Along with the development of high-throughput sequencing technologies, both sample size and SNP number are increasing rapidly in genome-wide association studies (GWAS), and the associated computation ...is more challenging than ever. Here, we present a memory-efficient, visualization-enhanced, and parallel-accelerated R package called “rMVP” to address the need for improved GWAS computation. rMVP can 1) effectively process large GWAS data, 2) rapidly evaluate population structure, 3) efficiently estimate variance components by Efficient Mixed-Model Association eXpedited (EMMAX), Factored Spectrally Transformed Linear Mixed Models (FaST-LMM), and Haseman-Elston (HE) regression algorithms, 4) implement parallel-accelerated association tests of markers using general linear model (GLM), mixed linear model (MLM), and fixed and random model circulating probability unification (FarmCPU) methods, 5) compute fast with a globally efficient design in the GWAS processes, and 6) generate various visualizations of GWAS-related information. Accelerated by block matrix multiplication strategy and multiple threads, the association test methods embedded in rMVP are significantly faster than PLINK, GEMMA, and FarmCPU_pkg. rMVP is freely available at https://github.com/xiaolei-lab/rMVP.
Although it has been widely applied in identification of genes responsible for biomedically, economically, or even evolutionarily important complex and quantitative traits, traditional candidate gene ...approach is largely limited by its reliance on the priori knowledge about the physiological, biochemical or functional aspects of possible candidates. Such limitation results in a fatal information bottleneck, which has apparently become an obstacle for further applications of traditional candidate gene approach on many occasions. While the identification of candidate genes involved in genetic traits of specific interest remains a challenge, significant progress in this subject has been achieved in the last few years. Several strategies have been developed, or being developed, to break the barrier of information bottleneck. Recently, being a new developing method of candidate gene approach, digital candidate gene approach (DigiCGA) has emerged and been primarily applied to identify potential candidate genes in some studies. This review summarizes the progress, application software, online tools, and challenges related to this approach.
Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, ...replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.
Implantation and placentation are critical steps for successful pregnancy. The pig has a non-invasive placenta and the uterine luminal epithelium is intact throughout pregnancy. To better understand ...the regulation mechanisms in functions of endometrium at three certain gestational stages that are critical for embryo/fetal loss in pigs, we characterized microRNA (miRNA) expression profiles in the endometrium on days 15 (implantation period), 26 (placentation period) and 50 (mid-gestation period) of gestation. The differentially expressed miRNAs across gestational days were detected and of which, 65 miRNAs were grouped into 4 distinct categories according to the similarities in their temporal expression patterns: (1) categories A and B contain majority of miRNAs (51 miRNAs, such as the miR-181 family) that were down- or up-regulated between gestational days 15 and 26, respectively; (2) categories C and D (14 miRNAs) consist miRNAs that were down- or up-regulated between gestational days 26 and 50, respectively. The expression patterns represented by eleven miRNAs were validated by qPCR. The majority of miRNAs were in categories A and B, suggesting that these miRNAs were involved in regulation of embryo implantation and placentation. The pathway analysis revealed that the predicted targets were involved in several pathways, such as focal adhesion, cell proliferation and tissue remolding. Furthermore, we identified that genes well-known to affect embryo implantation in pigs, namely SPP1, ITGB3 and ESR1, contain the miR-181a or miR-181c binding sites using the luciferase reporter system. The present study revealed distinctive miRNA expression patterns in the porcine endometrium during the implantation, placentation or mid-gestation periods. Additionally, our results suggested that miR-181a and miR-181c likely play important roles in the regulation of genes and pathways that are known to be involved in embryo implantation and placentation in pigs.
It is well known that the estrogen receptor alpha gene (
ESR1)
affects the reproductive traits of pigs; however, the immune role of
ESR1
gene has not yet been resolved. Here, we characterized the ...pleiotropic aspects of
ESR1
gene in immunity using the pig model. Tissue expression profile showed that the
ESR1
gene had a broad ectopic expression in multiple reproductive and immune-related tissues/organs, which provided the tissue-level spatial fundamental of
ESR1
gene that might function as a pleiotropic immune regulator. Using the peripheral blood cell model, a coupling transcriptome analytical strategy was proposed and verified that there existed strong positive or negative correlations of
ESR1
gene with hundreds of differentially expressed genes that were involved in the immune regulation, indicating that the
ESR1
gene might affect or be affected by, directly or indirectly, dozens of immune-related genes in the peripheral blood cells. Furthermore, the results of genetic association analysis showed that the
SmaI
-polymorphism of
ESR1
gene had significant or highly significant associations with multiple immune traits, including platelet (PLT), hematocrit (HCT), the number of CD4-CD8-CD3- cells, plateletcrit (PCT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC). Multiple evidences supported the immune pleiotropic roles of
ESR1
gene in pigs. The study advances our understanding of the cross-species immune pleiotropic landscape of
ESR1
gene and also provides a potential pleiotropic molecular marker for disease-resistant breeding in pigs.
The development and migration of T cells in the thymus and peripheral tissues are crucial for maintaining adaptive immunity in mammals. However, the regulatory mechanisms underlying T cell ...development and thymocyte identity formation in pigs remain largely underexplored.
Here, by integrating bulk and single-cell RNA-sequencing data, we investigated regulatory signatures of porcine thymus and lymph node T cells.
