Molecular Characterization of Five Porcine Candidate Genes for Drip Loss in Pork

Drip loss is the loss of fluid from a piece of meat without mechanical force and represents an important meat quality trait. Previous work revealed a quantitative trait locus (QTL) for drip loss in pork in an experimental Duroc x Piétrain (DUPI) F2 family on SSC 5. Based on functional data indicating their possible involvement in water holding capacity and their expression in skeletal muscle, we selected five positional candidates (ACO2, ADSL, CBY1, KCNJ4, PLA2AG6) out of 130 predicted genes in the QTL interval for further analysis. We performed a mutation analysis of all coding exons and discovered 204 polymorphisms. We genotyped 39 single nucleotide polymorphisms (SNPs) in 192 Piétrain pigs with extreme drip loss phenotypes and detected a possible association with drip loss for one non-coding SNP in the ADSL gene (ss107793818, p_raw = 0.021). Correspondingly, ADSL diplotypes were associated with drip loss and pH1 of M. longissimus dorsi. However, after correction for multiple testing, none of the tested SNPs were significantly associated with drip loss. One possible explanation for these results is that one of the QTL-alleles from the experimental DUPI family may be fixed or nearly fixed in the tested Piétrain population.     

Mapping QTL for production traits in segregating Piétrain pig populations using genome‐wide association study results of F2 crosses

In this study, genome‐wide association study (GWAS) results of porcine F2 crosses were used to map QTL in outcross Piétrain populations. For this purpose, two F2 crosses (Piétrain × Meishan, n = 304; Piétrain × Wild Boar, n = 291) were genotyped with the PorcineSNP60v2 BeadChip and phenotyped for the dressing yield, carcass length, daily gain and drip loss traits. GWASs were conducted in the pooled F2 cross applying single marker mixed linear models. For the investigated traits, between two and five (in total 15) QTL core regions, spanning 250 segregating SNPs around a significant trait‐associated peak SNP, were identified. The SNPs within the QTL core regions were subsequently tested for trait association in two outcross Piétrain populations consisting of 771 progeny‐tested boars and 210 sows with their own performance records. In the sow (boar) dataset, five (eight) of the 15 mapped QTL were validated. Hence, many QTL mapped in the F2 crosses (with Piétrain as a common founder breed) are still segregating in the current Piétrain breed. This confirms the usefulness of existing F2 crosses for mapping QTL that are still segregating in the recent founder breed generation. The approach utilizes the high power of an F2 cross to map QTL in a breeding population for which it is not guaranteed that they would be found using a GWAS in this population.     

Association of the melanocortin 4 receptor with feed intake and daily gain in F2 Mangalitsa x Piétrain pigs

The melanocortin 4 receptor (MC4R) is a key factor in the regulation of energy balance and body weight. Hence it is a candidate for feed intake and energy homeostasis-related traits. Studies in humans and swine have revealed several sequence variants in the gene that are associated with some of these traits. In pigs the coding non-synonymous missense variant Asp298Asn in MC4R has been associated with feed intake, fatness and growth. Here we confirm the association of this Piétrain-derived polymorphism with feed intake and daily gain in the F2 generation of a Mangalitsa x Piétrain cross. In one Piétrain founder animal, we detected an additional non-synonymous missense variant Arg236His. Thus, the MC4R gene could be a useful marker for increased growth in the relatively slow-growing Piétrain breed.     

