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Widely used antipig CD4 monoclonal antibodies (mAbs) fail to recognize CD4 alleles characteristic of miniature pig lines such as the National Institutes of Health (NIH) miniature pigs and microminipigs. We surveyed polymorphisms in the coding sequence of the porcine CD4 gene among Western and Oriental pig breeds and Japanese wild boars and investigated their distribution. Of the 13 alleles that we identified among the 47 animals, 2 in group I and 3 in group II were found exclusively in Western breed pigs. Group IV alleles, which included mAb-nonbinding alleles, were found frequently in Oriental breed pigs, suggesting that the mAb-nonbinding allele arose from the gene pool of Oriental pigs. Group IV alleles were also found in Duroc and Large White pigs, suggesting genetic inflow from Oriental pig breeds into Western breeds. Comparison of the CD4 sequences of species in Cetartiodactyla suggested that the group IV alleles in Sus scrofa occurred before the divergence of this species from the other artiodactyls. The different antibody specificities of the various CD4 alleles may facilitate the discrimination of T-cell populations in transplantation studies using miniature pigs. The significance of the preservation of CD4 polymorphisms to immune function in pigs warrants further investigation.

Because of increasing litter size in Western pig breeds, additional teats are desirable to increase the capacity for nursing offspring. We applied genome-wide SNP markers to detect QTL regions that affect teat number in a Duroc population. We phenotyped 1024 animals for total teat number. A total of 36 588 SNPs on autosomes were used in the analysis. The estimated heritability for teat number was 0.34 ± 0.05 on the basis of a genomic relationship matrix constructed from all SNP markers. Using a BayesC method, we identified a total of 18 QTL regions that affected teat number in Duroc pigs; 9 of the 18 regions were newly detected., (© 2015 Stichting International Foundation for Animal Genetics.)

The two-nucleotide deletion recently detected in the mannose-binding lectin 2 gene in purebred and crossbred domestic pigs was not found among 68 wild boars representing 4 populations from Europe and Asia. This suggests that the deletion is a result of breeding and/or genetic drift/bottle necks., (© 2015 John Wiley & Sons Ltd.)

Nucleotide-binding oligomerization domain 1 (NOD1) is a cytosolic pattern recognition receptor that recognizes γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), a component of bacterial peptidoglycan. NOD1 is thought to be involved in the immune homeostasis mediated by intestinal microbiota as well as the host defense against infection. In this study, we identified 12 synonymous and nine nonsynonymous single nucleotide polymorphisms (SNPs) in the coding sequence of porcine NOD1 within major commercial breeds in the swine industry. Among the nonsynonymous SNPs, two amino-acid alterations located in the leucine-rich repeats region, glycine to glutamic acid at position 641 (G641E) and aspartic acid to asparagine at position 918 (D918N), impaired iE-DAP-induced activation of nuclear factor-κB. These alleles showed the recessive mode of inheritance and therefore are likely to be maintained in pig populations at high frequencies. These results suggest the possibility for improvement in disease resistance by eliminating the G641E and D918N alleles of NOD1 from commercial pig populations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The single nucleotide polymorphism (SNP) G949T in the mannose-binding lectin ( MBL ) 1 gene has been associated with low MBL-A concentration in serum and detected at different frequencies in various European pig populations. However, the origin of this SNP is not known. Part of the MBL1 gene was sequenced in 12 wild boar/Large White crossbred pigs from the second backcross (BC 2 ) generation in a family material originating from two wild boar x Large White intercrosses. Also, MBL-A serum concentration was measured in the entire BC 2 generation (n = 45). Furthermore, the genotypes of 68 wild boars from Sweden, Austria, the Czech Republic, and Japan were determined in regard to five previously described SNPs in MBL1 . The T allele of G949T was present among the BC 2 animals. MBL-A serum concentration in the BC 2 animals showed a bimodal distribution, with one-third of the animals at levels between 0.7 and 1.6 μg mL(-1) and the remaining pigs at levels around 13 μg mL(-1) . There was a co-variation between the presence of the T allele and low MBL-A concentration in serum. The genotyping of the wild boars revealed differences between populations. The T allele of G949T was not detected in the Austrian and Japanese samples and is thus unlikely to be an original feature of wild boars. In contrast, it was present at high frequency (0.35) among the Swedish wild boars, probably representing a founder effect. Five MBL1 haplotypes were resolved. Only two of these were present among the Japanese wild boars compared to four in each of the European populations. This difference may reflect differences in selection pressure and population history., (© 2012 Blackwell Publishing Ltd.)

