Genomestudio software v2011

The genotype of the association panel was assayed using a Brassica 60K Illumina Infinium SNP array, according to the manufacturer's protocol (Infinium HD Assay Ultra Protocol Guide; Li et al., 2014 (link)). SNP alleles were called using GenomeStudio software v2011.1 (Illumina, Inc., San Diego, CA) with the Genotyping module (v1.9.4).
Only SNPs with a percentage of missing data of <10% across all genotypes and a minor allele frequency (MAF) of >0.05 were retained. From 52,157 SNPs in the array, 20,678 SNPs were filtered, and 31,839 were analyzed further. Physical localization of SNPs was assigned using BLASTN searches against the B. napus “Darmor-bzh” reference genome version 4.1, with an E-value cut-off of 1E-5 (Altschul et al., 1997 (link); Chalhoub et al., 2014 (link)). Only SNPs with a maximum bit-score were retained as unique SNPs, and subjected to further analysis.

Total genomic DNA was extracted from dried leaves collected from one plant of each accession using Plant DNeasy (Qiagen, Germany). Total DNA was quantified using a Quant-IT dsDNA Assay Kit (Thermo Fisher Scientific, USA). The 368 accessions were genotyped using the Cowpea iSelect Consortium Array containing 51,128 SNPs (Muñoz-Amatriaín et al. 2017 (link)). Genotyping was conducted at the University of Southern California Molecular Genomics Core facility (Los Angeles, California, USA). SNPs were called using GenomeStudio software V.2011.1 (Illumina, Inc. San Diego, CA). For GWAS, data were filtered by removing SNPs with more than 25% missing calls, and minor allele frequency (MAF) less than 0.05. The physical positions of these SNPs were determined using the IT97 K-499-35 reference genome (Lonardi et al. 2019 (link)).

Sample DNA (1 μg) was bisulphite-converted using the EZ DNA methylation kit (Zymo Research) and analysed using the Infinium HumanMethylation450 BeadChip (Illumina Inc.). The samples and 450K BeadChips were processed according to the manufacturer’s protocol at Barts and The London Genome Centre, UK. Pre-processing of the 450K dataset was performed using Genome studio software v.2011.1 (Illumina Inc.). Quality control of bisulphite conversion was performed by calculating the ratio of unmethylated probe to methylated probe. Samples that had incomplete conversion (a ratio >0.2) were removed. Methylation status for each probe is given as a Beta value (β-value). The β-value is the ratio of the methylated probe intensity and the overall intensity (sum of the methylated and unmethylated probe intensities). Pre-processing of the data was then performed using R (version 2.15.0). Peak correction was performed [39 (link)] and probes that contained a minor allele frequency of >5 % within 50 bp of the target site were removed [59 (link)]. The Illumina annotation [3 (link)] and an enhanced annotation ([42 (link)] were added to the peak-corrected datasets (Additional file 2: Table S2). We excluded probes located on the X- and Y- chromosomes from further analysis. The dataset generated in this study has been deposited in the Gene Expression Omnibus (GEO) under accession GSE77241.

RNA was isolated using the protocol described above. The concentration of the RNA samples was measured using the NanoDrop ND1000 spectrophotometer (Nanodrop Technologies, Delaware, USA). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., California, USA) and mRNA expression profiling was performed using the Illumina HumanHT‐12 v4 Expression BeadChip according to the manufacturer's protocol. Extraction of the data and quality control of the raw data was performed using Illumina's Genome studio software V2011.1. Heatmaps and clustering were performed in R.³²