Abstract
Glyphosate (N-phosphonomethyl-glycine) is the world’s most widely used broad spectrum, post-emergence herbicide. It inhibits the chloroplast-targeted enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), a component of the plant and microorganism-specific shikimate pathway and a key catalyst in the production of aromatic amino acids. Variants of EPSPS that are not inhibited by glyphosate due to particular amino acid alterations in the active site of the enzyme are known. Some of these variants have been identified in weed species that have developed resistance to glyphosate because of the strong selective pressure of continuous, heavy glyphosate use. We have used TILLING (Targeting Induced Local Lesions in Genomes), a non-transgenic, target-selected, reverse genetics, mutation breeding technique, and conventional genetic crosses, to identify and combine, through two rounds of mutagenesis, wheat lines having both T102I and P106S (so-called TIPS enzyme) mutations in both the A and the D sub-genome homoeologous copies of the wheat EPSPS gene. The combined effects of the T102I and P106S mutations are known from previous work in multiple species to minimize the binding of the herbicide while maintaining the affinity of the catalytic site for its native substrates. These novel wheat lines exhibit substantial tolerance to commercially relevant levels of glyphosate.
Highlights
Glyphosate (N-phosphonomethy-glycine) is the world’s most widely used herbicide [1, 2]
Non-transgenic glyphosate tolerant wheat were identified by KASP genotyping using probes developed to specific SNPs and genomic DNA isolated from seedling leaf tissue
Identification of novel enolpyruvylshikimate-3-phosphate synthase (EPSPS) TILLING alleles and glyphosate resistant phenotype At the commencement of this project, the wheat genome sequence was not available and only cDNA and EST sequences putatively encoding wheat EPSPS homoeologs were available in public databases
Summary
Glyphosate (N-phosphonomethy-glycine) is the world’s most widely used herbicide [1, 2] It forms the basis of the commercial Roundup ready system in which multiple crops such as maize (Zea mays L.), soybeans [Glycine max (L.) Merr.], cotton (Gossypium hirsutum L.), canola (Brassica napa L.), alfalfa (Medicago sativa L.) and sugar beets (Beta vulgaris L.) have been rendered resistant to the effects of glyphosate-containing herbicides by genetic transformation with herbicide insensitive forms of the glyphosate target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) [3]. Glyphosate is rapidly translocated to the meristems and kills these growing points by starving them of these essential amino acids [5].
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