Ectopic recombination: a novel mechanism of EPSPS gene amplification in glyphosate-resistant Chloris truncata

Amplification of 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene confers resistance to the herbicide glyphosate in the tetraploid Chloris truncata in Australia. To study the mechanism of amplification, the genomic organization of the EPSPS gene was investigated using fluorescence in situ hybridization (FISH) in one susceptible (Ct-S) and two resistant (Ct-R1 and Ct-R2) biotypes of C. truncata. FISH analysis revealed faint signals of the EPSPS gene on the telomeric regions of a single pair of homologous chromosomes in Ct-S plants. However, much brighter hybridization signals of the EPSPS gene were detected on three pairs of homologous chromosomes in Ct-R1 and on four pairs in the Ct-R2 plants. Thus, there was gene amplification on the native EPSPS locus as well as spread of EPSPS loci to additional chromosomes. All loci were detected in terminal regions which are hotspots of recombination. This local as well as ectopic EPSPS amplification to specific regions of chromosomes is a novel mechanism resistance to herbicides. We hypothesize that, during the bouquet stage of meiosis, telomeres come together forming a bouquet and this may provide an opportunity for ectopic recombination, supported by FISH analyses in interphase nuclei. Overall, the gene amplification appears to have occurred in two steps. First, there was tandem EPSPS amplification at the native locus, possibly via unequal recombination. Second, the amplified locus underwent ectopic recombination and spread to two additional chromosomes in Ct-R1 and three additional chromosomes in Ct-R2 plants.