Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics.

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

The genome variation and developmental transcriptome maps reveal genetic differentiation of skeletal muscle in pigs.

Natural and artificial directional selections have resulted in significantly genetic and phenotypic differences across breeds in domestic animals. However, the molecular regulation of skeletal muscle diversity remains largely unknown. Here, we conducted transcriptome profiling of skeletal muscle across 27 time points, and performed whole-genome re-sequencing in Landrace (lean-type) and Tongcheng (obese-type) pigs. The transcription activity decreased with development, and the high-resolution transcriptome precisely captured the characterizations of skeletal muscle with distinct biological events in four developmental phases: Embryonic, Fetal, Neonatal, and Adult. A divergence in the developmental timing and asynchronous development between the two breeds was observed; Landrace showed a developmental lag and stronger abilities of myoblast proliferation and cell migration, whereas Tongcheng had higher ATP synthase activity in postnatal periods. The miR-24-3p driven network targeting insulin signaling pathway regulated glucose metabolism. Notably, integrated analysis suggested SATB2 and XLOC_036765 contributed to skeletal muscle diversity via regulating the myoblast migration and proliferation, respectively. Overall, our results provide insights into the molecular regulation of skeletal muscle development and diversity in mammals.

CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.

Comprehensive Atlas of Alternative Splicing Reveals NSRP1 Promoting Adipogenesis through CCDC18.

Alternative splicing (AS) plays a crucial role in regulating gene expression, function, and diversity. However, limited reports exist on the identification and comparison of AS in Eastern and Western pigs. Here, we analyzed 243 transcriptome data from eight tissues, integrating information on transcription factors (TFs), selection signals, splicing factors (SFs), and quantitative trait loci (QTL) to comprehensively study alternative splicing events (ASEs) in pigs. Five ASE types were identified, with Mutually Exclusive Exon (MXE) and Skipped Exon (SE) ASEs being the most prevalent. A significant portion of genes with ASEs (ASGs) showed conservation across all eight tissues (63.21-76.13% per tissue). Differentially alternative splicing genes (DASGs) and differentially expressed genes (DEGs) exhibited tissue specificity, with blood and adipose tissues having more DASGs. Functional enrichment analysis revealed coDASG_DEGs in adipose were enriched in pathways associated with adipose deposition and immune inflammation, while coDASG_DEGs in blood were enriched in pathways related to immune inflammation and metabolism. Adipose deposition in Eastern pigs might be linked to the down-regulation of immune-inflammation-related pathways and reduced insulin resistance. The TFs, selection signals, and SFs appeared to regulate ASEs. Notably, ARID4A (TF), NSRP1 (SF), ANKRD12, IFT74, KIAA2026, CCDC18, NEXN, PPIG, and ROCK1 genes in adipose tissue showed potential regulatory effects on adipose-deposition traits. NSRP1 could promote adipogenesis by regulating alternative splicing and expression of CCDC18. Conducting an in-depth investigation into AS, this study has successfully identified key marker genes essential for pig genetic breeding and the enhancement of meat quality, which will play important roles in promoting the diversity of pork quality and meeting market demand.

How changes in landscape patterns affect the carbon emission: a case study in the Chengdu-Chongqing Economic Circle, China.

The construction of low-carbon cities is an optimal means to balance the competing interests of economic growth and carbon emission reduction. This study focuses on the optimization of land use patterns with a low carbon orientation, taking the Chengdu-Chongqing Economic Circle (CCEC), the fourth-largest economic growth pole in China, as an example. The panel data regression analysis is carried out to identify the dynamic correlations between the landscape changes and the carbon emission induced by land use and land cover change (LICE) of each city, each year, for the last 20 years. The results show that the CCEC has witnessed a 142.85% increase in carbon emissions during the period studied, with the growth of built-up land contributing 94% of total carbon emissions from 2000 to 2020. By constructing the panel regression model, this study finds that the intensity of carbon emissions increases significantly as the urban built-up land area and the agglomeration of artificial structures increase. The conversion of cropland, which dominates the landscape pattern, to built-up land has led to further fragmentation of the landscape pattern and a reduction in LPI, thus increasing carbon emissions. And a more complex regional landscape pattern will have a positive impact on carbon emission reduction. Based on the above findings, suggestions are articulated for carbon emission reduction.

A comprehensive epigenome atlas reveals DNA methylation regulating skeletal muscle development.

