Location: Reproduction Research2013 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Evidence of local adaptation in Porcine Toll-like receptor (TLR) genes in global pig populations. The aim of this study is to determine whether adaptation to local pathogenic environments of wild and domestic pig populations can be detected by signatures of selection within Toll-like receptor genes. Using sequences of selected porcine TLRs, we searched for 1) reduced within population nucleotide diversity relative to non-immunity loci as selection favoring particular alleles that confers adaptation to pathogens; 2) deviation from neutrality due to shift to a low frequency spectrum polymorphism (excess of rare polymorphisms in a population) resulting from new mutations arising after a selective sweep; 3) elevated (>1) ratio of non-synonymous substitution rate to synonymous substitution rate; 4) high genetic differentiation between populations as pathogen mediated environmental pressure acts within a population, resulting in population specific changes in allele frequencies and an increase in between population variation; and 5) locally adapted SNPs based on SNP with excess differences in frequencies among populations. The TLR genes were sequenced from a total of 111 animals (domestic pigs and wild boars) representing Asian wild boars, European wild boars, Chinese pigs, European commercial pigs and traditional European pigs were sequenced. Sequences of the extracellular domain of TLR1, TLR2, TLR3, TLR6, TLR7 and TLR8 on individual pigs from each population were extracted and aligned for data analysis. Suggestive evidence of population specific positive selection was observed in the N-terminal part and the C-terminal sub-domains of the extracellular domain within TLR2 of pigs within certain geographic locations. Moreover, TLR1 SNP 113 S/L was potentially under positive directional selection while non-synonymous TLR8 SNPs 178 E/D and 190 L/F within some pig populations displayed suggestive evidence of balancing selection. Innate immune genes with evidence of local adaptation will help target loci involved in resistance to naturally occurring infectious diseases in the past and may still be relevant to current pig populations. Genes and SNPs that were identified to be under positive or balancing selection may have been crucial in pig population adaptation to diverse global environments. Porcine methylome. DNA methylation patterns are tissue specific patterns that occur at CpG sites in the genome and are known to effect gene expression. These patterns are known to be influenced by environmental factors such as nutritional status or disease state. We have just begun analysis of several defined phenotypes. RNAseq libraries were constructed using the TruSeq Stranded RNA Sample Preparation Kit (Illumina San Diego, CA). The libraries were multiplexed and loaded onto flowcells for cluster formation and paired end sequenced on an Illumina HiSeq2000. The reduced representation genomic libraries were prepared from size selected DNA (30 to 160 bp) digested at the CCGG sites with the restriction enzyme MspI in order to target CpG islands throughout the genome. These libraries were then bisulfite-treated with the EpiTect Bisulfite Kit (Qiagen), multiplexed and loaded onto flowcells for cluster formation and single end sequenced on an Illumina HiSeq2000. Analysis of the RNAseq and genomic data is currently underway. Differential Gene Expression in Porcine Muscle. To examine differential gene expression that leads to significantly enhanced postnatal muscle growth, piglets with differing paternally inherited IGF2 alleles were produced by the mating of IGF2 intron 3-g3072 A/G boars to A/A sows. Six piglets of each sex for inheriting either the IGF2 Gpat or Apat alleles were harvested at 90 days gestation, birth, weaning (21 days), and harvest age (~250 days) and longissimus dorsi muscle collected. Total RNA has been isolated and two samples from each sex/age/genotype combination were pooled to generate sequencing libraries. This resulted in 24 indexed libraries that were prepared using the TruSeq Stranded Total RNA Sample Prep Kit. Eight libraries were multiplexed per sequencing run on the Illumina HiSeq2000 instrument. Gene expression data are currently being analyzed.