Development and characterisation of mutations conferring sulfonylurea resistance in forage kale and raphanobrassica

Seed mutagenesis using ethyl methanesulfonate was successfully used to develop mutations conferring resistance to the sulfonylurea herbicide, chlorsulfuron, in forage kale (<i>Brassica oleracea</i> var. <i>acephala</i>) and raphanobrassica (<i>Raphanus raphanistrum</i> subsp. sativus x <i>Brassica oleracea</i> var. <i>acephala</i>). Following field screening of open-pollinated M2 populations for chlorsulfuron resistance, several candidate seedlings were selected for further investigation. PCR amplification and DNA sequencing of regions from the acetohydroxyacid synthase gene identified individuals from both kale and raphanobrassica with a C to T point mutation (CCT to TCT) that gives rise to an amino acid substitution of proline-197 with serine. In two kale lines a CCT to CTT point mutation was found that results in the substitution of proline-197 with leucine. Based on the proline-197 to serine mutation, a high-resolution melting assay was developed as a breeding tool to determine whether individual plants were heterozygous or homozygous for the herbicide resistance allele. Five generations of self-pollination and inter-crossing between sibling plants, coupled with selection for plant performance after chlorsulfuron application, has resulted in the development of elite lines with homozygosity of the proline-197 to serine mutation for cultivar release.