Characterization of glyphosate-resistant mechanism(s) in spring wheat (Triticum aestivum L.) induced through mutagenesis
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Wheat susceptibility to glyphosate, a non-selective herbicide targeting the EPSPS enzyme, encouraged the development of glyphosate-resistant (GR) spring wheat through mutation breeding using ethyl methanesulfonate (EMS) to induce mutations. Several wheat mutants from spring wheat cultivars `Hollis', `Louise', `Macon', and `Tara2002' conferring increased glyphosate tolerance (GT) were isolated and investigated at the molecular and physiological levels. Three wheat EPSPS genes (TaEPSPS-7A1, TaEPSPS-4A1, and TaEPSPS-7D1) were cloned using PCR and inverse-PCR approaches and were located on chromosome 7AS, 4AL, and 7DS, respectively. EPSPS genes had high conservation at the cDNA sequence level, but showed more variation in intron sequences. The three wheat EPSPS genes were expressed at different levels with dominant expression from TaEPSPS-7A1 in wheat seedlings. Glyphosate resistance in six GT mutants appeared to result from different genetic mechanisms; GTM14 segregated as monogenic recessive, GTL1, GTL65, and GTT20 as monogenic dominant, and GTL33 and GTH9 as a single semi-dominant or multi-gene trait. Glyphosate at the rate of 420 g ae ha-1 effectively inhibited susceptible plants at tillering to jointing stages with an increased efficacy when fertilizer was applied to plants before the herbicide application. The Leucine239 to Phenylalanine (L239F) amino acid substitution, resulting from a C715T transition, was discovered in TaEPSPS-7D1 of the GTL1 mutant. However, the C715T mutation point was not significantly correlated with the glyphosate tolerance. The levels of TaEPSPS transcripts between wild type Louise (WTL) and GTL1 mutant were not significantly different at basal levels and at 1 h post-treatment with 530 g ae ha-1 glyphosate. The expression levels of TaEPSPS genes were induced by glyphosate in both WTL and GTL1 at 1 h after application, but did not differ from the nontreated controls.This research indicates that it is possible to isolate induced mutations providing varying degrees of glyphosate resistance. Glyphosate resistance may result from different resistance mechanisms in different GT wheat lines since resistance gene(s) were independently isolated and transmitted dissimilarly. Because no mutations linked to glyphosate resistance were discovered in the TaEPSPS genes of GTL1, GTL33, and GTH9, it is likely that resistance results from a non-target-site mechanism.