Gut microbiota affects the estrus return of sows by regulating the metabolism of sex steroid hormones

Background Sex hormones play important roles in the estrus return of post-weaning sows.Previous studies have demonstrated a complex and bi-directional regulation between sex hormones and gut microbiota.However,the extent to which the gut microbiota affects estrus return of post-weaning sows is largely unknown.Results In this study,we first screened 207 fecal samples from well-phenotyped sows by 16S rRNA gene sequencing and identified significant associations between microbes and estrus return of post-weaning sows.Using metagenomic sequencing data from 85 fecal samples,we identified 37 bacterial species that were significantly associated with estrus return.Normally returning sows were characterized by increased abundances of L.reuteri and P.copri and decreased abundances of B.fragilis,S.suis,and B.pseudolongum.The changes in gut microbial composition significantly altered the functional capacity of steroid hormone biosynthesis in the gut microbiome.The results were confirmed in a validation cohort.Significant changes in sex steroid hormones and related compounds were found between normal and non-return sows via metabolome analysis.An integrated analysis of differential bacterial species,metagenome,and fecal metabolome provided evidence that normal return-associated bacterial species L.reuteri and Prevotella spp.participated in the degradation of pregnenolone,progesterone,and testosterone,thereby promoting estrogen biosynthesis.Furthermore,the microbial metabolites related to sow energy and nutrient supply or metabolic disorders also showed relationships with sow estrus return.Conclusions An integrated analysis of differentially abundant bacterial species,metagenome,and fecal metabolome revealed the involvement of L.reuteri and Prevotella spp.in sow estrus return.These findings provide deep insight into the role of gut microbiota in the estrus return of post-weaning sows and the complex cross-talk between gut microbiota and sex hormones,suggesting that the manipulation of the gut microbiota could be an effective strategy to improve sow estrus return after weaning.

Genetic dissection and genomic prediction for pork cuts and carcass morphology traits in pig

Background As pre-cut and pre-packaged chilled meat becomes increasingly popular,integrating the carcasscutting process into the pig industry chain has become a trend.Identifying quantitative trait loci(QTLs)of pork cuts would facilitate the selection of pigs with a higher overall value.However,previous studies solely focused on evaluating the phenotypic and genetic parameters of pork cuts,neglecting the investigation of QTLs influencing these traits.This study involved 17 pork cuts and 12 morphology traits from 2,012 pigs across four populations genotyped using CC1 PorcineSNP50 BeadChips.Our aim was to identify QTLs and evaluate the accuracy of genomic estimated breed values(GEBVs)for pork cuts.Results We identified 14 QTLs and 112 QTLs for 17 pork cuts by GWAS using haplotype and imputation genotypes,respectively.Specifically,we found that HMGA1,VRTN and BMP2 were associated with body length and weight.Subsequent analysis revealed that HMGA1 primarily affects the size of fore leg bones,VRTN primarily affects the number of vertebrates,and BMP2 primarily affects the length of vertebrae and the size of hind leg bones.The prediction accuracy was defined as the correlation between the adjusted phenotype and GEBVs in the validation population,divided by the square root of the trait’s heritability.The prediction accuracy of GEBVs for pork cuts varied from 0.342 to 0.693.Notably,ribs,boneless picnic shoulder,tenderloin,hind leg bones,and scapula bones exhibited prediction accuracies exceeding 0.600.Employing better models,increasing marker density through genotype imputation,and pre-selecting markers significantly improved the prediction accuracy of GEBVs.Conclusions We performed the first study to dissect the genetic mechanism of pork cuts and identified a large number of significant QTLs and potential candidate genes.These findings carry significant implications for the breeding of pork cuts through marker-assisted and genomic selection.Additionally,we have constructed the first reference populations for genomic selection of pork cuts in pigs.

