Genome-wide detection of selection signatures in Jianli pigs reveals novel cis-regulatory haplotype in EDNRB associated with two-end black coat color.

BACKGROUND: Jianli pig, a renowned indigenous breed in China, has the characteristics of a two-end black (TEB) coat color, excellent meat quality, strong adaptability and increased prolificacy. However, there is limited information available regarding the genetic diversity, population structure and genomic regions under selection of Jianli pig. On the other hand, the genetic mechanism of TEB coat color has remained largely unknown. RESULTS: In this study, the whole genome resequencing of 30 Jianli pigs within a context of 153 individuals representing 13 diverse breeds was performed. The population structure analysis revealed that Jianli pigs have close genetic relationships with the Tongcheng pig breed, their geographical neighbors. Three methods (observed heterozygosity, expected heterozygosity, and runs of homozygosity) implied a relatively high level of genetic diversity and, a low inbreeding coefficient in Jianli compared with other pigs. We used Fst and XP-EHH to detect the selection signatures in Jianli pigs compared with Asian wild boar. A total of 451 candidate genes influencing meat quality (CREBBP, ADCY9, EEPD1 and HDAC9), reproduction (ESR1 and FANCA), and coat color (EDNRB, MITF and MC1R), were detected by gene annotation analysis. Finally, to fine-map the genomic region for the two-end black (TEB) coat color phenotype in Jianli pigs, we performed three signature selection methods between the TEB coat color and no-TEB coat color pig breeds. The current study, further confirmed that the EDNRB gene is a candidate gene for TEB color phenotype found in Chinese pigs, including Jinhua pigs, and the haplotype harboring 25 SNPs in the EDNRB gene may promote the formation of TEB coat color. Further ATAC-seq and luciferase reporter assays of these regions suggest that the 25-SNPs region was a strong candidate causative mutation that regulates the TEB coat color phenotype by altering enhancer function. CONCLUSION: Our results advanced the understanding of the genetic mechanism behind artificial selection, and provided further resources for the protection and breeding improvement of Jianli pigs.

N-Acetyl-L-Cysteine Ameliorates BPAF-Induced Porcine Sertoli Cell Apoptosis and Cell Cycle Arrest via Inhibiting the ROS Level.

Bisphenol AF (BPAF) is a newly identified contaminant in the environment that has been linked to impairment of the male reproductive system. However, only a few studies have systematically studied the mechanisms underlying BPAF-induced toxicity in testicular Sertoli cells. Hence, this study primarily aims to explore the toxic mechanism of BPAF on the porcine Sertoli cell line (ST cells). The effects of various concentrations of BPAF on ST cell viability and cytotoxicity were evaluated using the Counting Kit-8 (CCK-8) assay. The results demonstrated that exposure to a high concentration of BPAF (above 50 µM) significantly inhibited ST cell viability due to marked cytotoxicity. Flow cytometry analysis further confirmed that BPAF facilitated apoptosis and induced cell cycle arrest in the G2/M phase. Moreover, BPAF exposure upregulated the expression of pro-apoptotic markers BAD and BAX while downregulating anti-apoptotic and cell proliferation markers BCL-2, PCNA, CDK2, and CDK4. BPAF exposure also resulted in elevated intracellular levels of reactive oxygen species (ROS) and malondialdehyde (MDA), alongside reduced activities of the antioxidants glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). Furthermore, the ROS scavenger N-acetyl-L-cysteine (NAC) effectively blocked BPAF-triggered apoptosis and cell cycle arrest. Therefore, this study suggests that BPAF induces apoptosis and cell cycle arrest in ST cells by activating ROS-mediated pathways. These findings enhance our understanding of BPAF's role in male reproductive toxicity and provide a foundation for future toxicological assessments.

Character recognition competition for street view shop signs.

This paper presents the inaugural character recognition competition for street view shop signs, including the associated tasks, datasets, participating teams, the winning team's solution, and justification for the award.

ISG15 suppresses ovulation and female fertility by ISGylating ADAMTS1.

