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NC1037: Genetic and Functional Genomic Approaches to Improve Production and Quality of Pork

Annual/Termination Reports (SAES-422): [06/25/2008] [02/08/2010]

Date of Annual Report: 06/25/2008

Report Information:
  • Annual Meeting Dates: 05/14/08 to 05/15/08
  • Period the Report Covers: 10/2007 to 09/2008

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    8:00 am Dr. Joan K Lunney elcomed everyone to NC1037 annual meeting. As chair, Dr. Lunney presided over the sessions.

    8:15 am Business meeting and Election of NC1037 officers. A motion regarding the selection of officers for 2008 was approved. Dr. Jiang (Chair), Dr. Rekaya (Vice-Chair) and Ohio representative (secretary).

    8:30 am Dr. Jiang suggested Seattle (Washington State) as location for 2009 meeting. After some discussions, no decision was made. Also there have been some discussions regarding the schedule of the meeting for 2009. Two options were looked at. Regular schedule or at the same time as PAG-2009.

    8:50 am Dr. Rothschild , Iowa State University, gave an update on swine sequencing & SNP chip plans. He indicates that a purchase order has to be placed by August 22, 2008 where money will be committed but there is no guaranty on number of chips. He also indicated that an amount of $100 to $110K is already available. After some discussions, it was clear that some policies-rules are needed for the best use of such money. Drs. Rothschild, Hamernik, and Ernst agreed to form a committee to set some rules.

    9:30 am Break

    10:00 am Updates on USDA bovine genome research - Dr. Zimin gave an update on the assembly of the cow genome o He showed interest for collaboration on the pig genome assembly o Dr. Rothschild and Dr. Cassady suggested being in touch with Dr. Zimin for potential collaboration with the pig genome - Dr. Steven Schroeder from the Bovine Functional Genomics Laboratory (BFGL), BARC, gave an update on the cattle gene atlas - Dr. Matukumalli, BFGL, BARC gave some results on the application of BovineSNP50 for CNVs and genome-wide associations. - Dr. Paul VanRaden from Animal Improvement Programs Laboratory presented an application of Genomic selection in dairy cattle. - 12:00 pm Lunch break

    1:00 pm NC1037 Station Reports

    Chris Tuggle (Iowa state university) Jack Dekkers (Iowa State University) Max Rothschild (Iowa State University) Joe Cassady (North Carolina State University) Rick Barb and Romdhane Rekaya (ARS- University of Georgia) Cathy Ernst (Michigan State University) Le Ann Blomberg (BARC-USDA) Jiang (Washington State University)

    3:45 pm Administrative Reports: Deb Hamernik, CSREES, NPL, Animal Physiology; Bert Stromberg, Administrative Advisor; Max Rothschild, National Swine Genome Coordinator

    Thursday May 15, 2008

    8:00 am Review of NC1037 objectives/station reports

    1. Discussion of major areas of research in objective 1: Reproduction, growth and development, sow longevity, bone strength and behavior

    2. Discussion of major areas of research in objective 2: genetic mechanisms controlling animal health

    10:15 am Collaboration and possibility for collaboration - Meat Quality: potential collaboration between Iowa and Michigan. - Growth and Development: Potential collaboration between BARC and Michigan on fetal and neonatal development and between BARC and North Carolina on pre-weaning survival trial with Smithfield, Inc. - Feed efficiency/Intake: Extending the already existing collaboration between Georgia and Iowa on gene expression data analysis, follow up with the MSH/NPY treatment and the used of RFI resources at IA. - Reproduction: Potential collaboration between BARC and Iowa on comparing data for early embryonic development - Behavior: Possibility of joint analysis of chute scores (standardization of scoring systems)

    11:15 am Update on PRRS CAP project by J. Lunney - Great potential for collaboration between NC1037 members - Important issue for the industry - Discussion of the potential use of $100K as matching fund

    11:45 am Dr. Hamernik suggested adding impact statements to station reports as well as some discussions on CRIS reporting and station reports

    11:51 am Meeting adjourned at 11:51 a.m.


    URL: Copy of minutes

    Accomplishments:
    Objective 1. Further understand the dynamic genetic mechanisms that influence production efficiency and quality of pork.

    Genetic improvement of production efficiency and pork quality using traditional selection programs. Iowa State University reported a six-generation selection experiment for intramuscular fat (IMF) based on the average EBV estimated using real-time ultrasound. Of the 149 pigs harvested in generation six, the station found that line LS means for tenth rib backfat and loin muscle area were 16.63 mm and 45.45 cm2 in the control line, and 24.22 mm and 38.02 cm2 in the select line (P < 0.01), respectively. Analysis of STAGES data evaluated on all 621 pigs in generation six revealed significant differences between lines for days to 114 kg, backfat, and loin muscle area. Results through generation six indicate that selection for IMF resulted in slower growth, more tenth-rib backfat, and less LMA. Chemical analysis of a loin sample from pigs harvested revealed a significant phenotypic response in IMF (2.41% in the control line vs. 4.53% in the select line) similar to the difference between lines for IMF EBV. Line LS means for pigs harvested in generation six for 24 h Hunter L and Minolta were 47.00 and 22.17 in the control line, and 49.42 and 24.49 in the select line (P < 0.001), respectively. Subjective measures of marbling were significantly different between lines (2.50 in the control line vs. 4.89 in the select line). Evaluation of correlated response in litter traits after selection for IMF showed that dams in the control line farrowed litters that were 1.22 kg heavier at birth. Individual piglets in control line litters were 0.11 kg (P < 0.05) heavier at birth when compared to piglets farrowed by select line dams. Iowa State University also carried out a selection experiment for residual feed intake (RFI) in Yorkshire pigs, which produced generation 6 in summer 2008. Analysis of feed intake and growth curves showed that differences in RFI between the two lines occur after ~60 kg body weight. The slightly lower ADG observed in the select line also occured primarily after ~60 kg.

    Selection for litter size continues at the Nebraska State University. Two selection lines (Lines 2 and 45) and two contemporary control lines (Lines 1 and 6) have been maintained in the Station for 27 generations. Line 2 was derived from an Index line that had undergone 11 generations of selection for ovulation rate and embryo survival. This selection was followed by three generations of selection for total born per litter, six generations of selection for number of live pigs per litter, and seven generations of number of live pigs per litter and within litter selection for growth rate and backfat thickness. Line 1 has been randomly selected. The Station report indicated that selection lines 2 and 45 farrowed 5 additional fully formed pigs per litter than their respective controls, but also had greater incidence of stillborn piglets so that the difference in number of live pigs per litter is approximately 4 pigs per litter. Recent selection for growth is causing lines to differentiate in weight and backfat, but not in loin eye area. Improvement of growth appears to be greater in Line 45 than in Line 2.

