IWGSC Scaffold: This is the name of the IWGSC gDNA scaffold to which the mutation was mapped. The format is consistent with the original style used by EnsemblPlants. Note that the names of the IWGSC scaffolds in the URGI BLAST server are longer, but retain the same numerical identifier, e.g. IWGSC_CSS_4AS_scaff_5940868 (Ensembl! format) is identical to IWGSC_chr4AS_V2_ab_k71_contigs_longerthan_200_5940868 (URGI BLAST server format). For each mutation a hyperlink to the relevant IWGSC scaffold is included to download the original IWGSC gDNA scaffold.

Chromosome location: This indicates the chromosome to which the IWGSC scaffold was assigned to, based on the original flow sorted chromosome arm survey sequence of Chinese Spring.

NGS library: Name of the NGS library used for each individual mutant (Cadenza only). This is useful for cases where the same mutant was captured and sequenced twice.

Mutant line: Name of the mutant line, which consists of the name of the mutant population (Kronos or Cadenza) and a four digit number. This line name can then be used to request seeds (see section )

SNP position (IWGSC): The position of the EMS mutation relative to the start of the IWGSC scaffold.

SNP position (Ensembl!): The position of the EMS mutation relative to the POPSEQ ordered chromosome pseudomolecules hosted at EnsemblPlants (Release 27).

CS_Ref_base: This is the base call at that particular position in the reference genome of Chinese Spring (CS) published by the IWGSC.

WT_base: This is the base call at that particular position in the wild type control of either the tetraploid variety Kronos or the hexaploid variety Cadenza.

Mut_base: This is the base call at that particular position in the EMS-mutagenized individual from either the tetraploid variety Kronos or the hexaploid variety Cadenza.

Het/Hom: An automated call on whether the sequenced individual was heterozygous or homozygous for that particular SNP. If the WT_cov is 0, then a SNP is called as homozygous in that mutant line. Otherwise the SNP is called as heterozygous. Note that in cases were the WT_cov is very low compared to the Mut_cov, the mutation might be homozygous.

WT_cov: Number of reads with the wild type allele at that SNP position.

Mut_cov: Number of reads with the mutant allele at that SNP position.

Confidence: Assessment of the confidence of a mutant call. If 3-4 mutant alleles are discovered, the likelihood of the mutation being real is 62 - 85% and the mutations is being assigned a "Low" confidence. If 5 mutant alleles are discovered, the likelihood of the mutation being real is 95% and the mutations is being assigned a "Medium" confidence. If 6 or more mutant alleles are discovered, the likelihood of the mutation being real is 97% and the mutations is being assigned a "High" confidence.

Gene_model: This is the name of the IWGSC gene model that is present on that particular scaffold. More than one gene can be present on a single scaffold.

Transcript_model: This is the name of the representative IWGSC transcript variant for the Gene_model: as established by EnsemblPlants. Note that any gene model can have more than one transcript variant.

Consequence: Consequence of the mutation on the transcript model as predicted by the Variant Effect Predictor tool from Ensembl based on the IWGSC gene model. These include consequences such as: non-synonymous mutations, splice acceptor and splice donor mutations, premature stop codons, among others. The complete description of consequences can be found at http://useast.ensembl.org/info/genome/variation/predicted_data.html#consequences.

cDNA_pos: Position of the mutation relative to the cDNA of the predicted IWGSC gene model.

CDS_pos: Position of the mutation relative to the coding sequence (CDS) of the predicted IWGSC gene model.

Protein_pos: Position of the mutation relative to the translated protein sequence of the predicted IWGSC gene model. Note that mutations belonging to the same codon have the same position value.

AA change: Indicates whether a mutation leads to a change of an amino acid in the translated protein of the predicted gene model. The nomenclature follows standard IUPAC conventions, e.g. the change of a Valine residue at position 76 to an Isoleucine residue would be denoted as V76I. A synonymous change will appear with no amino acid after the number (e.g. V76 ).

Codon change: Indicates how a mutation affects a codon. The two unchanged nucleotides of the codon are written in lower case letters, whereas the affected nucleotide is in uppercase.

KASP specificity: KASP assay specificity is determined by PolyMarker based on multiple alignment of IWSGC scaffolds as outlined in Ramirez-Gonzalez et al. (2015, Bioinformatics 31, 2038).

KASP wildtype: Primer designed to amplify the wild type base at the SNP position. This primer has a wildtype nucleotide in the 3’ end of the primer and does not include the required KASP tail.

KASP mutant: Primer designed to amplify the mutant base at the SNP position. This primer has a mutant nucleotide in the 3’ end of the primer and does not include the required KASP tail.

KASP common:    This is the common primer of the KASP assay. Where possible, this primer is designed to specifically amplify the chromosome to which the mutation is assigned. This primer does not require the addition of a tail sequence before ordering.

Orientation: Forward or reverse orientation of the KASP assay with respect to the orientation of the IWGSC scaffold.