Transcriptome Profiling to Identify Genes Involved in Mesosulfuron-Methyl Resistance in Alopecurus aequalis

Abstract

Non-target-site resistance (NTSR) to herbicides is a worldwide concern for weed control. However, as the dominant NTSR mechanism in weeds, metabolic resistance is not yet well-characterized at the genetic level. For this study, we have identified a shortawn foxtail (Alopecurus aequalis Sobol.) population displaying both TSR and NTSR to mesosulfuron-methyl and fenoxaprop-P-ethyl, yet the molecular basis for this NTSR remains unclear. To investigate the mechanisms of metabolic resistance, an RNA-Seq transcriptome analysis was used to find candidate genes that may confer metabolic resistance to the herbicide mesosulfuron-methyl in this plant population. The RNA-Seq libraries generated 831,846,736 clean reads. The de novo transcriptome assembly yielded 95,479 unigenes (averaging 944 bp in length) that were assigned putative annotations. Among these, a total of 29,889 unigenes were assigned to 67 GO terms that contained three main categories, and 14,246 unigenes assigned to 32 predicted KEGG metabolic pathways. Global gene expression was measured using the reads generated from the untreated control (CK), water-only control (WCK), and mesosulfuron-methyl treatment (T) of R and susceptible (S). Contigs that showed expression differences between mesosulfuron-methyl-treated R and S biotypes, and between mesosulfuron-methyl-treated, water-treated and untreated R plants were selected for further quantitative real-time PCR (qRT-PCR) validation analyses. Seventeen contigs were consistently highly expressed in the resistant A. aequalis plants, including four cytochrome P450 monooxygenase (CytP450) genes, two glutathione S-transferase (GST) genes, two glucosyltransferase (GT) genes, two ATP-binding cassette (ABC) transporter genes, and seven additional contigs with functional annotations related to oxidation, hydrolysis, and plant stress physiology. These 17 contigs could serve as major candidate genes for contributing to metabolic mesosulfuron-methyl resistance; hence they deserve further functional study. This is the first large-scale transcriptome-sequencing study to identify NTSR genes in A. aequalis that uses the Illumina platform. This work demonstrates that NTSR is likely driven by the differences in the expression patterns of a set of genes. The assembled transcriptome data presented here provide a valuable resource for A. aequalis biology, and should facilitate the study of herbicide resistance at the molecular level in this and other weed species.

Trp‐1999‐Ser mutation of acetyl‐CoA carboxylase and cytochrome P450s‐involved metabolism confer resistance to fenoxaprop‐P‐ethyl in Polypogon fugax

“…ha −1 , and the remaining 20 plants were treated with water (herbicide solution = control). All herbicides were applied as described previously by using an air‐compressed, moving‐nozzle cabinet sprayer . Thereafter, all seedlings were returned to the greenhouse and grown for another 3 weeks.…”

Target site mutations and cytochrome P450s confer resistance to fenoxaprop‐P‐ethyl and mesosulfuron‐methyl in Alopecurus aequalis

“…Our previous RNA‐Seq study had identified 31 candidate genes that may have conferred mesosulfuron‐methyl resistance to A. aequalis , with eight of them annotated to the P450 family (see Supporting Information, Table S3 in File S1). Here, we compared the expression levels of these eight P450 genes between the R (AHTC‐06F1) and S (AHHN‐02) populations, by using real‐time quantitative reverse transcription polymerase chain reaction (qRT‐PCR), following the methods described in our prior study . Alopecurus aequalis seedlings at the three‐ to four‐leaf stage from each population were sprayed with herbicides, as outlined in Table S4 in File S1 in the Supporting Information.…”

“…Similarly, Gardin et al, reported five candidate genes putatively associated with NTSR in black‐grass ( Alopecurus myosuroides Huds.). In our previous study, TSR and NTSR were shown to co‐exist in a single resistant A. aequalis population, named AH‐18, for which 17 candidate genes were putatively involved in its NTSR to mesosulfuron‐methyl.…”

“…Fenoxaprop‐ P ‐ethyl (APP) and mesosulfuron‐methyl (SU) have been two of the main herbicides used for grass control in canola and wheat crops during the past 20 years in China. Unfortunately, many A. aequalis populations have since evolved a high‐level resistance to these two herbicides in eastern China, especially in the provinces of Anhui and Jiangsu …”

Optimizing RNA‐seq studies to investigate herbicide resistance

“…A general review of the literature (Tables and S1) reveals a wide array of RNA‐seq experimental set‐ups employed by weed researchers over the last few years . These initial set‐up decisions can drastically influence the number of transcripts that appear as significantly differentially expressed by the end of the analysis.…”