Construction of a transposase accessible chromatin landscape reveals chromatin state of repeat elements and potential causal variant for complex traits in pigs

Background:A comprehensive landscape of chromatin states for multiple mammalian tissues is essential for elucidating the molecular mechanism underlying regulatory variants on complex traits.However,the genome-wide chromatin accessibility has been only reported in limited tissue types in pigs.Results:Here we report a genome-wide landscape of chromatin accessibility of 20 tissues in two female pigs at ages of 6 months using ATAC-seq,and identified 557,273 merged peaks,which greatly expanded the pig regulatory ele-ment repository.We revealed tissue-specific regulatory elements which were associated with tissue-relevant biologi-cal functions.We identified both positive and negative significant correlations between the regulatory elements and gene transcripts,which showed distinct distributions in terms of their strength and distances from corresponding genes.We investigated the presence of transposable elements(TEs)in open chromatin regions across all tissues,these included identifications of porcine endogenous retroviruses(PERVs)exhibiting high accessibility in liver and homology of porcine specific virus sequences to universally accessible transposable elements.Furthermore,we prior-itized a potential causal variant for polyunsaturated fatty acid in the muscle.Conclusions:Our data provides a novel multi-tissues accessible chromatin landscape that serve as an important resource for interpreting regulatory sequences in tissue-specific and conserved biological functions,as well as regula-tory variants of loci associated with complex traits in pigs.

Mapping short tandem repeats for liver gene expression traits helps prioritize potential causal variants for complex traits in pigs

Background:Short tandem repeats(STRs)were recently found to have significant impacts on gene expression and diseases in humans,but their roles on gene expression and complex traits in pigs remain unexplored.This study investigates the effects of STRs on gene expression in liver tissues based on the whole-genome sequences and RNA-Seq data of a discovery cohort of 260 F6 individuals and a validation population of 296 F7 individuals from a heterogeneous population generated from crosses among eight pig breeds.Results:We identified 5203 and 5868 significantly expression STRs(eSTRs,FDR<1%)in the F6 and F7 populations,respectively,most of which could be reciprocally validated(π1=0.92).The eSTRs explained 27.5%of the cisheritability of gene expression traits on average.We further identified 235 and 298 fine-mapped STRs through the Bayesian fine-mapping approach in the F6 and F7 pigs,respectively,which were significantly enriched in intron,ATAC peak,compartment A and H3K4me3 regions.We identified 20 fine-mapped STRs located in 100 kb windows upstream and downstream of published complex trait-associated SNPs,which colocalized with epigenetic markers such as H3K27ac and ATAC peaks.These included eSTR of the CLPB,PGLS,PSMD6 and DHDH genes,which are linked with genome-wide association study(GWAS)SNPs for blood-related traits,leg conformation,growth-related traits,and meat quality traits,respectively.Conclusions:This study provides insights into the effects of STRs on gene expression traits.The identified eSTRs are valuable resources for prioritizing causal STRs for complex traits in pigs.

Unravelling the genetic basis for skeletal muscle mitochondrial DNA copy number variations in pigs

Dear Editor,Mitochondria are the center of cellular energy generation and free radical metabolism,with important functions in regulating intracellular calcium levels,cell apoptosis,cell cycle and epigenetic modifications.Mitochondrial DNA copy number(mtDNA-CN)is an indicator of mitochondrial biogenesis and has been associated with several diseases,such as cardiovascular disease,depression,chronic kidney disease and aging in humans(Clyde,2022).