DNA methylation is important for the epigenetic regulation of gene expression and plays a critical role in mammalian development. However, the dynamic regulation of genome-wide DNA methylation in skeletal muscle development remains largely unknown. Here, we generated the first single-base resolution DNA methylome and transcriptome maps of porcine skeletal muscle across 27 developmental stages. The overall methylation level decreased from the embryo to the adult, which was highly correlated with the downregulated expression of DNMT1 and an increase in partially methylated domains. Notably, we identified over 40 000 developmentally differentially methylated CpGs (dDMCs) that reconstitute the developmental trajectory of skeletal muscle and associate with muscle developmental genes and transcription factors (TFs). The dDMCs were significantly under-represented in promoter regulatory regions but strongly enriched as enhancer histone markers and in chromatin-accessible regions. Integrative analysis revealed the negative regulation of both promoter and gene body methylation in genes associated with muscle contraction and insulin signaling during skeletal muscle development. Mechanistically, DNA methylation affected the expression of muscle-related genes by modulating the accessibly of upstream myogenesis TF binding, indicating the involvement of the DNA methylation/SP1/IGF2BP3 axis in skeletal myogenesis. Our results highlight the function and regulation of dynamic DNA methylation in skeletal muscle development.

MicroRNA-200c-5p Regulates Migration and Differentiation of Myoblasts via Targeting Adamts5 in Skeletal Muscle Regeneration and Myogenesis.

Skeletal muscle, as a regenerative organization, plays a vital role in physiological characteristics and homeostasis. However, the regulation mechanism of skeletal muscle regeneration is not entirely clear. miRNAs, as one of the regulatory factors, exert profound effects on regulating skeletal muscle regeneration and myogenesis. This study aimed to discover the regulatory function of important miRNA miR-200c-5p in skeletal muscle regeneration. In our study, miR-200c-5p increased at the early stage and peaked at first day during mouse skeletal muscle regeneration, which was also highly expressed in skeletal muscle of mouse tissue profile. Further, overexpression of miR-200c-5p promoted migration and inhibited differentiation of C2C12 myoblast, whereas inhibition of miR-200c-5p had the opposite effect. Bioinformatic analysis predicted that Adamts5 has potential binding sites for miR-200c-5p at 3'UTR region. Dual-luciferase and RIP assays further proved that Adamts5 is a target gene of miR-200c-5p. The expression patterns of miR-200c-5p and Adamts5 were opposite during the skeletal muscle regeneration. Moreover, miR-200c-5p can rescue the effects of Adamts5 in the C2C12 myoblast. In conclusion, miR-200c-5p might play a considerable function during skeletal muscle regeneration and myogenesis. These findings will provide a promising gene for promoting muscle health and candidate therapeutic target for skeletal muscle repair.

Integration of multi-omics data reveals cis-regulatory variants that are associated with phenotypic differentiation of eastern from western pigs.

BACKGROUND: The genetic mechanisms that underlie phenotypic differentiation in breeding animals have important implications in evolutionary biology and agriculture. However, the contribution of cis-regulatory variants to pig phenotypes is poorly understood. Therefore, our aim was to elucidate the molecular mechanisms by which non-coding variants cause phenotypic differences in pigs by combining evolutionary biology analyses and functional genomics. RESULTS: We obtained a high-resolution phased chromosome-scale reference genome with a contig N50 of 18.03 Mb for the Luchuan pig breed (a representative eastern breed) and profiled potential selective sweeps in eastern and western pigs by resequencing the genomes of 234 pigs. Multi-tissue transcriptome and chromatin accessibility analyses of these regions suggest that tissue-specific selection pressure is mediated by promoters and distal cis-regulatory elements. Promoter variants that are associated with increased expression of the lysozyme (LYZ) gene in the small intestine might enhance the immunity of the gastrointestinal tract and roughage tolerance in pigs. In skeletal muscle, an enhancer-modulating single-nucleotide polymorphism that is associated with up-regulation of the expression of the troponin C1, slow skeletal and cardiac type (TNNC1) gene might increase the proportion of slow muscle fibers and affect meat quality. CONCLUSIONS: Our work sheds light on the molecular mechanisms by which non-coding variants shape phenotypic differences in pigs and provides valuable resources and novel perspectives to dissect the role of gene regulatory evolution in animal domestication and breeding.

Superhydrophobic Paper-Based Microfluidic Field-Effect Transistor Biosensor Functionalized with Semiconducting Single-Walled Carbon Nanotube and DNAzyme for Hypocalcemia Diagnosis.