BACKGROUND: ISGylation is a post-translational protein modification that regulates many life activities, including immunomodulation, antiviral responses, and embryo implantation. The exact contribution of ISGylation to folliculogenesis remains largely undefined. RESULTS: Here, Isg15 knockout in mice causes hyperfertility along with sensitive ovarian responses to gonadotropin, such as increases in cumulus expansion and ovulation rate. Moreover, ISG15 represses the expression of ovulation-related genes in an ISGylation-dependent manner. Mechanistically, ISG15 binds to ADAMTS1 via the ISG15-conjugating system (UBA7, UBE2L6, and HERC6), ISGylating ADAMTS1 at the binding sites Lys309, Lys593, Lys597, and Lys602, resulting in ADAMTS1 degradation via a 20S proteasome-dependent pathway. CONCLUSION: Taken together, the present study demonstrates that covalent ISG15 conjugation produces a novel regulatory axis of ISG15-ADAMTS1 that enhances the degradation of ADAMTS1, thereby compromising ovulation and female fertility.

Integration of ATAC-seq and RNA-seq analysis identifies key genes affecting intramuscular fat content in pigs.

Meat quality is one of the most important economic traits in pig breeding and production, and intramuscular fat (IMF) content is the major factor in improving meat quality. The IMF deposition in pigs is influenced by transcriptional regulation, which is dependent on chromatin accessibility. However, how chromatin accessibility plays a regulatory role in IMF deposition in pigs has not been reported. Xidu black is a composite pig breed with excellent meat quality, which is an ideal research object of this study. In this study, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analysis to identify the accessible chromatin regions and key genes affecting IMF content in Xidu black pig breed with extremely high and low IMF content. First, we identified 21,960 differential accessible chromatin peaks and 297 differentially expressed genes. The motif analysis of differential peaks revealed several potential cis-regulatory elements containing binding sites for transcription factors with potential roles in fat deposition, including Mef2c, CEBP, Fra1, and AP-1. Then, by integrating the ATAC-seq and RNA-seq analysis results, we found 47 genes in the extremely high IMF (IMF_H) group compared with the extremely low IMF (IMF_L) group. For these genes, we observed a significant positive correlation between the differential gene expression and differential ATAC-seq signal (r 2 = 0.42). This suggests a causative relationship between chromatin remodeling and the resulting gene expression. We identified several candidate genes (PVALB, THRSP, HOXA9, EEPD1, HOXA10, and PDE4B) that might be associated with fat deposition. Through the PPI analysis, we found that PVALB gene was the top hub gene. In addition, some pathways that might regulate fat cell differentiation and lipid metabolism, such as the PI3K-Akt signaling pathway, MAPK signaling pathway, and calcium signaling pathway, were significantly enriched in the ATAC-seq and RNA-seq analysis. To the best of our knowledge, our study is the first to use ATAC-seq and RNA-seq to examine the mechanism of IMF deposition from a new perspective. Our results provide valuable information for understanding the regulation mechanism of IMF deposition and an important foundation for improving the quality of pork.

Genome-Wide Assessment of Runs of Homozygosity and Estimates of Genomic Inbreeding in a Chinese Composite Pig Breed.

The primary purpose of the current study was to assess the genetic diversity, runs of homozygosity (ROH) and ROH islands in a Chinese composite pig and explore hotspot regions for traces of selection. First, we estimated the length, number, and frequency of ROH in 262 Xidu black pigs using the Porcine SNP50 BeadChip and compared the estimates of inbreeding coefficients, which were calculated based on ROHs (FROH) and homozygosity (FHOM). Our result shows that a total of 7,248 ROH exceeding 1Mb were detected in 262 pigs. In addition, Sus scrofa chromosome (SSC) 8 and SSC10, respectively, has the highest and lowest chromosome coverage by ROH. These results suggest that inbreeding estimation based on total ROH may be a useful method, especially for crossbreed or composite populations. We also calculated an inbreeding coefficient of 0.077 from the total ROH. Eight ROH islands were found in this study. These ROH islands harbored genes associated with fat deposition, muscular development, reproduction, ear shape, and adaptation, such as TRAF7, IGFBP7, XPO1, SLC26A8, PPARD, and OR1F1. These findings may help to understand the effects of environmental and artificial selection on the genome structure of composite pigs. Our results provide a basis for subsequent genomic selection (GS), and provides a reference for the hybrid utilization of other pig breeds.

Proteomic and lipidomic analyses reveal saturated fatty acids, phosphatidylinositol, phosphatidylserine, and associated proteins contributing to intramuscular fat deposition.

