[Show abstract][Hide abstract] ABSTRACT: Sterol regulatory element binding transcription factor (SREBF) is a key transcription regulator for lipid homeostasis. MicroRNA-33b (miR-33b) is embedded in intron 16 of porcine SREBF1 and is conserved among most mammals. Here, we investigated the effect of miR-33b on adipocyte differentiation and development in porcine subcutaneous pre-adipocytes (PSPA). PSPA were transiently transfected with miR-33b, and adipose differentiation was then induced. Delayed adipose differentiation and decreased lipid accumulation were observed in miR-33b-transfected PSPA. Computational predictions suggested that miR-33b may target early B cell factor 1 (EBF1), an adipocyte activator of lipogenesis regulators such as CCAAT-enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). Both gene and protein expression of EBF1 were downregulated in miR-33b-transfected PSPA, followed by considerable decreases in the expression of C/EBPα and PPARγ and their downstream lipogenic genes. However, miR-33b transfection did not markedly affect mRNA and protein expression of SREBF1. We also investigated differences in the expression of miR-33b and lipogenic genes in subcutaneous fat tissues between 5-month-old crossbred gilts derived from Landrace (lean-type) and Meishan (fatty-type) boars. Landrace-derived crossbred gilts expressed more miR-33b and less lipogenic genes than did gilts derived from Meishan. Our results suggest that miR-33b affected the differentiation and development of PSPA by attenuating the lipogenic gene expression cascade through EBF1 to C/EBPα and PPARγ. The differential expression of miR-33b observed in crossbred gilts may in part account for differences in lipogenic gene expression and the fat:lean ratio between pig breeds.
[Show abstract][Hide abstract] ABSTRACT: Vertnin (VRTN) is involved in the variation of vertebral number in pigs and it is located on Sus scrofa chromosome 7. Vertebral number is related to body size in pigs, and many reports have suggested presence of an association between body length (BL) and meat production traits. Therefore, we analyzed the relationship between the VRTN genotype and the production and body composition traits in purebred Duroc pigs. Intramuscular fat content (IMF) in the Longissimus muscle was significantly associated with the VRTN genotype. The mean IMF of individuals with the wild-type genotype (Wt/Wt) (5.22%) was greater than that of individuals with the Wt/Q (4.99%) and Q/Q genotypes (4.79%). In addition, a best linear unbiased predictor of multiple traits animal model showed that the Wt allele had a positive effect on the IMF breeding value. No associations were observed between the VRTN genotype and other production traits. The VRTN genotype was related to BL. The Q/Q genotype individuals (100.0 cm) were longer than individuals with the Wt/Q (99.5 cm) and Wt/Wt genotypes (98.9 cm). These results suggest that in addition to the maintenance of an appropriate backfat thickness value, VRTN has the potential to act as a genetic marker of IMF.
[Show abstract][Hide abstract] ABSTRACT: We performed a genome-wide association study using the porcine 60K SNP array to detect QTL regions for nine traits in a three-generational Duroc samples (n = 651), viz. generations 1, 2 and 3 from a population selected over five generations using a closed nucleus breeding scheme. We applied a linear mixed model for association mapping to detect SNP effects, adjusting for fixed effects (sex and season) and random polygenic effects (reflecting genetic relatedness), and derived a likelihood ratio statistic for each SNP using the efficient mixed-model association method. We detected a region on SSC6 for backfat thickness (BFT) and on SSC7 for cannon bone circumference (CANNON), with a genome-wide significance of P < 0.01 after Bonferroni correction. These regions had been detected previously in other pig populations. Six genes are located in the BFT-associated region, while the CANNON-associated region includes 66 genes. In the future, significantly associated SNPs, derived by sequencing the coding regions of the six genes in the BFT region, can be used in marker-assisted selection of BFT, whereas haplotypes constructed from the SSC7 region with strong LD can be used to select for the CANNON trait in our resource family.
