Additional file 1 of Reduced free asparagine in wheat grain resulting from a natural deletion of TaASN-B2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality
Creators: name: Oddy, Joseph
name: Alarcón-Reverte, Rocío
name: Wilkinson, Mark
name: Ravet, Karl
name: Raffan, Sarah
name: Minter, Andrea
name: Mead, Andrew
name: Elmore, J. Stephen
name: de Almeida, Isabel Moreira
name: Cryer, Nicholas C.
name: Halford, Nigel G.
name: Pearce, Stephen
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1 Fig. S1. Allelic diversity in TaASN1. Predicted amino acid sequence of the full-length ASN-B1 protein encoded by varieties Robigus, Julius, Norin 61, Mace and Spelt wheat (wild-type), compared to the truncated protein encoded by varieties CDC Landmark, Claire, Jagger, Cadenza, Paragon, Arina, CDC Stanley and Lancer (ASN-B1 truncation). In the latter varieties, a 16 bp deletion in exon 7 is predicted to introduce a premature stop codon at amino acid residue 375, indicated by *. The conserved GATase and ASN synthetase domains are highlighted. Fig. S2. PCR assay to distinguish presence and absence of TaASN-B2 in a collection of 24 global wheat varieties. a. Schematic diagram of the assay to show primer positions and expected amplicon sizes. Amplification of a 189 bp product with primers P1 and P2 indicates that TaASN B2 is deleted, while amplification of a 125 bp product with primers P3 and P4 indicates that TaASN-B2 is present. One amplified fragment is expected in each reaction. b. Agarose gel electrophoresis of PCR products from the assay. Varieties with TaASN-B2 deleted are highlighted in red, while varieties with the gene present are highlighted in green. A 100 bp ladder is shown in the first and last well of the gel for size comparison. Among the varieties are five carrying the TaASN-B2 deletion and four with TaASN-B2 present that were used to assay ASN expression in the grain. Full details of each variety are given in Table 2. Table S1. Natural variation in ASN proteins in wheat. For each protein, shades of green indicate that all amino acid substitutions are predicted to be tolerated and the encoded protein is predicted to be functional. Shades of yellow/orange indicate that at least one polymorphism is predicted to be disruptive for protein function based on SIFT analysis. Red indicates the gene is deleted in that variety. Full details of each protein type are provided in the key below the main table, where (T) indicates the amino acid substitution at that position is predicted to be tolerated, and (APF) indicates the change is predicted to affect protein function. Table S2. a. List of UK winter wheat (Triticum aestivum) varieties with TaASN-B2 present or absent, separated by market class. b. List of common wheat varieties with TaASN-B2 present or deleted among a panel of 24 global wheat varieties. ID, accession numbers and country of origin are provided. Table S3. Significance values for RT-qPCR and field analysis. a. ANOVA Analysis was performed using Timepoint*Variety*Homeologue as the treatment structure and Block/Subblock/Plot as the blocking structure. b. Significance values for factors in the ANOVA and REML analyses of field trial data. All analyses were performed on loge transformed data. ANOVA analyses were performed using Block/MainPlot/SplitPlot as the random model and (TaASN-B2/Variety)*Sulphur Treatment as the treatment model. REML analysis was performed using Year/Block/MainPlot/SplitPlot as the random model and Year*(TaASN-B2/Variety)*Sulphur Treatment as the treatment model. Table S4. Details of missing sequence data in ASN genes in some genome assemblies. Similarity among sequences was determined using all available sequence but some varieties had regions of ‘Ns’ within ASN genes, as indicated in the table below. Table S5. Primers used in this study.