Location: Reproduction Research
Project Number: 3040-31000-089-01-S
Project Type: Specific Cooperative Agreement
Start Date: Jun 1, 2012
End Date: Sep 30, 2014
PORCINE OBJECTIVES: Develop linkage disequilibrium (LD) haplotype maps of genes responsible for immune function and disease susceptibility in the exotic pig breeds. We will begin with Toll-like receptor genes. Toll-like receptors (TLR) 3, 7 and 8 recognize economically detrimental viruses including rotavirus and African swine fever virus. Gene specific resequencing of domestic as well as wild suids 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 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-g30nA mutation.
Genomic DNA samples of various exotic sus species including S.barbatus, S.verrucosus, S. celebenesis and Phachoerus african have been genotyped using the Porcine SNP60Beadchip. Genotype data will be used to construct haplotype maps of regions encompassing immune regulation genes. Resequencing of specific immune genes in both commercial and exotic swine populations will identify variations responsible for altering innate immunity and host susceptibility to infectious agents. Sequence analysis of the toll-like receptor genes has begun. We will follow those with major histocompatibility complex, interleukins and immunoglobulins and will be included to provide insight to phylogenetic divergence of 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 greater hyperplasia during fetal development and/or greater 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. These matings will result in offspring with 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 a real-time PCR allele discrimination assay. Both genotypes will be produced in each litter with identical sow genotypes. 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, if appropriate. Muscle fibers form in two waves of fusion with primary fiber formation occurring from 35 to 50 days 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. To target developmental events, Longissimus dorsi muscle will be collected from fetuses at gestational days 50 and 90. Samples from pigs will also be collected at birth, weaning (21 days) and when harvested at market weight (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. The W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois will perform deep-sequencing to determine differential expression of mRNA between IGF2 Gpat and IGF2 Apat animals. Based on body weight and loin eye area, four animals from each genotype/time point combination which are closest to the mean will be selected. When possible, littermates will be used.