Detection of non-reference porcine endogenous retrovirus loci in the Vietnamese native pig genome.

The Vietnamese native pig (VnP)-a porcine breed with a small body-has proven suitable as a biomedical animal model. Here, we demonstrate that, compared to other breeds, VnPs have fewer copies of porcine endogenous retroviruses (PERVs), which pose a risk for xenotransplantation of pig organs to humans. More specifically, we sought to characterize non-reference PERVs (nrPERVs) that were previously unidentified in the reference genome. To this end, we used whole-genome sequencing data to identify nrPERV loci with long terminal repeat (LTR) sequences in VnPs. RetroSeq was used to estimate nrPERV loci based on the most current porcine reference genome (Sscrofa11.1). LTRs were detected using de novo sequencing read assembly near the loci containing the target site duplication sequences in the inferred regions. A total of 21 non-reference LTR loci were identified and separated into two subtypes based on phylogenetic analysis. Moreover, PERVs within the detected LTR loci were identified, the presence of which was confirmed using conventional PCR and Sanger sequencing. These novel loci represent previously unknown PERVs as they have not been identified in the porcine reference genome. Thus, our RetroSeq method accurately detects novel PERV loci, and can be applied for development of a useful biomedical model.

Hamiltonian Monte Carlo method for estimating variance components.

A Hamiltonian Monte Carlo algorithm is a Markov chain Monte Carlo method, and the method has a potential to improve estimating parameters effectively. Hamiltonian Monte Carlo is based on Hamiltonian dynamics, and it follows Hamilton's equations, which are expressed as two differential equations. In the sampling process of Hamiltonian Monte Carlo, a numerical integration method called leapfrog integration is used to approximately solve Hamilton's equations, and the integration is required to set the number of discrete time steps and the integration stepsize. These two parameters require some amount of tuning and calibration for effective sampling. In this study, we applied the Hamiltonian Monte Carlo method to animal breeding data and identified the optimal tunings of leapfrog integration for normal and inverse chi-square distributions. Then, using real pig data, we revealed the properties of the Hamiltonian Monte Carlo method with the optimal tuning by applying models including variance explained by pedigree information or genomic information. Compared with the Gibbs sampling method, the Hamiltonian Monte Carlo method had superior performance in both models. We have provided the source codes of this method written in the Fortran language at https://github.com/A-ARAKAWA/HMC.

The phenotypic characteristics and relational database for Vietnamese native pig populations.

Although there are a number of Vietnamese native pig (VnP) populations, some are on the verge of extinction, and therefore adequate management and conservation are necessary. In this study, we conducted a field survey of VnP populations and analyzed interrelationships among their characteristics. We also established a relational database for management of field data on these populations. For data collection, we conducted interviews with farmers and visual inspection of 32 VnP populations in 22 provinces of Vietnam, as well as taking photographs of individual animals. Data on the characteristics of VnP populations were subjected to multiple correspondence analysis (MCA). For establishment of the database, normalization and table partitioning were performed to eliminate redundancy and ensure consistency of the collected data items. Passport data, characteristics data, and image data were collected from a total of 1,918 VnPs and entered as a normalized table. Upon MCA, most of the populations were not separated from each other, but the Mong Cai, O Lam, and Chu Prong populations were separated from the other populations. Thus, we have constructed a relational database from comprehensive information on the characteristics of VnP populations.

Characteristic features of porcine endogenous retroviruses in Vietnamese native pigs.

We aimed to clarify the genomic characteristics of porcine endogenous retroviruses (PERVs) in Vietnamese native pig (VnP) breeds. First, we investigated genetic polymorphisms in ß- and γ-like PERVs, and we then measured the copy numbers of infectious γ-like PERVs (PERV-A, B, and C). We purified genomic DNA from 15 VnP breeds from 12 regions all over the country and three Western pig breeds as controls, and investigated genetic polymorphisms in all known PERVs, including the beta (ß)1-4 and gamma (γ)1-5 groups. PERVs of ß1, ß2, ß3, and γ4 were highly polymorphic with VnP-specific haplotypes. We did not identify genetic polymorphisms in ß4, γ1, or γ2 PERVs. We then applied a real-time polymerase chain reaction-based method to estimate copy numbers of the gag, pol, and env genes of γ1 PERVs (defined as A, B, and C). VnP breeds showed significantly lower copy number of the PERV genes compared with the Western pig breeds (on average, 16.2 and 35.7 copies, respectively, p < .05). Two VnP breeds showed significantly higher copy number compared with the other VnPs (p < .05). Our results elucidated that VnPs have specific haplotypes and a low copy number of PERV genes.

