Identification of Reference Genes for Studying Herbicide Resistance Mechanisms in Japanese Foxtail (Alopecurus japonicus)

Abstract

Herbicide-resistant weeds pose a considerable threat to agriculture, but their resistance mechanisms are poorly understood. Differential gene expression analysis of a weed subjected to herbicide treatment is a key step toward more mechanistic studies. Such an analysis, often involving quantitative real-time PCR (qPCR), requires suitable reference genes as internal controls. In this study, we identified optimal reference genes in the noxious weed, Japanese foxtail. This weed has evolved resistance to acetyl-coenzyme A carboxylase (ACCase) inhibitors. We analyzed the stability of eight commonly used candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]; ubiquitin [UBQ]; capsine phosphatase [CAP]; beta-tubulin [TUB]; eukaryotic initiation factor 4a [EIF4A]; elongation factor-1 alpha [EF1]; 18S ribosomal RNA [18S]; 25S ribosomal RNA [25S]) from root, stem, and leaf tissue of plants that were either resistant or sensitive to ACCase inhibitors, with or without herbicide stress, using qPCR. The results were further ranked and analyzed using geNorm, NormFinder, and BestKeeper software. These analyses identified EF1 and UBQ in roots, EF1, TUB, CAP, and 18S in stems, and EF1, GAPDH, and 18S in leaves as suitable references for qPCR normalization. We have identified a set of reference genes that can be used to study herbicide resistance mechanisms in Japanese foxtail.

Enhanced metabolism and target gene overexpression confer resistance against acetolactate synthase‐inhibiting herbicides in Bromus sterilis

“…Relative copy number and expression was conducted with StepOne™ Real‐Time PCR System (Applied Biosystems) using genomic DNA (~10 ng), and complementary DNA (cDNA, ~10 ng) respectively as templates and gene‐specific primers (GSP) (ALS_ Forward primer and ALS_ Reverse primer). For selecting the suitable reference gene, cycle threshold (Ct) values before (water‐treated) and after herbicide treatment were compared with the four commonly used software: BestKeeper, NormFinder, geNorm and RefFinder 25,26 . Ubiquitin was chosen as an internal standard as it was the most stable reference gene based on the number of times a gene has secured top or second position (Supporting Information Fig.…”

“…For selecting the suitable reference gene, cycle threshold (Ct) values before (watertreated) and after herbicide treatment were compared with the four commonly used software: BestKeeper, NormFinder, geNorm and RefFinder. 25,26 Ubiquitin was chosen as an internal standard as it was the most stable reference gene based on the number of times a gene has secured top or second position (Supporting Information Fig. S1 and Table S1).…”

Fenoxaprop‐P‐ethyl resistance conferred by cytochrome P450s and target site mutation in Alopecurus japonicus

“…cDNAs were synthesized from 100 mg of young leaf tissues from each population of plants not treated with fenoxaprop‐ P ‐ethyl and plants 12 h after fenoxaprop‐ P ‐ethyl treatment. Elongation factor‐1 alpha ( EF‐1 ) was used as the internal control gene, as it is stable in A. japonicus , as described in our previous study . Based on the sequence of each P450 cloned, PCR primers were designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) and checked.…”

Identification of the Most Suitable Reference Gene for Gene Expression Studies With Development and Abiotic Stress Response in Bromus Sterilis

“…Even though suitable candidate genes under different experimental condition were identi ed in many weedy species, like Alopecurus sp. 20 , Eleusine sp. 8 , Avena sp.…”

“…The most commonly used references genes for normalisation of plant gene expression studies are ubiquitin (UBQ), β-tubulin (β-TUB), ribosomal RNA genes (18S rRNA and 25S rRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic elongation factor (eEF), eukaryotic initiation factor 1 (EIF1), actin (ACT), acetyl-CoA carboxylase (ACCase) etc 9,18 . Although these genes are known to have a stable expression in any given condition, several studies documented variability in their expression level between species of plants or different stress conditions or developmental stages [19][20][21] . As no gene can act as a universal reference, it is necessary to systematically select and identify the suitable reference gene for each species 22,23 .…”