ABSTRACT
Inbreeding can lead to the accumulation of homozygous single nucleotide polymorphisms (SNPs) in the genome, which can significantly affect gene expression and phenotype. In this study, we examined the impact of homozygous SNPs resulting from inbreeding on alternative polyadenylation (APA) site selection and the underlying genetic mechanisms using inbred Luchuan pigs. Genome resequencing revealed that inbreeding results in a high accumulation of homozygous SNPs within the pig genome. 3' mRNA-seq on leg muscle, submandibular lymph node, and liver tissues was performed to identify differences in APA events between inbred and outbred Luchuan pigs. We revealed different tissue-specific APA usage caused by inbreeding, which were associated with different biological processes. Furthermore, we explored the role of polyadenylation signal (PAS) SNPs in APA regulation under inbreeding and identified key genes such as PUM1, SCARF1, RIPOR2, C1D, and LRRK2 that are involved in biological processes regulation. This study provides resources and sheds light on the impact of genomic homozygosity on APA regulation, offering insights into genetic characteristics and biological processes associated with inbreeding.
ABSTRACT
BACKGROUND: The accurate identification of the functional elements in the bovine genome is a fundamental requirement for high-quality analysis of data informing both genome biology and genomic selection. Functional annotation of the bovine genome was performed to identify a more complete catalog of transcript isoforms across bovine tissues. RESULTS: A total of 160,820 unique transcripts (50% protein coding) representing 34,882 unique genes (60% protein coding) were identified across tissues. Among them, 118,563 transcripts (73% of the total) were structurally validated by independent datasets (PacBio isoform sequencing data, Oxford Nanopore Technologies sequencing data, de novo assembled transcripts from RNA sequencing data) and comparison with Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive data from different technologies such as whole transcriptome termini site sequencing, RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing. A large proportion of identified transcripts (69%) were unannotated, of which 86% were produced by annotated genes and 14% by unannotated genes. A median of two 5' untranslated regions were expressed per gene. Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as noncoding genes in fetal tissues but as protein-coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 annotated gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available quantitative trait loci data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. CONCLUSIONS: These validated results show significant improvement over current bovine genome annotations.
Subject(s)
Gene Expression Profiling , Genomics , Cattle/genetics , Animals , Sequence Analysis, RNA , Transcriptome , Quantitative Trait Loci , RNA , Protein Isoforms , Molecular Sequence AnnotationABSTRACT
Manipulation using alternative exon splicing (AES), alternative transcription start (ATS), and alternative polyadenylation (APA) sites are key to transcript diversity underlying health and disease. All three are pervasive in organisms, present in at least 50% of human protein-coding genes. In fact, ATS and APA site use has the highest impact on protein identity, with their ability to alter which first and last exons are utilized as well as impacting stability and translation efficiency. These RNA variants have been shown to be highly specific, both in tissue type and stage, with demonstrated importance to cell proliferation, differentiation and the transition from fetal to adult cells. While alternative exon splicing has a limited effect on protein identity, its ubiquity highlights the importance of these minor alterations, which can alter other features such as localization. The three processes are also highly interwoven, with overlapping, complementary, and competing factors, RNA polymerase II and its CTD (C-terminal domain) chief among them. Their role in development means dysregulation leads to a wide variety of disorders and cancers, with some forms of disease disproportionately affected by specific mechanisms (AES, ATS, or APA). Challenges associated with the genome-wide profiling of RNA variants and their potential solutions are also discussed in this review.
