A Modified TILLING Method for Wheat Breeding

The large genome and polyploidy of wheat (Triticum aestivum L.) makes it diffi cult to identify desirable genetic changes based on phenotypic screening due to gene redundancy. Forward genetics is, therefore, more diffi cult in wheat than in diploid plants. A modifi ed TILLING (Targeting Induced Local Lesions IN Genomes) method including the harvest of fi ve heads per M1 plant, storage of M 2 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 identifi cation 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 identifi ed 19 mutants. A hard grain variant of a soft cultivar was identifi ed 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.