Cytogenetic characterization of EPSPS gene amplification in glyphosate-resistant Hordeum glaucum and Bromus diandrus from Australia

As a result of extensive selection, two polyploid grass weeds, Hordeum glaucum (northern barley grass; 2n = 4x = 28) and Bromus diandrus (ripgut brome; 2n = 8x = 56), have evolved resistance to glyphosate, in Australia. Previous research suggested amplification of 5-enolpyruvylshikimate-3-Phosphate synthase (EPSPS) gene confers resistance in these two weed species. The objective of this research was to investigate the genomic organization of the EPSPS gene in these two species through molecular cytogenetic analyses of fluorescence in situ hybridization (FISH) to understand possible mechanism of amplification of this gene. EPSPS copy number of H. glaucum and B. diandrus plants was estimated via quantitative polymerase chain reaction. The susceptible plants of both species had one copy of EPSPS, whereas the resistant plants of H. glaucum and B. diandrus had 14-17 and 16-32 copies, respectively. FISH analysis of glyphosate-susceptible (Hg-RWS) H. glaucum, revealed four faint signals of the EPSPS gene in two pairs of homologous chromosomes, at the telomeric region. The glyphosate-resistant H. glaucum (Hg-YP1) also showed amplification of EPSPS gene at telomeric regions in two pairs of homologous chromosomes, but the signals were brighter and appeared as cluster of EPSPS genes. Similarly, the glyphosate-susceptible B. diandrus (Bd-S) plants showed faint signals of EPSPS gene on two homologous chromosomes, at the telomeric position. However, samples of two glyphosate-resistant, B. diandrus, Bd-SA988 and Bd-Vic showed much brighter hybridization signals of EPSPS gene, located at the telomere on two homologous chromosomes, suggesting an increase in EPSPS gene copies at this position. Overall, unequal crossover during meiosis may have triggered the initial EPSPS gene duplication sparking the evolution of glyphosate resistance.