Supplementary Figure 11: Negative PCR assays for putatively edited wheat haploids.

Successful amplification of unedited genes (Table S11) combined with multiple negative results for edited gene PCRs suggests that large deletions occurred. Each PCR reaction and gel was run twice with similar results. a, Negative PCR assay for the putative edited haploid 447-G8 compared to unedited control. This assay queried 384 nt upstream of the PCR binding site. In the Taqman qPCR assay, the VLHP2-2A well had strong signal for the control ADH1 assay (Ct = 24.5) and weak amplification of VLHP2-2A (Table S11); the score of 0.71 may be due to non-specific amplification of the VLHP2-2D allele, which has some overlap due to shared primers (see Table S13). A gel-based PCR for VLHP-2B and VLHP-2D produced bands, and direct sequencing showed that there was a WT sequence, but for the putatively edited -2A allele, there was no band (assay 2A-F with 2A-R, see Table S12). That first PCR assay queried -178 bp to +227 bp from the cut site. Additional PCR assays were designed. Primer 2A-F2 (+6 bp) and 2A-R (+227 bp) gave a band with WT sequence 3’ of the cut site. A PCR assay using primer 2A-F1 (-384 bp) through +50 bp (2A-R2) did not amplify in 447-G8 but did amplify WT in a positive control. This is shown here in Fig. S11a. This suggests a large deletion occurred 5’ of the cut site, leaving 3’ sequence intact. Ten forward primers (2A-F3 through 2A-F12) were designed upstream, spaced every few hundred bp, all the way until -3.9 kb upstream of the cut site. These were paired with the working 2A-R primer at +227 bp. No PCR products were obtained from any of these new pairs. Taken together, this suggests that a 5’ deletion has occurred, removing a large amount of DNA starting at the target site and proceeding upstream – leading to a deletion of at least several kb. b, Negative PCR assays for four haploids edited in VLHP2-2B, spanning the target site by ~1400 bp (383bp upstream and 1032bp downstream), compared to unedited control. In the Taqman qPCR assay, we saw positive (WT) amplification of VLHP-2A and VLHP-2D, as well as strong amplification for the in-well ADH1 control assays (Ct = 22-24, Table S11). The Gel-based PCR for VLHP2-2B and VLHP2-2D produced PCR bands and direct sequencing showed that there was a WT sequence, but VLHP2-2A did not produce a band. That assay queried -177 bp to +253 bp from the cut site (primers 2B-F and 2B-R). Two additional primer pairs were then designed to query either side of the editing site, to determine which of the primer binding sites were deleted. No bands were found in the gel for 440-D3, 440-A5, and 456-G9. A weak band was produced for the reaction amplifying 3’ of the cut site 440-B3. However, sequencing showed a 100% match to the D genome, indicating non-specific amplification. We then tried to amplify across what we presumed to be a large deletion, designing additional primer pairs up to 1415 bp flanking the target site. We did not recover any PCR product from these reactions, even though the assays successfully amplified the control (unedited) sample, shown below. We conclude that all four plants (440-B3, 440-D3, 440-A5, and 456-G9) have large deletions at the 2B target site.