    North Carolina State University has started a divergent selection for response to the backtest to study pig behavior, the extent to which it is genetically controlled, and its relationship with economically important traits. During their previous work the Station estimated the heritability of the backtest to be approximately 0.40. The long term goal will be to monitor correlated responses to selection for high and low total time spent struggling during the backtest. Between 7 and 21 days of age each pig will be evaluated using the backtest. A pig will be placed on a padded table in a supine position for 60 s and gently restrained. The experimenters right hand will be placed loosely on the pigs thorax with the right foreleg between the index and middle finger. The hind legs will be held with experimenters left hand and stretched and moved downward to begin the test and the hand will remain loosely on the hind legs throughout the test. Each bout of struggling with at least one hind leg will be recorded as an escape attempt. The number of escape attempts, duration of escape behavior, and latency to the first escape attempt will be recorded. It is expected that this test will lead to a better understanding of the relationship between pig behavior, growth, reproduction, and health.

    North Carolina Agricultural And Technical State University has recently received a SARE Grant entitled A Multi-Disciplinary Approach to Improve the Environmental Performance of Niche Pork Production Systems and Marketability of Heritage Swine Breeds. This project will investigate niche pork production systems that address market demands and natural resource conservation concerns, with a specific focus on maximizing vegetative ground cover and nutrient distribution in pastures and understanding marketability of heritage breeds produced in alternative production systems. Consumer interest in heritage breed pork continues to rise; howeve,r little is known about taste characteristics and production potential in alternative systems. The Station will also collaborate with an animal nutritionist in the department to develop a collaborative team that will integrate quantitative and molecular technologies for genetic improvement of pigs. The approach will utilize genomic science and bioinformatics tools to aid in the selection of pigs best fitted for outdoor swine production systems, by evaluating production characteristics such as, nutrient utilization, disease resistance and increased litter weights.

    Quantitative genomics of production efficiency and pork quality using genome scans and candidate gene approaches. Iowa State University has nearly completed a QTL study using the Berkshire x Yorkshire population. The group found QTL on many chromosomes with special emphasis on chromosomes 1, 17 and 18. From there, the station started to find the underlying genes responsible. Fine mapping of QTL on chromosomes 17 and 18 has continued with the addition of >45 genes in the relevant region on chromosome 17 and >10 on chromosome 18 and increasing the marker density to the linkage map. The Station also used comparative studies to examine candidate genes affecting longevity through reproduction and leg soundness. These include genes in the IGF pathway and genes associated with stress and muscle and bone pathways. Over 100 new genes have been mapped and analyses revealed that some of these genes have effects varying from 0.1 to nearly 1 parity more on average and also affect growth, reproduction and longevity. A NPB funded project is underway to look at 2000 sows (1000 old, 1000 young) to examine the effects of genes on leg traits. The group has identified over 100 genes, mapped 75 new ones and identified several genes that have large effects on many leg action and conformation traits. Using commercial populations they are also examining genes on chromosomes 2 and 12 for effects of scrotal hernia and have found several interesting associations.

    At Michigan State University, a Duroc x Pietrain resource population has been developed to map quantitative trait loci (QTL) affecting carcass composition and pork quality. In the report, they focused their work on confirmation of QTL on pig chromosome 6 by incorporation of marker genotype data for an additional 452 F2 pigs into the QTL analysis. A total of 962 F2 pigs were genotyped for the SSC6 markers S0087, SW122, SW1881 and SW322. Other SSC6 markers genotyped for the original genome scan (510 F2 pigs) included S0099, SW2406, SW2525, S0220 and SW2419. Data were analyzed with line cross least squares regression interval mapping methods using sex and litter as fixed effects and carcass weight or harvest age as covariates. QTL significant at the 5% chromosome-wise level were found for 24 h carcass temperature, Boston shoulder weight and marbling score. These results confirmed previously identified QTL and included four new QTL (carcass weight, first rib backfat, Boston shoulder weight and belly weight).

    Michigan State University has also targeted porcine insulin-like growth factor binding protein 2 (IGFBP2) for its association with economically important traits in pigs. For this study, 417 F2 pigs from their Duroc x Pietrain resource population were genotyped and the IGFBP2 was placed on SSC15 at 78.0 cM in a region previously found to contain significant QTL affecting meat color and tenderness. Pigs from litters segregating both alleles (N=226 F2 pigs) were used to determine potential associations between IGFBP2 genotype and growth, carcass and meat quality traits. Genotype effects (P<0.05) were found for loin muscle area (LMA) at 10, 19 and 22 wk of age, ADG, carcass length, ham wt, loin wt, 10th rib carcass LMA, 45 min pH, pH decline from 45 min to 24 h postmortem, CIE L*, CIE a*, subjective color score, Warner-Bratzler shear force, sensory panel muscle fiber tenderness and sensory panel overall tenderness.

    At Nebraska State University, fine mapping of QTL identified in previous scans was accomplished by using phenotypic and genotypic data from pigs of several generations. Phenotypic data were collected for birth weight (BWT, n = 1422), weaning weight (WWT, n = 1311), age at puberty (AP, n = 669), ovulation rate (OR, n = 797), number of fully formed pigs (FF, n = 841), number of pigs born alive (BA, n = 841), number of mummified pigs (MUM, n = 841), number of nipples (NN, n = 1434), incidence of pigs with splaylegs (n = 458), and number of stillborn pigs (SB, n = 841). Age at puberty was recorded in gilts of Lines 1 and 2 from Generation 2 through Generation 15 and in gilts of Lines 4, 5, and 6 through Generation 16. Ovulation rate was recorded in gilts of Lines 1 and 2 through Generation 11, and in gilts of Lines 4, 5, and 6 through Generation 16. Number of fully formed pigs, MUM, SB, BA were recorded in gilts within 24 h of parturition each generation.

    The Ohio State University has maintained both purebred Berkshire and Landrace populations of swine, which are available to evaluate the influence of pure and reciprocal crossbred lines on growth, carcass, and fresh and cooked pork quality traits of the loin, belly and ham. Data collected in previous years, including full pedigree and well defined contemporary groups are in place and DNA samples from all parent and progeny are available for use in collaborative gene, QTL discovery and or verification studies. The Station encouraged all interested collaborators within NC-1037 to inquire about potential collaboration opportunities. Litters of purebred and crossbred animals are produced in four farrowing groups annually with the ability to produce up to 60 litters per group. Specific aims of future generations will be discovery of mechanisms influencing variation in loin tenderness, a trait likely to have the greatest impact on consumer assessment of pork palatability and overall desirability.

    Washington State University has worked on the porcine mitochondrial transcription factor A (TFAM). The group determined both full-length cDNA and promoter sequences of the porcine TFAM gene, which were then used for comparative characterization of the TFAM gene with other species. Using Radiation Hybrid mapping the Station assigned this gene to porcine chromosome 14 (SSC14). A G557A substitution in intron 1 of porcine TFAM gene was detected and genotyped on a total of 252 animals, including 165 from seven Chinese and 87 from five Western pig breeds. Bayesian analysis via MCMC (Markov chain Monte Carlo) revealed that these two groups of pigs were well separated at this locus during the breed history; 95% of the posterior difference of TFAM allelic frequency between these two pig groups was greater than zero. As there are marked differences in fatness and lean meat production between Western and Chinese pig breeds, the TFAM gene deserves further investigation in order to evaluate its phenotypic effect on fat deposition and carcass traits in commercial pig populations. Washington Station has also worked on comparative assembly of Sus scrofa chromosome. The Station assigned more than 1,200 clones to their orthologous regions on human chromosomes 6, 9, 14, 15 and 18, which are consistent with the current comparative relationship between these two species. On the other hand, porcine chromosome 1 might have some small regions orthologous to human chromosomes 1, 10, 11 and 20. The comparative analysis provides information on candidate genes for QTL identified on the pig chromosome.

