Improving Genome-Specific KASP Markers for Hexaploid Wheat – Application of MAGIC BOX Automated Design Pipeline for Abiotic Stress QTL Dreb1

We have recently developed a KASP marker design pipeline called MAGIC BOX. This pipeline uses an internal BLAST output interpreter to design genome-specific KASP genotyping assay primers. Here we looked at hexaploid wheat (Triticum aestivum) in order to determine how well the pipeline functions. Initially we chose 65 test assays that functioned poorly because of target non-specificity. We were interested to know whether MAGIC BOX would be able to design targeted KASP marker assays for each, but also whether the pipeline would identify whether there were no suitable anchoring points and thus no way to make the assay specific. We tested the assays using a selection of global wheat accessions and nullasomic lines in order to view the effect on frequency and confirm that each of the assays discriminated between genomes correctly. We then looked at the published drought tolerance QTL Dreb1, part of an osmotic regulation pathway, to show how MAGIC BOX can be used to correctly assign unique bases for primer design so that assay Dreb1_JC could correctly target chromosome 5BL. We also showed the effect of anchoring to different genomes on the resulting assay frequencies to highlight the importance of correctly designing KASP marker assays. We show that MAGIC BOX can be used to quickly and effectively target the correct genomic region with minimum effort.