Subject(s)
Alternative Splicing , Polyadenylation , Humans , RNA, Messenger/genetics , Polyadenylation/genetics , Alternative Splicing/genetics , Exons , Cell DifferentiationABSTRACT
Since its discovery in 1991, genomic imprinting has been the subject of numerous studies into its mechanisms of establishment and regulation, evolution and function, and presence in multiple genomes. Disturbance of imprinting has been implicated in a range of diseases, ranging from debilitating syndromes to cancers to fetal deficiencies. Despite this, studies done on the prevalence and relevance of imprinting on genes have been limited in scope, tissue types available, and focus, by both availability and resources. This has left a gap in comparative studies. To address this, we assembled a collection of imprinted genes available in current literature covering five species. Here we sought to identify trends and motifs in the imprinted gene set (IGS) in three distinct arenas: evolutionary conservation, across-tissue expression, and health phenomics. Overall, we found that imprinted genes displayed less conservation and higher proportions of non-coding RNA while maintaining synteny. Maternally expressed genes (MEGs) and paternally expressed genes (PEGs) occupied distinct roles in tissue expression and biological pathway use, while imprinted genes collectively showed a broader tissue range, notable preference for tissue specific expression and limited gene pathways than comparable sex differentiation genes. Both human and murine imprinted genes showed the same clear phenotypic trends, that were distinct from those displayed by sex differentiation genes which were less involved in mental and nervous system disease. While both sets had representation across the genome, the IGS showed clearer clustering as expected, with PEGs significantly more represented than MEGs.
Subject(s)
Phenomics , Transcriptome , Humans , Animals , Mice , Transcriptome/genetics , Genomic Imprinting/genetics , Gene Expression Profiling , GenomicsABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious disease that affects the global pig industry. To understand mechanisms of susceptibility/resistance to PRRSV, this study profiled the time-serial white blood cells transcriptomic and serum metabolomic responses to PRRSV in piglets from a crossbred population of PRRSV-resistant Tongcheng pigs and PRRSV-susceptible Large White pigs. Gene set enrichment analysis (GSEA) illustrated that PRRSV infection up-regulated the expression levels of marker genes of dendritic cells, monocytes and neutrophils and inflammatory response, but down-regulated T cells, B cells and NK cells markers. CIBERSORT analysis confirmed the higher T cells proportion in resistant pigs during PRRSV infection. Resistant pigs showed a significantly higher level of T cell activation and lower expression levels of monocyte surface signatures post infection than susceptible pigs, corresponding to more severe suppression of T cell immunity and inflammatory response in susceptible pigs. Differentially expressed genes between resistant/susceptible pigs during the course of infection were significantly enriched in oxidative stress, innate immunity and humoral immunity, cell cycle, biotic stimulated cellular response, wounding response and behavior related pathways. Fourteen of these genes were distributed in 5 different QTL regions associated with PRRSV-related traits. Chemokine CXCL10 levels post PRRSV infection were differentially expressed between resistant pigs and susceptible pigs and can be a promising marker for susceptibility/resistance to PRRSV. Furthermore, the metabolomics dataset indicated differences in amino acid pathways and lipid metabolism between pre-infection/post-infection and resistant/susceptible pigs. The majority of metabolites levels were also down-regulated after PRRSV infection and were significantly positively correlated to the expression levels of marker genes in adaptive immune response. The integration of transcriptome and metabolome revealed concerted molecular events triggered by the infection, notably involving inflammatory response, adaptive immunity and G protein-coupled receptor downstream signaling. This study has increased our knowledge of the immune response differences induced by PRRSV infection and susceptibility differences at the transcriptomic and metabolomic levels, providing the basis for the PRRSV resistance mechanism and effective PRRS control.
Subject(s)
Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Animals , Swine , Porcine respiratory and reproductive syndrome virus/genetics , Porcine Reproductive and Respiratory Syndrome/genetics , Transcriptome , Immunity, Humoral , Adaptive Immunity/geneticsABSTRACT
Microplastic pollution in marine environments and organisms has received a great deal of international attention. However, the long-term field studies of microplastics are rare. Here, we assessed annual variation in microplastic abundance in the Maowei Sea, a classic mariculture bay in southern China, and analyzed the long-term accumulation in oyster tissues. U-shaped time trends of microplastics in water were observed from January to December in 2018 in the estuarine region, inner bay, and mouth bay sites, representing an inverse relationship with the local rainfall patterns. The common microplastic particles in Maowei Sea are PET/PE fibers, and polystyrene foams, which are mainly related to textile pollution and fishery activities. After one year of continuous monitoring, we did not find accumulation of microplastics in the whole soft tissues of oyster after 10% KOH digestion. No significant correlation of microplastic abundances between water and oysters was observed. The microplastic abundance in oyster was correlated with some environmental variables (i.e. salinity, pH, nutrients and total organic carbon) of the surrounding water following Spearman correlation analysis. The microplastic levels in oysters could probably be influenced by the environmental variables.