    Transcriptomics of production efficiency and pork quality using microarray analysis and real time PCR. University of Georgia has used transcriptional profiling to identify genetic mechanisms that respond to alpha-MSH, a MC3/4-R agonist. Three MC4R genotypes (2 homozygous and the heterozygous for MC4R) were selected from the UGA swine herd (PIC composite). Thirty-six pigs (6 per genotype per treatment) were randomly assigned to one of the following treatments: ICV administration of 150 ul 0.9% saline, or 10 µg NDP-MSH (agonist) in 150 ul of 0.9% saline. Feed intake was measure at 12 and 24 hous after treatment (time 0). All pigs were sacrificed 24 hours post-injection and hypothalamus, liver and middle layer of back fat was collected and mRNA was hybridized to 24,123 probe set Affymetrix Porcine Genome Arrays. The group found that MSH suppressed (P< 0.04) feed intake in all animals at 12 and 24 hr after treatment regardless of genotype with no treatment x genotype interaction detected (P > 0.8). There were 5070, 253 and 282 genes that were differentially expressed (P<0.07) in adipose, liver and hypothalamic tissue, respectively after central administration of MSH. The report concluded that the MC4R genotype did not effect the feeding behavior reponse to MSH but the greatest response to MSH was observed in adipose tissue gene expression.

    Iowa State University has worked on a USDA-NRICGP funded project to determine the genes expressed in both the endometrium and embryo/conceptus during the elongation and implantation phase of reproduction in both the Yorkshire and the Meishan breeds. using Affymetrix Genechip transcript profiling analysis. The endometrial results were analyzed and 47 genes were tested by Q-PCR across several contrasts; approximately 85% of expression patterns across > 200 pairwise comparisons were confirmed. Conceptus data analyzed in conjunction with similar data collected at Oklahoma State University determined that a joint analysis improves the power to detect differentially expressed genes. An analysis of the OSU data on embryonic gene expression during the critical time of elongation has been submitted for publication. In addition to the selection experiment for residual feed intake described above, Iowa State University also performed gene expression profiling for this project and initial analysis of gene expression data has been completed.

    Michigan State University has evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), liver, brain and uterine endothelium (UE). The extent of non-specific hybridization was evaluated by comparing intensities of 60 negative control oligonucleotides to background intensity and by estimating the distribution of intensities of non-control oligonucleotides. The study indicated that increasing temperature appreciably improved overall specificity while hybridizations performed at 40 °C showed unacceptable levels of non-specific hybridization. Diagnostics also utilized 60 perfect match/mismatch (PM/MM) oligonucleotide sets designed as part of the array elements (1, 2, 3, 5, 7 and 10 mismatched bases). A small number of negative controls showed consistently high signals suggesting they may not be suitable for use as negative controls. This work was supported in part by the USDA Pig Genome Coordination program. Michigan State University also used the Swine Protein-Annotated Long Oligonucleotide Microarray with pig longissimus dorsi (LD) samples for an expression QTL (eQTL) study. Microarrays have been completed for 176 LD samples, quality assessments of the arrays have been performed and data analysis is in progress. This work was supported by the USDA CSREES National Research Initiative.

    In collaboration with Dr. Chens Animal Genomics Laboratory at Nanjing Agricultural University, China, Washington State University has worked on porcine nuclear receptor coactivators 1 (NCOA1), 2 (NCOA2) and 3 (NCOA3) for their roles in adipogenesis. The group cloned these three porcine genes, identified their transcript variants and analyzed their expression level in relation to intramuscular fat (IMF) content in Longissimus Dorsi (LD) muscle. The station found that both NCOA1 transcript variant 2 (r=-0.554, p<0.01) and total NCOA1 (r=-0.516, p<0.01) expression levels were negatively correlated with IMF contents, while NCOA2 transcript variant 1 (r=0.605, p<0.01) and NCOA3 (r=0.435, p<0.05) were positively associated with IMF content in LD muscle. A similar study was also performed on porcine sterol regulatory element binding transcription factor 1 (SREBF1) for its role in intramuscular fat (IMF) deposition in pigs.

    Objective 2: Discover genetic mechanisms controlling animal health in pork production.

    Nomenclature and identification of new alleles for the SLA complex. USDA ARS BARC has focused their major efforts on updating the nomenclature for the SLA complex in collaboration with Michigan State University and other members of the ISAG SLA Nomenclature Committee. New SLA allele sequences and haplotypes have been designated: 74 new SLA alleles, including 18 SLA-1 alleles, 11 SLA-2 alleles, six SLA-3 alleles, two SLA-6 alleles, one SLA-DRA allele, 20 SLA-DRB1 alleles, three SLA-DQA alleles and 13 SLA-DQB1 alleles; in addition, 12 new SLA class I and 4 new class II haplotypes. These updates are now posted on the Immuno Polymorphism Database-Major Histocompatibility Complex (IPD-MHC) website (http://www.ebi.ac.uk/ipd/mhc/sla) which serves as the repository for maintaining a list of all recognized SLA genes and their allelic sequences.

    BARC, Japanese, and French scientists have worked on developing new methods for optimizing SLA allele genotyping. The analyses covered genetic polymorphisms in 23 SLA homozygous/heterozygous samples, proved that haplotype-specific patterns and variations of MS existed, and refined recombination points for several SLA haplotypes. These studies affirmed the existence of strong linkage disequilibrium (LD) in the entire SLA region; large haplotype blocks extended across >2-Mb segments. These MS markers constitute an alternative method to characterize the SLA haplotypes.

    BARC has also developed a simple and rapid method using the polymerase chain reaction (PCR)-sequence-specific primer (PCR-SSP) strategy for direct determination of SLA alleles, specifically, to type alleles at the multiple SLA class I genes and alleles at each of the three classical SLA class I loci (designated SLA-1, SLA-3 and SLA-2). The PCR-SSP typing system was designed with 47 discriminatory PCR primer pairs to amplify the current SLA class I alleles based on groups that have similar sequence motifs. Overall 24 class I allele groups corresponding to 56 class I genotypes were detected; additionally 23 low-resolution SLA class I haplotypes were identified in the 202 pig DNAs tested. This provides a reliable and unambiguous method for detecting SLA class I alleles. The MS and PCR-SSP typing methods provide important alternates to direct determination of the SLA alleles based on sequencing or SNP typing.

    Identification and mapping of genes responding to PRRS infection. BARC in collaboration with Kansas State and Iowa State has been coordinating the PRRS Host Genomic Consortium. This is a national effort to collect phenotype and genotype of 1000s of commercial pigs for their response to PRRSV infection. The PHGC database is being developed by Iowa State, Kansas State and BARC scientists to house the large amounts of phenotypic and genotypic data that will be collected across several research labs for the PHGC and stored for continued use by the PRRS research community. The schema for the database was originally designed based on data sets generated from the PRRS virus Big Pig project. This included data on pig, sex, birth date, infection details, weights, PRRS viral and serum antibody and cytokine levels. A new PRRS CAP2 grant will support BARC in collaboration with NC229 and PRRS CAP scientists to start SNP genotyping and whole genome association studies with this data. These analyses should reveal associations of PRRS disease resistance and growth traits with SNP alleles.