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

Background: Along with the draft sequencing of the pig genome, which has been completed by an international consortium, collection of the nucleotide sequences of genes expressed in various tissues and determination of entire cDNA sequences are necessary for investigations of gene function. The sequences of expressed genes are also useful for genome annotation, which is important for isolating the genes responsible for particular traits., Results: We performed a large-scale expressed sequence tag (EST) analysis in pigs by using 32 full-length-enriched cDNA libraries derived from 28 kinds of tissues and cells, including seven tissues (brain, cerebellum, colon, hypothalamus, inguinal lymph node, ovary, and spleen) derived from pigs that were cloned from a sow subjected to genome sequencing. We obtained more than 330,000 EST reads from the 5'-ends of the cDNA clones. Comparison with human and bovine gene catalogs revealed that the ESTs corresponded to at least 15,000 genes. cDNA clones representing contigs and singlets generated by assembly of the EST reads were subjected to full-length determination of inserts. We have finished sequencing 31,079 cDNA clones corresponding to more than 12,000 genes. Mapping of the sequences of these cDNA clones on the draft sequence of the pig genome has indicated that the clones are derived from about 15,000 independent loci on the pig genome., Conclusions: ESTs and cDNA sequences derived from full-length-enriched libraries are valuable for annotation of the draft sequence of the pig genome. This information will also contribute to the exploration of promoter sequences on the genome and to molecular biology-based analyses in pigs.

The number of immunoglobulin heavy chain (IGH) constant genes (IGHC) varies among mammals. To annotate the porcine IGHC genes, we sequenced the entire IGHC-containing genomic region from a single porcine haplotype. The resulting contiguous sequence included in 5' the IGH diversity (D) gene cluster and in 3' TMEM121, which flank the IGHC cluster in the human genome, suggesting that we had obtained the entire genomic region containing porcine IGHC. This region was about 190-kb long, in good agreement with those of other mammals. The porcine IGHC cluster contained 10 genes, IGHM, IGHD, six IGHG genes, IGHE and IGHA. The porcine IGHG genes formed a cluster between IGHD and IGHE, with IGHG3 considered as the most ancient IGHG gene, located at the beginning of the IGHG cluster. Furthermore, the porcine sequence contained two IGHG5 and two IGHG6 genes, but no IGHG genes for IgG2 and IgG4, suggesting flexibility within the IGHG cluster. We also recorded structural differences in the switch regions of the IGHC genes that may be important in their transcription. This haplotype can serve as a reference for future studies on other haplotypes and for functional analysis of porcine immunoglobulin (IG) isotypes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, we identified porcine single nucleotide polymorphisms (SNPs) by aligning eight sequences generated with two approaches: amplification of 665 intronic regions using one sample from each of eight breeds, including three East Asian pigs, and amplification of 289 3'-UTR regions using two samples from each of four major commercial breeds. The 1,760 and 599 SNPs were validated using two 384-sample DNA panels by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The phylogenetic tree and Structure analyses classified the pigs into two large clusters: Euro-American and East Asian populations. The membership proportions, however, differed between inferred clusters for K = 2 generated by the two approaches. With intronic SNPs, Euro-American breeds constituted about 100% of the Euro-American cluster, but with 3'-UTR SNPs, about 17% of the East Asian cluster comprised five Euro-American breeds. The differences in the SNP discovery panels may affect population structure found in study panels of large samples.

