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RESEARCH PRODUCT

Next-Generation Sequencing (NGS) application to the detection of mutations endowing herbicide resistance

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The Next Generation Sequencing (NGS) technologies offer tremendous possibilities for accurate detection of mutations endowing resistance. Yet, their use for this purpose has not emerged in crop protection. We aim at promoting NGS use for herbicide resistance diagnosis. In an exemplary study, we describe a simple procedure for this purpose, useable by virtually any scientist and implementing freely accessible programs for the analysis of NGS data. Three PCR amplicons encompassing seven codons of the ALS gene that are crucial for herbicide resistance were sequenced using non-quantified pools of crude DNA extracts from 40 plants in each of 28 field populations of two species of barnyard-grass: Echinochloa phyllopogon (= Echinochloa oryzicola), a tetraploid species, and the closely related hexaploid species Echinochloa crus-galli. The ALS codon positions targeted were 197, 205, 376, 377, 574, 653 and 654 (amplicons containing codon 122 could not reliably be amplified). A total of 63,959 quality NGS sequence runs were obtained using the 454 technology and subsequently analysed. Three herbicide resistance-endowing mutations (Pro-197-Ser, Pro-197-Leu and/or Trp-574-Leu) were identified in seven populations (one E. crus-galli and six E. phyllopogon populations). The NGS results were confirmed by individual plant Sanger sequencing. This work illustrates the potential of NGS-based detection of herbicide resistance, and its interest, compared to other molecular biology techniques, for analysing large numbers of individuals.