Brassica 60 k infinium array

The cluster file for B. napus was generated at AAFC through analysis of 437 genotypes and at TraitGenetics through the analysis of 432 genotypes. The cluster files for B. oleracea and B. rapa were generated with 129 and 121 samples, respectively. In both laboratories, DNA was extracted from young leaf tissue of greenhouse grown plants using a cetyltrimethylammonium bromide (CTAB)-based method (Murray and Thompson 1980 (link)). DNA was quantified and 200 ng were hybridised to the Brassica 60 K Infinium array as described in the manufacturer’s protocol (Illumina Inc., San Diego, CA). The arrays were scanned using an Illumina HiScan or BeadArray Reader, and SNP data were analysed using the Genotyping module of the GenomeStudio software package with the setting for the No Call threshold set to 0.05.

Genotyping was done using the Illumina (San Diego, CA, USA) Brassica 60K Infinium array at DNA Landmarks (Quebec, Canada). The final list of 1179 markers used in linkage map construction was selected based on GenTrain genotype scores above 0.75 as suggested by Illumina followed by selection for those which lack an inter-homeologous polymorphism (Trick et al., 2009 (link)). The genetic linkage map was constructed in JoinMap3 (Van Ooijen et al., 2001 ) using a threshold recombination frequency of <0.25 and a minimum logarithm of the odds ratio (LOD) score of 6 for grouping loci into linkage groups. The Kosambi function (Kosambi, 1944 ) was used to calculate genetic distances. Each linkage group was named based on the nomenclature recommended by the Multinational Brassica Genome Project steering committee (http://www.brassica.info/resource/maps/lg-assignments.php). The map was analysed further in the R/qtl program of the R statistical package (Broman et al., 2003 (link); Broman and Sen, 2009 ) to confirm marker orders and assess general map quality.

Genomic DNA of 119 rapeseed accessions was extracted from young leaf tissue of greenhouse-grown plants using a cetyltrimethylammonium bromide (CTAB)-based method (Murray and Thompson, 1980 (link)). The genomic DNA was quantified using a Nanodrop ND1000 Spectrophotometer (Nanodrop Technologies, Inc., Wilmington, DE, USA). The DNA samples were used for genotyping with the Brassica 60 K Infinium array as described in the manufacturer’s protocol (Illumina Inc., San Diego, CA, USA). The SNPs were filtered for site coverage (90%), minimum minor allele frequency (MAF) of 0.05 with only biallelic markers, and low rates of missing data (≤10%) using the TASSEL ver. 5 software (Bradbury et al., 2007 (link)). The SNPs with low quality and high rates of missing data were removed using a protocol described in Iquira et al. (2015) (link) and Kujur et al. (2015) (link). Analyses of polymorphic information content (PIC) were performed using the software PowerMarker version 3.25 (Liu and Muse, 2005 (link)).