Toll-like receptor 4 (TLR4) responds to lipid A, the active moiety of lipopolysaccharide from gram-negative bacteria, in cooperation with myeloid differentiation protein-2 and plays a vital role in innate immunity. Polymorphisms in TLR4 are associated with changes in susceptibility to various infectious diseases. We previously found seven amino acid polymorphisms in Sus scrofa TLR4. In this study, we showed by luciferase reporter assay that an alteration from cysteine to tryptophan at position 506 (C506W) caused loss of ability to induce nuclear factor-κB activation after lipid A stimulation. This polymorphism was found only in Japanese wild boar (JWB) populations of S. scrofa. Genotyping of TLR4 in different JWB populations revealed that C506W polymorphism was under pressure from purifying selection in a local population (Tajima's D=-0.98; p<0.05). However, in another population, this polymorphism existed at a frequency such that homozygous animals with the W506 alleles seldom appeared. These findings suggest that the C506W polymorphism is under different types of pressure by natural selection between populations, which may reflect differences in residential pathogens or demographic factors.

Salmonella enterica serovar Choleraesuis (SC) is a highly invasive pathogen that causes enteric and septicemic diseases in pigs. Although there have been some reports on gene expression profiles in the course of infection with SC in pigs, little is known about the genes involved in the infection. By measuring activation, as represented by nuclear factor-κB activity, after stimulation by the pathogen, we showed the involvement of Toll-like receptor (TLR) 5 and the TLR2-TLR1 heterodimer in the recognition of SC. We previously found single nucleotide polymorphisms (SNPs) in the TLRs of various pig populations. Here we demonstrated that the polymorphisms resulting in amino acid changes TLR5(R148L), TLR5(P402L), and TLR2(V703M) attenuated the responses to SC by the cells transfected with the TLR genes. Each of these three SNPs was differently restricted in distribution among breeds. These results suggest that there are differences in resistance to salmonellosis among breeds; these differences may be of great importance for the pig industry in terms of breeding and vaccine development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Nucleotide oligomerization domain 2 (NOD2) is a cytosolic pattern recognition receptor (PRR) that responds to muramyldipeptide (MDP), a component of peptidoglycans of gram positive and negative bacteria. NOD2 is involved in the modulation of signaling pathways for other PRRs, such as Toll-like receptors. Polymorphisms in NOD2 may evoke bowel disorders, and human Crohn's disease is significantly correlated with mis-sense insertion of the NOD2 gene. Such polymorphisms affecting ligand recognition in the NOD2 gene may also influence bowel flora in livestock, which is compromised by bowel diseases such as diarrhea. We investigated the functional variance of mis-sense polymorphisms in ligand recognition by porcine NOD2. The 1949T>C polymorphism, located in the region encoding the hinge domain of the molecule, notably diminished the functional response of porcine NOD2 to MDP. By comparison, the 2197A>C polymorphism, localized in the region corresponding to leucine-rich repeats, significantly augmented the response of porcine NOD2 to the ligand. The 1949C allele was rare among pig breeds, suggesting that this mutation is a disadvantage to pigs in their immune response to microbes. The 2197C allele, in contrast, was widely distributed among Western breeds and is most likely to be derived from wild boars in Asia. This is the first report of a causal relationship between molecular function and polymorphisms in PRRs in non-primate, non-rodent mammals. These findings suggest that the 2197C allele might confer an immune response advantage in modern pig breeds and may be a useful marker for breeding aimed at disease resistance in pigs.

