The linkage map underpinning the B

The linkage map underpinning the B. napus pseudomolecule arrangement was based on a mapping population of only 37 genotyped lines. This limited the resolution of the resource as the 21, 323 mapped markers defined only 527 recombination bins [7]. By increasing the size of the genotyped population to 75 lines, we aimed to increase the resolution of the linkage map, enabling improvement of the pseudomolecule resource (to version 4) by improving the determination of Tozadenant sequence scaffold order and orientation, and by detecting and splitting additional chimeric assemblies. To increase the resolution of the genetic linkage map of B. napus based on SNP markers scored in unigene sequences, we grew 38 additional lines of the TNDH population: seed of the Doubled Haploid (DH) lines was sown into Plantpak 9cm pots containing Scotts Levington F1 compost (Scotts, Ipswich, UK) and covered with a plastic propagator lid. The seeds were germinated and grown on under long day glass house conditions (16h photoperiod) at 15°C (400W HQI metal halide lamps). Plants were pricked out after 11 days into Plantpak P15 modules containing Scotts Levington M2 compost and arranged into a four block, one way randomized design with one plant of each of the DH lines per block and randomized within each block. Leaves were harvested 15 days after pricking out, 21 days after sowing. Leaf harvest was carried out as close to the midpoint of the light period as possible. The first true leaf of each plant was excised as close to the petiole as possible and the weight was recorded. Leaf samples for each Brassica line from each experimental block were pooled and frozen in liquid nitrogen, giving a final harvest of four pooled leaf samples per DH line. From these lines, we purified RNA: RNA was prepared by grinding tissue in liquid nitrogen and extracting the RNA using the E.Z.N.A. Plant RNA Kit (Omega Bio-Tek Inc.) according to the manufacturer׳s protocol. RNA concentration was measured using μl of each RNA sample on the NanoDrop ND-1000 Spectrophotometer. RNA quality was assessed by running μl of each RNA sample on an Agilent RNA 6000 Nano LabChip (Agilent Technology 2100 Bioanalyzer), samples with an RNA Integrity Number (RIN) value greater than 8 were deemed acceptable according to the Illumina mRNA-Seq protocol. From the purified RNA Illumina mRNAseq data was produced by The Genome Analysis Centre, Norwich on the Illumina HiSeq platform, with 100-base single end reads. The sequence reads were, trimmed to 80 bases and down-sampled to 28M reads per sample (to retain comparability with the earlier data), aligned to unigenes and scored for SNPs as described previously for an initial panel of 37 TNDH lines [7]. The scoring strings were used to extend those produced previously for 21, 323 SNP markers scored across the 37 TNDH lines used originally, and produce an updated linkage map. The data were used to refine the order of markers (Additional file 1 for the A genome, Additional file 2 for the C genome), increasing the number of recombination bins identified from 527 defined by the original 37 lines [7] to 887 defined by the full 75 lines. Consensus marker strings were defined for each of these recombination bins and a revised linkage map was constructed (Additional file 3). Analysis of best sequence similarity matches in the B. rapa and B. oleracea genome sequence scaffolds of the unigenes in which SNP scored indicated that 5 of those scaffolds were chimeric (i.e. markers scored either side of position representing a breakdown in collinearity with the A. thaliana genome mapped to different places in the genome) so could be split (Additional file 4). The order and orientation of further genome sequence scaffolds could also be confirmed, enabling the updating of the specification of scaffold order and orientation (Additional file 5) to establish an updated (version 4) pseudomolecule resource comprising 736,647,178bp of sequence data. These were then used to infer the order of unigenes and position the probe flanking sequences for the Brassica research community׳s Brassica 50K Illumina® Infinium SNP array, thus integrating these two genotyping resources. The result is an inferred order of 86,429 sequences (61,757 unigenes; 24,672 array probes) representing potential markers in the A genome (Additional file 6) and 87,426 sequences (64,293 unigenes; 23,133 array probes) representing potential markers in the C genome (Additional file 7).