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Weed control failures due to herbicide resistance are an increasing and worldwide problem significantly impacting crop yields. Herbicide resistance due to increased herbicide metabolism in weeds is not well characterized at the genetic level. An RNA-Seq transcriptome analysis was used to identify genes conferring metabolism-based herbicide resistance (MBHR) in a population (R) of a major global weed (Lolium rigidum), in which resistance to the herbicide diclofop-methyl was experimentally evolved through recurrent selection from a susceptible (S) progenitor population. A reference transcriptome of 19,623 contigs was assembled using 454 sequencing technology on a cDNA library and annotated using UniProt and Pfam databases. Transcriptomic-level gene expression was measured using Illumina 100 bp reads from untreated control, mock, and diclofop-methyl treatments of R and S. Due to the established importance of cytochrome P450 (CytP450), glutathione-S-transferase (GST), and glucosyltransferase (GT) genes in MBHR, 11 contigs with these annotations and higher constitutive expression in untreated R than in untreated S were selected as candidate genes for hypothesis testing, along with 17 additional differentially expressed contigs with annotations related to metabolism or signal transduction. In a forward genetics validation experiment, higher constitutive expression of nine contigs co-segregated with the resistance phenotype in an F2 population, including 3 CytP450, 3 GST, and 1 GT. At least nine genes with heritable increased constitutive expression are associated with MBHR trait. In a physiological validation experiment where 2, 4-D pre-treatment induced diclofop-methyl protection in S individuals due to increased metabolism, seven of the nine genetically-validated contigs were significantly induced. These data help explain accumulation of resistance-endowing genes and rapid evolution of MBHR, and provide the opportunity to improve diagnostics of MBHR using molecular tools such as transcriptional markers.

Background: We have previously demonstrated that an aldo‐keto reductase (AKR) from Echinochloa colona (EcAKR4‐1) can metabolize glyphosate and confers glyphosate resistance. This study aims to investigate if the EcAKR4‐1 orthologs from Lolium rigidum also play a role in glyphosate resistance in non‐target‐site based, glyphosate‐resistant (R) L. rigidum populations from Western Australia. Results: The full‐length L. rigidum AKR gene (LrAKR4C10) orthologous to EcAKR4‐1, together with a distinct LrAKR1, were cloned from plants of a glyphosate‐susceptible (S) (VLR1) and three glyphosate R L. rigidum populations (WALR50, WALR60 and WALR70). Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) results showed that basal expression levels of the two LrAKR genes did not differ between the R and S populations, but their expression was significantly induced by glyphosate (up to 4.3‐fold) or 2,4‐D treatment (up to 3.4‐fold) in R populations. Escherichia coli cells transformed respectively with LrAKR4C10 and LrAKR1 were more tolerant to glyphosate. Rice (Oryza sativa) seedlings overexpressing each of the two LrAKR gene survived glyphosate rates that were lethal to the green fluorescence protein (GFP) control plants. Structural modeling predicts a similar way of glyphosate binding and detoxification by LrAKR4C10 and EcAKR4‐1, but an alternative way of glyphosate binding by LrAKR1. Relatively lower capacity of the two LrAKRs in conferring glyphosate resistance than the known EcAKR4‐1 was discussed in relation to structural interaction. Conclusion: Glyphosate‐induced higher expression of the two LrAKR genes in L. rigidum populations contributes to a moderate level of glyphosate resistance likely through enhanced glyphosate metabolism. The herbicide 2,4‐D can also induce the LrAKR expression, indicating the potential antagonistic effect of 2,4‐D to glyphosate. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]