Differences in gene expression profiles for subcutaneous adipose, liver, and skeletal muscle tissues between Meishan and Landrace pigs with different backfat thicknesses.

Backfat thickness is one of the most important traits of commercially raised pigs. Meishan pigs are renowned for having thicker backfat than Landrace pigs. To examine the genetic factors responsible for the differences, we first produced female crossbred pig lines by mating Landrace (L) × Large White (W) × Duroc (D) females (LWD) with Landrace (L) or Meishan (M) boars (i.e., LWD × L = LWDL for Landrace offspring and LWD × M = LWDM for the Meishan offspring). We confirmed that LWDM pigs indeed had a thicker backfat than LWDL pigs. Next, we performed gene expression microarray analysis in both genetic lines to examine differentially expressed genes (DEGs) in energy metabolism-related tissues, subcutaneous adipose (fat), liver, and longissimus dorsi muscle tissues. We analyzed the annotation of DEGs (2-fold cutoff) to functionally categorize them by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The number of DEGs in muscle tissues of both lines was much less than that in fat and liver tissues, indicating that DEGs in muscle tissues may not contribute much to differences in backfat thickness. In contrast, several genes related to muscle (in fat tissue) and lipid metabolism (in liver tissue) were more upregulated in LWDM pigs than LWDL pigs, indicating that those DEGs might be responsible for differences in backfat thickness. The different genome-wide gene expression profiles in the fat, liver, and muscle tissues between genetic lines can provide useful information for pig breeders.

Effects of correcting missing daily feed intake values on the genetic parameters and estimated breeding values for feeding traits in pigs.

Daily feed intake (DFI) is an important consideration for improving feed efficiency, but measurements using electronic feeder systems contain many missing and incorrect values. Therefore, we evaluated three methods for correcting missing DFI data (quadratic, orthogonal polynomial, and locally weighted (Loess) regression equations) and assessed the effects of these missing values on the genetic parameters and the estimated breeding values (EBV) for feeding traits. DFI records were obtained from 1622 Duroc pigs, comprising 902 individuals without missing DFI and 720 individuals with missing DFI. The Loess equation was the most suitable method for correcting the missing DFI values in 5-50% randomly deleted datasets among the three equations. Both variance components and heritability for the average DFI (ADFI) did not change because of the missing DFI proportion and Loess correction. In terms of rank correlation and information criteria, Loess correction improved the accuracy of EBV for ADFI compared to randomly deleted cases. These findings indicate that the Loess equation is useful for correcting missing DFI values for individual pigs and that the correction of missing DFI values could be effective for the estimation of breeding values and genetic improvement using EBV for feeding traits.

Reproductive performance and expression of imprinted genes in somatic cell cloned boars.

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin-like growth factor -2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot-and-mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic.

Preservation and Reproduction of Microminipigs by Cloning Technology.

BACKGROUND/AIM: Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology. MATERIALS AND METHODS: Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded. RESULTS AND CONCLUSION: Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity.

Effect of candidate gene polymorphisms on reproductive traits in a Large White pig population.

The objective of this study was to test for association of candidate single nucleotide polymorphisms (SNPs) with sow prolificacy reproductive traits, such as litter size, ovulation rate and lifetime performance, in gilts of a Large White pig population. Preliminary research on 25 animals selected from the high- and low-performance groups of 347 animals with case-control studies indicated that seven genes were associated with total number of piglets born (TNB). Six of the seven genes were associated with reproductive traits, including TNB, number of piglets born alive (NBA) and average weight of piglet weaning (AWW). A MBL2 SNP was significantly associated with TNB and NBA in first parity. A CFB SNP was associated with TNB in first parity. An ACE SNP was associated with TNB in first and second parities. An EGF polymorphism was associated with TNB, NBA and AWW in second parity. A KCNC2 polymorphism was significantly associated with TNB and NBA in second parity. A SLC22A5 SNP was associated with TNB and NBA in second parity. Six candidate SNPs were associated with TNB; the only exception was a PRKAG3 polymorphism. A candidate gene approach enables some of these polymorphisms to be used in genetic improvement programs based on marker-assisted selection.

Variational bayesian method of estimating variance components.

We developed a Bayesian analysis approach by using a variational inference method, a so-called variational Bayesian method, to determine the posterior distributions of variance components. This variational Bayesian method and an alternative Bayesian method using Gibbs sampling were compared in estimating genetic and residual variance components from both simulated data and publically available real pig data. In the simulated data set, we observed strong bias toward overestimation of genetic variance for the variational Bayesian method in the case of low heritability and low population size, and less bias was detected with larger population sizes in both methods examined. The differences in the estimates of variance components between the variational Bayesian and the Gibbs sampling were not found in the real pig data. However, the posterior distributions of the variance components obtained with the variational Bayesian method had shorter tails than those obtained with the Gibbs sampling. Consequently, the posterior standard deviations of the genetic and residual variances of the variational Bayesian method were lower than those of the method using Gibbs sampling. The computing time required was much shorter with the variational Bayesian method than with the method using Gibbs sampling.