Hypocalcemia is caused by a sharp decline in blood calcium concentration after dairy cow calving, which can lead to various diseases or even death. It is necessary to develop an inexpensive, easy-to-operate, reliable sensor to diagnose hypocalcemia. The cellulose-paper-based microfluidic field-effect biosensor is promising for point-of-care, but it has poor mechanical strength and a short service life after exposure to an aqueous solution. Octadecyltrichlorosilane (OTS), as a popular organosilane derivative, can improve the hydrophobicity of cellulose paper to overcome the shortage of cellulose paper. In this work, OTS was used to produce the superhydrophobic cellulose paper that enhances the mechanical strength and short service life of MFB, and a microfluidic field-effect biosensor (MFB) with semiconducting single-walled carbon nanotubes (SWNTs) and DNAzyme was then developed for the Ca2+ determination. Pyrene carboxylic acid (PCA) attached to SWNTs through a non-covalent π-π stacking interaction provided a carboxyl group that can bond with an amino group of DNAzyme. Two DNAzymes with different sensitivities were designed by changing the sequence length and cleavage site, which were functionalized with SPFET/SWNTs-PCA to form Dual-MFB, decreasing the interference of impurities in cow blood. After optimizing the detecting parameters, Dual-MFB could determine the Ca2+ concentration in the range of 25 µM to 5 mM, with a detection limit of 10.7 µM. The proposed Dual-MFB was applied to measure Ca2+ concentration in cow blood, which provided a new method to diagnose hypocalcemia after dairy cow calving.

Landscape of tissue-specific RNA Editome provides insight into co-regulated and altered gene expression in pigs (Sus-scrofa).

RNA editing generates genetic diversity in mammals by altering amino acid sequences, miRNA targeting site sequences, influencing the stability of targeted RNAs, and causing changes in gene expression. However, the extent to which RNA editing affect gene expression via modifying miRNA binding site remains unexplored. Here, we first profiled the dynamic A-to-I RNA editome across tissues of Duroc and Luchuan pigs. The RNA editing events at the miRNA binding sites were generated. The biological function of the differentially edited gene in skeletal muscle was further characterized in pig muscle-derived satellite cells. RNA editome analysis revealed a total of 171,909 A-to-I RNA editing sites (RESs), and examination of its features showed that these A-to-I editing sites were mainly located in SINE retrotransposons PRE-1/Pre0_SS element. Analysis of differentially edited sites (DESs) revealed a total of 4,552 DESs across tissues between Duroc and Luchuan pigs, and functional category enrichment analysis of differentially edited gene (DEG) sets highlighted a significant association and enrichment of tissue-developmental pathways including TGF-beta, PI3K-Akt, AMPK, and Wnt signaling pathways. Moreover, we found that RNA editing events at the miRNA binding sites in the 3'-UTR of HSPA12B mRNA could prevent the miRNA-mediated mRNA downregulation of HSPA12B in the muscle-derived satellite (MDS) cell, consistent with the results obtained from the Luchuan skeletal muscle. This study represents the most systematic attempt to characterize the significance of RNA editing in regulating gene expression, particularly in skeletal muscle, constituting a new layer of regulation to understand the genetic mechanisms behind phenotype variance in animals.Abbreviations: A-to-I: Adenosine-to-inosine; ADAR: Adenosine deaminase acting on RNA; RES: RNA editing site; DEG: Differentially edited gene; DES: Differentially edited site; FDR: False discovery rate; GO: Gene Ontology; KEGG: Kyoto Encyclopaedia of Genes and Genomes; MDS cell: musclederived satellite cell; RPKM: Reads per kilobase of exon model in a gene per million mapped reads; UTR: Untranslated coding regions.

Comparison of skeletal muscle miRNA and mRNA profiles among three pig breeds.

The pig is an important source of animal protein, and is also an ideal model for human disease. There are significant differences in growth rate, muscle mass, and meat quality between different breeds. To understand the molecular mechanisms underlying porcine skeletal muscle phenotypes, we performed mRNA and miRNA profiling of muscle from three different breeds of pig, Landrace (lean-type), Tongcheng (obese-type), and Wuzhishan (mini-type) by Solexa sequencing. Forty-three genes and 106 miRNAs were differentially expressed between Landrace and Tongcheng pigs, 92 genes and 151 miRNAs were differentially expressed between Tongcheng and Wuzhishan pigs, and 145 genes and 156 miRNAs were differential expressed between Landrace and Wuzhishan pigs. Gene ontology analysis suggested that genes differentially expressed between Landrace and Tongcheng pigs were mainly involved in the biological processes of oxidative stress and muscle organ development. Meanwhile, for Tongcheng vs Wuzhishan and Landrace vs Wuzhishan pigs, the differentially expressed genes were involved in fatty acid metabolism, oxidative stress, muscle contraction, and muscle organ development, processes that are closely related to meat quality. To investigate the molecular mechanisms underlying meat quality diversity based on differentially expressed genes and miRNAs, interaction networks were constructed, according to target prediction results and integration analysis of up-regulated genes with down-regulated miRNAs or down-regulated genes with up-regulated miRNAs. Our findings identify candidate genes and miRNAs associated with muscle development and indicate their potential roles in muscle phenotype variance between different pig breeds. These results serve as a foundation for further studies on muscle development and molecular breeding.

