Fingerprinting and end-sequencing BAC clones to contribute to the international effort of building a bovine and porcine physical (BAC) map

Grant: AG 58-5438-2-313
Award Amount: $1,158,037
PI: L.B. Schook, J.E. Beever, H.A. Lewin
Start Date: June 1, 2002
End Date: May 31, 2007

The goal of this project was to refine the porcine Bacterial Artificial Chromosome (BAC) physical map in localized regions that contain loci affecting traits of economic importance, and to increase the number of Single Nucleotide Polymorphisms (SNPs) contributing to the bovine HAPMAP project.

The porcine BAC physical map was refined in localized regions known to harbor loci of economic importance. These regions include genes influencing ovulation rate, pre-weaning growth rate, pork tenderness, lean yield and arthrogryposis multiplex congentia. The refined maps were used to select a minimal tile path (MTP) of BAC clones within the regions. The MTP BAC clones served as templates for skim sequencing of these regions and generation of sequence information for single nucleotide polymorphism (SNP) discovery. SNP panels were developed for each region and used to estimate linkage disequilibrium and define population haplotypes. SNPs available for the Bovine HAPMAP project came from those identified by the University of Illinois at Urbana-Champaign (UIUC) and Baylor College of Medicine as part of the Bovine Genome Sequencing Project.

UIUC researchers provided 100,000 cattle BAC-end sequences (BESs) to construction of a cattle fingerprint physical map. Maps of cattle chromosomes 15 and 29 covered 85% of human chromosome 11 sequence, revealing a complex patchwork shuffling of segments not explained by a simple translocation followed by internal rearrangements. Overlay of the mouse conserved syntenies onto human chromosome 11 revealed that segmental boundaries appear to be conserved in all three species. This resource provided a foundation for the evolutionary analysis of mammalian karyotypes and for sequencing of the cattle genome.

A set of cattle BESs (~300,000) was used for selection of markers to create a high-density comparative map of human and cattle genomes. To facilitate detection of chromosomal regions influencing important traits, 2,914 SNPs were assigned to cattle chromosomes. A total of 192 cattle from 18 cattle breeds were genotyped for a subset of these SNPs. All genotyped SNPs were submitted to the NCBI dbSNP database and became a part of the SNP panel used by the Bovine Genome Sequencing Consortium and the International Bovine HapMap Consortium.

Tibial hemimelia is a genetic defect that has been reported in Galloway and Shorthorn breeds of cattle. Affected calves have multiple skeletal deformities and are born dead or fail to thrive and die shortly after birth. Two mutations causing tibial hemimelia were identified and result in a complete loss-of-function of ALX4. A DNA-based diagnostic test was developed and has been used on more than 16,000 cattle to accurately determine an individual's genotype.

A swine physical map was constructed and the mapped loci were used to assemble a comparative map of human and pig genomes containing 2,274 loci assigned to 34 linkage groups. This physical map has the highest resolution of any porcine map to date and its integration with the porcine linkage map will greatly facilitate the positional cloning of genes influencing complex traits of both agricultural and biomedical interest. The swine physical map covers the 18 pig autosomes and the X chromosome in 176 contigs. Chromosome 13 is represented by a single contig. The level of map contiguity is higher than that achieved using similar approaches in other species.