168,624 deletions). Additionally, we found genes associated with growth, immunity, and digestive functions. CONCLUSION: Our findings help in explaining the unique genetic and phenotypic characteristics of Wannan Black pigs, which in turn can be informative for future breeding programs of Wannan Black pigs.

Relationship among porcine lncRNA TCONS_00010987, miR-323, and leptin receptor based on dual luciferase reporter gene assays and expression patterns.

OBJECTIVE: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. METHODS: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiR-RB-REPORTTM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. RESULTS: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_ 00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). CONCLUSION: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays.

OBJECTIVE: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. METHODS: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3'-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3'-UTR and pGL3 Control LDLR mutant-3'-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3'-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. RESULTS: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3'-UTR-driven (p<0.01), but not mutant LDLR-3'-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = -0.656, p<0.05). CONCLUSION: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3'-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

Transcriptome analysis of divergent residual feed intake phenotypes in the M. longissimus thoracis et lumborum of Wannan Yellow rabbits.

Introduction: Feed efficiency is an important economic trait in rabbit meat production. The identification of molecular mechanisms and candidate genes for feed efficiency may improve the economic and environmental benefits of the rabbit meat industry. As an alternative to the conventional feed conversion ratio, residual feed intake (RFI) can be used as an accurate indicator of feed efficiency. Methods: RNA sequencing was used to identify the differentially expressed genes (DEGs) in the M. longissimus thoracis et lumborum of eight Wannan Yellow rabbits with excessively high or low RFIs (HRFI or LRFI, respectively). Thereafter, Gene Ontology (GO) analysis, enrichment using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) network analysis was conducted. Results: In total, 445 DEGs were identified in the M. longissimus thoracis et lumborum of rabbits with high and low RFIs. The significantly enriched GO terms identified in these two groups were primarily involved in energy and mitochondrial metabolism and oxidation-reduction processes. KEGG analysis identified 11 significantly enriched pathways, including oxidative phosphorylation, PI3K-Akt signaling, and extracellular matrix-receptor interaction pathways. According to GSEA, the expressions of genes and pathways related to mitochondrial function were upregulated in HRFI rabbits, whereas genes with upregulated expressions in LRFI rabbits were related to immune response and energy metabolism. Additionally, PPI network analysis revealed five potential candidate genetic markers. Conclusion: Comparative analysis of the M. longissimus thoracis et lumborum transcriptomes in HRFI and LRFI rabbits revealed FOS, MYC, PRKACB, ITGA2, and FN1 as potential candidate genes that affect feed efficiency in rabbits. In addition, key signaling pathways involved in oxidative phosphorylation and PI3K-Akt and ECM-receptor interaction signaling impact rabbit feed efficiency. These findings will aid in breeding programs to improve feed efficiency and optimize RFI selection of rabbits for meat production.

Hair Follicle Transcriptome Analysis Reveals Differentially Expressed Genes That Regulate Wool Fiber Diameter in Angora Rabbits.

Wool fiber diameter (WFD) is an important index of wool traits and the main determinant of wool quality and value. However, the genetic determinants of fiber diameter have not yet been fully elucidated. Here, coarse and fine wool of Wan strain Angora rabbits and their hair follicle traits were characterized. The results indicated significant differences in the diameters of wool fibers and their hair follicles. The RNA sequencing (RNA-Seq) technique was used to identify differences in gene expression in hair follicles between coarse and fine wool. In total, 2574 differentially expressed genes (DEGs) were found between the two hair follicle groups. Transcription factors, keratin-associated protein (KAP) and keratin (KRT) families, and ECM-related genes may control the structure of fine fibers in rabbits. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that skin development, epidermal cell and keratinocyte differentiation, epithelium development, and Notch and ribosome signaling pathways were significantly enriched, respectively. GSEA further filtered six important pathways and related core genes. PPI analysis also mined functional DEGs associated with hair structure, including LEF1, FZD3, SMAD3, ITGB6, and BMP4. Our findings provide valuable information for researching the molecular mechanisms regulating wool fiber and could facilitate enhanced selection of super-fine wool rabbits through gene-assisted selection in the future.

Identification of Signatures of Selection by Whole-Genome Resequencing of a Chinese Native Pig.

Identification of genomic signatures of selection that help reveal genetic mechanisms underlying traits in domesticated pigs is of importance. Anqing six-end-white pig (ASP), a representative of the native breeds in China, has many distinguishing phenotypic characteristics. To identify the genomic signatures of selection of the ASP, whole-genome sequencing of 20 ASPs produced 469.01 Gb of sequence data and more than 26 million single-nucleotide polymorphisms. Combining these data with the available whole genomes of 13 Chinese wild boars, 157 selected regions harboring 48 protein-coding genes were identified by applying the polymorphism levels (θπ) and genetic differentiation (F ST ) based cross approaches. The genes found to be positively selected in ASP are involved in crucial biological processes such as coat color ( MC1R ), salivary secretion ( STATH ), reproduction ( SPIRE2 , OSBP2 , LIMK2 , FANCA , and CABS1 ), olfactory transduction ( OR5K4 ), and growth ( NPY1R , NPY5R , and SELENOM ). Our research increased the knowledge of ASP phenotype-related genes and help to improve our understanding of the underlying biological mechanisms and provide valuable genetic resources that enable effective use of pigs in agricultural production.

MiR-222 inhibits apoptosis in porcine follicular granulosa cells by targeting the THBS1 gene.

Apoptosis of granulosa cells affects follicular atresia and reproduction and is regulated by miRNAs and the expression of certain genes. For the present study, we investigated the regulatory relationship between microRNA-222 (miR-222) and THBS1 in porcine follicular granulosa cells (pGCs) and its effects on apoptosis to provide empirical data for developing methods to improve pig fecundity. Results revealed that miR-222 promotes the proliferation of pGCs. MiRNA mimics and luciferase reporter assays revealed that miR-222 functions as an anti-apoptotic factor in pGCs. MiR-222 mimics in pGCs result in the upregulation of the anti-apoptotic BCL-2 gene, down-regulation of the proapoptotic caspase-3 gene, and inhibition of apoptosis. MiR-222 inhibitors reduced BCL-2 and had no significant effect on caspase-3. MiR-222 mimics promoted estrogen levels. Inhibition of THBS1 inhibited pGC apoptosis. Transfection of THBS1-siRNA reduced the proapoptotic BAX gene. MiR-222 can directly target the 3'-untranslated region of the THBS1 gene. MiR-222 mimics suppressed THBS1 mRNA and proteins, but these were upregulated by the miR-222 inhibitor. Transfection of THBS1-siRNA resulted in the inhibition of the miR-222 inhibitor, which suggests that miR-222 inhibits pGC apoptosis by targeting THBS1. These findings suggest that miR-222 and THBS1 play important roles in follicular atresia, ovarian development, and female reproduction.