title: An assessment of heavy ion irradiation mutagenesis for reverse genetics in wheat (Triticum aestivum L.).
abstract: Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed.
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation, Agriculture Flagship, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, Brisbane, QLD, 4067, Australia.
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Fitzgerald
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation, Agriculture Flagship, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, Brisbane, QLD, 4067, Australia; Queensland Alliance for Agriculture and Food Innovation, University of Queensland, St. Lucia, QLD, 4072, Australia.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Powell
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation, Agriculture Flagship, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, Brisbane, QLD, 4067, Australia.
authors.forename: Jiri
authors.initials: J
authors.lastname: Stiller
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation, Agriculture Flagship, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, Brisbane, QLD, 4067, Australia.
authors.forename: Terri L
authors.initials: TL
authors.lastname: Weese
authors.affiliation: RIKEN Nishina Center for Accelerator-Based Science, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Abe
authors.affiliation: National Key Facility for Crop Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Guangyao
authors.initials: G
authors.lastname: Zhao
authors.affiliation: National Key Facility for Crop Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Jizeng
authors.initials: J
authors.lastname: Jia
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation, Agriculture Flagship, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, Brisbane, QLD, 4067, Australia.
authors.forename: C Lynne
authors.initials: CL
authors.lastname: McIntyre
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation, Agriculture Flagship, Black Mountain Laboratories, Clunies Ross St, Acton, ACT, 2601, Australia.
authors.forename: Zhongyi
authors.initials: Z
authors.lastname: Li
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation, Agriculture Flagship, Black Mountain Laboratories, Clunies Ross St, Acton, ACT, 2601, Australia.
authors.forename: John M
authors.initials: JM
authors.lastname: Manners
authors.affiliation: Commonwealth Scientific and Industrial Research Organisation, Agriculture Flagship, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, Brisbane, QLD, 4067, Australia; Queensland Alliance for Agriculture and Food Innovation, University of Queensland, St. Lucia, QLD, 4072, Australia.
authors.forename: Kemal
authors.initials: K
authors.lastname: Kazan
mesh_terms: D001483:Base Sequence; D017353:Gene Deletion; D017343:Genes, Plant; D020450:Heavy Ions; D006720:Homozygote; D008969:Molecular Sequence Data; D057865:Plant Immunity / Q000235:genetics; D059386:Reverse Genetics / Q000379:methods; D014908:Triticum / Q000235:genetics* / Q000528:radiation effects