Fibroblast growth factor receptor 2 (FGFR2): genomic sequence and variations

Fibroblast growth factor receptors (FGFRs) play an important role in development and tumorigenesis. Mutations in FGFR2 cause more than five craniosynostosis syndromes. The FGFR2 genomic structure is the largest of the FGFR family. We have refined and extended the genomic organization of the FGFR2 gene by sequencing more than 119 kb of PACs, cosmids, and PCR products and assembling a region of approximately 175 kb. Although the gene structure has been reported to include only 20 exons, we have verified the presence of at least 22 exons, some of which are alternatively spliced. The sizes of six exons differed from those reported previously. Comparison of our sequence and those in the NCBI database detected more than 300 potential single nucleotide polymorphisms (SNPs). However, sequencing regions containing 52 of these potential SNPs verified only 14 in PCR products generated from 16 CEPH alleles. In contrast, direct sequencing of the CEPH DNAs revealed 21 other polymorphisms. Only one SNP was found in the 2,926 bp of coding sequence. Twenty-seven SNPs, two insertion polymorphisms and five microsatellite polymorphisms are contained in approximately 16.6 kb of non-coding sequence. These data yield an average of one polymorphism for approximately 488 bp of non-coding sequence examined. This collection of SNP, insertion, and repeat polymorphisms will aid future association studies between the FGFR2 gene and human disease and will enhance mutation detection.     

SNP genotypes reveal breed substructure,selection signatures and highly inbred regions in Piétrain pigs

The Piétrain pig originates from the Belgian village Piétrain some time between 1920 and 1950. Owing to its superior conformation, the Piétrain has spread worldwide since the 1960s. As initial population sizes were limited and close inbreeding was commonplace, the breed’s genetic diversity has been questioned. Therefore, this study examines Piétrain breed substructure, diversity and selection signatures using SNP data in comparison with Duroc, Landrace and Large White populations. Principal component analysis indicated three subpopulations, and F_ST analysis showed that US Piétrains differ most from European Piétrains. Average inbreeding based on runs of homozygosity (ROH) segments larger than 4 Mb ranged between 16.7 and 20.9%. The highest chromosomal inbreeding levels were found on SSC8 (42.7%). ROH islands were found on SSC8, SSC15 and SSC18 in all Piétrain populations, but numerous population-specific ROH islands were also detected. Moreover, a large ROH island on SSC8 (34–126 Mb) appears nearly fixed in all Piétrain populations, with a unique genotype. Chromosomal ROH patterns were similar between Piétrain populations. This study shows that Piétrain populations are genetically diverging, with at least three genetically distinct populations worldwide. Increasing genetic diversity in local Piétrain populations by introgression from other Piétrain populations seems to be only limited. Moreover, a unique 90 Mb region on SSC8 appeared largely fixed in the Piétrain breed, indicating that fixation was already present before the 1960s. We believe that strong selection and inbreeding during breed formation fixed these genomic regions in Piétrains. Finally, we hypothesize that independent coat color selection may have led to large ROH pattern similarities on SSC8 between unrelated pig breeds.     

Genome‐wide association analysis for growth,muscularity and meat quality in Piétrain pigs

Improvement in growth and meat quality is one of the main objectives in sire line pig breeding programmes. Mapping quantitative trait loci for these traits using experimental crosses and a linkage‐based approach has been performed frequently in the past. The Piétrain breed often was involved as a founder breed to establish the experimental crosses. This breed was selected for muscularity and leanness but shows relatively poor meat quality. It is frequently used as a sire line breed. With the advent of genome‐wide and dense SNP chips in pig genomic research, it is possible to also conduct genome‐wide association studies within the Piétrain breed. In this study, around 500 progeny‐tested sires were genotyped with 60k SNPs. Data filtering showed that around 48k SNPs were useable in this sample. These SNPs were used to conduct a genome‐wide association study for growth, muscularity and meat quality traits. Because it is known that a mutation in the RYR1 gene located on chromosome 6 shows a major effect on meat quality, this mutation was included in the models. Single‐marker and multimarker association analyses were performed. The results revealed between zero and eight significant associations per trait with P < 5 × 10^?5. Of special interest are SNPs located on SSC6, SSC10 and SSC15.     