An atlas of expression quantitative trait loci of microRNAs in longissimus muscle of eight-way crossbred pigs

MicroRNAs(miRNAs)are key regulators of myocyte development and traits,yet insight into the genetic basis of variation in miRNA expression is still limited.Here,we present a systematic analysis of expression quantitative trait loci(eQTL)for miRNA profiling in longissimus muscle of pigs from an eight-breed crossed heterogeneous population.By integrating the whole-genome sequencing and miRNAomics data,we map 54 cis-and 292 trans-e QTLs at high resolution that are associated with the expression of 54 and 92miRNAs,respectively.Twenty-three trans-acting loci are identified to affect the expression of nine myomi Rs(known muscle-specific miRNAs).MiRNAs in mammalian conserved miRNA clusters are found to be subjected to regulation by shared cis-e QTLs,while the expression of mature miRNA-5p/-3p counterparts is more likely to be regulated by different cis-e QTLs.Fine mapping and bioinformatics analyses pinpoint the peak cis-e SNP of mi R-4331-5p,rs344650810,which is located in its seed region,as a causal variant for the changes in expression and function of this miRNA.Additionally,rs344650810 is significantly(P<0.01)correlated with the density and percentage of type I muscle fibers.Altogether,this study provides a comprehensive atlas of miRNA-e QTLs in porcine skeletal muscle and new insights into regulatory mechanisms of miRNA expression.

Developmental dynamics of the single nucleus regulatory landscape of pig hippocampus

The hippocampus is a brain region associated with memory,learning and spatial navigation,its aging-related dysfunction is a common sign of Alzheimer’s disease.Pig is a good model for human neurodegenerative disease,but our understanding of the regulatory program of the pig hippocampus and its cross-species conservation in humans remains limited.Here,we profiled chromatin accessibility in 33,409 high-quality nuclei and gene expression in 8,122 high-quality nuclei of the pig hippocampus at four postnatal stages.We identified 510,908 accessible chromatin regions(ACRs)in 12 major cell types,among which progenitor cells such as neuroblasts and oligodendrocyte progenitor cells showed a dynamic decrease from early to later developmental stages.We revealed significant enrichment of transposable elements in cell type-specific ACRs,particularly in neuroblasts.We identified oligodendrocytes as the most prominent cell type with the greatest number of genes that showed significant changes during the development.We identified ACRs and key transcription factors underlying the trajectory of neurogenesis(such as POU3F3 and EGR1)and oligodendrocyte differentiation(RXRA and FOXO6).We examined 27 Alzheimer’s disease-related genes in our data and found that 15 showed cell type-specific activity(TREM2,RIN3 and CLU),and 15 genes displayed age-associated dynamic activity(BIN1,RABEP1 and APOE).We intersected our data with human genome-wide association study results to detect neurological disease-associated cell types.The present study provides a single nucleusaccessible chromatin landscape of the pig hippocampus at different developmental stages and is helpful for the exploration of pigs as a biomedical model in human neurodegenerative diseases.

The combined genotypes effect of ESR and FSHβ genes on litter size traits in five different pig breeds

Estrogen receptor (ESR) and Follicular-stimula-ting hormone beta subunit (FSHβ) genes were chosen as candidates to determine whether they control litter size and some other reproductive traits in swine. 269 sows from five different pig breeds were genotyped by an established PCR-RFLPs protocol at both ESR and FSHβ loci. The effects of both ESR and FSHβ on pig reproductive traits, including total number born (TNB) and number born alive (NBA), are analyzed by SAS software (version 6.12). These computation results demonstrated that both ESR locus and FSHβ locus are the major genes influencing litter size in pigs. The sows of BBBB combined genotype of ESR and FSHβ loci generally produce 1.85-3.01 TNB and 2.0-3.0 NBA more than those of ABAA combined genotype. The notable effect of ESR locus and FSHβ locus on litter size of pigs have made it possible to improve the pig reproduction by Marker-assisted selection (MAS). Moreover, introgression of the beneficial alleles into commercial pig breeding lines,

Subcutaneous and intramuscular fat transcriptomes show large differences in network organization and associations with adipose traits in pigs

