Conyza bonariensis (hairy fleabane) is one of the most problematic and widespread glyphosate-resistant weeds in the world. This highly competitive weed species significantly interferes with crop growth and substantially decreases crop yield. Despite its agricultural importance, the molecular mechanisms of glyphosate resistance are still unknown. The present RNA-Seq study was performed with the goal of identifying differentially expressed candidate transcripts (genes) related to metabolism-based non-target site glyphosate resistance in C. bonariensis. The whole-transcriptome was de novo assembled from glyphosate-resistant and -sensitive biotypes of C. bonariensis from Southern Brazil. The RNA was extracted from untreated and glyphosate-treated plants at several timepoints up to 288 h after treatment in both biotypes. The transcriptome assembly produced 90,124 contigs with an average length of 777 bp and N50 of 1118 bp. In response to glyphosate treatment, differential gene expression analysis was performed on glyphosate-resistant and -sensitive biotypes. A total of 9622 genes were differentially expressed as a response to glyphosate treatment in both biotypes, 4297 (44.6%) being up- and 5325 (55.4%) down-regulated. The resistant biotype presented 1770 up- and 2333 down-regulated genes while the sensitive biotype had 2335 and 2800 up- and down-regulated genes, respectively. Among them, 974 up- and 1290 down-regulated genes were co-expressed in both biotypes. In the present work, we identified 41 new candidate target genes from five families related to herbicide transport and metabolism: 19 ABC transporters, 10 CYP450s, one glutathione S-transferase (GST), five glycosyltransferases (GT), and six genes related to antioxidant enzyme catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). The candidate genes may participate in metabolic-based glyphosate resistance via oxidation, conjugation, transport, and degradation, plus antioxidation. One or more of these genes might ‘rescue’ resistant plants from irreversible damage after glyphosate treatment. The 41 target genes we report in the present study may inform further functional genomics studies, including gene editing approaches to elucidate glyphosate-resistance mechanisms in C. bonariensis.

Italian ryegrass (Lolium multiflorum; LOLMU) is one of the most troublesome weeds in temperate regions in the world. This weed species interfere with wheat, corn, rye, and oat, causing significant crop yield losses. This species has evolved glyphosate resistance, making it difficult to control. The mechanisms of glyphosate resistance are still unknown, and an understanding thereof will favor the development of new strategies of management. The present study is the first transcriptome study in LOLMU using glyphosate-resistant and -sensitive biotypes, aiming to identify and to provide a list of the candidate target genes related to glyphosate resistance mechanism. The transcriptome was assembled de novo, producing 87,433 contigs with an N50 of 740 bp and an average length of 575 bp. There were 92 and 54 up- and down-regulated genes, respectively, in the resistant biotype, while a total of 1683 were differentially expressed in the sensitive biotype in response to glyphosate treatment. We selected 14 highly induced genes and seven with repressed expression in the resistant biotype in response to glyphosate. Of these genes, a significant proportion were related to the plasma membrane, indicating that there is a barrier making it difficult for glyphosate to enter the cell.

Core Ideas UBQ5 was the most stable reference gene for gene expression analysis in rice treated with herbicides. The herbicides increased gene expression associated with defense against oxidative stress. OsHO‐1 showed the greatest change in expression in response to herbicide treatment. ABSTRACT Herbicides are stressors that can have negative effects on plants. In Oryza sativa (L.), differential gene expression may be evaluated through real‐time reverse transcription quantitative polymerase chain reaction (RT‐qPCR). The aim of this study was to evaluate the stability of candidate reference genes and to quantify the relative expression of oxidative stress genes at different times (12, 24, 48, and 96 hours after treatment [HAT]) with penoxsulam, cyhalofop‐butyl, and bentazon herbicides. NormFinder, BestKeeper, and GeNorm software and the comparative ∆Ct method were used to assess expression stability and to determine the RT‐qPCR threshold values of the candidate reference genes. The UBQ5 gene was the most stable among the reference genes analyzed. The gene expression results showed upregulation of OsCAT and OsMnSOD1 genes at all times after herbicide application. The OsAPX2 and OsGST3 genes showed increased gene expression at 12 and 96 HAT for all herbicides. The OsHO‐1 gene had the most significant expression changes, with maximum expression levels at 24 HAT with bentazon and at 96 HAT with penoxsulam and cyhalofop‐butyl. Overall, antioxidant system gene expression increased after the application of bentazon, penoxsulam, and cyhalofop‐butyl in rice.

