Sequencing and Assembling a Reference Sequence of the 1 Gb Wheat Chromosome 3B

In order to assemble a high quality reference sequence of the 1 Gb wheat chromosome 3B, we sequenced 8452 BACs comprising the Minimal Tiling Path using Roche/454 8 kb long paired-end reads. BACs were pooled by 10 while promoting the pooling of overlapping BACs. In total, 931 BAC-pool libraries were produced. First, read assembly and scaffolding led to generate 16136 scaffolds accounting for 1 Gb. A curation of the scaffolding was then applied. In addition, Illumina reads from sorted 3B DNA were mapped to fill gaps within scaffolds and to correct 454-derived consensus sequences. Bioinformatics tools were developed to integrate physical contig information with sequencing data, detecting overlapping sequences between BAC pools and, finally, merging scaffolds while removing redundancies. This has led to decrease the number of scaffolds to around 3700 while increasing the N50 to 640 kb. The construction of a pseudomolecule is now underway through the integration of genotyping data of mapping populations. Such reference sequence enables direct application in gene cloning, marker development and genomics breeding. It also allows us to study the organization, composition and evolution of a giant polyploid genome at a scale never reached before.