Background: Viral genomic RNA-both single-stranded (ss) and double-stranded (ds)-is recognized by RNA-sensing Toll-like receptors (TLRs), notably TLR3 (dsRNA), TLR7 (ssRNA), and TLR8 (ssRNA). However, our knowledge of the roles of porcine TLR3, 7, and 8 in antiviral immunity is inadequate., Methods: From information on exon-intron boundaries obtained through comparisons of the genomic and cDNA sequences, polymorphisms in the coding sequences of each gene were detected in 84 male pigs of 11 breeds., Results: Genomic structures are conserved between pigs and humans. The RNA-sensing TLR genes had fewer polymorphisms causing amino acid alterations than did the cell-surface TLR genes, but the alterations were distributed with a similar bias toward ectodomains., Conclusions: The low level of diversity of substitutive polymorphisms in RNA-sensing TLRs than cell-surface ones implies that polymorphisms severely affecting function have been eliminated by selection pressure during longstanding pig breeding., General Significance: Recognition of virus-derived RNA is critical in host defense against infection. These results should provide a useful clue to analysis of the association between polymorphisms in RNA-sensing TLRs and disease resistance.

Pathogens localized extracellularly or incorporated into endosomes are recognized mainly by Toll-like receptors, whereas pathogens and pathogen-derived molecules that invade into the cytoplasm of host cells typically are recognized by intracellular pattern recognition receptors (PRRs), such as retinoic acid-inducible gene (RIG)-like helicases (RLHs) and nucleotide-binding oligmerization domain (NOD)-like receptors (NLRs). RIG-I and melanoma differentiation-associated gene 5 (MDA5), which belong to the RLH family, recognize viral genomic RNA, whereas NOD2, a member of the NLR family, responds to microbial peptidoglycans. These receptors may play an important role in pig opportunistic infectious diseases, such as pneumonia and diarrhea, which markedly impair livestock productivity, such that polymorphisms of these receptor genes are potential targets of pig breeding to increase disease resistance. Here, we report single nucleotide polymorphisms (SNPs) in porcine DDX58, IFIH1, and NOD2, which encode RIG-I, MDA5, and NOD2, respectively. Interestingly, compared with DDX58 and IFIH1, NOD2 abounded in nonsynonymous SNPs both throughout the coding sequence and in sequences encoding domains important for ligand recognition, such as helicase domains for RIG-I and MDA5 and leucine-rich repeats in NOD2. These differences in the distribution of SNPs in intracellular PRRs may parallel the diversity of their ligands, which include nucleic acids and peptidoglycans.

Our aim was to investigate microsatellite (MS) diversity and find crossover regions at 42 polymorphic MS loci in the swine leukocyte antigen (SLA) genomic region of 72 pigs with different well-defined homozygous and heterozygous SLA haplotypes. We analyzed the genetic polymorphisms of 42 MS markers in 23 SLA homozygous-heterozygous, common pig breeds with 12 SLA serological haplotypes and 49 National Institutes of Health (NIH) and Clawn homozygous-heterozygous miniature pigs with nine SLA serological or genotyped haplotypes including four recombinant haplotypes. In comparing the same and different haplotypes, both haplospecific patterns and allelic variations were observed at the MS loci. Some of the shared haplotype blocks extended over 2 Mb suggesting the existence of strong linkage disequilibrium (LD) in the entire SLA region. Crossover regions were easily defined by the MS markers within the class I and/or III region in the NIH and Clawn recombinant haplotypes. The present haplotype comparison shows that our set of MS markers provides a fast and cost-efficient alternative, or complementary, method to the serological or sequence-based determination of the SLA alleles for the characterization of SLA haplotypes and/or the crossover regions between different haplotypes.

