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Reference genes for gene expression studies targeting sugarcane infected with Sugarcane mosaic virus (SCMV).

ABSTRACT:

Objective

The selection of reference genes in sugarcane under Sugarcane mosaic virus (SCMV) infection has not been reported and is indispensable to get reliable reverse transcription quantitative PCR (RT-qPCR) results for validation of transcriptome analysis. In this regard, seven potential reference genes were tested by RT-qPCR and ranked according to their stability using BestKeeper, NormFinder and GeNorm algorithms, and RefFinder WEB-based software in an experiment performed with samples from two sugarcane cultivars contrasting for SCMV resistance, when mechanically inoculated with a severe SCMV strain and using mock inoculated plant controls.

Results

The genes Uridylate kinase (UK) and Ubiquitin-conjugating enzyme 18 (UBC18) were the most stable according to GeNorm algorithm and the Pearson correlation coefficients with the BestKeeper index. On the other hand, ribosomal protein L35-4 (RPL1), Actin (ACT) and Ubiquitin1 (UBQ1) were the least stable genes for all algorithms tested.

SUBMITTER: da Silva MF

PROVIDER: S-EPMC6423880 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Reference genes for gene expression studies targeting sugarcane infected with Sugarcane mosaic virus (SCMV).

da Silva Marcel Fernando MF Gonçalves Marcos Cesar MC Dos Santos Brito Michael M Nóbile Paula Macedo PM de Andrade Larissa Mara LM Medeiros Cibele Nataliane CN Creste Silvana S Pinto Luciana Rossini LR

BMC research notes 20190318 1

<h4>Objective</h4>The selection of reference genes in sugarcane under Sugarcane mosaic virus (SCMV) infection has not been reported and is indispensable to get reliable reverse transcription quantitative PCR (RT-qPCR) results for validation of transcriptome analysis. In this regard, seven potential reference genes were tested by RT-qPCR and ranked according to their stability using BestKeeper, NormFinder and GeNorm algorithms, and RefFinder WEB-based software in an experiment performed with samp ...[more]

PMID: 30885257

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Project description:BackgroundViruses are infectious pathogens, and plant virus epidemics can have devastating consequences to crop yield and quality. Sugarcane mosaic virus (SCMV, belonging to family Potyviridae) is one of the leading pathogens that affect the sugarcane crop every year. To combat the pathogens' attack, plants generate reactive oxygen species (ROS) as the first line of defense whose sophisticated balance is achieved through well-organized antioxidant scavenging pathways.ResultsIn this study, we investigated the changes occurring at the transcriptomic level of ROS associated and ROS detoxification pathways of SCMV resistant (B-48) and susceptible (Badila) sugarcane genotypes, using Saccharum spontaneum L. genome assembly as a reference genome. Transcriptomic data highlighted the significant upregulation of ROS producing genes such as NADH oxidase, malate dehydrogenase and flavin-binding monooxygenase, in Badila genotype after SCMV pathogenicity. To scavenge the ROS, the Badila genotype illustrated a substantial enhancement of antioxidants i.e. glutathione s-transferase (GST), as compared to its resistant counterpart. GST is supposed to be a key indicator of pathogen attacks on the plant. A remarkably lower GST expression in B-48, as compared to Badila, indicated the development of resistance in this genotype. Additionally, we characterized the critical transcription factors (TFs) involved in endowing resistance to B-48. Among these, WRKY, AP2, NAC, bZIP, and bHLH showed enhanced expression in the B-48 genotype. Our results also confirmed the linkage of transcriptomic data with the enzymatic and qPCR data. The estimation of enzymatic activities for superoxide dismutase, catalase, ascorbate peroxidase, and phenylalanine ammonia-lyase supported the transcriptomic data and evinced higher resistance in B-48 genotype.ConclusionThe current study supported the efficiency of the B-48 genotype under SCMV infection. Moreover, comparative transcriptomic data has been presented to highlight the role of significant transcription factors conferring resistance to this genotype. This study provides an in-depth knowledge of the expression profiling of defense mechanisms in sugarcane.

Project description:Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.

Dataset Information

Reference genes for gene expression studies targeting sugarcane infected with Sugarcane mosaic virus (SCMV).

Objective

Results

Publications

Reference genes for gene expression studies targeting sugarcane infected with Sugarcane mosaic virus (SCMV).

Similar Datasets

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