The large genome and polyploidy of wheat (Triticum aestivum L.) makes it difficult to identify desirable genetic changes based on phenotypic screening due to gene redundancy. Forward genetics is, therefore, more difficult in wheat than in diploid plants. A modified TILLING (Targeting Induced Local Lesions IN Genomes) method including the harvest of five heads per M1 plant, storage of M2 seeds, using unlabeled primers and agarose gels for mutation detection, and crossing of useful mutants for desired grain quality was explored in this report. A soft wheat cultivar, QAL2000, and a hard wheat cultivar, Ventura, were mutagenized with ethyl methanesulfonate (EMS). Screening of the waxy genes Wx‐A1 and Wx‐D1 in 2348 EMS‐treated M2 plants allowed identification of 121 mutants, including silent, missense, and knockout (truncation) mutations. A complete waxy wheat was successfully bred in 18 mo by crossing two truncation mutants (Wx‐A1‐truncation and Wx‐D1‐truncation; Wx‐B1 is naturally null in both mutants). Screening of two puroindoline genes (Pina and Pinb) in QAL2000 identified 19 mutants. A hard grain variant of a soft cultivar was identified due to a mutation in Pinb caused by a premature stop codon. Background mutations were observed and further self‐fertilization or crossing with a wild type was performed to eliminate deleterious mutations. With the rapid accumulation of wheat genomics information, many potential target genes of interest can be screened for mutations in these TILLING populations.

BackgroundTILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes.ResultsWe demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation.ConclusionPolyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.