Single‐nucleotide polymorphism discovery in Leptographium longiclavatum,a mountain pine beetle‐associated symbiotic fungus,using whole‐genome resequencing

Single‐nucleotide polymorphisms (SNPs) are rapidly becoming the standard markers in population genomics studies; however, their use in nonmodel organisms is limited due to the lack of cost‐effective approaches to uncover genome‐wide variation, and the large number of individuals needed in the screening process to reduce ascertainment bias. To discover SNPs for population genomics studies in the fungal symbionts of the mountain pine beetle (MPB), we developed a road map to discover SNPs and to produce a genotyping platform. We undertook a whole‐genome sequencing approach of Leptographium longiclavatum in combination with available genomics resources of another MPB symbiont, Grosmannia clavigera. We sequenced 71 individuals pooled into four groups using the Illumina sequencing technology. We generated between 27 and 30 million reads of 75 bp that resulted in a total of 1, 181 contigs longer than 2 kb and an assembled genome size of 28.9 Mb (N₅₀ = 48 kb, average depth = 125x). A total of 9052 proteins were annotated, and between 9531 and 17 266 SNPs were identified in the four pools. A subset of 206 genes (containing 574 SNPs, 11% false positives) was used to develop a genotyping platform for this species. Using this roadmap, we developed a genotyping assay with a total of 147 SNPs located in 121 genes using the Illumina^® Sequenom iPLEX Gold. Our preliminary genotyping (success rate = 85%) of 304 individuals from 36 populations supports the utility of this approach for population genomics studies in other MPB fungal symbionts and other fungal nonmodel species.     

Linkage disequilibrium pattern and genome‐wide association mapping for meat traits in multiple porcine F2 crosses

In the present study, data from four F2 crosses were analysed and used to study the linkage disequilibrium (LD) structure within and across the crosses. Genome‐wide association analyses (GWASes) for conductivity and dressing out meat traits were conducted using single‐marker and Bayesian multi‐marker models using the pooled data from all F2 crosses. Porcine F2 crosses generated from the distantly related founder breeds Wild Boar, Piétrain and Meishan, as well as from a porcine F2 cross from the closely related founder breed Piétrain and an F1 Large White × Landrace cross were pooled. A total of 2572 F2 animals were genotyped using a 62K SNP chip. The positions of the SNPs were based on genome assembly Sscrofa11.1. After post‐alignment and genotype filtering, approximately 50K SNPs were usable for LD studies and GWASes. The main findings of the present study are that the breakdown of LD was faster in crosses from closely related founder breeds compared to crosses from distantly related founders. The fastest breakdown of LD was observed by pooling the data. Based on the single‐marker results and LD structure, clusters and windows were built for 1‐Mb intervals. For conductivity and dressing out, 183 and 191 nominal significant associations respectively and six and five clusters respectively were found. Dominance was important for conductivity, and considering dominance in GWASes improved the mapping signals. Most clear signals were found for conductivity on SSC6, 8 and 15 and for dressing out on SSC2 and 7. Considering dominance might contribute to the accuracy of genomic selection and serve as a guide for choosing mating pairs with good combining abilities. However, further research is needed to investigate if dominance is also important in crossbreed pig breeding schemes.     

Malaria severity and human nitric oxide synthase type 2 (NOS2) promoter haplotypes

Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.     

Porcine OGN and ASPN: mapping, polymorphisms and use for quantitative trait loci identification for growth and carcass traits in a Meishan x Piétrain intercross

The porcine orthologues of human chromosome HSA9q22.31 genes osteoglycin (OGN) and asporin (ASPN) were mapped to porcine chromosome SSC3 using linkage analysis and a somatic cell hybrid panel. This mapping was refined to SSC3q11 using fluorescence in situ hybridization. These results confirm the existence of a small conserved synteny group between SSC3 and HSA9. Polymorphisms were revealed in both genes, including a pentanucleotide microsatellite (SCZ003) in OGN and two single nucleotide polymorphisms (AM181682.1:g.780G>T and AM181682.1:g.825T>C) in ASPN. The two genes were included in a set of markers for quantitative trait loci (QTL) mapping on SSC3 in the Hohenheim Meishan x Piétrain F2 family. Major QTL for growth and carcass traits were centred in the ASPN-SW902 region.     

