Genome-wide detection of changes in allelic frequency in Landrace pigs selected for resistance to mycoplasma pneumonia of swine.

Closed-pig line breeding could change the genetic structure at a genome-wide scale because of the selection in a pig breeding population. We investigated the changes in population structure among generations at a genome-wide scale and the selected loci across the genome by comparing the observed and expected allele frequency changes in mycoplasma pneumonia of swine (MPS)-selected pigs. Eight hundred and seventy-four Landrace pigs, selected for MPS resistance without reducing average daily gain over five generations, had 37,299 single nucleotide polymorphisms (SNPs) and were used for genomic analyses. Regarding population structure, individuals in the first generation were the most widely distributed and then converged into a specific group, as they were selected over five generations. For allele frequency changes, 96 and 14 SNPs had higher allele frequency changes than the 99.9% and 99.99% thresholds of the expected changes, respectively. These SNPs were evenly spread across the genome, and a few of these selected regions overlapped with previously detected quantitative trait loci for MPS and immune-related traits. Our results indicated that the considerable changes in allele frequency were identified in many regions across the genome by closed-pig line breeding based on estimated breeding value.

Polymorphisms in Pattern Recognition Receptor Genes Are Associated with Respiratory Disease Severity in Pig Farms.

Reduced productivity caused by infections, particularly respiratory diseases, is a serious problem in pig farming. We have previously reported polymorphisms in porcine pattern recognition receptor genes affecting molecular functions and demonstrated that the 2197A/C polymorphism in the nucleotide-binding oligomerization domain containing 2 (NOD2) gene influences porcine circovirus 2-induced mortality. Here, we investigated how these polymorphisms affect respiratory disease-induced lesions, using samples from a slaughterhouse dealing with pigs from two farms. Lung lesions were evaluated using two scoring systems, Goodwin (GW) and slaughterhouse pleuritis evaluation system (SPES), to determine the influence of Mycoplasma hyopneumoniae (Mhp) and Actinobacillus pleuropneumoniae (App), respectively. SPES scores were significantly higher when the 1205T allele of Toll-like receptor 5 (TLR5-1205T), rather than TLR5-1205C, was present. On the farm with more severe Mhp invasion, lower GW lesion scores were significantly associated with the presence of the NOD-like receptor family pyrin domain containing 3 (NLRP3)-2906G allele; where App invasion was worse, lower SPES scores were significantly associated with the presence of the NOD2-2197C allele. Combinations of polymorphisms in pattern recognition receptor genes can therefore be utilized for breeding for resistance against respiratory diseases in pigs. DNA markers of these polymorphisms can thus be used to improve productivity by reducing respiratory diseases due to bacterial pathogens in pig livestock.

NOD2 Genotypes Affect the Symptoms and Mortality in the Porcine Circovirus 2-Spreading Pig Population.

The nucleotide oligomerization domain (NOD)-like receptor 2 (NOD2) is an intracellular pattern recognition receptor that detects components of peptidoglycans from bacterial cell walls. NOD2 regulates bowel microorganisms, provides resistance against infections such as diarrhea, and reduces the risk of inflammatory bowel diseases in humans and mice. We previously demonstrated that a specific porcine NOD2 polymorphism (NOD2-2197A > C) augments the recognition of peptidoglycan components. In this study, the relationships between porcine NOD2-2197A/C genotypes affecting molecular functions and symptoms in a porcine circovirus 2b (PCV2b)-spreading Duroc pig population were investigated. The NOD2 allele (NOD2-2197A) with reduced recognition of the peptidoglycan components augmented the mortality of pigs at the growing stage in the PCV2b-spreading population. Comparison of NOD2 allele frequencies in the piglets before and after invasion of PCV2b indicated that the ratio of NOD2-2197A decreased in the population after the PCV2b epidemic. This data indicated that functional differences caused by NOD2-2197 polymorphisms have a marked impact on pig health and livestock productivity. We suggest that NOD2-2197CC is a PCV2 disease resistant polymorphism, which is useful for selective breeding by reducing mortality and increasing productivity.

Genome-wide association studies for production, respiratory disease, and immune-related traits in Landrace pigs.

Identification of a quantitative trait locus (QTL) related to a chronic respiratory disease such as Mycoplasmal pneumonia of swine (MPS) and immune-related traits is important for the genetic improvement of disease resistance in pigs. The objective of this study was to detect a novel QTL for a total of 22 production, respiratory disease, and immune-related traits in Landrace pigs. A total of 874 Landrace purebred pigs, which were selected based on MPS resistance, were genotyped using the Illumina PorcineSNP60 BeadChip. We performed single nucleotide polymorphism (SNP)-based and haplotype-based genome-wide association studies (GWAS) to detect a novel QTL and to evaluate the possibility of a pleiotropic QTL for these traits. SNP-based GWAS detected a total of six significant regions in backfat thickness, ratio of granular leucocytes to lymphatic cells, plasma concentration of cortisol at different ages, and complement alternative pathway activity in serum. The significant region detected by haplotype-based GWAS was overlapped across the region detected by SNP-based GWAS. Most of these detected QTL regions were novel regions with some candidate genes located in them. With regard to a pleiotropic QTL among traits, only three of these detected QTL regions overlapped among traits, and many detected regions independently affected the traits.