ABSTRACT
Because of their relatively short lifespan (<4 years), rats have become the second most used model organism to study health and diseases in humans who may live for up to 120 years. First-, second- and third-generation sequencing technologies and platforms have produced increasingly greater sequencing depth and accurate reads, leading to significant advancements in the rat genome assembly during the last 20 years. In fact, whole genome sequencing (WGS) of 47 strains have been completed. This has led to the discovery of genome variants in rats, which have been widely used to detect quantitative trait loci underlying complex phenotypes based on gene, haplotype, and sweep association analyses. DNA variants can also reveal strain, chromosome and gene functional evolutions. In parallel, phenome programs have advanced significantly in rats during the last 15 years and more than 10 databases host genome and/or phenome information. In order to discover the bridges between genome and phenome, systems genetics and integrative genomics approaches have been developed. On the other hand, multiple level information transfers from genome to phenome are executed by differential usage of alternative transcriptional start (ATS) and polyadenylation (APA) sites per gene. We used our own experiments to demonstrate how alternative transcriptome analysis can lead to enrichment of phenome-related causal pathways in rats. Development of advanced genome-to-phenome assays will certainly enhance rats as models for human biomedical research.
Subject(s)
Disease Models, Animal , Phenotype , Rats/genetics , Animals , Whole Genome SequencingABSTRACT
Accurate quantification of species fractions is critical to determine meat adulteration. This study aimed to develop a novel quantitative real-time PCR (qRT-PCR) method for detection of mammalian and poultry DNA. A shared single-copy nuclear DNA sequence derived from the first exon of the LcoR gene was identified as a multi-species universal reference for a qRT-PCR assay. The conservation and copy number of the LcoR gene were evaluated among different species. The limit of detection was 0.01 ng DNA or 0.01% meat ingredient, and the limit of quantification was 0.01 ng DNA or 0.05% meat ingredient. Both the relative error (R.E.) and relative standard deviation (R.S.D.) were ≤ 25%. Moreover, modified coefficient k was introduced into this quantitative system to improve the accuracy and reliability of results, with maximum R.E. improved from 19.43% to 16.16%. The quantitative method would contribute to fighting against meat adulteration and maintaining a fair market.
Subject(s)
Food Contamination/analysis , Mammals/genetics , Poultry/genetics , Real-Time Polymerase Chain Reaction/methods , Animals , Base Sequence , Cell Nucleus/genetics , DNA/analysis , DNA Primers , DNA, Complementary , Food Analysis/methods , Gene Dosage , Limit of Detection , Meat Products/analysis , Proteins/genetics , Repressor Proteins/genetics , Reproducibility of ResultsABSTRACT
Simple sequence repeats (SSRs) are commonly used as molecular markers in research on genetic diversity and discrimination among taxa or breeds because polymorphisms in these regions contribute to gene function and phenotypically important traits. In this study, we investigated genome-wide characteristics, repeat units, and polymorphisms of SSRs using sequencing data from SSR-enriched libraries created from Wuzhishan (WZS), Bama (BM), inbred Luchuan (LC) and Zangxiang (ZX) miniature pig breeds. The numbers and types of SSRs, distributions of repeat units and polymorphic SSRs varied among the four breeds. Compared to the Duroc pig reference genome, 2518 polymorphic SSRs were unique and common to all four breeds and functional annotation revealed that they may affect the coding and regulatory regions of genes. Several examples, such as FGF23, MYF6, IGF1R, and LEPROT, are associated with growth and development in pigs. Three of the polymorphic SSRs were selected to confirm the polymorphism and the corresponding alleles through fluorescence polymerase chain reaction (PCR) and capillary electrophoresis. Together, this study provides useful insights into the discovery, characteristics and distribution of SSRs in four pig breeds. The polymorphic SSRs, especially those common and unique to all four pig breeds, might affect associated genes and play important roles in growth and development.