We constructed a 5000-rad comprehensive radiation hybrid (RH) map of the porcine (Sus scrofa) genome and compared the results with the human genome. Of 4475 typed markers, 4016 (89.7%) had LOD >5 compared with the markers used in our previous RH map by means of two-point analysis and were grouped onto the 19 porcine chromosomes (SSCs). All mapped markers had LOD >3 as determined by RHMAPPER analysis. The current map comprised 430 microsatellite (MS) framework markers, 914 other MS markers, and 2672 expressed sequence tags (ESTs). The whole-genome map was 8822.1 cR in length, giving an average marker density of 0.342 Mb/cR. The average retention frequency was 35.8%. Using BLAST searches of porcine ESTs against the RefSeq human nucleotide and amino acid sequences (release 22), we constructed high-resolution comparative maps of each SSC and each human chromosome (HSA). The average distance between ESTs in the human genome was 1.38 Mb. SSC contained 50 human chromosomal syntenic groups, and SSC11, SSC12, and SSC16 were only derived from the HSA13q, HSA17, and HSA5 regions, respectively. Among 38 porcine terminal regions, we found that at least 20 regions have been conserved between the porcine and human genomes; we also found four paralogous regions for the major histocompatibility complex (MHC) on SSC7, SSC2, SSC4, and SSC1.

CD1 is an MHC class I-like protein that presents lipid antigens to T cell receptors. We determined 470,187 bp of the genomic sequence encompassing the region encoding porcine CD1 genes. We identified 16 genes in this region and newly identified CD1A2, CD1B, CD1C, CD1D, and CD1E. Porcine CD1 genes were located in clusters between KIRREL and olfactory receptor (OR) genes, as observed in humans, although they were divided into two regions by a region encoding OR genes. Comparison of the genomic sequences of CD1 gene loci in pigs with other mammals showed that separation of the CD1 gene cluster by ORs was observed only in pigs. CD1A duplication in the porcine genome was estimated to have occurred after the divergence of the human and porcine. This analysis of the genomic sequence of the porcine CD1 family will contribute to our understanding of the evolution of mammalian CD1 genes.

Toll-like receptors (TLRs) recognize various microbial components and play key roles in activating the innate immune system. Hence, their function is important in swine infectious diseases. We completely determined 173,804 bp of nucleotide sequence of a genomic region including porcine TLR6 and the newly identified porcine TLR homologues TLR1 and TLR10. The porcine genomic structure of these genes was highly conserved in comparison with the corresponding region in humans. Analysis of their expression in porcine tissues showed differences in expression patterns between porcine TLR10 and TLR1 or TLR6. Moreover, phylogenetic analysis of the cytoplasmic regions of TLR genes suggested that the signal transduction pathway of TLR10 was different from those of TLR1 and TLR6. We also developed six polymorphic microsatellite markers within this genomic region; these markers will be valuable for association studies between TLR genes and resistance or susceptibility to infectious diseases in swine.

Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.

Several quantitative trait loci (QTL) have been detected on SSC1qter (Sus scrofa chromosome 1qter), including QTL for the number of vertebrae, as reported in our previous study. To provide the tools for analysis of QTLs on SSC1qter, we constructed a comparative map of swine and human. In addition, we identified 26 swine STSs and mapped 16 of them on SSC1qter using the INRA - University of Minnesota porcine radiation hybrid (IMpRH) panel. We screened a BAC library using these swine STSs and developed 35 new polymorphic microsatellite markers from the BAC clones, of which 26 were informative in our reference family. We also mapped nine microsatellite markers we had isolated previously. Consequently a total of 44 new polymorphic microsatellite markers were located within a 60-cM region of SSC1qter, spanning from SW1092 to the telomere.

A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 microg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identity to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22-->q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-gamma production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs.

Comparative mapping studies facilitate the identification of genes located in quantitative trait locus (QTL) regions in domestic animals by utilizing information from the human genome. Radiation hybrid (RH) mapping is effective for this purpose because of its high resolution in ordered gene mapping on chromosomes. We constructed an RH map of pig chromosome 7, by adding 23 markers associated with genes. This RH map clearly demonstrated the mosaic of homology between pig chromosome 7 (SSC7) and human chromosomes 6, 14 and 15 at a 'gene' level, and was confirmed by linkage analysis. Clarification of the homology of SSC7 to human chromosomes will contribute to the elucidation of the gene(s) responsible for QTL detected on this chromosome.