Subcutaneous fat(SCF)and intramuscular fat(IMF)deposition is relevant to health in humans,as well as meat production and quality in pigs.In this study,we generated RNA sequence data for 122 SCF,120 IMF,and 87 longissimus dorsi muscle(LDM)samples using 155 F6 pigs from a specially designed heterogeneous population generated by intercrossing four highly selected European commercial breeds and four indigenous Chinese pig breeds.The phenotypes including waist back fat thickness and intramuscular fat content were also measured in the 155 F6 pigs.We found that the genes in SCF and IMF differed largely in both expression levels and network connectivity,and highlighted network modules that exhibited strongest gain of connectivity in SCF and IMF,containing genes that were associated with the immune process and DNA double-strand repair,respectively.We identified 215 SCF genes related to kinase inhibitor activity,mitochondrial fission,and angiogenesis,and 90 IMF genes related to lipolysis and fat cell differentiation,displayed a tissue-specific association with back fat thickness and IMF content,respectively.We found that cis-expression QTL for trait-associated genes in the two adipose tissues tended to have tissuedependent predictability for the two adipose traits.Alternative splicing of genes was also found to be associated with SCF or IMF deposition,but the association was much less extensive than that based on expression levels.This study provides a better understanding of SCF and IMF gene transcription and network organization and identified critical genes and network modules that displayed tissue-specific associations with subcutaneous and intramuscular fat deposition.These features are helpful for designing breeding programs to genetically improve the two adipose traits in a balanced way.

Mapping and analysis of a spatiotemporal H3K27ac and gene expression spectrum in pigs

The limited knowledge of genomic noncoding and regulatory regions has restricted our ability to decipher the genetic mechanisms underlying complex traits in pigs. In this study, we characterized the spatiotemporal landscape of putative enhancers and promoters and their target genes by combining H3K27ac-targeted Ch IP-Seq and RNA-Seq in fetal(prenatal days 74–75) and adult(postnatal days 132–150) tissues(brain, liver, heart, muscle and small intestine) sampled from Asian aboriginal Bama Xiang and European highly selected Large White pigs of both sexes. We identified 101,290 H3K27ac peaks, marking 18,521promoters and 82,769 enhancers, including peaks that were active across all tissues and developmental stages(which could indicate safe harbor locus for exogenous gene insertion) and tissue-and developmental stage-specific peaks(which regulate gene pathways matching tissue-and developmental stage-specific physiological functions). We found that H3K27ac and DNA methylation in the promoter region of the XIST gene may be involved in X chromosome inactivation and demonstrated the utility of the present resource for revealing the regulatory patterns of known causal genes and prioritizing candidate causal variants for complex traits in pigs. In addition, we identified an average of 1,124 super-enhancers per sample and found that they were more likely to show tissue-specific activity than ordinary peaks. We have developed a web browser to improve the accessibility of the results(http://segtp.jxau.edu.cn/pencode/?genome=sus Scr11).

An imputed whole-genome sequence-based GWAS approach pinpoints causal mutations for complex traits in a specific swine population

Sequencing-based genome-wide association studies(GWAS) have facilitated the identification of causal associations between genetic variants and traits in diverse species. However, it is cost-prohibitive for the majority of research groups to sequence a large number of samples. Here, we carried out genotype imputation to increase the density of single nucleotide polymorphisms in a large-scale Swine F;population using a reference panel including 117 individuals, followed by a series of GWAS analyses. The imputation accuracies reached 0.89 and 0.86 for allelic concordance and correlation, respectively. A quantitative trait nucleotide(QTN) affecting the chest vertebrate was detected directly, while the investigation of another QTN affecting the residual glucose failed due to the presence of similar haplotypes carrying wild-type and mutant allelesin the reference panel used in this study. A high imputation accuracy was confirmed by Sanger sequencing technology for the most significant loci. Two candidate genes,CPNE5 and MYH3, affecting meat-related traits were proposed. Collectively, we illustrated four scenarios in imputation-based GWAS that may be encountered by researchers, and our results will provide an extensive reference for future genotype imputation-based GWAS analyses in the future.