ABSTRACT
RNA alternative polyadenylation contributes to the complexity of information transfer from genome to phenome, thus amplifying gene function. Here, we report the first X. tropicalis resource with 127,914 alternative polyadenylation (APA) sites derived from embryos and adults. Overall, APA networks play central roles in coordinating the maternal-zygotic transition (MZT) in embryos, sexual dimorphism in adults and longitudinal growth from embryos to adults. APA sites coordinate reprogramming in embryos before the MZT, but developmental events after the MZT due to zygotic genome activation. The APA transcriptomes of young adults are more variable than growing adults and male frog APA transcriptomes are more divergent than females. The APA profiles of young females were similar to embryos before the MZT. Enriched pathways in developing embryos were distinct across the MZT and noticeably segregated from adults. Briefly, our results suggest that the minimal functional units in genomes are alternative transcripts as opposed to genes.
Subject(s)
Amphibian Proteins/genetics , Genome , RNA, Messenger/genetics , Sex Characteristics , Transcriptome , Xenopus/genetics , Amphibian Proteins/metabolism , Animals , Embryo, Nonmammalian , Embryonic Development , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Ontology , Male , Molecular Sequence Annotation , Polyadenylation , RNA, Messenger/metabolism , Sex Factors , Exome Sequencing , Xenopus/growth & development , Xenopus/metabolism , Zygote/growth & development , Zygote/metabolismABSTRACT
Alternative polyadenylation is an essential RNA processing event that contributes significantly to regulation of transcriptome diversity and functional dynamics in both animals and plants. Here we review newly developed next generation sequencing methods for genome-wide profiling of alternative polyadenylation (APA) sites, bioinformatics pipelines for data processing and both wet and dry laboratory approaches for APA validation. The library construction methods LITE-Seq (Low-Input 3'-Terminal sequencing) and PAC-seq (PolyA Click sequencing) tag polyA+ cDNA, while BAT-seq (BArcoded, three-prime specific sequencing) and PAPERCLIP (Poly(A) binding Protein-mediated mRNA 3'End Retrieval by CrossLinking ImmunoPrecipitation) enrich polyA+ RNA. Interestingly, only WTTS-seq (Whole Transcriptome Termini Site sequencing) targets both polyA+ RNA and polyA+ cDNA. Varieties of bioinformatics pipelines are well established to pursue read quality control, mapping, clustering, characterization and pathway analysis. The RHAPA (RNase H alternative polyadenylation assay) and 3'RACE-seq (3' rapid amplification of cDNA end sequencing) methods directly validate APA sites, while WTSS-seq (whole transcriptome start site sequencing), RNA-seq (RNA sequencing) and public APA databases can serve as indirect validation methods. We hope that these tools, pipelines and resources trigger huge waves of interest in the research community to investigate APA events underlying physiological, pathological and psychological changes and thus understand the information transfer events from genome to phenome relevant to economically important traits in both animals and plants.
