Using next-generation sequencing to detect mutations endowing resistance to pesticides: application to acetolactate-synthase (ALS)-based resistance in barnyard grass, a polyploid grass weed.
BACKGROUND
Next-generation sequencing (NGS) technologies offer tremendous possibilities for accurate detection of mutations endowing pesticide resistance, yet their use for this purpose has not emerged in crop protection. This study aims at promoting NGS use for pesticide resistance diagnosis. It describes a simple procedure accessible to virtually any scientist and implementing freely accessible programs for the analysis of NGS data.


RESULTS
Three PCR amplicons encompassing seven codons of the acetolactate-synthase gene crucial for herbicide resistance were sequenced using non-quantified pools of crude DNA extracts from 40 plants in each of 28 field populations of barnyard grass, a polyploid weed. A total of 63,959 quality NGS sequence runs were obtained using the 454 technology. Three herbicide-resistance-endowing mutations (Pro-197-Ser, Pro-197-Leu and/or Trp-574-Leu) were identified in seven populations. The NGS results were confirmed by individual plant Sanger sequencing.


CONCLUSIONS
This work demonstrated the feasibility of NGS-based detection of pesticide resistance, and the advantages of NGS compared with other molecular biology techniques for analysing large numbers of individuals. NGS-based resistance diagnosis has the potential to play a substantial role in monitoring resistance, maintaining pesticide efficacy and optimising pesticide applications.
INRA, UMR1347 Agroécologie, Dijon, France.
Christophe
C
Délye
Romain
R
Causse
Véronique
V
Gautier
Charles
C
Poncet
Séverine
S
Michel
D000095:Acetolactate Synthase / Q000235:genetics*; D003062:Codon; D031747:Echinochloa / Q000201:enzymology / Q000235:genetics*; D053209:Herbicide Resistance / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D057685:Plant Weeds / Q000235:genetics*; D010944:Plants / Q000235:genetics*; D011123:Polyploidy; D017422:Sequence Analysis, DNA
D003062:Codon; D000095:Acetolactate Synthase
Echinochloa; diagnosis; herbicide; next-generation sequencing; pesticide; resistance