    BARC has used the new Pigoligoarray to identify immune regulatory and protective pathways in samples from swine infected with virulent PRRS virus with comparison to samples from vaccinated pigs. In their initial PRRS experiment animals were divided into three groups: (1) pigs infected with a virulent PRRSV isolate MNW2B; (2) pigs vaccinated using a contemporary PRRS ATP vaccine (Ingelvac®); and (3) control pigs. Different tissues were collected between days 3-6 post infection/vaccination. Results obtained for cranial lung tissues affirmed 923, 619 and 747 putatively differentially expressed genes for vaccinated versus control, vaccinated versus infected, and control versus infected, respectively. As expected, pathways involving interferons and other cytokines, as well as chemokines, have been identified as critical for differentiating infected from vaccinated pigs. Final statistical and pathway analyzes are underway to identify the biological functions and regulatory pathways that are most significant.

    At the University of Nebraska, two replications of a PRRSV challenge experiment with a total of 400 pigs have been conducted. Replication 1 occurred during summer 2002, and Replication 2 in winter of 2003. In each replicate a total of 100 PRRSV-negative, SEW pigs of the NE Index line (Line 2) and 100 pigs of a commercial Duroc-Hampshire (DH) line were used. Line 2, described above, is an inbred Large White-Landrace population that, at the time of the experiment, had been selected for 24 generations for increased litter size. It was expected to have low resistance to disease because of its relatively high inbreeding (~25%). Line DH is a non-inbred, terminal sire line that excels in growth and leanness and was expected to have greater disease resistance than Line 2. Two pigs from each of 200 litters by 163 dams and 83 sires were sampled to ensure genetic diversity within lines to maximize the chance that genes for both resistance and susceptibility to PRRSV existed in the sample. RNA was extracted from lung and lymph tissue of these same pigs and differences in gene expression between resistant and susceptible pigs was determined using microarray gene expression analyses.

    Identification and mapping of genes responding to Salmonella infection. Iowa State University reported their first study on Affymetrix-based transcriptional profiling in mesenteric lymph node for response to Salmonella infection, as well as a second paper on comparing the expression of genes responding to S. enterica serovar Cholerasuis (SC) versus S. enterica serovar Typhimurium (ST). The group has completed an Affymetrix-based transcriptional profiling study comparing the host response to infection by ST, and planned to submit a manuscript for publication in January 2008. They have identified genes that specifically respond to only one of these two infections, and they have evidence that many genes that respond to SC do not respond to ST infection. The Station has confirmed the microarray data for >20 genes tested by Q-PCR for both SC and ST infections, and has identified many regulatory pathways affected by Salmonella that have been shown to be involved in pathogen response in other species, as well as novel genes that indicates our understanding of pathogen response pathways needs to be expanded significantly.

    Characterization of the porcine circovirus associated disease complex caused by porcine circovirus type 2. The condition exists at the University of Nebraskas research farm. During the last three generations, the Station has scored all pigs of different lines from weaning through approximately 180 d of age for symptoms of PCVAD. Samples of pigs showing symptoms were submitted for necropsy to confirm typical symptoms in lymph and other tissues. PCR was conducted to confirm presence of circovirus. Pigs of Lines 1 and 2 from Generations 25-27 and from Lines 6 and 45, also from Generations 25-27, were scored for PCVAD at weaning (18 d), and at 60, 90, and 130 d of age. At each age, pigs were weighed and blood samples were collected. Serum collected at each age from all pigs scored positive for PCVAD and from a littermate or penmate without symptoms of PCVAD was submitted to the Iowa State University Veterinary Diagnostic Laboratory where PCV2 ELISA (antibody) and PCR (viremia) diagnostics were conducted. Analyses are complete for 3-4 samples from approximately 1000 pigs. Association of diagnostic data with PCVAD scores and pig weights will be used to determine degree of genetic influence in response to virus.

    Impact Statements:
    1. We have identified QTL and candidate genes in different pig resource populations, which will facilitate fine mapping to identify quantitative trait nucleotides controlling growth, carcass composition, meat quality, residual feed intake, reproduction and longevity. Such new information of important genetic variation will be useful in marker-assisted selection for genetic improvement of U.S. swine herds.
    2. Long-term selection experiments performed by the research group can lead to substantial advances in production efficiency and product quality, including increased litter size, improved growth rate and feed efficiency, improved product quality, and improved resistance to disease. These will result in the availability of high quality pork at the lowest possible price to the U.S. consumer and for international trade.
    3. The working group has assessed and validated the new swine Protein-Annotated Oligonucleotide Microarray, which demonstrates its utility for a variety of porcine tissue types and is useful for other researchers who plan to use the array for their experiments. Such studies will allow other researchers and industry to capitalize on developed information, methods and databases.
    4. Utilization of pigs as a model organism might provide new opportunities for alternate uses of swine through a greater understanding of the biological aspects of swine. These opportunities could create new businesses within the U.S. pork sector and increase profit potential for those participating in this unique industry.
    5. Understanding the genetic complexity of resistance to disease would provide opportunities to reduce the use of antibiotics or allow for more effective use of antibiotics in swine production. This would likely lead to greater consumer acceptance of pork and improve U.S. pork profit potential by increasing the demand for pork and decreasing expenses associated with unhealthy pigs.
    Last Modified: 03-Aug-2009

    Date of Annual Report: 02/08/2010

    Report Information:
  • Annual Meeting Dates: 01/09/10 to 01/10/10
  • Period the Report Covers: 10/2008 to 09/2009

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    January 9, 2010

    8:00 am - 12:00 pm The morning of January 9 was a joint symposium with NRSP8 cattle/swine Subcommittees. Both Penny Riggs (Texas A&M University) and Cathy Ernst (Michigan State University) presided over the morning session. Four speakers were invited to speak and their topics are listed below.

    8:00-8:15 am Penny Riggs (Texas A&M University, riggs@tamu.edu) and Cathy Ernst (Michigan State University, ernstc@msu.edu) "Introduction" 8:15-9:00 am Erdogan Memili, Mississippi State University (em149@ads.msstate.edu) "Molecular determinants of fertility in bovine gametes & embryos and regulation of developmental competency" 9:00-9:45 am Martien Groenen, Wageningen University (martien.groenen@wur.nl) "The Porcine Hapmap Project: Genome-wide assessment of nucleotide diversity, haplotype diversity and footprints of selection in the pig" 9:45-10:15 am "Break" 10:15-11:00 am Chris Tuggle, Iowa State University (cktuggle@iastate.edu) "Developing predictive models for identifying pigs with superior immune response and improved food safety" 11:00 am - 11:45 pm David Threadgill, NC State (threadgill@ncsu.edu) "Moving from QTL to systems: the role of genetic interactions in driving phenotypes"

    1:30 pm - 6:30 pm The afternoon of January 9 was a joint workshop with NRSP8 swine Subcommittee. Dr. Cathy Ernst (Michigan State University) presided over the afternoon session. Three people were invited to speak, covering pig models for human disease, eQTL for identification of candidate genes and application of porcine 50KSNP chips. Elisabetta Guiffra, a visiting scientist from Italy also discussed and updated her PRRSv QTL study. The topics are listed below.

