RNA-Seq transcriptome analysis to identify genes involved in metabolism-based diclofop resistance in Lolium rigidum

Weed control failures due to herbicide resistance are an increasing and worldwide problem significantly impacting crop yields. Metabolism-based herbicide resistance (referred to as metabolic resistance) in weeds is not well characterized at the genetic level. An RNA-Seq transcriptome analysis was used to find candidate genes conferring metabolic resistance to the herbicide diclofop in a diclofop-resistant population (R) of the major global weed Lolium rigidum. A reference cDNA transcriptome (19,623 contigs) was assembled and assigned putative annotations. Global gene expression was measured using Illumina reads from untreated control, adjuvant-only control, and diclofop treatment of R and susceptible (S). Contigs showing constitutive expression differences between untreated R and untreated S were selected for further validation analysis, including 11 contigs putatively annotated as cytochrome P450 (CytP450), glutathione transferase (GST), or glucosyltransferase (GT), and 17 additi onal contigs with annotations related to metabolism or signal transduction. In a forward genetics validation experiment, nine contigs had constitutive up-regulation in R individuals from a segregating F2 population, including 3 CytP450, one nitronate monooxygenase (NMO), 3 GST, and 1 GT. Principal component analysis using these nine contigs differentiated F2-R from F2-S individuals. In a physiological validation experiment where 2,4-D pre-treatment induced diclofop protection in S individuals due to increased metabolism, seven of the nine genetically-validated contigs were significantly induced. Four contigs (2 CytP450, NMO, and GT) were consistently highly expressed in nine field-evolved metabolic resistant L. rigidum populations. These four contigs were strongly associated with the resistance phenotype and are major candidates for contributing to metabolic diclofop resistance.

Plant Journal, 78: 865-876