Characterization of the porcine transferrin gene (TF ) and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infection

Transferrin (TF)‐mediated provision of iron is essential for a productive infection by many bacterial pathogens, and iron‐depletion of TF is a first line defence against bacterial infections. Therefore, the transferrin (TF) gene can be considered a candidate gene for disease resistance. We obtained the complete DNA sequence of the porcine TF gene, which spans 40 kb and contains 17 exons. We identified polymorphisms on a panel of 10 different pig breeds. Comparative intra‐ and interbreed sequence analysis revealed 62 polymorphisms in the TF gene including one microsatellite. Ten polymorphisms were located in the coding sequence of the TF gene. Four SNPs (c.902A>T, c.980G>A, c.1417A>G, c.1810A>C) were predicted to cause amino acid exchanges (p.Lys301Ile, p.Arg327Lys, p.Lys473Glu, p.Asn604His). We performed association analyses using six selected TF markers and 116 pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 7. The analysis showed breed‐specific TF allele frequencies. In German Landrace, we found evidence for a possible association of the severity of A. pleuropneumoniae infection with TF genotypes.     

A two-nucleotide deletion renders the mannose-binding lectin 2 (MBL2) gene nonfunctional in Danish Landrace and Duroc pigs

The mannose-binding lectins (MBLs) are central components of innate immunity, facilitating phagocytosis and inducing the lectin activation pathway of the complement system. Previously, it has been found that certain single-nucleotide polymorphisms (SNPs) in porcine MBL1 and MBL2 (pMBL1, pMBL2) affect mRNA expression, serum concentration, and susceptibility to disease, but the combinatory effect of pMBL1 and pMBL2 genotypes needs further elucidation. In the present study, pMBL1 and pMBL2 alleles, combined pMBL haplotypes, and MBL-A concentration in serum were analyzed in purebred Landrace (N?=?30) and Duroc (N?=?10) pigs. Furthermore, the combined pMBL haplotypes of 89 Piètrain × (Large White × Landrace) crossbred pigs were studied, and the genotypes of 67 crossbreds challenged with Escherichia coli were compared to their individual disease records. In the purebred animals, three non-synonymous SNPs and a two-nucleotide deletion were detected in the coding sequence of pMBL2. The two-nucleotide deletion was present at a frequency of 0.88 in the Landrace pigs and 0.90 in the Duroc pigs, respectively. In the crossbreds, the T allele of the SNP G949T in pMBL1—previously shown to have profound effect on MBL-A concentration even in the heterozygote condition—was detected in 47 % of the animals. Finally, an association was found between low-producing MBL genotypes and low body weight on the day of weaning in the same animals.     

ARG2 impairs endothelial autophagy through regulation of MTOR and PRKAA/AMPK signaling in advanced atherosclerosis

Impaired autophagy function and enhanced ARG2 (arginase 2)-MTOR (mechanistic target of rapamycin) crosstalk are implicated in vascular aging and atherosclerosis. We are interested in the role of ARG2 and the potential underlying mechanism(s) in modulation of endothelial autophagy. Using human nonsenescent “young” and replicative senescent endothelial cells as well as Apolipoprotein E-deficient (apoe^?/?Arg2^+/+) and Arg2-deficient apoe^?/? (apoe^?/?arg2^?/?) mice fed a high-fat diet for 10 wk as the atherosclerotic animal model, we show here that overexpression of ARG2 in the young cells suppresses endothelial autophagy with concomitant enhanced expression of RICTOR, the essential component of the MTORC2 complex, leading to activation of the AKT-MTORC1-RPS6KB1/S6K1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) cascade and inhibition of PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit). Expression of an inactive ARG2 mutant (H160F) had the same effect. Moreover, silencing RPS6KB1 or expression of a constitutively active PRKAA prevented autophagy suppression by ARG2 or H160F. In senescent cells, enhanced ARG2-RICTOR-AKT-MTORC1-RPS6KB1 and decreased PRKAA signaling and autophagy were observed, which was reversed by silencing ARG2 but not by arginase inhibitors. In line with the above observations, genetic ablation of Arg2 in apoe^?/? mice reduced RPS6KB1, enhanced PRKAA signaling and endothelial autophagy in aortas, which was associated with reduced atherosclerosis lesion formation. Taken together, the results demonstrate that ARG2 impairs endothelial autophagy independently of the L-arginine ureahydrolase activity through activation of RPS6KB1 and inhibition of PRKAA, which is implicated in atherogenesis.     

