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.
