A selected set of 136 rapeseed accessions were genotyped using the Brassica 60K Illumina Infinium@ SNP array (Illumina) as described previously (Liu et al., 2016 (link)). After quality control (minor allele frequency>0.05 and missing data<20%) (Liu and Muse, 2005 (link)), a total of 21426 SNPs were selected for subsequent GWAS analysis. SNP density map was drawn using the CMplot package (Yin et al., 2021 (link)) in R3.3.3 (Team RC, 2013 ) to gauge the marker coverage across the B. napus genome. Principal component analysis was performed using Tassel v5.0 software (Bradbury et al., 2007 (link)). Only the first two principal components were plotted by the ggplot2 package (Wickham, 2011 (link)) in R3.3.3 (Team RC, 2013 ). MEGA-X software (Kumar et al., 2018 (link)) was used to draw the phylogenetic tree and show the genetic relationship among GWAS accessions. Both principal component analysis and phylogenetic tree are used to reveal the distribution of population structure. LD (linkage disequilibrium) attenuation was estimated to demonstrate the degree of genetic linkage, and it is drawn by PopLDdecay software (max decay distant = 2500, break = 6000bp, bin1 = 1000, bin2 = 6000) as described previously (Zhang et al., 2019 (link)).
Brassica 60k infinium snp array
Manufactured by Illumina
Sourced in United States, Germany
The Brassica 60K Illumina Infinium SNP array is a high-throughput genotyping platform designed for the analysis of single nucleotide polymorphisms (SNPs) in Brassica species. It provides a comprehensive and efficient tool for genome-wide genetic analysis, enabling researchers to explore genetic diversity, identify markers associated with traits of interest, and conduct genomic studies.
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Lab products found in correlation
Beadstudio genotyping software, by Illumina (7 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Genomestudio software v2011, by Illumina (1 mentions)
Spss version 19, by IBM (1 mentions)
Hiscan, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Genomestudio software, by Illumina (1 mentions)
Genomestudio genotyping software, by Illumina (1 mentions)
27 protocols using brassica 60k infinium snp array
1
Genotyping and Population Structure Analysis of Rapeseed Accessions
2
Genotyping of Brassica Diversity Panel
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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.
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.
3
SNP Genotyping of Rapeseed Accessions
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Single-nucleotide polymorphism (SNP) genotyping had been implemented in previous reports (Li F. et al., 2014 (link); Li et al., 2016 (link)). Briefly, genomic DNA of 472 rapeseed accessions was extracted from the young leaf tissue and was then hybridized to the Brassica 60K Illumina® Infinium SNP array, which contains 52,157 SNP markers, according to manufacturer’s protocol. The SNPs with minor allele frequency (MAF) < 5% or call frequency <80% were filtered.
In order to mapping SNPs to the physical position of genome, a local BLAST program (Altschul et al., 1990 (link)) was performed. The sequences of retained SNPs were queried against the B. napus genome sequences (Chalhoub et al., 2014 (link)). The top and unique blast-hits were selected for the further analysis.
In order to mapping SNPs to the physical position of genome, a local BLAST program (Altschul et al., 1990 (link)) was performed. The sequences of retained SNPs were queried against the B. napus genome sequences (Chalhoub et al., 2014 (link)). The top and unique blast-hits were selected for the further analysis.
4
High-throughput SNP Genotyping for Brassica Crops
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Genomic DNA for genotyping was extracted from leaf tissues collected from three plants of each accession by a modified cetyltrimethylammonium bromide method.27 Genotyping was performed using the Brassica 60K Illumina Infinium SNP array. The SNP data were clustered and called automatically using Illumina BeadStudio genotyping software. The standards of quality control for SNP data were as follows: call frequency ≥0.8, minor allele frequency (MAF) ≥0.05 and homozygous genotype frequency cannot be zero. The probe sequences of the SNP array were used to perform a BLAST search against the B. napus Genomes Browser (http://www.genoscope.cns.fr/brassicanapus/ , 20 November 2015, date last accessed). They were regarded as non-specific markers when BLAST matched to two or more locations in the reference genome. SNP markers used for LD analysis and association analysis were filtered with the following steps: (i) non-specific markers were excluded; (ii) markers that were not up to these standards of quality control for SNP data were eliminated; (iii) markers that did not have specific physical location information were excluded. SNPs that were selected for assessing population structure and relative kinship were filtered using the above first two steps.