[Show abstract][Hide abstract] ABSTRACT: Respiratory disease is the most important health concern for the swine industry. Genetic improvement for disease resistance is challenging because of the difficulty in obtaining good phenotypes related with disease resistance; however, identification of genes or markers associated with disease resistance can help in the genetic improvement of pig health. The purpose of our study was to investigate whether quantitative trait loci (QTL) associated with disease resistance were segregated in a purebred population of Landrace pigs that had been selected for meat production traits and mycoplasmal pneumonia of swine (MPS) scores over five generations. We analysed 1395 pigs from the base to the fifth generation of this population. Two respiratory disease traits [MPS scores and atrophic rhinitis (AR) scores] and 11 immune-capacity traits were measured in 630-1332 animals at 7 weeks of age and when the animal's body weight reached 105 kg. Each of the pigs, except sires in the base population, was genotyped using 109 microsatellite markers, and then, QTL analysis of the full-sib family population with a multi-generational pedigree structure was performed. Variance component analysis was used to detect QTL associated with MPS or AR scores, and the logarithm of odds (LOD) score and genotypic heritability of the QTL were estimated. Five significant (LOD > 2.51) and 18 suggestive (LOD > 1.35) QTL for respiratory disease traits and immune-capacity traits were detected. The significant QTL for Log-MPS score, located on S. scrofa chromosome 2, could explain 87% of the genetic variance of this score in this analysis. This is the first report of QTL associated with respiratory disease lesions.
[Show abstract][Hide abstract] ABSTRACT: We have collected more than 190000 porcine expressed sequence tags (ESTs) from full-length complementary DNA (cDNA) libraries and identified more than 2800 single nucleotide polymorphisms (SNPs). In this study, we tentatively chose 222 SNPs observed in assembled ESTs to study pigs of different breeds; 104 were selected by comparing the cDNA sequences of a Meishan pig and samples of three-way cross pigs (Landrace, Large White, and Duroc: LWD), and 118 were selected from LWD samples. To evaluate the genetic variation between the chosen SNPs from pig breeds, we determined the genotypes for 192 pig samples (11 pig groups) from our DNA reference panel with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Of the 222 reference SNPs, 186 were successfully genotyped. A neighbor-joining tree showed that the pig groups were classified into two large clusters, namely, Euro-American and East Asian pig populations. F-statistics and the analysis of molecular variance of Euro-American pig groups revealed that approximately 25% of the genetic variations occurred because of intergroup differences. As the F(IS) values were less than the F(ST) values(,) the clustering, based on the Bayesian inference, implied that there was strong genetic differentiation among pig groups and less divergence within the groups in our samples.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The number of vertebrae in pigs varies and is associated with body size. Wild boars have 19 vertebrae, but European commercial breeds for pork production have 20 to 23 vertebrae. We previously identified two quantitative trait loci (QTLs) for number of vertebrae on Sus scrofa chromosomes (SSC) 1 and 7, and reported that an orphan nuclear receptor, NR6A1, was located at the QTL on SSC1. At the NR6A1 locus, wild boars and Asian local breed pigs had the wild-type allele and European commercial-breed pigs had an allele associated with increased numbers of vertebrae (number-increase allele).
Here, we performed a map-based study to define the other QTL, on SSC7, for which we detected genetic diversity in European commercial breeds. Haplotype analysis with microsatellite markers revealed a 41-kb conserved region within all the number-increase alleles in the present study. We also developed single nucleotide polymorphisms (SNPs) in the 450-kb region around the QTL and used them for a linkage disequilibrium analysis and an association study in 199 independent animals. Three haplotype blocks were detected, and SNPs in the 41-kb region presented the highest associations with the number of vertebrae. This region encodes an uncharacterized hypothetical protein that is not a member of any other known gene family. Orthologs appear to exist not only in mammals but also birds and fish. This gene, which we have named vertnin (VRTN) is a candidate for the gene associated with variation in vertebral number. In pigs, the number-increase allele was expressed more abundantly than the wild-type allele in embryos. Among candidate polymorphisms, there is an insertion of a SINE element (PRE1) into the intron of the Q allele as well as the SNPs in the promoter region.
Genetic diversity of VRTN is the suspected cause of the heterogeneity of the number of vertebrae in commercial-breed pigs, so the polymorphism information should be directly useful for assessing the genetic ability of individual animals. The number-increase allele of swine VRTN was suggested to add an additional thoracic segment to the animal. Functional analysis of VRTN may provide novel findings in the areas of developmental biology.
