Development of a multiplex amplicon‐sequencing assay to detect low‐frequency mutations in poinsettia (Euphorbia pulcherrima) breeding programmes

Poinsettia is an economically important ornamental potted plant in which certain bract colour variants are often obtained by mutation breeding. Previously, in poinsettia, we identified Bract1, a GST gene involved in the sequestration and transport of anthocyanins to the vacuole. This gene carries a short, highly mutable 4‐bp repeat in its coding region. Loss of one repeat unit leads to a loss of function for Bract1, and in homozygous mutants, anthocyanin‐based coloration is absent, resulting in white or cream‐coloured bracts. Although mutation induction through ionizing radiation leads to a high frequency of mutations in Bract1, mutants are difficult to obtain from homozygous red genotypes. In this study, we used Bract1‐specific amplicon sequencing as a tool to identify mutations in pools of tissues, which enabled the detection of mutations in dilutions of up to one mutant in 50 nonmutated samples. This approach enabled efficient screening of recalcitrant homozygous genotypes for mutated alleles and the reduction of the mutation load in the application of ionizing radiation in mutation breeding programmes.