Project Number: 5438-31000-083-04
Project Type: Specific Cooperative Agreement

Start Date: Jun 01, 2007
End Date: May 31, 2012

Objective:
Develop linkage disequilibrium (LD) haplotype maps of genes responsible for immune function and disease susceptibility in the exotic pig breeds. Results will direct gene specific resequencing which will be used to determine the influence of selective pressures on immune function during the domestication and selection of commercial swine populations. Use RNAseq to examine differential expression (DE) of genes within the muscle of animals that differ in genotype at the paternally expressed IGF2 locus. Results from previous transcriptome sequencing (see FY2010 Objectives) will be used in conjunction with deep-sequencing of the muscle transcriptome from pigs of differing genotypes and developmental time points to examine how genotype influences the transcriptome and to determine molecular phenotypes resulting from the presence or absence of the IGF2 intron 3-g3072A mutation.

Approach:
Genomic DNA samples of various exotic sus species including Sus barbatus, Sus verrucossus, Sus celebenesis and P. africanus will be genotyped. Genotype data will be used to construct maps of regions encompassing immune regulation genes. Resequencing of specific immune genes in both commercial and exotic swine populations will be used to identify variations responsible for altering innate immunity and host susceptibility to infectious agents. We will target the immune function genes such as toll-like receptors, major histocompatibility complex, interleukins and immunoglobulins. The may be included to provide insight to phylogenetic divergence of TLR and the other gene families. Though several groups have established the presence and increased prevalence of the IGF2 intron 3-g3072A mutation in the US commercial swine population, it is unclear whether increased muscling results from increased hyperplasia during fetal development and/or by increased postnatal hypertrophy. Furthermore, the molecular impact of increased IGF2 expression levels remains unclear. For this objective, IGF2 mutant and wild-type animals will be produced with a common genetic background. First, a complete growth curve of body weight and length will be established from fetal development through market weight. Muscle hyperplasia, hypertrophy and fiber type will be investigated. Finally, global gene expression, throughout the life span of market animals will be established. Boars having the genotype G/A at the IGF2 locus will be mated to commercial sows which are A/A at the IGF2 locus. From our previous work and other unpublished data, we have demonstrated that both alleles are present in the US Berkshire population with the wild-type G allele having the highest frequency. These matings will result in offspring with either a paternal G for the IGF2 locus (lighter muscled) or a paternal A at the IGF2 locus (heavier muscled). IGF2 genotype will be determined by real-time PCR allele discrimination assay. This breeding scheme will allow for both genotypes to be produced in each litter and hold the sow genotype constant. Key events in fetal and postnatal development will be targeted to determine changes in gene expression, muscle cell size and number and muscle fiber type where appropriate. Muscle fibers form in two waves of fusion with primary fiber formation occurring between day 35 and 50 of gestation and secondary hyperplasia beginning between day 50 and 60 of gestation. Fiber formation is thought to be complete by day 90 of gestation. All further growth of muscle occurs through hypertrophy as muscle fiber number is set at this point. To target these developmental events, Longissimus dorsi (LD) muscle will be collected from fetuses at gestational days 50 and 90. Samples from pigs will also be collected at birth, weaning (21 days old) and at the time of harvest at market weight (approximately 25 weeks). These samples will be used to determine gene expression levels, muscle fiber hyperplasia and hypertrophy and fiber type in IGF2 mutant and wild type pigs.