[Show abstract][Hide abstract] ABSTRACT: While testing a quantitative trait locus (QTL) for pork color in a cross population of pigs from the mating of Large White dams to a Japanese wild boar, our laboratory discovered a candidate gene (NUDT7) that might affect heme biosynthesis in porcine muscle. Therefore, this experiment was designed to test the effect of NUDT7 on heme biosynthesis in cultured myoblasts. Rat L6 myoblasts were transfected with a mammalian expression vector for pig NUDT7 immediately after the induction of cell differentiation, and samples were harvested at 2, 4, 6, and 8 days. Expression of exogenous NUDT7 mRNA was highest on day 4, when the heme content was substantially lower (P<0.01) than that of the control (14.2 vs. 63.9 pmol/10(5) cells). These results suggest that overexpression of pig NUDT7 may be associated with heme biosynthesis downregulation in skeletal muscle, which may partially explain differences in meat color among breeds of livestock.
[Show abstract][Hide abstract] ABSTRACT: In the livestock industry, meat color has become important because consumer acceptance is subject to the appearance of the product in the marketplace. Our previous analyses of a whole genome QTL scan for various meat qualities using 2 F(2) families from Japanese wild boar (known as a red meat) x Large White and from Duroc x Chinese Jinhua suggested that a meat color (heme content) QTL is located on SSC6. The objective of this study was to fine-map this SSC6 meat color QTL and subsequently investigate positional candidate genes for polymorphisms that may cause changes in meat color. Therefore, we conducted interval mapping on SSC6 using an additional 9 gene markers through combined analyses of the 2 F(2) families of Japanese wild boar x Large White (353 progeny) and Duroc x Chinese Jinhua (204 progeny). Comparative analysis with humans, mice, and cattle suggested that there were 10 functional genes in the region. Among these genes, we suggested that a novel pig gene encoding a nudix (nucleoside diphosphate linked moiety X)-type motif 7 (NUDT7, a member of the nudix hydrolases) is a strong candidate for the QTL because the mouse Nudt7 is reported to hydrolyze succinyl-CoA, a substrate of the reaction limiting the rate of heme biosynthesis. We therefore determined the pig NUDT7 gene sequence including the 5' promoter region and explored genetic polymorphisms between Japanese wild boar and Large White. We identified 116 polymorphisms within the NUDT7 CDS or in the 5' region. None of the AA substitutions were associated with the meat color QTL; however, 3 polymorphisms were found in putative transcription factor recognition sites. We then investigated the differential expression of NUDT7 in Japanese wild boar and Large White by allele-specific quantitative real-time PCR. The expression level of the Large White type allele was greater than that of the Japanese wild-boar-type allele. Consequently, we speculated that the difference in meat color between Japanese wild boar and Large White is caused partly by differential expression of this candidate gene. Upregulation of NUDT7 expression in muscle may reduce succinyl-CoA content and thus reduce the level of heme biosynthesis.
Journal of Animal Science 09/2009; 88(1):23-31. · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herein, we report the variability among 57 porcine homologs of murine coat colour-related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F(2) family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F(2) family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig.
[Show abstract][Hide abstract] ABSTRACT: Mx, an interferon-inducible protein, is found in various vertebrates and confers resistance to several RNA viruses. At least two Mx proteins occur in vertebrates, and these proteins are key components of innate defense against viral infection. In mice and humans, the two Mx genes have different antiviral activities. Both Mx1 and Mx2 have also been detected in pigs, although only a partial sequence of porcine Mx2 has been reported, and there is no information on its antiviral activity. Here, we report the structure of the intact porcine Mx2 gene having an open reading frame of 2136 bp. We also determined the sequence of the genomic region containing the entire porcine Mx2 gene in addition to Mx1 gene. A weak constitutive expression of porcine Mx2 mRNA and endogenous Mx2 protein was observed in interferon-untreated cells. Porcine endogenous Mx2 protein showed nuclear localization. Furthermore, assays using NIH3T3 cells transfected with Mx genes showed that porcine Mx2 possessed antiviral activity against influenza, although this activity was lower than that of human MxA. This report is the first to describe the intact porcine Mx2 gene, which is a functional gene that may play a key role in the clearance of viruses in pigs.