SMAD7, an antagonist of TGF-beta signaling, is a candidate of prenatal skeletal muscle development and weaning weight in pigs.

SMAD7 promotes and enhances skeletal muscle differentiation by inhibiting transforming growth factor beta (TGF-ß)/activin signaling and bone morphogenetic protein (BMP) pathways. However, its function, the mechanism regulating its translation, and its association with production meat traits remain unclear in pigs. In this study, we explored SMAD7 gene spatio-temporal and tissue distribution, conducted a single nucleotide polymorphism association analysis, and examined regulation of its expression during skeletal muscle development. We found that SMAD7 was positively related to TGF-ß pathway genes and mainly expressed in prenatal developing muscle, and dual luciferase and western blot assays demonstrated that SMAD7 expression was regulated by miRNA-21 at the protein level via inhibition of mRNA translation. Finally, the association analysis showed that a single nucleotide mutation (Exon 4_28816;C/A) was significantly associated with the weaning weight of piglets among Yorkshire pigs. These data indicate that SMAD7 plays a potentially important role in mammalian prenatal skeletal muscle development and is a candidate gene for promoting greater weaning weight in pig breeding.

Wnt antagonist, secreted frizzled-related protein 1, is involved in prenatal skeletal muscle development and is a target of miRNA-1/206 in pigs.

BACKGROUND: The Wnt signaling pathway is involved in the control of cell proliferation and differentiation during skeletal muscle development. Secreted frizzled-related proteins (SFRPs), such as SFRP1, function as inhibitors of Wnt signaling. MicroRNA-1/206(miRNA-1/206) is specifically expressed in skeletal muscle and play a critical role in myogenesis. The miRNA-mRNA profiles and bioinformatics study suggested that the SFRP1 gene was potentially regulated by miRNA-1/206 during porcine skeletal muscle development. METHODS: To understand the function of SFRP1 and miRNA-1/206 in swine myogenesis, we first predicted the targets of miRNA-1/206 with the TargetScan and PicTar programs, and analyzed the molecular characterization of the porcine SFRP1 gene. We performed a temporal-spatial expression analysis of SFRP1 mRNA and miRNA-206 in Tongcheng pigs (a Chinese indigenous breed) by quantitative real-time polymerase chain reaction, and conducted the co-expression analyses of SFRP1 and miRNA-1/206. Subsequently, the interaction between SFRP1 and miRNA-1/206 was validated via dual luciferase and Western blot assays. RESULTS: The bioinformatics analysis predicted SFRP1 to be a target of miRNA-1/206. The expression level of the SFRP1 was highly varied across numerous pig tissues and it was down-regulated during porcine skeletal muscle development. The expression level of the SFRP1 was significantly higher in the embryonic skeletal compared with postnatal skeletal muscle, whereas miR-206 showed the inverse pattern of expression. A significant negative correlation was observed between the expression of miR-1/206 and SFRP1 during porcine skeletal muscle development (p <0.05). Dual luciferase assay and Western-blot results demonstrated that SFRP1 was a target of miR-1/206 in porcine iliac endothelial cells. CONCLUSIONS: Our results indicate that the SFRP1 gene is regulated by miR-1/206 and potentially affects skeletal muscle development. These findings increase understanding of the biological functions and the regulation of the SFRP1 gene in mammals.

A genome-wide scan for signatures of selection in Chinese indigenous and commercial pig breeds.

