2007 Annual Report 1a.Objectives (from AD-416) To develop 60 single nucleotide polymorphism (SNP) markers that will serve as a universal identification (ID) marker set for U.S. beef and dairy breeds and a similarly informative set of 40 SNP markers for U.S. sheep. These markers will be developed for DNA diagnostics to be publicly available for use by livestock researchers, livestock producers, and regulatory agencies. A related project will validate, characterize, and develop multiplex matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assays for SNPs in genes essential for virus replication in swine. 1b.Approach (from AD-416) In cattle, the 60 SNP markers will be derived from a selected pool of 90 candidates contained on 90 amplicons that have been screened for a number of criteria by MARC and have applications in animal ID and parentage. In sheep, the 40 SNP markers will be derived from a selected pool of 60 candidates contained on 60 amplicons also screened for a number of criteria by MARC and have applications in animal ID and parentage. Efficient multiplex assays will be developed for high-throughput MALDI-TOF MS for use in the livestock industry. In swine, amplicons containing host genes essential for virus replication will be resequenced through critical animal populations to identify and genotype SNPs and develop efficient MALDI-TOF assays. Validation of the swine SNPs will be accomplished utilizing the MALDI-TOF assays to genotype the MARC swine mapping families for inheritance patterns consistent with mendelian genetics. Additional genotyping of DNA from phenotypically distinct swine will also be performed. 3.Progress Report This report serves to document research conducted under a Specific Cooperative Agreement between ARS and CoGenics, Inc. (formerly Genaissance Pharmaceuticals, Inc.). Additional research details can be found in the report for the in-house CRIS 5438-32000-029-00D, Genetic and Biological Determinants of Respiratory Disease Sucptibility. In cattle, 61 new highly-informative single nucleotide polymorphisms (SNPs) in U.S. beef and dairy populations are publicly available in GenBank. This represents 122 total files viewable at GenBank and exceeds the original goal of 90. These 121 SNP parentage markers are presently being used by companies in the U.S., Canada, and EU. The 121 parentage SNPs and the surrounding genomic DNA were sequenced through 216 diverse beef and prominent dairy sires (126,229 bp per sire, 27,000,000 bp total). There were > 2000 flanking polymorphisms annotated from this data set including 3,500,000 genotypes (important for assay B designs). The 121 were markers selected for high minor allele frequencies, genome-wide distribution, accuracy in genotyping, and high-throughput "multiplexibility". All 121 files and the corresponding 136,573 tracefiles are publicly viewable at NCBI’s dbTrace Archive and at the University of Louisville Center for Genetics and Molecular Medicine. We annotated and deposited 1,221,538 total bp of sequence in GenBank. The annotation includes 259 amplicons, 258 exons, 183 CDSs, and 2004 bovine repetitive elements. The average minor allele frequency of the 121 traceback SNPs is 0.425 (CI95 0.009,USMARC beef) and 0.436 (CI95 0.011, BARC dairy) and average spacing within chromosome of 27 cM (CI95 2.7 cM). With these 121 parentage markers, the average probability that the genotypes from two unrelated individuals are identical by state is < 1E-50. The estimated average probability of paternity exclusion is < 0.9999999999 and 0.999999 for all 121 SNPs with one or zero parents known, respectively. This lends significant power to parentage-based traceback if needed. Through this agreement, the results are being made publicly available at University of Louisville by the Center for Genetics and Molecular Medicine: http://cgemm.louisville.edu/usmarc/MARC_web_page/traceback.html. This public website is important because it allows unrestricted access for any company or institution to the key genetic information required to develop their own DNA testing assays. Consequently, a number of companies have used results from this website to develop commercial DNA testing in cattle. Required documentation of monitoring activities included, but were not limited to: emails, site visits, meetings, and conference calls.