The Bovine Genome Sequence: potential resources and practical uses. Nicola Hastings, Andy Law and John L. Williams * * Department of Genetics and Genomics, Roslin Institute, Roslin, Midlothian, Scotland EH25 9PS Introduction We are currently characterising Quantitative Trait Loci (QTL) involved in resistance to Mastitis. This EC-funded study requires new microsatellite markers specifically targeted to the QTL regions on bovine chromosomes. One potential resource of new microsatellites is the bovine genome sequence. The first assembly of the bovine genome sequence has been released and is comprised of short contig assemblies. However, the preliminary assembly does not assign contig sequences to chromosomes. In order to develop the targeted microsatellites we combined the publicly-available trace files from the bovine genome sequencing project along with the full assembly of the human genome sequence and radiation hybrid mapping data using a series of in silico steps. The technique described below allows the discovery of new microsatellite markers in targeted bovine chromosomes in a timely and cost-effective manner using only bioinformatic tools. Identification of microsatellite-containing sequences Six million bovine genomic sequences were downloaded from the Ensembl Trace Archive on 5th May 2004 from which we constructed a blast-able database which was searched using a sequence file containing all possible microsatellite motifs. The blast output files were processed using a filter script written in python to identify 'good' microsatellite hits. To be classified as a 'good' microsatellite, the sequence hit had to be at least 13 bases long or at least 4 multiples of the motif, whichever was longer e.g. (A)13, (CA)7, (GCG)5, (AATG)4 would all qualify. Multiple hits to the same sequence separated by gaps of less than 35 base pairs were merged into a single record. This process resulted in sequences being identified as containing microsatellite sequences. Results This approach yielded approximately 4000 new microsatellite sequences per bovine chromosome. Sequences that had multiple high-scoring hits against the human genome, and those sequences that corresponded with previously defined bovine microsatellites were removed from the list, leaving 471 di-, 315 tri-, 157, tetra and 382 penta-nucleotide repeats (see Table 1). The quality of new markers was assessed manually. To date, approximately 25 markers have been mapped using radiation hybrid mapped to confirm marker position and the polymorphic information content assessed in a panel of cattle. 70% of the new microsatellites were successfully mapped onto the predicted bovine chromosomes, 25% did not map to their predicted chromosomes and 15% proved difficult to map. This means that approximately 10,000 new markers are potentially available across the genome for fine mapping studies. Conclusions 1. The publicly available bovine genome sequence trace files are a rich source of potential microsatellite sequences. 2. It is possible to use the human genome sequence to selectively extract microsatellites targeted to specific bovine chromosome regions based on previous knowledge of conservation of synteny between the two species. 3. The rate limiting steps in this procedure are: a. the radiation hybrid mapping required to confirm the targeted chromosomal location. b. the confirmation of usable polymorphisms in the population under study. 4. This method successfully allowed the project to develop microsatellite markers from available in silico resources well in advance of a ‘full’ genome build. Chromosome targeting To localise the markers to specific chromosomes we chose to follow a comparative genomics-based strategy. The genomic sequence of regions of the human chromosomes corresponding to the bovine chromosomes known to contain putative QTLs for mastitis resistance were extracted from Ensembl. These human sequences were then combined into a blast-searchable database and the bovine sequences identified as containing apparent microsatellites were searched against the human sequence, having first been masked using xnun to prevent false matches against the microsatellite motifs (Figure 1). Type of Repeat Di-nucleotideTri-nucleotideTetra-nucleotidePenta-nucleotide Total No No. of single hits Removal of known acc. no Table 1. Total number of sequences containing di, tri, tetra and penta nucleotides. No of single hits refers to the total number once multiple hits were removed. The bottom row refers to total number left after the removal of multiple hits and removal of known accession numbers as these may have already been used for microsatellite discovery, this in turn is also the total number of filtered usable microsatellite sequences. Figure 1: Diagram describing the method involved to discover new microsatellites on any chosen bovine chromosome using both the human genome sequence and the newly sequenced bovine genome. References: Human genome sequence; Bovine genome sequence; ftp://ftp.ensembl.org/pub/traces/bos_taurus QTL Chromosome with mapped markers Problem: Gap without markers Bovine Genome Sequence Extract trace files into a single database (DB1) Search database for all sequences containing microsatellite motifs Create database with homologous human chromosome (DB3) Human Genome Sequence Blast search DB2 against DB3 Extract sequences and create database (DB2) Output: sequences containing microsatellites to targeted bovine chromosome Funded by EC QLK5-CT : New breeding tools for improving mastitis resistance in Euro pean dairy cattle