    1:30-2:00 Melissa Ashwell, North Carolina State University (melissa_ashwell@ncsu.edu) Chondrocyte gene and protein expression in a porcine osteoarthritis model  Early detection of tissue degenerative factors 2:00-2:30 Juan Steibel, Michigan State University (steibelj@msu.edu) Genome-wide linkage analysis of gene expression of loin muscle tissue identifies candidate genes in pigs 2:30-3:00 Max Rothschild, Iowa State University (mfrothsc@iastate.edu) Applications of new porcine genomic tools to trait discovery and understanding genomic architecture 3:00-3:10 Elisabetta Giuffra, Italy Update on GWAS for PRRS viremia in Italian commercial pigs

    3:10-3:45 NC-1037 Planning and Organizing Session After the talks, there was a break and the meeting then reconvened as the NRSP8/NC-1037 membership group. Initially, there was informal discussion on the level of participation of members of both NRSP8 Swine subcommittee and NC-1037, and what could be done to encourage greater participation from absent members, especially NC-1037 members. There are two general groups of individuals who are members of NC-1037; quantitative geneticists/breeders and genome mappers/molecular geneticists. Attendance at PAG meetings is less attractive to the former group members, who also have a Quantitative Genetics Multi-state project meeting available to them. It was decided that as Georgia had agreed to host the next meeting they would be consulted for methods to improve attendance; one possibility was to have the meeting in conjunction with the National Swine Improvement Federation meeting in December, which usually is held in Tennessee.

    3:45-3:55 NRSP-8 Swine Coordination Report Max Rothschild as NRSP8 Swine Genome Coordinator presented his report and indicated he was asking for community ideas on how to spend support funds for advancing the community, indicating that recently a large amount of such funds had gone to purchase of SNPchips for the PHGC project; Joan Lunney indicated that support was invaluable in developing this important Multi-State project. Max indicated that two projects requesting funds for SNPchips had been brought to his attention, a meat quality project from S. Lonergan at Iowa State and a PCV2 project from Daniel Ciobanu at University of Nebraska-Lincoln. Daniel also introduced himself to the group as a new member and described the project as a collaboration with several faculty members at UNL including Rodger Johnson, as well as Max R. at ISU and Montse Torremorell at University of Minnesota. Subsequent to this discussion, Jack Dekkers indicated he would request funds for maintaining his residual feed intake lines so that members of NRSP8 would have access to these resources, and several members indicated they would request support for pig genome annotation efforts. Please send additional ideas to Max.

    3:55-4:05 NRSP-8 Bioinformatics Coordination Report Jim Reecy provided a report on Bioinformatics coordination team efforts, including several tools of value to the NRSP8/NC-1037 subcommittees, including Animal Trait Ontology, animal QTLdb, genome annotation efforts, and the PHGC database development.

    4:05-4:15 NRSP-8/NC-1037 Administrative Reports Peter Burfening provided information in place of Muquarrab Quereshi (NRSP8 Administrator), indicating that 2009 was a year of significant advancement but also of change much of which was not beneficial as of yet to animals genome research support.

    Bert Stromberg (NC-1037 Administrator) indicated that a new project will be due to NIMS in December 2011 to start in October 2012, and that the NC-1037 group should be considering how to both provide evidence of collaboration across stations and what the direction of any renewal should be. He indicated there was concern expressed within Experiment station administration because NC-1037 did not physically meet during the previous year. Joan Lunney asked whether the numerous conference calls to develop and implement the PHGC should be documented as evidence of significant inter-station collaboration. Bert indicated he would check into that. At any rate, the next meeting, to be held after October 1, 2010, should have a major focus on the future and a possible re-write.

    4:15-4:25 Harvey Blackburn, National Animal Germplasm Program, DNA Repository Discussion Harvey Blackburn of the National Animal Germplasm Program provided details on his group and invited scientists to explore storage of genetic material and germplasm there. Such services are free to both send and receive samples.

    4:25-4:30 NC-1037 Officer Elections NC-1037 members unanimously agreed to the following changes in our rotating leadership schedule: Chair V Chair Sec 2010 R. Rekaya GA Jon Beever IL NC rep 2011 Jon Beever IL NC rep MI rep 2012 NC rep MI rep IA rep

    4:30-5:30 NRSP-8 Swine Committee/NC-1037 Station Reports Additional members then presented updates on their research, including Jack Dekkers at ISU, who discussed his RFI selection lines, Chris Tuggle who discussed the functional genomics and endocrinological analysis of these lines, Jiang Zhihua who discussed two mapping projects on genetic networks for economically important trait and HAAPY mapping with new generation sequencing. Written station reports were received from BARC, Georgia, Iowa, Michigan, Nebraska, Penn State and Washington.

    5:30-6:30 Joan Lunney, USDA-BARC, Update on PRRS Host Consortium Joan Lunney introduced members and updated everyone on currently funded projects (PAG PHGC slide 18) and their current progress in terms of PHGC phenotyping and genotyping. The group further discussed traits that are important to use for GWAS and results of Steibels statistical analyses to identify pigs in high/low (H/L) virus/weight groups. Gene expression plans were also reviewed at the meeting. Several industry representatives were in attendance, including Archie Clutter (Newsham) and Alan Mileham (Genus).

    Thanks to Dr. Tuggle for providing materials for preparation of the minutes.

    Accomplishments:
    Objective 1. Further understand the dynamic genetic mechanisms that influence production efficiency and quality of pork.

    Georgia Station worked on identification of genes and pathways that respond to intracerebroventricular injection of melanocortin-4 receptor (MC4R) agonist, NDP-MSH, in pigs homozygous for MC4R, D298 allele (n= 12), N298 allele (n = 12) or heterozygous (n = 12). Although a small number of animals were used in each combination of experimental factors (genotype and treatment), every effort was made to balance the design by assigning animals with similar weight and gender distribution to each group in order to avoid potential confounding with genotype by treatment interaction. A total of 1724, 40 and 2 genotype x treatment interactions were detected for adipose tissue, liver and hypothalamus, respectively. As Nesfatin-1 is a secreted factor and expressed in adipose tissue, the station hypothesized that it plays an important role in regulating feed intake of the pig. To test this hypothesis, the group fitted prepubertal gilts with intracerebroventricular (i.c.v.) cannulas. Cumulative feed intake for each pig was monitored for 48 h following i.c.v. injection of 100 g of either recombinant human leptin or nesfatin-1 in 150 _l of phosphate buffered saline (n = 5 per treatment). Control animals received i.c.v. injection of 150 _l of phosphate buffered saline alone (n = 5). The station found that Nesfatin-1 suppressed (P < 0.01) feed intake for 48 h after i.c.v. injection in an almost identical fashion as leptin. The availability of reliable and inexpensive genotyping platforms for single nucleotide polymorphism (SNP) markers has made the possibility for genomic breeding value estimation in several livestock species a reality. Unfortunately, at around two hundred dollars per animal this technology is still too expensive for its massive use at the commercial level. Currently, this technology is mainly used for genotyping top animals and then used in a two-step procedure for estimating genomic breeding values. For its use at the population level, the SNPs genotypes of non-typed animals have to be inferred somehow from the already genotyped animals and their relationships. Therefore, the station proposed combining genotyping information from high and low density SNP panels with the latter being used for large scale genotyping. This low density and low cost chip will provide an additional source of information, linkage disequilibrium, in inferring genotypes of non-typed animals. The group found that their proposed method is able to increase the accuracy of the estimated breeding values by 6 to 13% depending on the number of SNPs in the low-density panel and the number of genotyped animals.

