Plant & Animal Genome IX Conference

Workshop: Cattle/Sheep
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DISCOVERY AND MAPPING OF SNPS IN BOVINE GENES: HIGH THROUGHPUT EST SEQUENCING, PRIMER DEVELOPMENT, AND GENOTYPING

USDA, ARS, U.S. Meat Animal Research Center, PO Box 166, Clay Center, NE, USA 68933-0166

Application of functional genomics approaches and QTL analyses would be significantly enhanced by production of cDNA libraries with low redundancy, collection of cattle EST data, and development of markers amenable to high-throughput genotyping systems. Furthermore, identification of genes underlying observed QTL effects would be substantially more efficient if dense comparative maps between cattle and the human genome were available. A program to produce specialized libraries for sequence collection, collect EST sequence, identify SNPs targeted to ESTs, and set up high-throughput genotyping has been established. Four pooled-RNA, normalized libraries were produced, representing tissues with known effects on traits such as carcass composition, animal health, and reproduction. Analysis of >20,000 sequences from each of the libraries suggests that within-library sequence redundancy has only reached 37%, demonstrating the success of this approach in producing libraries of low redundancy. An automated system for designing PCR amplification primers from the data has been developed, with approximately 80% success in production of primers that successfully amplify across introns in bovine genomic DNA. Sequencing of amplicons from these primers has a 72% success rate in identification of SNPs in the mapping families. Over 200 ESTs have been placed on the linkage map using a primer extension-mass spectrometry genotyping system. The results of this program will enhance comparative genetic linkage maps of the bovine genome to other mammals, provide information and clones for intelligent design of microarrays for functional genomics, and produce a database of SNP suitable for the latest high-throughput genotyping technologies.