Whole-genome sequence-based association study for immune cells in an eight-breed pig heterogeneous population

Pigs are an important domestic animal for meat production and animal models for biomedical research such as research on infectious diseases(Meurens et al.,2012).Immune cells,including T cells,B cells,monocytes,and macrophages,are among the major indicators of immune status in health and disease.Revealing the genetic basis of immune cell phenotypes would be helpful for improving the immunity of pigs genetically and reducing the disease occurrence,antibiotic usage.

Dissection of the genetic mechanisms underlying congenital anal atresia in pigs

Anal atresia is an innate disorder in the development of the distal hindgut in infants,which is caused by the failure of anal cloacal membrane to rupture due to the dysplasia of posterior intestine(Cuschieri,2001).The disorder shows a prevalence of0.03%-0.05%,of which patients with a family history account for 1%-9%of cases(Wood and Levitt,2018).

Revisiting the Evolutionary History of Pigs via De Novo Mutation Rate Estimation in A Three-generation Pedigree

The mutation rate used in the previous analyses of pig evolution and demographics was cursory and hence invited potential bias in inferring evolutionary history.Herein,we estimated the de novo mutation rate of pigs as 3.6×10-9 per base per generation using high-quality whole-genome sequencing data from nine individuals in a three-generation pedigree through stringent filtering and validation.Using this mutation rate,we re-investigated the evolutionary history of pigs.The estimated divergence time of~10 kiloyears ago(KYA)between European wild and domesticated pigs was consistent with the domestication time of European pigs based on archaeological evidence.However,other divergence events inferred here were not as ancient as previously described.Our estimates suggest that Sus speciation occurred~1.36 million years ago(MYA);European wild pigs split from Asian wild pigs only~219 KYA;and south and north Chinese wild pigs split~25 KYA.Meanwhile,our results showed that the most recent divergence event between Chinese wild and domesticated pigs occurred in the Hetao Plain,northern China,approximately 20 KYA,supporting the possibly independent domestication in northern China along the middle Yellow River.We also found that the maximum effective population size of pigs was~6 times larger than estimated before.An archaic migration from other Sus species originating~2 MYA to European pigs was detected during western colonization of pigs,which may affect the accuracy of previous demographic inference.Our de novo mutation rate estimation and its consequences for demographic history inference reasonably provide a new vision regarding the evolutionary history of pigs.

Detection of genomic signatures for pig hairlessness using high-density SNP data

Hair provides thermal regulation for mammals and protects the skin from wounds,bites and ultraviolet(UV)radiation,and is important in adaptation to volatile environments.Pigs in nature are divided into hairy and hairless,which provide a good model for deciphering the molecular mechanisms of hairlessness.We conducted a genomic scan for genetically differentiated regions between hairy and hairless pigs using 60K SNP data,with the aim to better understand the genetic basis for the hairless phenotype in pigs.A total of 38405 SNPs in 498 animals from 36 diverse breeds were used to detect genomic signatures for pig hairlessness by estimating between-population(FST)values.Seven diversifying signatures between Yucatan hairless pig and hairy pigs were identified on pig chromosomes(SSC)1,3,7,8,10,11 and 16,and the biological functions of two notable genes,RGS17 and RB1,were revealed.When Mexican hairless pigs were contrasted with hairypigs,strong signatures were detected on SSC1 and SSC10,which harbor two functionally plausible genes,REV3L and BAMBI.KEGG pathway analysis showed a subset of overrepresented genes involved in the T cell receptor signaling pathway,MAPK signaling pathway and the tight junction pathways.All of these pathways may be important in local adaptability of hairless pigs.The potential mechanisms underlying the hairless phenotype in pigs are reported for the first time.RB1 and BAMBI are interesting candidate genes for the hairless phenotype in Yucatan hairless and Mexico hairless pigs,respectively.RGS17,REV3L,ICOS and RASGRP1 as well as other genes involved in the MAPK and T cell receptor signaling pathways may be important in environmental adaption by improved tolerance to UV damage in hairless pigs.These findings improve our understanding of the genetic basis for inherited hairlessness in pigs.