Efficient isolation of ion beam-induced mutants for homoeologous loci in common wheat and comparison of the contributions of Glu-1 loci to gluten functionality

Abstract

Ion beam mutations can be efficiently isolated and deployed for functional comparison of homoeologous loci in polyploid plants, and Glu - 1 loci differ substantially in their contribution to wheat gluten functionality. To efficiently conduct genetic analysis, it is beneficial to have multiple types of mutants for the genes under investigation. Here, we demonstrate that ion beam-induced deletion mutants can be efficiently isolated for comparing the function of homoeologous loci of common wheat (Triticum aestivum). Through fragment analysis of PCR products from M2 plants, ion beam mutants lacking homoeologous Glu-A1, Glu-B1 or Glu-D1 loci, which encode high molecular weight glutenin subunits (HMW-GSs) and affect gluten functionality and end-use quality of common wheat, could be isolated simultaneously. Three deletion lines missing Glu-A1, Glu-B1 or Glu-D1 were developed from the original mutants, with the Glu-1 genomic regions deleted in these lines estimated using newly developed DNA markers. Apart from lacking the target HMW-GSs, the three lines all showed decreased accumulation of low molecular weight glutenin subunits (LMW-GSs) and increased amounts of gliadins. Based on the test data of five gluten and glutenin macropolymer (GMP) parameters obtained with grain samples harvested from two environments, we conclude that the genetic effects of Glu-1 loci on gluten functionality can be ranked as Glu-D1 > Glu-B1 > Glu-A1. Furthermore, it is suggested that Glu-1 loci contribute to gluten functionality both directly (by promoting the formation of GMP) and indirectly (through keeping the balance among HMW-GSs, LMW-GSs and gliadins). Finally, the efficient isolation of ion beam mutations for functional comparison of homoeologous loci in polyploid plants and the usefulness of Glu-1 deletion lines for further studying the contribution of Glu-1 loci to gluten functionality are discussed.

Genome-wide analysis of complex wheat gliadins, the dominant carriers of celiac disease epitopes

“…Our previous study has shown that a mutant population of Xiaoyan 81 created by ion beam mutagenesis is useful for isolating the deletion mutants of important wheat chromosomal loci30. Thus, in this work, we screened this mutant population for isolating the deletion mutants lacking individual Gli loci ( Gli-A1, -B1, -D1, -A2, -B2 or -D2 ) by MALDI-TOF-MS analysis.…”

“…Xiaoyan 81 and the six Gli locus deletion lines were cultivated in the field3038. At 25 DAF, the grains of Xiaoyan 81 were collected, and used for total RNA purification as described previously38.…”

“…The mature grains of the seven lines were harvested, cleaned, and stored at 4 °C until use. The ion beam mutant population of Xiaoyan 81 used for isolating Gli locus deletion lines was reported in our previous study30. The six NT lines (N1AT1D, N1BT1D, N1DT1A, N6AT6D, N6BT6A and N6DT6B) and its WT progenitor CS were also described previously6364.…”

“…The assessment of gliadins was accomplished with reverse phase high performance liquid chromatography (RP-HPLC) following the method described in our previous work30. The levels of CD epitopes in the flour samples were determined using an AgraQuant ® Gluten G12 Assay kit (Romer Labs UK Ltd., Cheshire, UK), with some modifications in preparing the gluten sample.…”

“…Because of the high complexity of gliadins in bread wheat, multidisciplinary investigations combining transcriptomic, proteomic, mutagenetic and bioinformatic approaches were used. Important to our efforts was the preparation of six unique mutants each lacking one of the six Gli loci in our model variety Xiaoyan 81, an elite winter type bread wheat cultivar30. To efficiently identify the mRNAs of transcribed gliadin genes, the newly emerged third generation sequencing platform PacBio RSII, which is effective for obtaining full-length transcript sequences3132333435, was adopted.…”