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

Start Date: Jun 01, 2007
End Date: Sep 30, 2009

Objective:
Generate 5¿ and 3¿ cDNA tag libraries from various tissues and developmental stages of the pig to assist the annotation of the porcine genome sequence. Libraries will be deep-sequenced using the Solexa sequencing platform. Sequence tags will be quantified and mapped to their respective locations within the genome. Develop linkage disequilibrium (LD) haplotype maps of genes responsible for innate immunity in the commercially relevant pig breeds.

Approach:
A diverse set of tissues have been collected from the sow chosen as the template for the complete genome sequence of the pig, University of Illinois sow 2-14. Furthermore, cell lines and subsequent cloned offspring corresponding to the same individual have been developed for collection of tissues throughout various stages of development in the pig. RNA will be isolated from these tissues and used to 5¿ and 3¿ cDNA tag libraries. Each constructed library will be first assessed for quality (# of tags containing 5¿ transcriptional start sites) by sequencing of 384 individual clones for each library. High-quality libraries will be subjected to deep-sequencing using the high-throughput Solexa sequencing platform. Quality assessment will be performed by comparison with current Unigene assemblies and available porcine genome sequence as well as cross-species comparisons to completed genomes and known transcripts. Genomic DNA samples corresponding to pigs used in constructing the high density pig SNP chip will be used to construct haplotype maps of the toll-like receptor genes (TLRs) that are associated with innate immunity in pigs. Haplotype maps of the TLRs will then be used to identify SNPs and be used in association studies with respect to PRRSV resistance. TLR genetic diversity will be assess within commercial breeds as well as across exotic populations to assess the restriction of diversity associated with innate immunity resulting through selective breeding. The SNPs will be identified by sequencing Holstein BACs from the RPCI-42 library using 454 technology. Overlapping BAC clones from the two haplotypes represented in the RPCI-42 library will be identified by genotyping clones with an existing panel of public domain SNPs. Thus, a BAC-based haplotype reconstruction of the BTA3 QTL-containing region of RPCI-42 library will be accomplished, and the SNPs used for detailed analysis of known QTL heterozygotes. Resequencing of the region may facilitate identification of the gene(s) responsible for the QTL and yield useful information on copy number polymorphism and breed-specific differences in haplotype structure.