5
Genotyping Brassica Crops using SNP Array
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Genotyping for association mapping was performed using the Brassica 60 K Illumina® Infinium SNP array [32 (link)] according to the manufacturer’s protocol (https://www.illumina.com/techniques/microarrays.html ) in the National Key Laboratory of Crop Genetic Improvement, National Subcenter of Rapeseed Improvement in Wuhan, Huazhong Agricultural University, 430070 Wuhan, China. Illumina BeadStudio genotyping software was used for SNP data clustering and calling according to a previously described protocol [31 (link)]. SNPs with a call frequency of <0.9 and a minor allele frequency (MAF) of ≤0.05 were excluded in this research. The remaining SNPs were scrutinized visually and those SNPs that were resolved as three clearly separated clusters (AA, AB, and BB) in the tested B. napus material were used for further research. In addition, to identify the physical position of SNP markers, the source sequences for designing SNP probes of the Brassica 60 K SNP arrays were used to perform a BlastN search against the B. napus ‘Darmor-bzh’ reference genome (version 4.1, http://www.genoscope.cns.fr/brassicanapus/data/ ) [26 (link)]. Only the top BLAST hits (E values of < “1.0 E−10”) were considered to be mapped in the genome, while BLAST matches to multiple loci with the same top identity were not considered to be mapped [31 (link), 35 ].
6
Genotyping and Phenotyping of Brassica Inbred Lines
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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 − με|/ σε > 3) by QQ plot (Quantile-Quantile plot) for each trait (FigureS1 ).
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 − με|/ σε > 3) by QQ plot (Quantile-Quantile plot) for each trait (Figure
7
Genotyping Brassica Accessions Using SNP Array
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Genomic DNA was extracted from the bulked young leaves of 24 plants of each genotype using the TIANGEN® plant genomic extraction kit (DP305-03) (Beijing, China). Accessions were genotyped using the Brassica 60K Illumina Infinium® SNP array (Edwards et al., 2013 (link)), according to the Infinium® HD Assay Ultra Protocol Guide. Using a local BLASTn search, the SNPs of the array were aligned to the B. napus reference genome assembly v4.1 (Chalhoub et al., 2014 (link)). Only the top BLAST hits against the reference genome were considered, allowing no less than 50 bp overlap, 90% sequence identity, and no gaps (Clarke et al., 2016 (link)).
8
Genetic Mapping of Brassica Traits
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Genotyping data for the BnDYDH population included an analysis of SNPs from the Brassica 60K Illumina Infinium SNP array (Clarke et␣al., 2016 (link)), the 20K Illumina Infinium SNP array (Chalhoub et␣al., 2014 (link)) and the 8K Illumina Infinium SNP array (Delourme et␣al., 2013 (link)). The genetic map contained 28 000 loci that represented 3592 unique loci, and covered 2128.2 cM (Laperche et␣al., 2017 (link)). The phenotypic data were mapped as quantitative traits with the R/qtl package (Broman et␣al., 2003 (link)) or as a qualitative traits where scores of 1 to 3 were considered to indicate plant resistance and scores of 4 to 6 were considered to indicate susceptibility.
9
Genotyping and Statistical Analysis of Inbred Lines
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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).
The data for the five traits were tested by analysis of variance (ANOVA) using SPSS version 19.0 (IBM Corp., Armonk, NY, USA).
10
Brassica SNP Genotyping with Illumina Assay
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SNP genotyping was performed using the Brassica 60K Illumina® Infinium SNP array by Emei Tongde Co. (Beijing) according to the manufacture's protocol (http://www.illumina.com/technology/infinium_hd_assay.ilmn ). The SNP data were clustered and called automatically using the Illumina BeadStudio genotyping software. Those SNPs with AA or BB frequency equal to zero, call frequency <0.8 or minor frequency <0.05 were excluded. The remaining SNPs were scrutinized visually and those SNPs that did not show three clearly defined clusters representing the three possible genotypes (AA, AB and BB) were also excluded.
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