Fig. 1

KASP assay primer design rationale for ALVE integration sites. Primer 1 (wildtype) and primer 2 (ALVE) are fluorophore-labelled primers and their amplification enables genotyping direct from solution. The starting sequence for the genotype-specific primers is often the same, but they differ when they cross the target site duplication, with primer 1 continuing through the host genome sequence and primer 2 entering the ALVE insertion. Rounds of elongation are short, so amplification between primer 2 and 1r would be unlikely, even with short insertions. Such short amplification required a four-primer approach rather than the single reverse primer typical in SNP genotyping KASP assays