    A group of researchers at Iowa Station performed a variety of projects in order to improve intramuscular fat, sow longevity, feet and leg soundness, and residual feed intake in pigs. The Station developed methods, models, and software to predict intramuscular fat (IMF) on the live pig using real-time ultrasound, which increased IMF by 2.1% after six generations of experimental selection, with slightly more backfat and slower growth, but little or no effect on other meat quality traits. A new selection line based on an index of composition and meat quality, including IMF, has been initiated. The station continued their research in the sow longevity utilizing a commercial sow population and identified early predictors of longevity (leg score and early reproduction) using commercially available lines. The group reported low heritability estimates for the majority of the feet and leg soundness traits and for other visually evaluated body conformation traits. Using information from the new DNA sequence that is now available, the porcine single nucleotide polymorphism (SNP) 60K chip is being used to examine associations with reproduction, feet and leg soundness and growth and performance traits. These efforts have resulted in several genetic markers that may be useful in the industry in the near future. The station completed a sixth generation selection experiment for residual feed intake and evaluated pigs from the efficient and control lines to get insight into the biological basis of the line differences. Selection for efficiency has resulted in a strong correlated response in serum IGF-1 concentration measured at an early age. The group used the 60k high density array to genotype 750 pigs from these lines and identified several regions with effects on feed intake, growth, backfat, and efficiency. Microarray gene expression profiling in these lines identified several transcription factors that regulate gene expression in back fat and liver tissues, in response to fasting. Genes that are differentially expressed during trophoblastic elongation in porcine embryos were identified. To facilitate integrated analysis of gene expression data, a pig gene expression database (www.anexdb.org) has been developed for the Affymetrix platform and all 1.6 million porcine ESTs were used to create improved annotation of the Affymetrix porcine Genechip.

    Michigan Station focused on four research areas: 1) Assessment of the swine protein-annotated oligonucleotide microarray; 2) Genome-wide expression QTL (eQTL) analysis of loin muscle tissue for identification of candidate genes in pigs, 3) Transcriptional profiling during fetal skeletal muscle development of Piau and commercial pigs, and 4) Factors affecting measures of longevity and stayability in Yorkshire sows. The station evaluated the specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org) by profiling the expression of transcripts from four porcine tissues. Of the total 20,400 oligonucleotides on the Pigoligoarray, the group found 12,429 transcripts that were putatively differentially expressed (DE). Analyses for tissue-specific expression [over-expressed in one tissue with respect to all the remaining three tissues (q<0.01)] identified 958 DE transcripts in liver, 726 in muscle, 286 in uterine endothelium and 1027 in brain stem. These hybridization results were confirmed by quantitative PCR (QPCR) expression patterns for a subset of genes after affirming that cDNA and amplified antisense RNA (aRNA) exhibited similar QPCR results. The station performed a first comprehensive genome-wide eQTL scan on loin muscle tissue from 176 F2 Duroc X Pietrain pigs. Based on 124 microsatellite markers and measured transcript abundance of 20,400 oligonucleotides, the group detected 62 unique eQTL, with 40 oligonucleotides physically aligned to the pig genome including 24 that mapped to the chromosome where their linkage peak occurs. In order to identify differentially expressed genes in longissimus dorsi (LD) of pigs at 40 and 70 d of gestation, the group isolated total RNA from fetuses obtained from gilts at each gestational age (n=3 commercial gilts; n=4 Piau gilts) for transcriptional profiling using the Pigoligoarray (www.pigoligoarray.org). Interestingly, the station found that only 226 (24%) of these genes were common to the two breed types, whereas 422 (45%) were preferential to the Piau breed and 270 (29%) were preferential to the commercial pigs. In order to assess the relationship of developmental performance factors with longevity and to determine the genetic variation within six different descriptions of longevity, the Michigan Station collected 14,262 records from Yorkshire females with at least one farrowing record, from both nucleus and multiplication herds across 21 farms. The group found that 1) within a contemporary group, fatter, slower growing gilts had a decreased risk of being culled; 2) sows that had more pigs born alive, fewer stillborn, and heavier litters at weaning in their first litter had a decreased risk of being culled; and 3) sows from nucleus herds experienced a greater risk of being culled. These results indicated that there are early indicators that can be monitored to provide insight to the future productivity or length of productive life of Yorkshire females. Additionally, sufficient genetic variation exists, regardless of definition, to improve sow longevity.

    Nebraska Station also reported their work on sow longevity, but focused on examination whether restricting energy intake during gilts developmental period would increase their longevity and lifetime productivity. Two lines, L45X, a cross of the NE litter selection Line 45, and an industry Large White Landrace cross (LWxLR) were used. Project gilts of both lines were sired by boars of an industry maternal line and thus were half sibs. The lines differed in fertility, litter size, and rate of lean growth and were managed with ad libitum access to feed to breeding age or with 25% restriction of energy from 123 d of age to breeding. The group found that both line and treatment significantly affect the probability that gilts expressed pubertal estrus by 230 d of age; 95% of L45X gilts reached puberty compared with 88% of LW x LR gilts (P = 0.01), and 96% of gilts developed with ad libitum intake reached puberty compared with 86% of gilts developed with energy restriction (P = 0.0001). Overall, energy restriction decreased the proportion of gilts that expressed estrus by 230 d of age and increased the age at puberty. Thereafter, females developed with both regimens had similar reproductive performance at each parity and similar lifetime production. DNA was extracted from tissue of each pig and SNP genotypes using the 60K SNP chip were determined and will be used to identify regions of the genome associated with sow longevity and reproduction traits.