BACKGROUND: Modern breeding and artificial selection play critical roles in pig domestication and shape the genetic variation of different breeds. China has many indigenous pig breeds with various characteristics in morphology and production performance that differ from those of foreign commercial pig breeds. However, the signatures of selection on genes implying for economic traits between Chinese indigenous and commercial pigs have been poorly understood. RESULTS: We identified footprints of positive selection at the whole genome level, comprising 44,652 SNPs genotyped in six Chinese indigenous pig breeds, one developed breed and two commercial breeds. An empirical genome-wide distribution of Fst (F-statistics) was constructed based on estimations of Fst for each SNP across these nine breeds. We detected selection at the genome level using the High-Fst outlier method and found that 81 candidate genes show high evidence of positive selection. Furthermore, the results of network analyses showed that the genes that displayed evidence of positive selection were mainly involved in the development of tissues and organs, and the immune response. In addition, we calculated the pairwise Fst between Chinese indigenous and commercial breeds (CHN VS EURO) and between Northern and Southern Chinese indigenous breeds (Northern VS Southern). The IGF1R and ESR1 genes showed evidence of positive selection in the CHN VS EURO and Northern VS Southern groups, respectively. CONCLUSIONS: In this study, we first identified the genomic regions that showed evidences of selection between Chinese indigenous and commercial pig breeds using the High-Fst outlier method. These regions were found to be involved in the development of tissues and organs, the immune response, growth and litter size. The results of this study provide new insights into understanding the genetic variation and domestication in pigs.

Genome-wide scans to detect positive selection in Large White and Tongcheng pigs.

Due to the direction, intensity, duration and consistency of genetic selection, especially recent artificial selection, the production performance of domestic pigs has been greatly changed. Therefore, we reasoned that there must be footprints or selection signatures that had been left during domestication. In this study, with porcine 60K BeadChip genotyping data from both commercial Large White and local Chinese Tongcheng pigs, we calculated the extended haplotype homozygosity values of the two breeds using the long-range haplotype method to detect selection signatures. We found 34 candidate regions, including 61 known genes, from Large White pigs and 25 regions comprising 57 known genes from Tongcheng pigs. Many selection signatures were found on SSC1, SSC4, SSC7 and SSC14 regions in both populations. According to quantitative trait loci and network pathway analyses, most of the regions and genes were linked to growth, reproduction and immune responses. In addition, the average genetic differentiation coefficient FST was 0.254, which means that there had already been a significant differentiation between the breeds. The findings from this study can contribute to further research on molecular mechanisms of pig evolution and domestication and also provide valuable references for improvement of their breeding and cultivation.

Exploring Multi-Tissue Alternative Splicing and Skeletal Muscle Metabolism Regulation in Obese- and Lean-Type Pigs.

Alternative splicing (AS) is a crucial mechanism in post-transcriptional regulation, contributing significantly to the diversity of the transcriptome and proteome. In this study, we performed a comprehensive AS profile in nine tissues obtained from Duroc (lean-type) and Luchuan (obese-type) pigs. Notably, 94,990 AS events from 14,393 genes were identified. Among these AS events, it was observed that 80% belonged to the skipped exon (SE) type. Functional enrichment analysis showed that genes with more than ten AS events were closely associated with tissue-specific functions. Additionally, the analysis of overlap between differentially alternative splicing genes (DSGs) and differentially expressed genes (DEGs) revealed the highest number of overlapped genes in the heart and skeletal muscle. The novelty of our study is that it identified and validated three genes (PYGM, MAPK11 and CAMK2B) in the glucagon signaling pathway, and their alternative splicing differences were highly significant across two pig breeds. In conclusion, our study offers novel insights into the molecular regulation of diverse tissue physiologies and the phenotypic differences between obese- and lean-type pigs, which are helpful for pig breeding.

Transcription Factor SATB2 Regulates Skeletal Muscle Cell Proliferation and Migration via HDAC4 in Pigs.

Skeletal muscle development remarkably affects meat production and growth rate, regulated by complex regulatory mechanisms in pigs. Specific AT sequence-binding protein 2 (SATB2) is a classic transcription factor and chromatin organizer, which holds a profound effect in the regulation of chromatin remodeling. However, the regulation role of SATB2 concerning skeletal muscle cell fate through chromatin remodeling in pigs remains largely unknown. Here, we observed that SATB2 was expressed higher in the lean-type compared to the obese-type pigs, which also enriched the pathways of skeletal muscle development, chromatin organization, and histone modification. Functionally, knockdown SATB2 led to decreases in the proliferation and migration markers at the mRNA and protein expression levels, respectively, while overexpression SATB2 had the opposite effects. Further, we found histone deacetylase 4 (HDAC4) was a key downstream target gene of SATB2 related to chromatin remodeling. The binding relationship between SATB2 and HDAC4 was confirmed by a dual-luciferase reporter system and ChIP-qPCR analysis. Besides, we revealed that HDAC4 promoted the skeletal muscle cell proliferation and migration at the mRNA and protein expression levels, respectively. In conclusion, our study indicates that transcription factor SATB2 binding to HDAC4 positively contributes to skeletal muscle cell proliferation and migration, which might mediate the chromatin remodeling to influence myogenesis in pigs. This study develops a novel insight into understanding the molecular regulatory mechanism of myogenesis, and provides a promising gene for genetic breeding in pigs.