Intramuscular fat (IMF) content is an important factor in porcine meat quality. Previous studies have screened multiple candidate genes related to IMF deposition, but the lipids that affect IMF deposition and their lipid-protein network remain unknown. In this study, we performed proteomic and lipidomic analyses of the longissimus dorsi (LD) muscle from high-IMF (IMFH) and low-IMF (IMF-L) groups of Xidu black pigs. Eighty-eight proteins and 143 lipids were differentially abundant between the groups. The differentially abundant proteins were found to be involved in cholesterol metabolism, the PPAR signaling pathway, and ferroptosis. The triacylglycerols (TAGs) upregulated in the IMF-H group were mainly shown to be synthesized by saturated fatty acids (SFAs), while the downregulated TAGs were mainly synthesized by polyunsaturated fatty acids (PUFAs). All differentially abundant phosphatidylinositols (PIs) and phosphatidylserines (PSs) were found to be upregulated in the IMF-H group. A correlation analysis of the proteomic and lipidomic revealed candidate proteins (APOA4, VDAC3, PRNP, CTSB, GSPT1) related to TAG, PI, and PS lipids. These results revealed differences in proteins and lipids between the IMF-H and IMF-L groups, which represent new candidate proteins and lipids that should be investigated to determine the molecular mechanisms controlling IMF deposition in pigs. SIGNIFICANCE: Intramuscular fat (IMF) is a key factor affecting meat quality, and meat with a higher IMF content can have a better flavor. In this study, proteomic results show that the ferroptosis pathway, including the PRNP, VDAC3 and CP proteins, affects IMF deposition. Lipid composition is the key factor affecting IMF deposition, but there are few reports on this. In this study, through lipidomic analysis, we suggest that saturated fatty acid (SFA), phosphatidylinositol (PI), and phosphatidylserine (PS) may contribute to IMF deposition. A correlation analysis reveals the potential regulatory network between lipids and proteins. This study clarifies the difference in protein and lipid compositions in longissimus dorsi (LD) muscle with high and low IMF contents. This information suggests that it would be beneficial to increase the intramuscular fat content of pork not only from a genetic perspective but also from a nutritional perspective.

Genome-wide analysis of long noncoding RNA and mRNA profiles in PRRSV-infected porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is one of the most globally devastating swine diseases. It is essential to develop new strategy to control PRRS via an understanding of mechanisms that PRRSV utilizes to interfere with the host's innate immunity. In this study, we deeply sequenced and analyzed long noncoding RNA (lncRNA) and mRNA expression profiles of the porcine alveolar macrophages (PAMs) after PRRSV infection. 126 lncRNAs and 753 mRNAs were differentially expressed between PRRSV-infected and control PAMs. The co-expressed genes of down-regulated lncRNAs were significantly enriched within NF-kappa B and toll-like receptor signaling pathways. Co-expression network analysis indicated that part of the dysregulated lncRNAs associated with the interferon-induced genes. These dysregulated lncRNAs may play an important role in the host's innate immune responses to PRRSV infection. However, further research is required to characterize the function of these lncRNAs.

MicroRNA transcriptome analysis of poly I:C-stimulated and PRRSV-infected porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe reproductive failure in sows, respiratory diseases, and high mortality in piglets, which results in serious economic losses to the swine industry worldwide. Previous studies have described that PRRSV could suppress the host immune system and had antiapoptotic activity in its initial phase of infection. Polyinosinic-polycytidylic acid (poly I:C), a synthesized analogue of viral double-strand RNA, activates innate immunity responses and induces apoptosis in cells. Therefore, we performed miRNA transcriptome analysis of poly I:C-stimulated and PRRSV-infected porcine alveolar macrophages (PAMs) using deep sequencing technology, to compare the different miRNA profiles between the statuses of innate immune activation and inactivation. After sequencing, 267 known mature miRNAs and 64 novel miRNAs were observed in PAMs, and a total of 197 miRNAs were significantly differently expressed in poly I:C-stimulated PAMs, compared with mock control cells. Thirty-three of them were also significantly alerted in PRRSV-infected PAMs. This indicated that PRRSV only slightly alerted the miRNA expression profile of host cells compared with poly I:C-stimulated PAMs, which confirmed that PRRSV could suppress host innate immune responses during the early stages of infection. Among the differentially expressed miRNAs, we found that ssc-miR-27b-3p could significantly inhibit PRRSV RNA and protein replication in MARC-145 cells and PAMs. Its antiviral mechanism needs further research in the future.

Transcription elements AREB6 and miR-34a affect apoptosis of PAMs by regulating the expression of SS2-related gene PPP1R11.