    Penn Station reported development of over 10,000 markers on the pig IMNpRH212,000rad panel to obtain 10,030 mapping vectors, including 2635 MSs, 2530 ESTs, 2759 genes, 2043 BESs and 63 SNPs. The station merged these with ~50% of those markers typed on the IMpRH-7000rad panel to construct parallel framework maps at LOD >=10, thus integrating the porcine genetic-RH-FPC sequence maps and improving the pig-human comparative map. The group observed a ~3.0-fold increase in map resolution in the IMNpRH212,000rad panel over the IMpRH7000rad panel. In addition, Penn Station also built an initial high-resolution comprehensive radiation hybrid (RH) map of the pig chromosome (SSC) 4 that integrates the corresponding RH12,000rad, RH7000rad, genetic, BAC finger-printed contig (FPC) maps, pig RNA-seq data, and a comparative map to human chromosome (HSA) 1 and 8. The map contains 573 markers, including 71 microsatellites (MS), 168 genes and ESTs, and 89 BESs, within three linkage groups. Two linkage groups (62 and 39 markers) cover the entire SSC4p with an accumulated map distance of 1999.7 cR7000 and 3790.8 cR12,000. The other group (227 markers) covers the whole SSC4q with a map distance of 3508.1.8 cR7000 and 7416.5 cR12,000. The group also used transcriptomic data developed from RNA-seq, the NCBI Ssc UniGene (Build 37), and the Sanger gene annotation (September, 2009) within Build 9 of the Sus scrofa genome to identify conserved regions in the human and swine genomes. Approximately 42 million Illumina RNA-Seq reads were aligned to Build 9 of the Sus scrofa genome using GSNAP. RNA-seq contigs were assembled de novo using a hybrid protocol incorporating ABySS-P and PCAP, and previously-unpublished EST data was assembled into contigs using CAP3. The contigs were aligned to the swine and human genomes, integrated into the Alpheus database, and compared in a series of permutations designed to identify regions that were 1) expressed in swine, 2) not present in the porcine UniGene assembly, 3) identified in the human genome, and 4) not currently represented in the annotation of swine genome.

    The major activities at the Washington Station included molecular characterization of adipocytes, association analysis of functional genes and in silico construction of QTLs/candidate gene maps for pork quality. In molecular characterization of adipocytes, the station obtained mature (lipid-filled) pig-derived adipocytes from the perirenal adipose depot after routine slaughter at the Washington State University meats laboratory. The isolated mature adipocytes were purified by using serial differential plating methods to prevent any contamination of mature adipocyte cultures by fibroblast-like cells. At the end of the procedure, clonal mature adipocytes were marked using a fine-tip pen on the bottom of the flask, and photomicrographs of cell transitions were then captured for these marked adipocytes during dedifferentiation. The group observed that clonal cultures of pig-derived mature adipocytes are capable of dedifferentiating and forming proliferative-competent progeny cells in vitro. Mature pig adipocytes extrude lipid before proliferation, whereas beef-derived adipocytes divide without expelling lipid. In collaboration with Dr. Chen at Nanjing Agricultural University and Dr. Liu at Huazhong Agricultural University, China, the group also reported candidate genes for pork quality, such as splicing factor serine-arginine rich protein (SFRS18) for intramuscular fat content and cardiomyopathy associated 1 (CMYA1) for backfat in pigs. Furthermore, the Washington Station pursued an in silico construction of a pork quality QTL/candidate gene map based on the current PigQTLdb. The database contains a total of 1831 QTLs from 113 publications representing 316 different pig traits. Among them, at least 520 significant or suggestive QTLs were identified for 86 traits related to fat deposition and fatty acid composition in pigs, such as abdominal fat, backfat (average) thickness, backfat at shoulder, backfat at first rib, backfat between 3rd and 4th rib, backfat at tenth rib, backfat at last rib, backfat at last lumbar, backfat weight, fat percentage in carcass, intermuscular fat percentage, marbling, fat androstenedione level and fatty acid composition. The group found that these QTLs for fat deposition and composition are not evenly distributed in the porcine genome, ranging from 2 on SSC16 to 68 on SSC7. Among these 14 clusters of traits, 8 are related to backfat measurements. Of them, most QTL data refers to average backfat. Although both marbling and intramuscular fat content measure fat stored in muscle and they are well correlated, their genetic backgrounds are quite different. So far, at least 30 candidate genes have been identified that affect fat deposition in pigs.

    Objective 2: Discover genetic mechanisms controlling animal health in pork production.

    In collaboration with Kansas Station and Iowa Station, BARC reported progress on the PRRS Host Genomic Consortium (PHGC) to use a nursery pig infection model to determine the role of host genetics in resistance to PRRS and in effects on pig health and related growth effects. Crossbred pigs from high health farms were donated by 4 different commercial sources (PIC/Genus, Newsham, Fast Genetics, Genetiporc). To date 6 trials of 200 have been completed or started. The project uses a Nursery Pig Model to assess pig resistance/ susceptibility to primary PRRSV infection. After acclimation, the pigs were infected with PRRSV and followed for 42 days post infection (dpi). Blood samples were collected at 0,4,7,10,14,21,28,35 and 42 dpi and weekly weights recorded. Results from the first 5 trials of 200 pigs each have affirmed that all pigs become PRRSV infected; some pigs clear virus from serum quicker and weight effects are variable. Multivariate analyses of viral load and weight data have identified PHGC pigs in different virus/weight categories, so that ongoing serum cytokine and gene expression studies can compare data from PRRS resistant/maximal growth pigs to PRRS susceptible/reduced growth pigs. Genomic DNA from PHGC pigs has been prepared. The first 850 samples have been genotyped with the PorcineSNP60 Genotyping BeadChip (containing over 60K single nucleotide polymorphisms or SNPs). The next 450 DNAs will be genotyped in the spring 2010.

    Scientists at Michigan Station and BARC performed analyses to validate the utility of the new Swine Protein-Annotated Oligonucleotide Microarray (www.pigoligoarray.org), a second generation porcine 70-mer oligonucleotide-microarray comprised of 20,400 oligos. BARC and NCSU scientists then used the Pigoligoarray to probe immune regulatory and protective pathways in samples from swine infected with virulent PRRS virus and compare them to samples from vaccinated pigs as part of the national PRRS CAP grant. Animals were divided into groups: pigs infected with 2 different virulent PRRSV isolates, NC Powell and MNW2B and compared to control pigs and pigs vaccinated using a contemporary PRRS ATP vaccine. Tissues [cranial lung, distal lung, tracheobronchial lymph nodes (TBLN) and tonsils] were collected between days 3-6 post infection/vaccination. Total RNA was isolated and labeled using Alexa Fluor® 555 and Alexa Fluor® 647 dyes (Invitrogen). A microarray loop design was applied to compare gene expression between individuals from all three groups and additionally to investigate differences due to day post infection within the PRRSV infected group. Analyzes were carried out using R and SAS software. Results obtained for cranial lung tissues affirmed 923, 619 and 747 putatively differentially expressed genes for vaccinated versus control, vaccinated versus infected, and control versus infected, respectively. As expected, pathways involving interferons and other cytokines, as well as chemokines, have been identified as critical for differentiating infected from vaccinated pigs. More detailed statistical and Ingenuity pathway analyses are underway to identify the biological functions and regulatory pathways that are most significant.

    BARC, ISU and NADC scientists also used real-time expression studies to affirm results using the NRSP8-Qiagen long oligo arrays to determine gene expression in tissues collected from Salmonella enterica serovar Choleraesuis infected porcine lung and lymph nodes. These results demonstrated key genes regulating responses from control, 24 hour (hr), and 48 hr Salmonella enterica serovar Choleraesuis infected porcine lung tissue and implicated NFkB regulated immune pathways. Separate studies are underway with reproductive endothelium tissues from early pregnant and cycling Yorkshire and Meishan gilts using the Affymetrix porcine GeneChipÔ.