A specific promoter-type in ribonuclease L gene is associated with phagocytic activity in pigs.

We have previously generated Large White pigs with high immune competence using a selection strategy based on phagocytic activity (PA), capacity of alternative complement pathway, and antibody response after vaccination against swine erysipelas. In this study, to identify the genetic changes caused by the immune selection pressure, we compared gene expression and polymorphisms in the promoter region between pigs subjected to the immune selection (immune-selected pigs) and those that were not (non-selected pigs). After lipid A stimulation, using a microarray analysis, 37 genes related to immune function and transcription factor activity showed a greater than three-fold difference in expression between macrophages derived from immune-selected and non-selected pigs. We further performed a polymorphic analysis of the promoter region of the differentially expressed genes, and elucidated the predominant promoter-types in the immune-selected and non-selected pigs, respectively, in the genes encoding ribonuclease L (RNASEL), sterile α motif and histidine-aspartate domain containing deoxynucleoside triphosphate triphosphohydrolase 1, signal transducer and activator of transcription 3, and tripartite motif containing 21. Analysis of the association between these promoter genotypes and the immune phenotypes revealed that the immune-selected promoter-type in RNASEL was associated with increased PA and was inherited recessively. Considering that RNASEL has been reported to be involved in antimicrobial immune response of mice, it may be possible to enhance the PA of macrophages and improve disease resistance in pig populations using RNASEL promoter-type as a DNA marker for selection.

Tracing founder haplotypes of Japanese apple varieties: application in genomic prediction and genome-wide association study.

Haplotypes provide useful information for genomics-based approaches, genomic prediction, and genome-wide association study. As a small number of superior founders have contributed largely to the breeding history of fruit trees, the information of founder haplotypes may be relevant for performing the genomics-based approaches in these plants. In this study, we proposed a method to estimate 14 haplotypes from 7 founders and automatically trace the haplotypes forward to apple parental (185 varieties) and breeding (659 F1 individuals from 16 full-sib families) populations based on 11,786 single-nucleotide polymorphisms, by combining multiple algorithms. Overall, 92% of the single-nucleotide polymorphisms information in the parental and breeding populations was characterized by the 14 founder haplotypes. The use of founder haplotype information improved the accuracy of genomic prediction in 7 traits and the resolution of genome-wide association study in 13 out of 27 fruit quality traits analyzed in this study. We also visualized the significant propagation of the founder haplotype with the largest genetic effect in genome-wide association study over the pedigree tree of the parental population. These results suggest that the information of founder haplotypes can be useful for not only genetic improvement of fruit quality traits in apples but also for understanding the selection history of founder haplotypes in the breeding program of Japanese apple varieties.

Full-scale simultaneous partial nitrification, anammox, and denitrification process for treating swine wastewater.

A full-scale swine-wastewater activated sludge treatment plant that contains naturally enriched anammox biofilms was investigated for 2 years. Red biofilm in this system included Planctomycetes at a maximum of 62.5% of the total bacteria diversity, including Candidatus Jettenia and Candidatus Brocadia. The plant was operated with an influent containing 1,104 ± 513 mg/L biochemical oxygen demand (BOD) and 629 ± 198 mg/L total nitrogen (TN) (BOD/N of 1.78 ± 0.58) at a volumetric BOD loading rate of 0.32 ± 0.12 kg/m3/d. Notwithstanding drastically varying influent concentrations, BOD removal efficiency was stable at 95 ± 4%. However, TN removal fluctuated at 75 ± 14%. Dissolved oxygen (DO) concentrations in the aeration tank were 0.06-2.0 mg/L. DO concentration greatly affected nitrogen removal, e.g. when DO was lower than 0.3 mg/L, total inorganic nitrogen removal was 61 ± 14% (≤20 °C), 78 ± 16% (20-30 °C), and 75 ± 12% (≥30 °C), whereas at higher DO concentrations, removal rates were 47 ± 13%, 55 ± 16%, and 68%, respectively. As BOD concentration in the influent was limited compared to nitrogen concentration, nitrogen was likely removed by simultaneous nitrification, anammox, and denitrification (SNAD) under microaerobic conditions. Maintaining low DO concentrations would therefore be a simple method to improve nitrogen removal during SNAD processes for swine-wastewater treatment with fluctuating influent.

Effects of thiosulfate addition on ammonia and nitrogen removal in biofilters packed with Oyaishi (pumice tuff).

