Mapping Locations and Investigating Target Sequence in Glyphosate-tolerant Spring Wheat

Glyphosate, a non-selective herbicide, potently inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A point mutation of EPSPS at position 106 of proline to serine, threonine, alanine, or leucine (P106S/T/A/L) has been documented to confer glyphosate resistance in several weed populations. Glyphosate-tolerant spring wheat cv. Louise (AABBDD) was developed by mutagenesis using ethyl methanesulfonate (EMS). To determine whether the glyphosate tolerant phenotype of these mutants results from mutations in the P106 codon, epsps sequence and gene location were investigated and mapped. Mutant spring wheat lines designated as ORI-Louise and Louise 33-6-1-4, wild-type Louise, wheat progenitors, and Nullitetras were used in this study. Synthesized cDNA from wheat species and generated specific primers were used in PCR amplification. The PCR products were cloned and sequenced. The open reading frame (ORF) of epsps gene was 1533 bp and contained GC content - approximately 57.5% - and predicts for a polypeptide of 511 amino acid residues. The partial epsps sequence of hexaploid wheat can be divided into D and AB consensus sequences. Location of epsps gene was mapped to chromosome 7A, 5B, and 7D using Nullitetra lines. Alignment of EPSPS sequences among wild type and mutant lines indicated that P106 is conserved. Within D consensus, ORI-Louise contained the point mutation of nucleotide C715T resulting in an amino acid substitution of L239F. No point mutation was found in the mutated Louise 33-6-1-4 line. Mutagenized spring wheat lines ORI-Louise and Louise 33-6-1-4 do not have the mutation at P106 in the target site, therefore, as a mechanism for glyphosate resistance.