Targeted sequencing of chromosomal regions containing genes of economic importance to the livestock industry.
In the new millennium, the demands of a rapidly growing world population will continue to put pressure on the U.S. animal agriculture industry. The industry must develop new products that create value within he agricultural system, and as a result, increase the profitability of agriculture and revitalize rural America. These challenges come at a time when many current agricultural technologies are being questioned, when key productivity enhancers (such as medicated feeds) are in jeopardy, and when waste management constrains the formation of economically viable units. At the same time, the safety of the food supply is in question because of the incidence of BSE (Mad Cow Disease) and foot and mouth disease in Europe as well as frequent outbreaks of food-borne pathogens here in the U.S. The Livestock Genome Sequencing Initiative directly addresses three major challenges. Results of this project to date have provided producers and the breeding industry with genetic tests to reduce the incidence of genetic and infectious diseases, to trace the origin of meat and dairy products, and to increase productivity and swine and cattle. These genomically-based tools thus provide for the sustainable and secure production of meat and dairy products for American consumers and the world markets.
A. Completion of the human genome sequence provides a foundation for understanding genetic complexity and how it contributes to diverse phenotypes and disease. It is clear that model organisms will continue to play an invaluable role in the synthesis of this understanding. The pig represents an evolutionary clade distinct from primates or rodents and thus, provides considerable power in the analysis of DNA sequence and phenotypic diversity. The pig, a domesticated eutherian mammal, has co-evolved with humans and represents a taxum with diverse selected phenotypes (Bidanel and Rothschild, 2002). The pig has played a central role in the scientific and medical communities, thus providing scientific justification for understanding the porcine genome with respect to physiological models of growth and development, health, and reproduction (Tumbelson and Schook 1996; Schook et al. 2005a).
Today, a genome sequence is an essential precursor for biological studies of an organism. The aim of the current proposal is to generate a draft sequence of the porcine genome that maximizes the genome coverage and fragment ordering that can be achieved with 3-fold sequence coverage. The assembled draft genome will be annotated automatically and displayed in Ensembl, where data can be downloaded, queried and compared with other genomes. Both the sequence assembly and annotation will also be available to NCBI and UCSC. The draft will also provide a framework for future improvements that can be generated by incorporating either additional whole genome shotgun data or high quality finished regions. During the past 5 years, the USDA CREES has provided significant resources to support the swine genomics in anticipation of actual genomic sequencing. This has included the development of a BAC library, RH panels and maps, and the construction of a BAC contig physical map. These resources provide a unique opportunity that is exploited in this proposal. The ability to select a minimal tiling path of overlapping BAC clones to support a clone-by-clone sequencing initiative. Thus, unlike other projects (cattle, chicken and honey bee) that were initiated prior to the construction of a complete physical map, we will be able to avoid assembly and annotation issues associated with those projects by this BAC skimming approach. This will a high quality 3X draft that can be readily used by the international community. We believe that this approach provides a superior product that what would be achieved through whole genome shotgun sequencing.
To develop a pre-finish level (>6-X coverage) quality assemble of the porcine genome sequence. The genome sequence assembly will be an invaluable.
The overall goals of this Tools and Reagents proposal are to create the discovery platform to support the subsequent development of a porcine high-density 50K single nucleotide polymorphism (SNP) chip and the development of a high density SNP radiation hybrid (RH) map. This project ensures that a subsequent partnership with an industrial partner will lead to the rapid production of a low cost, publicly available genotyping platform. The pig represents the only major agricultural animal that does not yet have an international SNP Consortium or SNP map. One of the sub-aims of this project is to use this discovery process to engage and focus industry and other SNP end-users towards the development of a porcine high-density SNP chip. This SNP chip would permit genome scans for the identification of quantitative trait loci (QTL), the characterization of germplasm diversity and elucidation of linkage disequilibrium (LD) within commercially relevant populations. This project represents an integrated activity of the global pig genomics community and will create a public SNP Repository that includes the current private and public SNPs as well as de novo SNPs discovered in this project. As indicated in the letters of support, we have secured access to SNPs that have been privately generated in the US, Japan, Italy, Denmark, China and the Netherlands. Moreover, we have secured the support of the US swine, pork, and genotyping industries as represented by the National Swine Improvement Federation, Monsanto Choice Genetics, MMI Genomics, GeneSeek, Genus and through the US Pig Genome Coordinator, the USDA and US researchers. This Tools and Reagents grant has also been designed to complement parallel SNP development efforts in the EU (SABRE and INRA projects).
The proposed strategy leverages innovative approaches with proven utility in the bovine and chicken SNP discovery efforts and incorporates new cost effective Solexa second generation sequencing technologies. Thus, the outlined approach is both cost effective and integrates innovative strategies and emerging technologies. The objectives of this proposal include the:
We expect this project to provide a minimum of 12,000 validated and characterized SNPs that are uniformly distributed across the pig genome with high MAF (>0.05) that supports within breed/line selection. Furthermore, in addition to demonstrating the utility of our innovative approach, the constructed libraries and sequencing strategy are estimated to allow the selection of at least 400K SNPs for future incorporation into a high-density genotyping platform. Finally, we have estimated that there are currently 20,000 validated SNPs in the private sector. The US and EU private sectors have indicated their willingness to share these SNPs with the Consortium (see letters of support) to ensure that we can develop an integrated, single porcine SNP chip that ensures high MAFs and uniform genome spacing in a resultant 50K SNP assay.
Generate DNA sequences corresponding to full-length mRNA transcripts of the pig to assist the annotation of the porcine genome sequence and develop a catalog of corresponding cDNA clones as a community resource for functional analysis of porcine genes.
The regeneration of cells and tissue after injury or trauma is critical to medical and civilian communities. Cardiac injuries and regenerative engineering poses specific challenges since cardiac myocytes, the primary cells responsible for the mechanical beating of the heart muscle, do not regenerate. The cardiac system poses a significantly challenging problem in tissue engineering due to the complex 3-dimensional mechano-actuation properties of the cardiac cells. A grand challenge in cardiology since early 50s is the development of an artificial heart that can replace a failing heart. Until today, artificial heart is used only for the temporary use (hrs) until a healthy donor heart is found. The latter is difficult to get, and is often rejected by the body after successful replacement. This very limited success in heart replacement, in spite of considerable effort and resources invested so far, calls for a new paradigm in the approach of heart replacement.