Structural and functional genomics tools for cattle research

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BAC contigs with individual clones that have end sequence, or with markers embedded in them, can be used to aid the positioning and orientation of sequences within the sequence assembly. A total of 1520 ESTs was screened on the BAC pools and 802 were unambiguously assigned to BAC clones. The CHORI 240 library was used within the international consortium to create the minimum tiling path used for shotgun sequencing and alignment of the bovine sequences with the FPC map and hence aid the ordering of sequence scaffolds. Inclusion of CHORI 240 clones within the INRA FPC map allows the alignment of both FPC maps and the INRA map with the minimum tiling path of clones which has contributed to the merging of BAC contigs and sequence scaffolds BAC physical resource are not for commercial exploitation, but for the creation of pre-competitive resources for the Bovine Genomics research community. The data has been published (see below) and raw data has been made available to the International Bovine Genome Sequencing Project to aid the assembly of the sequence. Furthermore, a Web interface is available to the scientific community to access the data of the project that have been integrated in the global data obtained by INRA with the BAC library: http://locus.jouy.inra.fr/fpc/cattle_bac_map.html

Sequence confirmed cDNA clones were used to design a bovine macro-array. The most representative clone from each of the 14,989 unique cDNA sequence clusters produced in this project plus additional clones and sequence information from immune cell and gut associated cDNA libraries were selected. Sample sequencing was carried out on a sub-set of clones to confirm their identity. The cDNA inserts amplified by PCR to produce the targets that were spotted on to glass slides to create a high-density expression micro-array, which is available to the research community.

PCR primers 2480 loci that could be located within the human genome sequence, and were used to type the RH panel used in the earlier EC funded project (Bio 2 CT 98 0277). A total 1772 marker produced good typing vectors, which could be used for RH map construction. Additional ESTs were genotyped by laboratories collaborating with the BovGen project. A further set of typing vectors was produced using the amplification fragment length polymorphism technique (AFLP) such that 2735 markers were added to those on the first-generation RH map of Williams et al produced by the earlier funded EC project. The majority of the new markers, 1999, are within genes, 1072 are microsatellite loci, 262 AFLP markers, 376 BAC end sequences and 257 are from ESTs sequences that do not show convincing similarity to the annotated bovine sequence. RH maps were created for all 29 autosomes and X and Y chromosomes. The total length of the whole genome RH map was 760 Rays (R). The average marker interval, over the whole genome, is 19 cR ranging between 12 cR (BTA 29) to 29 cR (BTA 20). Distance comparisons between common markers on the RH map, MARC 2004 linkage map and the bovine sequence suggests, on average, 1 cR on the BovGen RH map is equivalent to 0.04 cM and 23 Kbp, respectively, although this varies considerably across the genome. This data has been published In addition to the ESTs, micro-satellite and AFLP markers the high throughput 'Bead Station' genotyping system from Illumina was tested to type sequences containing SNP polymorphisms on the RH panel. A total of 5131 of these Illumina markers were successfully typed in a 4-week period. The markers were assigned to chromosomes by two point linkage analysis against existing RH vector sets, however, as the data had been created using a technique with significantly high sensitivity the two data sets could not be merged to create integrated chromosome maps. An independent set of RH maps was therefore constructed for the Illumina data and have been published.

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