Physical Mapping, Survey Sequencing and BAC-to-BAC Sequencing of Wheat Chromosome 6B

The International Wheat Genome Sequencing Consortium (IWGSC) makes efforts to obtain the reference sequence of bread wheat genome.  Japan is in charge for the chromosome 6B.  We are now making physical maps for both chromosome arms, 6BS and 6BL, and also are sequencing those utilizing a minimum tiling path (MTP) BAC-by-BAC sequencing strategy.  Here we present our current status for the physical mapping, survey sequencing and reference genome sequencing of chromosome 6B.  Finger printing and further data trimming generated 689 BAC contigs which covered 91% of the entire physical length of both arms, and from these contigs we selected 7633 MTP clones which should be sequenced.  We also conducted survey sequencing of 6B by massively parallel Roche GS FLX pyrosequencing, which produced more than 10 times volume of nucleotides from estimated size of each arm and those reads were assembled to 508 Mb corresponding to 56% of chromosome 6B.  We are now sequencing the MTP BACs to complete our work for wheat 6B.  For that, we developed highly reliable and high throughput sequencing system.  BAC DNAs were prepared in 96 deepwell box, constructed tagged library with 96 MIDs, sequenced by GS FLX, and then assembled.  For over 90% BAC clones tested, the sequence reads were more than x20 coverage, and the bias in distribution of reads of BAC clone was not affected the results largely.  This sequencing system is useful for sequencing lots of BACs at once, so we are continuing to read the MTP BACs now.