Subject(s)
Plants/genetics , Polyadenylation/physiology , 3' Untranslated Regions/genetics , Animals , DNA, Complementary/genetics , DNA, Complementary/metabolism , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Poly A/metabolism , Polyadenylation/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA , Transcriptome/geneticsABSTRACT
Evidence has shown that triphenyltin (TPT) triggers severe malformations in Xenopus tropicalis embryos, partly due to activation of PPARγ (peroxisome proliferator activated receptor γ) protein. In the present study, we investigated how abundance of pparγ and TPT exposure interact and affect X. tropicalis embryonic development. We observed pparγ expression signals appeared in the neural crest and neural fold, as well as in the brain, eyes and spinal cord organs. Both pparγ overexpression and its Morpholino (MO) knockdown inhibited pax6 (paired box 6) expression, a marker of eye development, and significantly up- and down-regulated lipid and glucose homeostasis related genes, such as lpl (lipoprotein lipase), slc2a4 (solute carrier family 2 (facilitated glucose transporter), member 4) and pck1 (phosphoenolpyruvate carboxykinase 1, cytosolic), thus inducing eye phenotypes. Overexpression of pparγ induced small eye phenotype, while pparγ MO induced small eye plus turbid eye lens microencephaly and enlarged trunk. In contrast, 5-20µgSn/L (stannum/L) TPT exposure reversed some impacts induced by pparγ overexpression, i.e., no small eye, up-regulation of pax6 and down-regulation of pparγ, lpl, slc2a4 and pck1. Meanwhile, microinjection of pparγ MO combined with exposure to 20µgSn/L TPT caused 85% mortality. In brief, our work clearly indicates that pparγ is essential to eye development and inhibition of its expression combined with TPT exposure can be extremely harmful to X. tropicalis embryo.
Subject(s)
Embryo, Nonmammalian/drug effects , Embryonic Development/drug effects , Mutagens/toxicity , Organotin Compounds/toxicity , PPAR gamma/metabolism , Xenopus/embryology , Animals , Embryo, Nonmammalian/physiology , Embryonic Development/physiologyABSTRACT
Currently available mouse knockout (KO) lines remain largely uncharacterized for genome-to-phenome (G2P) information flows. Here we test our hypothesis that altered myogenesis seen in AMPKα1- and AMPKα2-KO mice is caused by use of alternative polyadenylation sites (APSs). AMPKα1 and AMPKα2 are two α subunits of adenosine monophosphate-activated protein kinase (AMPK), which serves as a cellular sensor in regulation of many biological events. A total of 56,483 APSs were derived from gastrocnemius muscles. The differentially expressed APSs (DE-APSs) that were down-regulated tended to be distal. The DE-APSs that were related to reduced and increased muscle mass were down-regulated in AMPKα1-KO mice, but up-regulated in AMPKα2-KO mice, respectively. Five genes: Car3 (carbonic anhydrase 3), Mylk4 (myosin light chain kinase family, member 4), Neb (nebulin), Obscn (obscurin) and Pfkm (phosphofructokinase, muscle) utilized different APSs with potentially antagonistic effects on muscle function. Overall, gene knockout triggers genome plasticity via use of APSs, completing the G2P processes. However, gene-based analysis failed to reach such a resolution. Therefore, we propose that alternative transcripts are minimal functional units in genomes and the traditional central dogma concept should be now examined under a systems biology approach.
Subject(s)
AMP-Activated Protein Kinases/genetics , Polyadenylation/genetics , Transcriptome/genetics , AMP-Activated Protein Kinases/metabolism , Adenosine Monophosphate/metabolism , Animals , Genome/genetics , Genotype , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Development/genetics , Muscle, Skeletal/metabolism , Phenotype , Polyadenylation/physiologyABSTRACT
Porcine respiratory and reproductive syndrome (PRRS), caused by PRRS virus (PRRSV), is one of the most serious infectious diseases in the swine industry worldwide. Indigenous Chinese Tongcheng (TC) pigs reportedly show strong resistance to PRRSV infection. The miRNA expression profiles of porcine alveolar macrophages (PAMs) of control TC pigs and those infected with PRRSV in vivo were analyzed by high-throughput sequencing to explore changes induced by infection. A total of 182 known miRNAs including 101 miRNA-5p and 81 miRNA-3p were identified with 23 up-regulated differentially expressed miRNAs (DEmiRNAs) and 25 down-regulated DEmiRNAs. Gene Ontology analysis showed that predicted target genes for the DEmiRNAs were enriched in immune response, transcription regulation, and cell death. The integrative analysis of mRNA and miRNA expression revealed that down-regulated methylation-related genes (DNMT1 and DNMT3b) were targeted by five up-regulated DEmiRNAs. Furthermore, 35 pairs of miRNAs (70 miRNAs) were co-expressed after PRRSV infection and six pairs were co-expressed differently. Our results describe miRNA expression profiles of TC pigs in response to PRRSV infection and lay a strong foundation for developing novel therapies to control PRRS in pigs.