The comparison of T cell subpopulations derived from porcine thymus and lymph nodes revealed that their transcriptomic differences were influenced more by tissue origin than by T cell phenotypes, and that lymph node cells exhibited greater transcriptional diversity than thymocytes. Through weighted gene co-expression network analysis (WGCNA), we identified the key modules and candidate hub genes regulating the heterogeneity of T cell subpopulations. Further, we integrated the porcine thymocyte dataset with peripheral blood mononuclear cell (PBMC) dataset to systematically compare transcriptomic differences between T cell types from different tissues. Based on single-cell datasets, we further identified the key transcription factors (TFs) responsible for maintaining porcine thymocyte identity and unveiled that these TFs coordinately regulated the entire T cell development process. Finally, we performed GWAS of cell type-specific differentially expressed genes (DEGs) and 30 complex traits, and found that the DEGs in thymus-related and peripheral blood-related cell types, especially CD4_SP cluster and CD8-related cluster, were significantly associated with pig productive and reproductive traits.
Our findings provide an insight into T cell development and lay a foundation for further exploring the porcine immune system and genetic mechanisms underlying complex traits in pigs.
Improvement of the growth rate is a challenge in the pig industry, the Average Daily Gain (ADG) and Days (AGE) to 100 kg are directly related to growth performance. We performed genome-wide ...association study (GWAS) and genetic parameters estimation for ADG and AGE using the genomic and phonemic from four breed (Duroc, Yorkshire, Landrace, and Pietrain) populations. All analyses were performed by a multi-loci GWAS model, FarmCPU. The GWAS results of all four breeds indicate that five genome-wide significant SNPs were associated with ADG, and the nearby genomic regions explained 4.08% of the genetic variance and 1.90% of the phenotypic variance, respectively. For AGE, six genome-wide significant SNPs were detected, and the nearby genomic regions explained 8.09% of the genetic variance and 3.52% of phenotypic variance, respectively. In total, nine candidate genes were identified to be associated with growth and metabolism. Among them,
was reported to associate with pig growth,
were reported to associate with growth traits in human and mouse. The newly detected candidate genes will advance the understanding of growth related traits and the identification of the novel variants will suggest a potential use in pig genomic breeding programs.
Hematologic and biochemical data are useful for indicating disease diagnosis and growth performance in swine. However, the assessment of these parameters in healthy commercial pigs is rare in China. ...Thus, blood samples were collected from 107 nursery pigs and 87 sows and were analyzed for 25 hematologic and 14 biochemical variables. After the rejection of the outliers and the detection of the data distribution, the correlations between the blood parameters were analyzed and the hematologic/biochemical RIs were preliminarily established using the 95% percentile RI. Correlation analysis showed that albumin was the hub parameter among the blood parameters investigated, and genes overlapping with key correlated variables were discovered. Most of the hematologic and biochemical parameters were significantly different between nursery pigs and sows. The 95% RIs of white blood cells and red blood cells were 7.18–24.52 × 109/L and 5.62–7.84 × 1012/L, respectively, for nursery pigs, but 9.34–23.84 × 109/L and 4.98–8.29 × 1012/L for sows. The 95% RIs of total protein and albumin were 43.16–61.23 g/dL and 19.35–37.86 g/dL, respectively, for nursery pigs, but 64.96–88.68 g/dL and 31.91–43.28 g/dL for sows. In conclusion, our study highlights the variability in blood parameters between nursery pigs and sows and provides fundamental data for the health monitoring of commercial pigs in China.
Pigs are the most important source of meat and valuable biomedical models. However, the porcine immune system, especially the heterogeneity of CD8 T cell subtypes, has not been fully characterized. ...Here, using single-cell RNA sequencing, we identified 14 major cell types from peripheral blood circulating cells of pigs and observed remarkable heterogeneity among CD8 T cell types. Upon re-clustering of CD8
T cells, we defined four CD8 T cell subtypes and revealed their potential differentiation trajectories and transcriptomic differences among them. Additionally, we identified transcription factors with potential regulatory roles in maintaining CD8 T cell differentiation. The cell-cell communication analysis inferred an extensive interaction between CD8 T cells and other immune cells. Finally, cross-species analysis further identified species-specific and conserved cell types across different species. Overall, our study provides the first insight into the extensive functional heterogeneity and state transitions among porcine CD8 T cell subtypes in pig peripheral blood, complements the knowledge of porcine immunity, and enhances its potential as a biomedical model.
Heterosis has been extensively used for pig genetic breeding and production, but the genetic basis of heterosis remains largely elusive. Crossbreeding between commercial and native breeds provides a ...good model to parse the genetic basis of heterosis. This study uses Duhua hybrid pigs, a crossbreed of Duroc and Liangguang small spotted pigs, as materials to explore the genetic basis underlying heterosis related to growth traits at the genomic level. The mid-parent heterosis (MPH) analysis showed heterosis of this Duhua offspring on growth traits. In this study, we examined the impact of additive and dominance effects on 100 AGE (age adjusted to 100 kg) and 100 BF (backfat thickness adjusted to 100 kg) of Duhua hybrid pigs. Meanwhile, we successfully identified SNPs associated with growth traits through both additive and dominance GWASs (genome-wide association studies). These findings will facilitate the subsequent in-depth studies of heterosis in the growth traits of Duhua pigs.
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