Ammonia removal is achieved partly by absorption and nitrification in biofilters, resulting in the accumulation of nitrogen and the necessity of treating the effluent water. We investigated the effects of thiosulfate addition to a biofilter containing pumice tuff for ammonia and nitrogen removal in a laboratory-scale experiment. The addition of thiosulfate to the circulating water led to a decreased nitrate and nitrite along with an increase of sulfate. The inorganic nitrogen in the circulating water decreased by up to 44% with thiosulfate addition compared to without thiosulfate. Batch experiments revealed that denitrification activity decreased exponentially along with increases in dissolved oxygen; however, approximately 30% of denitrification activity was maintained at dissolved oxygen concentration of 3.3 mg/L. Metabarcoding of 16S rRNA genes indicated that the genus Thiobacillus had a relative abundance of 0.002%-0.016% of total bacteria in the biofilter packing material. The circulating water pH was decreased below 5 with sulfur oxidation, and ammonium was accumulated without pH control resulting in a decrease in the relative abundance of the family Nitrosomonadaceae. Its relative abundance increased with control of pH to near neutral, indicating that ammonia-oxidizing activity could be maintained by adjusting pH. Thiosulfate addition could stimulate nitrogen removal by sulfur-dependent denitrification in biofiltration systems.

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.

Anammox biofilm in activated sludge swine wastewater treatment plants.

We investigated anammox with a focus on biofilm in 10 wastewater treatment plants (WWTPs) that use activated sludge treatment of swine wastewater. In three plants, we found red biofilms in aeration tanks or final sedimentation tanks. The biofilm had higher anammox 16S rRNA gene copy numbers (up to 1.35 × 1012 copies/g-VSS) and higher anammox activity (up to 295 µmoL/g-ignition loss/h) than suspended solids in the same tank. Pyrosequencing analysis revealed that Planctomycetes accounted for up to 17.7% of total reads in the biofilm. Most of them were related to Candidatus Brocadia or Ca. Jettenia. The highest copy number and the highest proportion of Planctomycetes were comparable to those of enriched anammox sludge. Thus, swine WWTPs that use activated sludge treatment can fortuitously acquire anammox biofilm. Thus, concentrated anammox can be detected by focusing on red biofilm.

Genomic dissection of a 'Fuji' apple cultivar: re-sequencing, SNP marker development, definition of haplotypes, and QTL detection.

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'. Through the re-sequencing of 'Fuji' genome, total of 2,820,759 variants, including single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) were detected between 'Fuji' and 'Golden Delicious' reference genome. We selected mapping-validated 1,014 SNPs, most of which were heterozygous in 'Fuji' and capable of distinguishing alleles inherited from the parents of 'Fuji' (i.e., 'Ralls Janet' and 'Delicious'). We used these SNPs to define the haplotypes of 'Fuji' and trace their inheritance in relatives, which were shown to have an average of 27% of 'Fuji' genome. Analysis of variance (ANOVA) based on 'Fuji' haplotypes identified one quantitative trait loci (QTL) each for harvest time, acidity, degree of watercore, and mealiness. A haplotype from 'Delicious' chr14 was considered to dominantly cause watercore, and one from 'Ralls Janet' chr1 was related to low-mealiness.

Parental diagnosis of satsuma mandarin (Citrus unshiu Marc.) revealed by nuclear and cytoplasmic markers.

Satsuma mandarins (Citrus unshiu Marc.) are the predominant cultivated citrus variety in Japan. Clarification of its origin would prove valuable for citrus taxonomy and mandarin breeding programs; however, current information is limited. We applied genome-wide genotyping using a 384 citrus single nucleotide polymorphism (SNP) array and MARCO computer software to investigate the satsuma mandarin parentage. Genotyping data from 206 validated SNPs were obtained to evaluate 67 citrus varieties and lines. A total of five parent-offspring relationships were newly found by MARCO based on the 206 SNP genotypes, indicating that 'Kishuu mikan' type mandarins (Citrus kinokuni hort. ex Tanaka accession 'Kishuu mikan' and 'Nanfengmiju') and 'Kunenbo' type mandarins (Citrus nobilis Lour. var. kunip Tanaka accession 'Kunenbo' and 'Bendiguangju') are possible parents of the satsuma mandarin. Moreover, cleaved amplified polymorphic sequences analysis showed that the genotypes of four regions in chloroplast DNA of 'Kishuu mikan' type mandarins were identical to that of the satsuma mandarin. Considering the historical background, satsuma mandarins may therefore derive from an occasional cross between a 'Kishuu mikan' type mandarin seed parent (derivative or synonym of 'Nanfengmiju') and a 'Kunenbo' type mandarin pollen parent (derivative or synonym of 'Bendiguangju').

Porcine NOD1 polymorphisms with impaired ligand recognition and their distribution in pig populations.

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.

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.

Population structure of pigs determined by single nucleotide polymorphisms observed in assembled expressed sequence tags.

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.

Porcine single nucleotide polymorphism (SNP) development and population structure of pigs assessed by validated SNPs.

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.

Influence of polymorphisms in porcine NOD2 on ligand recognition.

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.

Differences in distribution of single nucleotide polymorphisms among intracellular pattern recognition receptors in pigs.

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.