Genetic variation in <Emphasis Type="Italic">IGFBP2</Emphasis> and <Emphasis Type="Italic">IGFBP5</Emphasis> is associated with breast cancer in populations of African descent

The insulin-like growth factor (IGF) signaling pathway is thought to play a major role in the etiology of breast cancer. Although incidence rates of breast cancer overall are lower in African Americans than in Caucasians, African-American women have a higher incidence under age 40 years, are diagnosed with more advanced disease, and have poorer prognosis. We investigated the association of breast cancer and genetic variants in genes in the IGF signaling pathway in a population-based case–control study of African-American women. We found significant associations at a locus encompassing parts of the IGFBP2 and IGFBP5 genes on chromosome 2q35, which we then replicated in a case–control study of Nigerian women. Based on those initial findings, we genotyped a total of 34 single nucleotide polymorphisms (SNPs) across the region in both study populations. Statistically significant associations with breast cancer were observed across approximately 50 kb of DNA sequence encompassing three exons in the 3′ end of IGFBP2 and three exons in the 3′ end of IGFBP5. SNPs were associated with breast cancer risk with P values as low as P = 0.0038 and P = 0.01 in African-Americans and Nigerians, respectively. This study is the first to report associations between genetic variants in IGFBP2 and IGFBP5 and breast cancer risk.     

Sequence variation in two lignin biosynthesis genes,cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase 2 (CAD2)

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase 2 (CAD2) are genes which may influence variation in lignin content and composition within plants. Sequence variation within these genes may be responsible for changes in enzyme activity and/or specificity, which could cause variation in lignin content or composition. This study examines sequence variation within these two genes in Eucalyptus globulus, an important species used in pulp and paper-making. Twenty-one single nucleotide polymorphisms (SNPs) were identified in the exons of CCR, of which nine were neutral mutations and 12 were missense mutations. Six of the missense mutations affected highly conserved amino acids within the protein sequence of CCR. Eight SNPs were identified in the CAD2 exons, six of which were neutral mutations and two which were missense mutations. One of the missense mutations affected a highly conserved amino acid within the protein sequence. In addition, 32 SNPs were identified in the CCR introns along with four insertion/deletions and two polyA length variation regions. Polymorphism affecting highly conserved amino acids may alter enzyme function and this molecular variation may be linked to variation in lignin profiles. Selecting positive alleles which produce favourable lignin profiles would be advantageous in tree breeding programs.     

Single-nucleotide polymorphisms in porcine mannan-binding lectin A

The MBL1 and MBL2 genes encode mannan-binding lectins (MBL) A and C, respectively, that are collagenous lectins (collectins) produced mainly by the liver. Several single-nucleotide polymorphisms (SNPs) in the human MBL2 gene are responsible for various innate immune dysfunctions due to abnormal structure or expression of human MBL-C. The MBL1 gene encodes MBL-A, which has bacteria-binding properties in pigs and rodents but is mutated to a pseudogene in humans and chimpanzees. In these studies, we surveyed both porcine MBL genes for SNPs that might impair disease resistance. Single-strand conformational polymorphism (SSCP) analysis of MBL cDNAs from porcine liver revealed three SNPs within the coding region of MBL1 in various breeds of pigs. One nonsynonymous SNP that substituted cysteine for glycine in the collagen-like domain of pig MBL-A was found by a multiplex PCR test in all European pig breeds examined, with allele frequencies ranging from 1.4 to 46.4%. No SNPs were identified in the coding region of porcine MBL2 but the expression of MBL-C in the liver was widely variable in comparison to the expression of MBL-A, GAPDH, PigMAP, and haptoglobin. These results indicate that some pigs have a miscoding defect in MBL-A and a possible expression defect in MBL-C, which are analogous to coding and promoter polymorphisms that affect human MBL-C.