Subject(s)
Gene Expression Regulation/genetics , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/virology , MicroRNAs/genetics , Porcine Reproductive and Respiratory Syndrome/genetics , Animals , Gene Expression Profiling/methods , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/pathogenicity , SwineSubject(s)
Breeding , Fertility/genetics , Sheep, Domestic/genetics , Animals , Egypt , Female , Genetic Association Studies , Polymorphism, Single NucleotideABSTRACT
Construction of next-generation sequencing (NGS) libraries involves RNA manipulation, which often creates noisy, biased, and artifactual data that contribute to errors in transcriptome analysis. In this study, a total of 19 whole transcriptome termini site sequencing (WTTS-seq) and seven RNA sequencing (RNA-seq) libraries were prepared from Xenopus tropicalis adult and embryo samples to determine the most effective library preparation method to maximize transcriptomics investigation. We strongly suggest that appropriate primers/adaptors are designed to inhibit amplification detours and that PCR overamplification is minimized to maximize transcriptome coverage. Furthermore, genome annotation must be improved so that missing data can be recovered. In addition, a complete understanding of sequencing platforms is critical to limit the formation of false-positive results. Technically, the WTTS-seq method enriches both poly(A)+ RNA and complementary DNA, adds 5'- and 3'-adaptors in one step, pursues strand sequencing and mapping, and profiles both gene expression and alternative polyadenylation (APA). Although RNA-seq is cost prohibitive, tends to produce false-positive results, and fails to detect APA diversity and dynamics, its combination with WTTS-seq is necessary to validate transcriptome-wide APA.
Subject(s)
Gene Expression Profiling/methods , RNA, Messenger/chemistry , Transcriptome , Animals , Female , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , Sensitivity and Specificity , Sequence Analysis, RNA/methods , XenopusABSTRACT
Genetic polymorphisms, particularly single nucleotide polymorphisms (SNPs), have been widely used to advance quantitative, functional and evolutionary genomics. Ideally, all genetic variants among individuals should be discovered when next generation sequencing (NGS) technologies and platforms are used for whole genome sequencing or resequencing. In order to improve the cost-effectiveness of the process, however, the research community has mainly focused on developing genome-wide sampling sequencing (GWSS) methods, a collection of reduced genome complexity sequencing, reduced genome representation sequencing and selective genome target sequencing. Here we review the major steps involved in library preparation, the types of adapters used for ligation and the primers designed for amplification of ligated products for sequencing. Unfortunately, currently available GWSS methods have their drawbacks, such as inconsistency in the number of reads per sample library, the number of sites/targets per individual, and the number of reads per site/target, all of which result in missing data. Suggestions are proposed here to improve library construction, genotype calling accuracy, genome-wide marker density and read mapping rate. In brief, optimized GWSS library preparation should generate a unique set of target sites with dense distribution along chromosomes and even coverage per site across all individuals.
