High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner？ system we were able to detect mutations in heterozygous and homozygous states for both genes.Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.The identification and analysis of gene mutations in plants is fundamental to the investigation of gene function. One approach, Targeted Induced Loci Lesions IN Genomes (TILLING) was originally developed in Arabidopsis [1] and has subsequently been successful in a range of crop plants [2,3]. In this reverse genetic approach, an ethyl methane sulfonate (EMS) mutagenised population is screened for SNPs within target genes [4]. EMS mutagenesis generates multiple alleles within each gene, including nonsense, missense, splicing and cis-regulatory mutants, in comparison to T-DNA and transposon mutagenesis that generate only knockout mutants [5,6]. Analysis of an allelic series can provide information on important domains or amino acids within the protein of interest. A number of TILLING populations have been developed for a variety of crops of different genome size, including rice [2], wheat [7,8], Brassica rapa [3], Brassica napus [9], Lotus japonicus [10], Medicago trunculata [11], Arachis hypogaea [12], and Solanum lycopersicum [13]. These populations have subsequently been used to isolate mutations in a variety of genes, including those involved in starch metabolism in Lotus japonicas [14], peanut allergens [12] and a gene encoding a fatty acid elongas