Genomic structural analysis of porcine fatty acid desaturase cluster on chromosome 2.

Fatty acid composition is an economically important trait in meat-producing livestock. To gain insight into the molecular genetics of fatty acid desaturase (FADS) genes in pigs, we investigated the genomic structure of the porcine FADS gene family on chromosome 2. We also examined the tissue distribution of FADS gene expression. The genomic structure of FADS family in mammals consists of three isoforms FADS1, FADS2 and FADS3. However, porcine FADS cluster in the latest pig genome assembly (Sscrofa 10.2) containing some gaps is distinct from that in other mammals. We therefore sought to determine the genomic structure, including the FADS cluster in a 200-kbp range by sequencing gap regions. The structure we obtained was similar to that in other mammals. We then investigated the porcine FADS1 transcription start site and identified a novel isoform named FADS1b. Phylogenetic analysis revealed that the three members of the FADS cluster were orthologous among mammals, whereas the various FADS1 isoforms identified in pigs, mice and cattle might be attributable to species-specific transcriptional regulation with alternative promoters. Porcine FADS1b and FADS3 isoforms were predominantly expressed in the inner layer of the subcutaneous adipose tissue. Additional analyses will reveal the effects of these functionally unknown isoforms on fatty acid composition in pig fat tissues.

MicroRNA-33b downregulates the differentiation and development of porcine preadipocytes.

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.

Evaluation of effects of multiple candidate genes (LEP, LEPR, MC4R, PIK3C3, and VRTN) on production traits in Duroc pigs.

We evaluated multiple effects of genetic variations of five candidate loci (LEP, LEPR, MC4R, PIK3C3 and VRTN) on four production traits (average daily weight gain (ADG); backfat thickness (BFT); loin eye muscle area (EMA); and intramuscular fat content (IMF)) in a closed nucleus herd of pure Duroc pigs. Polymorphisms in LEPR, MC4R and PIK3C3 had significant single gene effects on ADG and BFT. The additive genetic variance in ADG and BFT (16.99% and 22.51%, respectively) was explained by genetic effects of these three loci. No correlations were observed between the LEP genotype and production traits in this study. Although we detected marginally epistatic interactions between LEPR and PIK3C3 on the eye muscle area, there were no significant epistatic effects on any traits among all loci pairs. These results suggest that LEPR, MC4R, PIK3C3 and VRTN may independently influence growth rate and fat deposition. Furthermore, the statistical models for predicting the breeding values of each trait had the lowest Akaike's information criterion values when considering the effect of the MC4R, LEPR, PIK3C3 and VRTN genotype simultaneously. These results suggest that LEPR, MC4R, PIK3C3 and VRTN are useful markers for accurately predicting breeding values in Duroc pigs.

Genomic regions affecting backfat thickness and cannon bone circumference identified by genome-wide association study in a Duroc pig population.

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.

Association of swine vertnin (VRTN) gene with production traits in Duroc pigs improved using a closed nucleus breeding system.

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.

Il2rg gene-targeted severe combined immunodeficiency pigs.

A porcine model of severe combined immunodeficiency (SCID) promises to facilitate human cancer studies, the humanization of tissue for xenotransplantation, and the evaluation of stem cells for clinical therapy, but SCID pigs have not been described. We report here the generation and preliminary evaluation of a porcine SCID model. Fibroblasts containing a targeted disruption of the X-linked interleukin-2 receptor gamma chain gene, Il2rg, were used as donors to generate cloned pigs by serial nuclear transfer. Germline transmission of the Il2rg deletion produced healthy Il2rg(+/-) females, while Il2rg(-/Y) males were athymic and exhibited markedly impaired immunoglobulin and T and NK cell production, robustly recapitulating human SCID. Following allogeneic bone marrow transplantation, donor cells stably integrated in Il2rg(-/Y) heterozygotes and reconstituted the Il2rg(-/Y) lymphoid lineage. The SCID pigs described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies.

Identification of a second gene associated with variation in vertebral number in domestic pigs.

BACKGROUND: 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). RESULTS: 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. CONCLUSIONS: 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.

Overexpression of NUDT7, a candidate quantitative trait locus for pork color, downregulates heme biosynthesis in L6 myoblasts.

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.

Molecular cloning and characterization of porcine Mx2 gene.

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.

Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1).

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.