Subject(s)
Polymorphism, Single Nucleotide/genetics , Sequence Analysis, DNA/methods , Animals , Genotype , High-Throughput Nucleotide Sequencing , HumansABSTRACT
Copy number variations (CNVs) refer to large insertions, deletions and duplications in the genomic structure ranging from one thousand to several million bases in size. Since the development of next generation sequencing technology, several methods have been well built for detection of copy number variations with high credibility and accuracy. Evidence has shown that CNV occurring in gene region could lead to phenotypic changes due to the alteration in gene structure and dosage. However, it still remains unexplored whether CNVs underlie the phenotypic differences between Chinese and Western domestic pigs. Based on the read-depth methods, we investigated copy number variations using 49 individuals derived from both Chinese and Western pig breeds. A total of 3,131 copy number variation regions (CNVRs) were identified with an average size of 13.4 Kb in all individuals during domestication, harboring 1,363 genes. Among them, 129 and 147 CNVRs were Chinese and Western pig specific, respectively. Gene functional enrichments revealed that these CNVRs contribute to strong disease resistance and high prolificacy in Chinese domestic pigs, but strong muscle tissue development in Western domestic pigs. This finding is strongly consistent with the morphologic characteristics of Chinese and Western pigs, indicating that these group-specific CNVRs might have been preserved by artificial selection for the favored phenotypes during independent domestication of Chinese and Western pigs. In this study, we built high-resolution CNV maps in several domestic pig breeds and discovered the group specific CNVs by comparing Chinese and Western pigs, which could provide new insight into genomic variations during pigs' independent domestication, and facilitate further functional studies of CNV-associated genes.
Subject(s)
Chromosomes, Mammalian/genetics , DNA Copy Number Variations/genetics , Genome , Sus scrofa/genetics , Animals , Base Pairing/genetics , Breeding , Genetic Loci , Repetitive Sequences, Nucleic Acid , Reproducibility of ResultsABSTRACT
Whole transcriptome analysis plays an essential role in deciphering genome structure and function, identifying genetic networks underlying cellular, physiological, biochemical and biological systems and establishing molecular biomarkers that respond to diseases, pathogens and environmental challenges. Here, we review transcriptome analysis methods and technologies that have been used to conduct whole transcriptome shotgun sequencing or whole transcriptome tag/target sequencing analyses. We focus on how adaptors/linkers are added to both 5' and 3' ends of mRNA molecules for cloning or PCR amplification before sequencing. Challenges and potential solutions are also discussed. In brief, next generation sequencing platforms have accelerated releases of the large amounts of gene expression data. It is now time for the genome research community to assemble whole transcriptomes of all species and collect signature targets for each gene/transcript, and thus use known genes/transcripts to determine known transcriptomes directly in the near future.
Subject(s)
Gene Expression Profiling/methods , Sequence Analysis, RNA/methods , Animals , Genome/genetics , Humans , RNA, Messenger/genetics , Transcriptome/geneticsABSTRACT
Our previous microarray study revealed that S100A6 was significantly upregulated in porcine alveolar macrophages (PAMs) infected with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). In the present study, we cloned both cDNA and genomic DNA sequences of the gene. Transient transfection indicated that the porcine S100A6 protein was located in the nucleus and cytoplasm. Reverse transcription polymerase chain reaction (RT-PCR) revealed that the porcine S100A6 gene was highly expressed in the kidney and subcutaneous fat. Polyinosinic-polycytidylic acid [poly (I:C)] induced porcine S100A6 gene expression in PK-15 cells. Quantitative real-time PCR (Q-PCR) analysis further showed that the porcine S100A6 gene was upregulated in different cells and tissues of Tongcheng pigs infected with HP-PRRSV. Chromosome walking obtained the porcine S100A6 promoter region and then luciferase reporter assays confirmed its regulatory activities. We observed a putative NF-κB binding site in the core promoter region, which may explain the upregulation of porcine S100A6 in response to PRRSV. Transfection of NF-κB (p65 subunit) intensely induced the promoter activity of the porcine S100A6 gene, while an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), inhibited this activity. Furthermore, compared to its wild type, the promoter activity was significantly reduced when it contained a mutant NF-κB binding site. All these results provide a solid foundation to further investigate how S100A6 is involved in PRRSV infection.