Toward the Reference Genome Sequence of Wheat Chromosome 6B; BAC-By-BAC Sequencing and Assembling

The International Wheat Genome Sequencing Consortium (IWGSC) makes efforts to obtain the high quality reference sequence of 17GB bread wheat genome.  As part of the Japanese-funded “Genomics-based Technology for Agricultural Improvement” project, we are working to sequence the chromosome 6B short (6BS) and long (6BL) arm utilizing a minimum tiling path (MTP) BAC-by-BAC sequencing strategy.  Here we present our current status for the 6B sequencing and assembling.  Using the chromosome arm-specific BAC libraries, we selected 3076 and 4557 BAC clones comprising the minimal tiling path established during the construction of the physical map for 6BS and 6BL, respectively, by Whole Genome Profiling (WGP; Amplicon Express, Keygene).  As sequencing each BAC individually would have been too costly and labor-intensive, we developed highly reliable and high throughput sequencing system.  BAC DNAs were prepared in 96 deep-well box, were tagged with 48 or 96 MIDs, were sequenced by Roche GS FLX, and then were assembled.  We obtained about 70Gb sequence data from 8,428 of 8,544 applied BAC clones (x52.5 /BAC clone).  The number of assembled contigs per one clone is 9.1 on an average, 4.0 on a mode.  Also we checked the overlapping status of 192 MTP clones for 30 BAC contigs on our physical map.  About 94% of overlapping region showed more than 95% sequence homology.  These results clearly indicated that our sequencing strategy with the tagged library and GS FLX is reliable and quite useful for sequencing of BAC clones on a larger scale.  We are currently working to fill gaps and make longer scaffolds using these BAC-by-BAC sequences in combination with the survey sequencing and mate-pair sequencing data for generation of a reference sequence of chromosome 6B of wheat.