Haplotype-resolved assembly of a pig genome using single-sperm sequencing.

Single gamete cell sequencing together with long-read sequencing can reliably produce chromosome-level phased genomes. In this study, we employed PacBio HiFi and Hi-C sequencing on a male Landrace pig, coupled with single-sperm sequencing of its 102 sperm cells. A haplotype assembly method was developed based on long-read sequencing and sperm-phased markers. The chromosome-level phased assembly showed higher phasing accuracy than methods that rely only on HiFi reads. The use of single-sperm sequencing data enabled the construction of a genetic map, successfully mapping the sperm motility trait to a specific region on chromosome 1 (105.40-110.70 Mb). Furthermore, with the assistance of Y chromosome-bearing sperm data, 26.16 Mb Y chromosome sequences were assembled. We report a reliable approach for assembling chromosome-level phased genomes and reveal the potential of sperm population in basic biology research and sperm phenotype research.

Regulation of myo-miR-24-3p on the Myogenesis and Fiber Type Transformation of Skeletal Muscle.

Skeletal muscle plays critical roles in providing a protein source and contributing to meat production. It is well known that microRNAs (miRNAs) exert important effects on various biological processes in muscle, including cell fate determination, muscle fiber morphology, and structure development. However, the role of miRNA in skeletal muscle development remains incompletely understood. In this study, we observed a critical miRNA, miR-24-3p, which exhibited higher expression levels in Tongcheng (obese-type) pigs compared to Landrace (lean-type) pigs. Furthermore, we found that miR-24-3p was highly expressed in the dorsal muscle of pigs and the quadriceps muscle of mice. Functionally, miR-24-3p was found to inhibit proliferation and promote differentiation in muscle cells. Additionally, miR-24-3p was shown to facilitate the conversion of slow muscle fibers to fast muscle fibers and influence the expression of GLUT4, a glucose transporter. Moreover, in a mouse model of skeletal muscle injury, we demonstrated that overexpression of miR-24-3p promoted rapid myogenesis and contributed to skeletal muscle regeneration. Furthermore, miR-24-3p was found to regulate the expression of target genes, including Nek4, Pim1, Nlk, Pskh1, and Mapk14. Collectively, our findings provide evidence that miR-24-3p plays a regulatory role in myogenesis and fiber type conversion. These findings contribute to our understanding of human muscle health and have implications for improving meat production traits in livestock.

Characterizing core microbiota and regulatory functions of the pig gut microbiome.

Domestic pigs (Sus scrofa) are the leading terrestrial animals used for meat production. The gut microbiota significantly affect host nutrition, metabolism, and immunity. Hence, characterization of the gut microbial structure and function will improve our understanding of gut microbial resources and the mechanisms underlying host-microbe interactions. Here, we investigated the gut microbiomes of seven pig breeds using metagenomics and 16S rRNA gene amplicon sequencing. We established an expanded gut microbial reference catalog comprising 17 020 160 genes and identified 4910 metagenome-assembled genomes. We also analyzed the gut resistome to provide an overview of the profiles of the antimicrobial resistance genes in pigs. By analyzing the relative abundances of microbes, we identified three core-predominant gut microbes (Phascolarctobacterium succinatutens, Prevotella copri, and Oscillibacter valericigenes) in pigs used in this study. Oral administration of the three core-predominant gut microbes significantly increased the organ indexes (including the heart, spleen, and thymus), but decreased the gastrointestinal lengths in germ-free mice. The three core microbes significantly enhanced intestinal epithelial barrier function and altered the intestinal mucosal morphology, as was evident from the increase in crypt depths in the duodenum and ileum. Furthermore, the three core microbes significantly affected several metabolic pathways (such as "steroid hormone biosynthesis," "primary bile acid biosynthesis," "phenylalanine, tyrosine and tryptophan biosynthesis," and "phenylalanine metabolism") in germ-free mice. These findings provide a panoramic view of the pig gut microbiome and insights into the functional contributions of the core-predominant gut microbes to the host.