In our previous work, gene PPP1R11 (protein phosphatase 1 regulatory subunit 11) was significantly expressed in pigs after Streptococcus suis 2 (SS2) challenged. This study firstly confirmed that SS2 induced significant expression of PPP1R11 gene in porcine alveolar macrophage (PAM) cells, and apoptosis of PAM cells were observed. After that, the core promoter of porcine PPP1R11 was identified and its transcription factor AREB6 which significantly regulated PPP1R11. We also characterized that the PPP1R11 gene is a target of miR-34a. Further, we found that PPP1R11 helped to inhibit apoptosis of PAM cells under SS2 infecting, through transcription factor AREB6 was negatively correlated with apoptosis whereas miR-34a was positively correlated. Those findings provide a functional connection among the transcription factor AREB6, miR-34a, PPP1R11 gene and apoptosis of PAM cells in the pathogenesis of the SS2 infection.

Autophagy in Ovarian Follicular Development and Atresia.

Autophagy is a mechanism that exists in all eukaryotes under a variety of physiological and pathological conditions. In the mammalian ovaries, less than 1% of follicles ovulate, whereas the remaining 99% undergo follicular atresia. Autophagy and apoptosis have been previously found to be involved in the regulation of both primordial follicular development as well as atresia. The relationship between autophagy, follicular development, and atresia have been summarized in this review with the aim to obtain a more comprehensive understanding of the role played by autophagy in follicular development and atresia.

Genetic effect and combined genotype effect of ESR, FSHß, CTNNAL1 and miR-27a loci on litter size in a Large White population.

To select new Large White line with high number of piglets born, genotypes of estrogen receptor (ESR), the follicle stimulating hormone ß subunit (FSHß), catenin alpha like 1 (CTNNAL1) and miR-27a were tested in 472 Large White sows. The associations of different genotypes with litter size traits were also studied. The results showed ESRBB and FSHßBB sows produced 0.41-1.49 more pigs per litter (p < .05) for total number born (TNB) and number born alive (NBA) than did other corresponding genotypes. TNB of CTNNAL1CG sows is 0.50 more pigs per litter (p < .05) than that of CTNNAL1GG sows with the dominance effect of 0.25 pigs per litter (p < .05). miR-27aBB sows had a less estimated breeding value (EBV) to TNB and had a more number of mummified pigs (NM) than did miR-27aAA or miR-27aAB sows (p < .05). Therefore, ESRB, FSHßB, CTNNAL1G, miR-27aA allele was favorable for litter size traits. Furthermore, combined genetic effect analysis showed ESRAAFSHßBB, ESRAACTNNAL1CG, ESRAAmiR-27aAA, FSHßBBCTNNAL1CC, FSHßBBmiR-27aAA and CTNNAL1CG miR-27aAB was the favorable combined genotype for litter size traits. These results identified favorable alleles and genotypes for litter size traits and suggested a potential selection scheme for litter size in Large White pigs.

ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome.

Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications.

MicroRNA-144 is regulated by CP2 and decreases COX-2 expression and PGE2 production in mouse ovarian granulosa cells.

Mammalian folliculogenesis is a complex process in which primordial follicles develop into pre-ovulatory follicles, followed by ovulation to release mature oocytes. In this study, we explored the role of miR-144 in ovulation. miR-144 was one of the differentially expressed microRNAs, which showed 5.59-fold changes, in pre-ovulatory ovarian follicles between Large White and Chinese Taihu sows detected by Solexa deep sequencing. We demonstrated that overexpression of miR-144 significantly decreased the luciferase reporter activity under the control of the cyclooxygenase-2 (COX-2) or mothers against decapentaplegic homologue 4 (Smad4) 3'-untranslated region (3'-UTR) and suppressed COX-2 and Smad4 expression. In contrast, a miR-144 inhibitor increased COX-2 and Smad4 expression in mouse granulosa cells (mGCs). Meanwhile, Smad4 upregulated COX-2 expression, but this effect was abolished when the mGCs were treated with the transforming growth factor beta signalling pathway inhibitor SB431542. Moreover, luciferase reporter, chromatin immunoprecipitation and electrophoretic mobility shift assay results showed that the transcription factor CP2 upregulated miR-144 expression, which partially contributed to the suppression of COX-2 in mGCs. Both CP2 and miR-144 alter prostaglandin E2 (PGE2) production by regulating COX-2 expression. In addition, miR-144 regulated mGC apoptosis and affected follicular atresia, but these activities did not appear to be through COX-2 and Smad4. Taken together, we revealed an important CP2/miR-144/COX-2/PGE2/ovulation pathway in mGCs.

Phylogenomic analysis of transferrin family from animals and plants.