[Show abstract][Hide abstract] ABSTRACT: Here, we report the variability in the KIT tyrosine kinase receptor and its ligand KITLG genes by determining single nucleotide polymorphisms (SNPs) in 384 individuals including 11 pig breeds, two synthetic-line cross pigs, two cross breeds, and one Japanese wild boar. SNPs and indels within the coding sequence regions of KITLG and KIT and their 5′-flanking regions were detected by aligning sequences from eight pigs, and subsequently the SNPs were genotyped using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS). Principal component analysis using allele frequencies in the SNP locus showed a distant relationship between Asian and Euro-American pig groups, except for Berkshire and Tokyo X breeds. These breeds were located within the mid-portion of the distribution in the first principal component. The Hampshire breed was distant from the other pig groups on the axis of the second principal component. Haplotype frequencies that were deduced using non-synonymous substitutions of the KIT gene revealed the uniqueness of Landrace, Large White, Middle White, and three-way cross pigs (LWD) and of the Hampshire breed. On the other hand, the haplotypes of KITLG and KIT detected in the Berkshire breed were prevalent in Asian pig groups. This tendency is different from that observed in other Euro-American pig breeds.
[Show abstract][Hide abstract] ABSTRACT: Dopachrome tautomerase (DCT) is an enzyme that isomerizes the melanogenic intermediate 3,4-dihydroxyphenylalanine-chrome (DOPAchrome) to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in the biosynthesis of melanin. In this study, we extracted the porcine bacterial artificial chromosome (BAC) clone containing the entire DCT gene and then sequenced it completely to determine its genomic structure. The BAC clone sequence was 127 098 bp in length. Compared to the DCT gene coding sequence determined in our previous study, the DCT gene structure determined in the current study comprised eight exons spanning 50.3 kbp of the genomic DNA, and the sequences from exon 7–9 of the glypican 6 (GCP6) gene were also detected in this clone. The gene order and the transcriptional orientation of the porcine genomic region in this BAC clone were identical to those of the corresponding human genomic region. In addition, we cloned three transcripts by using the 5′-rapid amplification of cDNA ends (RACE) method in combination with alternative promoter usage and alternative splicing. Two 3′-RACE products differed in the poly(A) addition site. Three 5′-RACE clones differed in exon 1 that codes for a signal peptide that is important for the cytoplasmic trafficking of premature DCT; however, the relationship between the alternative transcripts and pig coat color is unclear.
[Show abstract][Hide abstract] ABSTRACT: Genomic imprinting is interpreted as a phenomenon, in which some genes inherited from one parent are not completely expressed due to modification of the genome caused during gametogenesis. Subsequently, the expression level of an allele at the imprinted gene is changed dependent on the parental origin, which is referred to as the parent-of-origin effect. In livestock, some QTL for reproductive performance and meat productivity have been reported to be imprinted. So far, methods detecting imprinted QTL have been proposed on the basis of interval mapping, where only a single QTL was tested at a time. In this study, we developed a Bayesian method for simultaneously mapping multiple QTL, allowing the inference about expression modes of QTL in an outbred F2 family. The inference about whether a QTL is Mendelian or imprinted was made using Markov chain Monte Carlo estimation by comparing the goodness-of-fits between models, assuming the presence and the absence of parent-of-origin effect at a QTL. We showed by the analyses of simulated data sets that the Bayesian method can effectively detect both Mendelian QTL and imprinted QTL.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The number of vertebrae in pigs varies and is associated with meat productivity. Wild boars, which are ancestors of domestic pigs, have 19 vertebrae. In comparison, European commercial breeds have 21-23 vertebrae, probably owing to selective breeding for enlargement of body size. We previously identified two quantitative trait loci (QTL) for the number of vertebrae on Sus scrofa chromosomes (SSC) 1 and 7. These QTL explained an increase of more than two vertebrae. Here, we performed a map-based study to define the QTL region on SSC1. By using three F2 experimental families, we performed interval mapping and recombination analyses and defined the QTL within a 1.9-cM interval. Then we analyzed the linkage disequilibrium of microsatellite markers in this interval and found that 10 adjacent markers in a 300-kb region were almost fixed in European commercial breeds. Genetic variation of the markers was observed in Asian local breeds or wild boars. This region encoded an orphan nuclear receptor, germ cell nuclear factor (NR6A1, formerly known as GCNF), which contained an amino acid substitution (Pro192Leu) coincident with the QTL. This substitution altered the binding activity of NR6A1 to its corepressors, nuclear receptor-associated protein 80 (RAP80) and nuclear receptor corepressor 1 (NCOR1). In addition, somites of mouse embryos demonstrated expression of NR6A1 protein. Together, these results suggest that NR6A1 is a strong candidate for one of the QTL that influence number of vertebrae in pigs.
Genome Research 06/2007; 17(5):586-93. · 14.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.