Validation of Reference Genes for Quantitative PCR in Johnsongrass (Sorghum halepense L.) under Glyphosate Stress

Abstract

Weeds are one of the main causes of the decrease in crop yields, with Johnsongrass (Sorghum halepense L.) being one of the most significant. Weeds can be controlled by herbicides, but some have developed resistance. Quantitative PCR is the technique of choice for studying gene expression related to herbicide resistance because of its high sensitivity and specificity, although its quantitative accuracy is highly dependent on the stability of the reference genes. Thus, in this study we evaluated the stability of different reference genes of glyphosate-resistant S. halepense. Nine genes frequently used as reference genes were selected: MDH, ADP, PP2A, EIF4α, ACT, ARI8, DnaJ, Hsp70, and ALS1, and their expression analyzed in susceptible and resistant biotypes at 0, 24 and 72 h post-application of glyphosate. The stability was analyzed with the geNorm, NormFinder, and BestKeeper software programs and using the ΔCt method. RefFinder was used to generate a comprehensive stability ranking. The results showed that PP2A and ARI8 were the most stable genes under the test conditions. EPSPS expression was also verified against the best two and the worst two reference genes. This study provides useful information for gene expression analysis under glyphosate stress and will facilitate resistance mechanism studies in this weed species.

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Centipedegrass under Different Abiotic Stress

“…For example, the level of expression for HSP70 exceeds that observed in heat-sensitive genotypes, indicating its superior protective effect against heat stress [60], However, its expression level may not be well reflected under other stress conditions. Furthermore, the expression of HSP70 was unstable under herbicide stress of Johnsongrass (Sorghum halepense) [7], and similar results were found in this study. Upon analyzing all samples, the expression of HSP70 was found to be the most unstable, and this pattern was consistent under both cold and drought stress.…”

“…This technique plays a crucial role in investigating plant gene expression patterns under various conditions. Traditionally, certain housekeeping genes like ACT, TUB, UBC, etc., are commonly employed as reference genes [5][6][7]. However, it is important to note that the expression levels of these genes may vary under various experimental settings.…”

Selection and Validation of Reference Genes in Sudan Grass (Sorghum sudanense (Piper) Stapf) under Various Abiotic Stresses by qRT-PCR

“…Palakolanu et al found that PP2A and CYP were the reference genes with the best performance under abiotic stress in sorghum [35]. María et al found that ARI 8 and PP2A were the top ranks under glyphosate stress in sorghum [36]. Otherwise, eIF4α and PP2A always express stably in Larix olgensis, Vaccinium corymbosum × angustifolium, Chenopodium quinoa Willd, oat, Suaeda glauca L., and so on, especially in salt stress and drought stress [37][38][39][40][41].…”

Selection of Suitable Reference Genes for Gene Expression Normalization Studies in Dendrobium huoshanense

“…Selecting out the stable reference genes of plant species is very important in gene expression research on the biosynthetic pathway of their main components [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ]. In this study, based on the traditional housekeeping gene identity and the reports of plant stable internal reference genes in the literature [ 40 , 41 , 42 ], 15 candidate reference genes from D. huoshanense , namely GAPDH , eIF , EF-1α , PP2A , UBCE , RPL5 , TBP , APT1 , MDH , PTBP3 , PEPC , CYP71 , NCBP2 , TIP41 , and F-box , were assessed their expression stability by qPCR under abiotic stresses, namely oxidative (H 2 O 2 ), drought (PEG), cold (4 °C), UV radiation (UV), and hormone treatment (methyl jasmonate (MeJA)), as well as in different tissues (root, stem, and leaf). Next, geNorm [ 43 ], NormFinder [ 44 ], and BestKeeper [ 45 ] were preliminarily employed to analyze the gene expression stability, and RefFinder ( (accessed on 1 August 2021)) was then used to comprehensively reassess the candidate genes.…”