Transferrins have been identified in animals and green algae, and they consist of a family of evolutionarily related proteins that play a central role in iron transport, immunity, growth and differentiation. This study assessed the transferrin genes among 100 genomes from a wide range of animal and plant kingdoms. The results showed that putative transferrins were widespread in animals, but their gene quantity and type differ greatly between animal groups. Generally, Mammalia possess abundant transferrin genes, whereas Trematoda contain few ones. Melanotransferrin and serotransferrin are widely distributed in vertebrates, while melanotransferrin-like and transferrin-like 1 are frequent in invertebrates. However, only a few plant species detected putative transferrins, and a novel transferrin member was first uncovered in Angiospermae and Pteridophyta. The structural comparison among transferrin family members revealed seven very well-repeated and conserved characteristic motifs, despite a considerable variation in the overall sequences. The phylogenetic analysis suggested that gene duplication, gene loss and horizontal transfer contributed to the diversification of transferrin family members, and their inferred evolutionary scenario was proposed. These findings help to the understanding of transferrin distribution, characteristic motifs and residues, and evolutionary process.

The transcription factor ccaat/enhancer binding protein ß (C/EBPß) and miR-27a regulate the expression of porcine Dickkopf2 (DKK2).

Using Affymetrix porcine Gene-Chip analyses, we found that Dickkopf2 (DKK2), a WNT antagonist, is differentially expressed in pre-ovulatory follicles between Large White and Chinese Taihu sows. This study aims to identify the regulatory factors responsible for DKK2 expression. Deletion fragment and mutation analyses identified DKK2-D3 as the porcine DKK2 core promoter. There were four C/EBPß binding sites within the DKK2 core promoter. The C allele that results from a spontaneous alteration (DKK2 c.-1130 T > C) in the core promoter was associated with a higher total number born (TNB) and a higher number born alive (NBA) in all parities in a synthetic pig population. This was possibly the result of a change in C/EBPß binding ability, which was confirmed using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA). Moreover, C/EBPß specifically bound to and activated the DKK2 promoter, as revealed by mutation analysis, overexpression and RNA interference (RNAi) experiments. We also confirmed that miR-27a is a negative regulator of the DKK2 gene using miR-27a overexpression and inhibition experiments and mutation analyses. RTCA xCELLigence experiments showed that miR-27a suppressed Chinese hamster ovary (CHO) cell proliferation by down-regulating DKK2 gene expression. Taken together, our findings suggest that C/EBPß and miR-27a control DKK2 transcription.

Genome-wide identification of allele-specific expression in response to Streptococcus suis 2 infection in two differentially susceptible pig breeds.

Although allele expression imbalance has been recognized in many species, and strongly linked to diseases, no whole transcriptome allele imbalance has been detected in pigs during pathogen infections. The pathogen Streptococcus suis 2 (SS2) causes serious zoonotic disease. Different pig breeds show differential susceptibility/resistance to pathogen infection, but the biological insight is little known. Here we analyzed allele-specific expression (ASE) using the spleen transcriptome of four pigs belonging to two phenotypically different breeds after SS2 infection. The comparative analysis of allele specific SNPs between control and infected animals revealed 882 and 1096 statistically significant differentially expressed allele SNPs (criteria: ratio ≧ 2 or ≦ 0.5) in Landrace and Enshi black pig, respectively. Twenty nine allelically imbalanced SNPs were further verified by Sanger sequencing, and later six SNPs were quantified by pyrosequencing assay. The pyrosequencing results are in agreement with the RNA-seq results, except two SNPs. Looking at the role of ASE in predisposition to diseases, the discovery of causative variants by ASE analysis might help the pig industry in long term to design breeding programs for improving SS2 resistance.

MiR-506 inhibits PRRSV replication in MARC-145 cells via CD151.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine, which is caused by PRRS virus (PRRSV). CD151, one of PRRSV entry mediators, determines the cell susceptibility for PRRSV. Emerging evidence indicates that the host microRNAs (miRNAs) play key roles in modulating virus infection and viral pathogenesis. In the present study, targeting porcine CD151 miRNAs were identified, and their function during PRRSV infection in MARC-145 cells was further verified. We found that miR-506 could directly target porcine CD151 3'-UTR mRNA by luciferase reporter assay. Overexpression of miR-506 significantly decreased CD151 expression at both mRNA and protein levels. Furthermore, overexpression of miR-506 reduced cellular PRRSV replication and virus release in MARC-145 cells. Our results suggested that miR-506 could inhibit PRRSV replication by directly targeting PRRSV receptor of CD151 in MARC-145 cells. However, the molecular mechanisms of miR-506 and its function in vivo need further investigation.