-Real time reverse transcription polymerase chain reaction (RT-qPCR)is an important technique to analyze differences in gene expression due to its sensitivity, accuracy, and specificity. However, before analyzing the expression of the target gene, it is necessary to identify and evaluate the stability of candidate reference genes for the proper normalization. This study aimed at evaluating the stability of candidate reference genes in order to identify the most appropriate genes for the normalization of the transcription in rice and red rice in competition under different nitrogen levels, as well as to demonstrate the effectiveness of the reference gene selected for the expression of the cytosolic ascorbate peroxidase (OsAPX2). Eleven candidate reference genes were assessed using the RefFinder which integrates the four leading software: geNorm, NormFinder, Bestkeeper, and the comparative delta-Ct method in addition to the analysis of variance to identify genes with lower standard deviation and coefficient of variation values. Eight out of the eleven genes have shown the desired effectiveness and, among them, the gene UBC-E2 has the highest stability according to RefFinder and the analysis of variance. The expression of the gene OsAPX2 has proven to be effective in validating the candidate reference gene. This study is the first survey on the stability of candidate reference genes in rice and red rice in competition, providing information to obtain more accurate results in RT-qPCR. Keywords:Oryza sativa, gene expression, normalization. RESUMO -Reação em cadeia da polimerase da transcrição reversa em tempo real (RT qPCR) é uma técnica importante para analisar a expressão diferencial dos genes, devido à sua sensibilidade, exatidão e especificidade. No entanto, antes da análise da expressão do gene alvo, é necessário identificar e avaliar a estabilidade dos genes candidatos a referência para a normalização apropriada. O objetivo deste trabalho foi avaliar a estabilidade de genes candidatos a genes de referência, a fim de identificar os genes mais adequados para normalização da transcrição em arroz e arroz-vermelho em competição, sob diferentes doses de nitrogênio, e demonstrar a eficácia do gene de referência selecionado pela expressão da ascorbato peroxidase citosólica (OsAPX2

The hairy fleabane (Conyza bonariensis (L.) Cronq.) is among the most problematic glyphosate-resistant weeds to manage around the world. In weed science, molecular approaches such as RNA sequencing (RNA-Seq) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR) have been employed to study molecular responses to glyphosate treatment in Conyza species. Glyphosate treatment leads to reactive oxygen species (ROS) production in plants which could damage the RNA. Degraded RNA is an issue and can compromise further molecular analysis. Thus, the objective of this study was to evaluate whether glyphosate treatment interferes negatively on RNA integrity of glyphosate-resistant andsensitive hairy fleabane biotypes. Two experiments were performed using glyphosate doses from 0 to 11,840 g a.e. ha -1 and evaluated in a time-course until 288 hours after treatment. The total of 86 RNA samples were evaluated. The RNA integrity was evaluated in a Bioanalyzer 2100 equipment according to RNA integrity number (RIN) scores and electrophoresis gel. The RIN scores ranged from 5.1 to 9.0. Glyphosate doses do not reduce the RIN scores in both glyphosate-resistant and -sensitive biotypes of hairy fleabane. Visual and automatic analysis of electrophoresis gel show suitable results for all RNA samples, with well-defined bands at 28S and 18S positions and no degradation. The results of the analysis indicate that glyphosate treatment does not affect the RNA integrity of glyphosate-resistant and -sensitive biotypes of hairy fleabane until 288 and 192 hours after glyphosate treatment, respectively. The RNA integrity analysis provides useful results to evaluate the RNA condition for further analysis. However, the costs were around US$ 14.25 per sample, considering only reagents. These results are useful for planning future timecourse experiments in Conyza spp. after glyphosate treatment.Keywords: hairy fleabane, molecular analysis, RNA integrity number (RIN). (Conyza bonariensis (L.) Cronq.) está entre as plantas daninhas resistentes ao glifosato mais difíceis de serem manejadas em todo o mundo. Na ciência das plantas daninhas, abordagens moleculares, como o sequenciamento de RNA (RNA-Seq) e a reação da transcriptase reversa da polimerase em tempo real (RT-qPCR), têm sido empregadas para estudar as respostas moleculares ao tratamento com glifosato em espécies de Conyza. No entanto, o tratamento com glifosato leva à produção de espécies reativas de oxigênio (ROS) em plantas, que podem danificar o RNA. A degradação do RNA é um problema e pode comprometer futuras análises moleculares. Assim, o objetivo deste estudo foi avaliar se o tratamento com glifosato interfere negativamente na integridade do RNA de biótipos de buva resistente e sensível ao glifosato. Dois experimentos foram realizados I D I D I D Planta Daninha 2019; v37:e019217909 PIASECKI, C. et al. Does the glyphosate treatment interfere negatively on RNA integrity in glyphosate-resistant and -sensitive ... 2 utilizando doses de glifosato de 0 a 11.840 g e.a. ha -1 e av...