Supported by "Genomics for Agricultural Innovation" Project, Ministry of Agriculture, Forestry and Fisheries of Japan
Our goal is the construction of the highly accurate physical map linked with the genetic map and the reliable reference genome sequence of the wheat chromosome 6B.
Using the chromosome arm-specific BAC libraries from 6BL and 6BS, we fingerprint our BAC clones by the Whole Genome Profiling (WGP™, KeyGene), and then we exclude low quality BAC clones from the contig building step in order to construct a more accurate physical map. After filtering of BAC clones, we perform a contig assembly using Finger Printed Contigs (FPC) software. For that, we set a chromosome 6B-specific parameters for the assembly, such as number of tags and the distance between the tags, according to the previous report (Philippe et al., 2012) and the guideline for physical map assembly released from the IWGSC (Scalabrin et al.). Then we construct BAC contigs for both arms, 6BL and 6BS and further more, we select minimal tiling path clones (MTP) which are subjected to the genome sequencing.
BAC nucleotide sequencing is performed using the next-generation sequencers (Roche 454 GS-FLX Titanium or plus, and Illumina GAIIx). To be more efficient and stabilize the whole experimental process, we develop multi-tagging technology for the sequencing of 96 BAC clones at once and also improve the isolation method of BAC DNA to maximize yield and to minimize contamination of E. coli genomic DNA.