Ordering Draft Genome Sequence of Flax (Linum usitatissimum)

Flax (Linum usitatissimum L., 2n = 2x =30) is a self-pollinated annual species of the Linaceae family. It is a dual purpose crop grown for its seed oil (linseed) and/or its stem fibres from which linen and other products are made. Linseeds are rich in omega-3 fatty acids (55-57%) essential for humans and ideal for the production of biodegradable products such as linoleum flooring and paints. The genome size of the flax variety CDC Bethune was estimated at ~373Mb based on flow cytometry (Wang et al. 2012). This genome was sequenced using a whole genome shotgun (WGS) sequencing strategy on the Illumina sequencing platform (Wang et al. 2012). A total of 302 Mb in 116,602 contigs or 318 Mb in 88,384 scaffolds was obtained, representing ~81% of the flax genome. The remaining ~70 Mb were gaps within and between scaffolds. All contigs or scaffolds were unordered and some of them, especially long scaffolds, were mis-assembled. In order to refine the genome assembly, we identified mis-assembly breakpoints of the WGS assembly using the WGS Illumina resequencing data of 90 flax genotypes. A total of 100 out of 668 largest scaffolds representing 95% of the assembled genome were thus broken into 227 smaller refined sequences.  The 416 FPC contigs of the physical map of CDC Bethune (Ragupathy et al. 2011) were also analyzed for weak assemblies and 6 FPC contigs were sub-divided each into two contigs. We then ordered the scaffold sequences into 15 linkage groups by integrating the physical map, its associated BAC end sequences, the SSR-based consensus genetic map (Cloutier et al. 2012), and the high-density SNP maps (over 10,000 SNP markers) from three mapping populations which were created using a genotyping-by-sequencing (GBS) strategy and the Illumina HiSeq 2000 sequencing platform. To date, a total of 271 Mb of scaffold sequences have been anchored to ~345 Mb FPC contigs, covering 95% of all predicted genes in the WGS assembly. The estimated chromosome size based on FPC contigs ranged from 15.1 Mb to 32.8 Mb in 15 linkage groups. Based on the ordered flax sequences within the 15 chromosomes, sequence gaps of variable length were found to be distributed along all chromosomes. To fill those gaps and to further solve the assembly errors due to short reads of Illumina and repeats, we applied WGS resequencing strategy combining second generation sequencing (Roche 454) and third generation sequencing (PacBio) technologies  to generate genome-wide long sequences (up to ~700 bp in Roche 454 and ~2.3kb in PacBio) for hybrid assembly with ordered scaffolds. An improved, ordered and annotated flax genome reference sequence is expected. This reference sequence of flax will enhance genomic research and molecular breeding in flax and aid in genome sequencing, assembly and comparative analysis of other species in the Linaceae family.