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

• Published in: The Plant journal : for cell and molecular biology 2024-12, Vol.120 (6), p.2553-2562
• Main Authors: Islam, Md Mazharul, Gill, Bikram S., Malone, Jenna M., Preston, Christopher, Jugulam, Mithila
• Format: Article
• Language: English
• Subjects: 3-Phosphoshikimate 1-Carboxyvinyltransferase - genetics; 5‐enolpyruvylshikimate‐3‐phosphate synthase; Australia; Bromus diandrus; Chromosomes; Chromosomes, Plant - genetics; Copy number; Cytogenetics; EPSPS gene; Fluorescence in situ hybridization; Gene Amplification; Gene Dosage; Gene duplication; Glycine - analogs & derivatives; Glycine - pharmacology; Glyphosate; glyphosate resistance; Grasses; Herbicide resistance; Herbicide Resistance - genetics; Herbicides - pharmacology; Hordeum; Hordeum - genetics; Hybridization; In Situ Hybridization, Fluorescence; Meiosis; Plant Proteins - genetics; Plant Proteins - metabolism; Polymerase chain reaction; Polyploidy; Telomeres; unequal crossover; Weeds
• ISSN: 0960-7412; 1365-313X; 1365-313X
• DOI: 10.1111/tpj.17128

SUMMARY 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. Significance Statement This study uncovers for the first time that unequal crossover during meiosis may have triggered the initial gene duplication sparking the evolution of glyphosate resistance in Hordeum glaucum (northern barley grass) and Bromus diandrus (ripgut brome) are two problem weeds in South Australia. Hordeum glaucum (northern barley grass) and Bromus diandrus (ripgut brome) are two problem weeds in South Australia that evolved resistance to glyphosate, a widely used herbicide, as a result of amplification of 5‐enolpyruvylshikimate‐3‐Phosphate synthase (EPSPS) gene (the molecular target of glyphosate). In this research, w