    BARC developed a new bead based assay and optimized it with South Dakota Station using the BioRad Bio-Plex (Luminex) platform. This multiplex assay will detect innate inflammatory [interleukin-1beta (IL-1 ²), IL-6, IL-8, interferon-alpha (IFN ±), TNF ±]; regulatory (IL-10), Th1 (IL-12, IFN ³) and Th2 (IL-4) cytokines. A 9-plex cytokine assay has been developed and its use verified comparing cytokine levels in sera from PRRSV vaccinated and challenged pigs. Further evaluation and collaborative utilization of swine cytokine multiplex assays are being targeted including assays for oral fluids and detection of different infectious agents.

    The PRRS Host Genomic Consortium (PHGC) Database was developed by Iowa Station, Kansas Station and BARC scientists to house large amounts of phenotypic and genotypic data that will be collected across several research labs for the PRRS research community. The schema for the database was originally designed based on data sets generated from the PRRS virus Big Pig project. This included data on pig, sex, birth date and infection details, PRRS viral levels in serum over time and tissue levels at kill, anti-PRRSV antibody (ELISA and neutralizing antibody) responses, serum cytokine levels and tissue gene expression results, and SLA alleles. This internet accessible relational database was designed to allow for the addition of new data types as they are generated over the course of the project. This flexibility will allow real-time data updates and sharing among users from geographically different locations. Access to the PHGC database is controlled through a Cooperative Research and Development Agreement (CRADA) Material Transfer Agreement (MTA); the core data is restricted to PHGC members prior to publication.

    Nebraska Station conducted two replications (211 and 220 pigs) of a PRRSV infection experiment. At 34 ± 5 days of age (8.2 ± 1.8 kg body weight), weaned pigs free of PRRSV were transported from their farm of origin to the wean-to-finish barn at the Haskell Agricultural Laboratory and allotted to one of 16 pens. After a 7-day (Rep1) or 19-day (Rep2) adjustment period, pigs were weighed, blood samples were collected, and they were inoculated with PRRSV FL12 (104.8 TCID50/2 mL). Blood was drawn at 4, 7, and 14 d post-inoculation, weight was recorded at 4, 7, 14, and 35 d post-inoculation and every 2-wk after d 35. Blood samples were analyzed for viremia and interleukin 8 (IL8). An index of serum viremia and body weight changes were used to describe response. Levels of IL8 were related to viremia and body weight. The station found that mean viremia for Rep 1 and 2 was similar at 4 d (5.76 and 5.59 viremia, log10) and 7 d (6.15 and 5.67) post-infection, dropped sharply at 14 d in Rep 2 (3.82), but not in Rep 1. Correlations among weights at 0, 4, 7, 14 and 35 d after inoculation with PRRSV, viremia at 4, 7 and 14 d after inoculation, and pre-inoculation levels of IL8 were low. Weight gain from 0 to 4, 4 to 7, 7 to 14, and 14 to 35 d after inoculation, viremia at 4, 7, and 14 d after inoculation, and pre-inoculation levels of IL8 were negatively correlated. Tissue, blood samples, and data from these pigs and from 400 pigs infected in previous experiments will be used for SNP associations studies to identify markers associated with resistance.

    Nebraska Station also examined genetic and environmental variance/co-variances for incidence of Porcine Circovirus Associated Disease (PCVAD) and immune responses to the PCV2 virus. Incidence of PCVAD was recorded in 3,440 pigs, 14.4% showed definitive symptoms, and blood samples at several ages were drawn. Based on these data, additional experiments in which pigs are infected with PCV2 virus were planned. In the first replication of that experiment, 229 weaning age pigs were transported to the wean-to-finish barn at the Haskell Agricultural Laboratory. Serial weights and blood samples were collected through 90 d of age. Pigs were not vaccinated for PRCV2 and came from a herd positive for this virus. Pigs showed symptoms of natural infection at approximately 90 d of age. They were euthanized at approximately 110 d of age and blood, lung, spleen, and lymph tissue was collected. The station found that heritable variation existed for incidence of PCVAD, body weights, PCV2 viremia and PCV2 antibody titers. Incidence of PCVAD was negatively correlated genetically with post-weaning weights and antibody titers, and positively correlated with viremia levels. Post-weaning weight was negatively correlated with viremia and positively correlated with antibody titers, and viremia and antibody titers were negatively correlated. Males had a greater incidence of PCVAD than females (P < 0.001). Additional replications of this experiment are planned for 2010. When sufficient replication occurs, gene expression and SNP association analyses will be performed.

    Impact Statements:
    1. The PRRS Host Genomic Consortium coordinated by BARC is a multi-year project that is funded by a US consortium representing the US National Pork Board (NPB), USDA, universities and private companies; it represents the first-of-its-kind approach to food animal infectious disease research. Overall, the PHGC project will enable researchers to verify important genotypes and phenotypes that predict resistance/ susceptibility to PRRSV infection.
    2. The finding on the large number of significant interactions between MSH treatment and MC4R genotype observed at Georgia Station for adipose tissue demonstrates its dynamic and complex role in the regulation of feed intake. Gene expression analysis supports the hypothesis that it is a signal to the central nervous system regarding metabolic state. Results from combining low and high density SNP panels suggest that for some livestock industries, the proposed procedure could offer a practical and cost effective tool for large scale use of genomic information in the genetic evaluation.
    3. QTL mapping projects conducted at Iowa Station have led to a better understanding of the genetic basis of economic traits in the pig and in methods that can be used in genetic selection to improve the efficiency and quality of pork production. For example, by using three genetic markers, a farrow-to-finish producer can increase average parities/sow from e.g. 3.4 to 3.5, which increases profit $0.23 for every market hog sold. Likewise, a farrow-to-wean producer can realize a return of $0.13 for every pig sold for the same increase of 0.1 average parities.
    4. Assessment and evaluation of the new Swine Protein-Annotated Oligonucleotide Microarray conducted at Michigan Station and BARC demonstrates its utility for a variety of porcine tissue types and is useful for other researchers who plan to use the array for their experiments. Genome-wide expression QTL (eQTL) analysis of loin muscle tissue for identification of candidate genes in pigs performed at Michigan Station represents the first comprehensive genome-wide expression quantitative trait loci (eQTL) study reported for a livestock species.
    5. Experiments conducted at Nebraska Station improve our understanding of genetic basis for resistance to two important disease pathogens (PRRSV, and PCV2 virus) and to genetic variation in sow longevity. SNP association analyses applied to data collected in these experiments will identify procedures to enhance genetic selection response for these important economic traits.
    6. The high-resolution integrated IMNpRH2-12,000rad and IMpRH-7000rad maps as well as the genetic and BAC FPC maps developed at Penn Station provide an inclusive comparative information/knowledge between pig and human genome (or other mammalian genomes). The maps should be useful for the swine genome sequencing, genome annotation and QTL fine mapping.
    7. The cell culture system and results observed at Washington Station suggest that this in vitro system will aid in our understanding of lipid metabolism, regulation of single cells, processes involved in characteristics of putative stem cells residing in adipose tissue. Identification of QTL and QTN for economically important traits will faciliate marker-assisted selection to optimize production, quality, nutritional value and resistance to diseases in pigs.
    Last Modified: 26-Feb-2010
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