Beadstudio genotyping software

Genotyping of the entire inbred line population was performed commercially by Emei Tongde Co. (Beijing) using the Brassica 60 K Illumina® Infinium SNP array (http://www.illumina.com/technology/infinium_hd_assay.ilmn) according to the manufacturer's protocol. The SNP data were clustered and called automatically using Illumina BeadStudio genotyping software. SNPs with QQ or qq frequencies equal to zero, call frequency <0.9, or minor frequency <0.05 were excluded. The remaining SNPs were scrutinized visually, and those SNPs not showing three clearly defined clusters representing the three possible genotypes (QQ, Qq, qq) were also excluded. A total of 33,689 SNP markers among the 367 lines and hybrids that met the QC criteria were used for the association analyses.
The phenotypes of 8 traits were filtered based on the following two criteria: (1) the frequency of missing data for each subject <0.1; (2) outliers were discarded according to a distribution-based outlier detection of residuals (|ε − μ_ε|/ σ_ε > 3) by QQ plot (Quantile-Quantile plot) for each trait (Figure S1).

In previous reports, detailed descriptions about the process of SNP genotyping and mapping are provided, as are analyses of population structure and linkage disequilibrium (LD) (Li et al., 2014 (link); Wang et al., 2016a (link)).
In brief, the raw SNP data generated from the Brassica 60K Illumina® Infinium SNP array were clustered and automatically called using Illumina BeadStudio genotyping software. Subsequently, 26,841 high-quality SNPs with minor allele frequency (MAF) of more than 0.05 were retained for further analysis. In order to mapping the SNP to an exact position of the reference genome, a BLAST search (Altschul et al., 1990 (link)) was performed against B. napus genome sequences (Chalhoub et al., 2014 (link)) using the SNP sequences. Only the top and unique blast-hits were reserved.
Eventually, 19,945 SNPs were selected for principal component analysis (PCA), and a relative kinship and population structure analysis. The GCTA tool was used to construct a P matrix of PCA (Yang et al., 2011 (link)), SPAGedi software was served to build a K matrix of relative kinship (Hardy and Vekemans, 2002 (link)), STRUCTURE v2.3.4 was employed to infer a Q matrix of population structure (Pritchard et al., 2000 (link)) and TASSEL 5.0 was used to calculate LD (Bradbury et al., 2007 (link)).

The whole population of inbred lines was genotyped using the Brassica 60 K Illumina® Infinium SNP array by Emei Tongde Co. (Beijing) according to the manufacturer’s protocol (http://www.illumina.com/technology/beadarray-technology/infinium-hd-assay.html). The SNP data were clustered and called automatically using Illumina BeadStudio genotyping software. Those SNPs with either AA or BB frequency equal to zero (i.e., monomorphic), call frequency < 0.9, or minor frequency < 0.05 were excluded.
The data for the five traits were tested by analysis of variance (ANOVA) using SPSS version 19.0 (IBM Corp., Armonk, NY, USA).

Genotyping of the 520 B. napus accessions was carried out in Huazhong Agricultural University, Wuhan, China, using the Brassica 60 K Illumina Infinium SNP array. The SNP data were analyzed by Illumina BeadStudio genotyping software, as previously described11 (link). SNPs with minor allele frequencies (MAFs) of <0.05, or lacking a call higher than 0.9 were excluded from analysis. After processing, 31,839 SNP markers were retained for further analysis. To determine the physical localization of SNPs, flanking sequences were subjected to BLASTN searches against the B. napus genome, with an E-value cut-off of 1E-514 (link)47 (link). SNPs with a maximum Bit-Score were retained as unique SNPs, and subjected to further analysis.

The Brassica 60 K Illumina^® Infinium consortium SNP array [82 (link)] (http://www.illumina.com/technology/beadarray-technology/infinium-hd-assay.html) was used for accessions genotype. SNP data were analyzed using Illumina BeadStudio genotyping software (http://www.illumina.com/) with parameters set as a missing rate ≤ 0.2, heterozygous rate ≤ 0.2, and minor allele frequency (MAF) > 0.05. BLAST was performed to search the probe sequences of these SNPs against the B.napus Darmor-bzh reference genome [34 (link)] with an threshold of e⁻¹⁰. SNPs with merely one matched position in reference genome were used for further analysis. The population structure and relative kinship of the 280 B. napus accessions were analyzed using STRUCTURE v. 2.3.4 and SPAGeDi software, respectively [83 (link)]. The linkage disequilibrium (LD) decay between all SNPs was assessed by TASSEL 4.0 [84 ]. The trait–SNP association was analyzed using mixed linear model (MLM) for both the single repetition and the BLUP [85 (link)]. Marker haplotypes at each associated locus were identified using the four-gamete rule with Haploview software [86 (link)].

DNA that had been used for SNP genotyping was isolated from the young leaves of ~10 plants (pooled) for each of the 448 accessions (plant material was harvested from accessions grown in Wuhan 2013), according to a traditional large-scale CTAB method (Richards et al., 2001 (link)). Protocols for SNP genotyping, filtering and location were similar to those of (Li et al., 2014 (link)) and they were derived as part of a recently completed study (Wang et al., 2014 (link)). In brief, the Brassica 60 K Illumina® Infinium SNP array was used to obtain genotype data according to the manufacturer's protocols. SNP data were clustered and called up automatically with the Illumina BeadStudio genotyping software. Approximately 25,000 SNPs that did not show three clearly defined clusters that represent the three possible genotypes (AA, AB, and BB) were excluded. The probe sequences of each SNP were used to BLAST against B. napus genome sequence V4.1 (Chalhoub et al., 2014 (link)). The location of each SNP on 19 B. napus chromosomes was obtained by extracting the top hit positions of its corresponding probe sequence on the genome. Finally, SNPs that showed a minor allele frequency (MAF) of less than 0.05 were excluded from further analyses.