Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses

Müller, Oliver A.; Grau, Jan; Thieme, Sabine; Prochaska, Heike; Adlung, Norman; Sorgatz, Anika; Bonas, Ulla

doi:10.1371/journal.pone.0136499

Cited by 42 publications

(46 citation statements)

References 75 publications

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“…NormFinder enables grouping of the samples, and calculates the intra-and intergroup variation of candidate genes, and suggests suitable internal control gene. Our data indicated that all candidate genes displayed stability values below 1.5, suggesting for the housekeeping functions of the selected genes (Mascia et al, 2010;Lacerda et al, 2015;Müller et al, 2015) (Figure 2A, B). Meanwhile, the stability values output by geNorm and NormFinder analyses were distinctive for each candidate.…”

Section: Schemes To Select Stable Internal Control Gene Candidatesmentioning

confidence: 84%

“…Then, two representative algorithms, geNorm and NormFinder, were applied (Andersen et al 2004; Vandesompele et al ). These algorithms are used to rate the constancy of expressions among samples (Lacerda et al 2015;Mascia et al 2010;Müller et al 2015). The geNorm analysis run by qBase+ software computes an expression stability values (M) for tested genes, as the average pairwise variation among candidates (Vandesompele et al 2002).…”

Section: Schemes To Select Stable Internal Control Gene Candidatesmentioning

confidence: 99%

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Evaluation of internal control genes for quantitative realtime PCR analyses for studying fruit development of dwarf tomato cultivar ‘Micro-Tom’

Choi

Hoshikawa

Fujita

et al. 2018

Plant Biotechnology

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Quantitative real-time PCR (qRT-PCR) is widely used to analyze the expression profiles of the genes of interest. In order to obtain accurate quantification data, normalization by using reliable internal control genes is essential. In this study, we evaluated the stability and applicability of eight internal control gene candidates for analyzing gene expression during fruit development in dwarf tomato cultivar Micro-Tom. We collected seventeen different samples from flowers and fruits at different developmental stages, and estimated the expression stability of the candidate genes by two statistical algorithms, geNorm and NormFinder. The combined ranking order and qRT-PCR analyses for expression profiles of SlYABBY2a, SlYABBY1a, FRUITFULL1 and APETALA2c suggested that EXPRESSED was the most stable and reliable internal control gene among the candidates. Our analysis also suggested that RPL8 was also suitable if the sample group is limited to fruits at different maturation stages. In addition to EXPRESSED, GAPDH was also applicable for relative quantitation to monitor gene expression profiles through fruit development from pistil to pericarp.

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Section: Schemes To Select Stable Internal Control Gene Candidatesmentioning

confidence: 84%

Section: Schemes To Select Stable Internal Control Gene Candidatesmentioning

confidence: 99%

Evaluation of internal control genes for quantitative realtime PCR analyses for studying fruit development of dwarf tomato cultivar ‘Micro-Tom’

Choi

Hoshikawa

Fujita

et al. 2018

Plant Biotechnology

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“…For example, in Xanthomonas campestris pv. vesicatoria –tomato leaf infection experiments, the gene amplified by the IMP‐β primer set has previously been reported to be more stably expressed than those amplified by primer sets LSM7, COX and EF1‐α (Müller et al., ). This could not be confirmed herein where the IMP‐β primer set was outperformed by LSM7 primer set in terms of both expression stability and smaller variations when sample subsets were analysed.…”

Section: Discussionmentioning

confidence: 99%

“…NormFinder uses a model‐based approach allowing intra‐ and inter‐group comparisons of the obtained Ct values, and it provides a ranking of the candidate reference genes based on the estimated standard deviations. Recent studies have proposed different reference genes for expression studies during tomato ( Solanum lycopersicum ) development (Expósito‐Rodríguez, Borges, Borges‐Pérez, & Pérez, ) and tomato–pathogen interactions (Fradin et al., ; Müller et al., ; Pombo et al., ). Primer pairs for ten of these genes—four classical housekeeping genes, such as elongation factor EF1‐ α and ATPase (Expósito‐Rodríguez et al., ; Pombo et al., ), and six newly suggested ones (Müller et al., ; Pombo et al., )—were selected and evaluated in tomato–grey mould interaction.…”

Section: Introductionmentioning

confidence: 99%

Expression of tomato reference genes using established primer sets: Stability across experimental set‐ups

Rezzonico

Nicot²,

Fahrentrapp

2017

Journal of Phytopathology

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Reliable reference genes are critical for relative quantification using quantitative real‐time PCR (qPCR). Ten tomato genes (Solanum lycopersicum) and their respective primer sets, which have been used over the last 6 years as references in expression studies, were evaluated for their performance using leaf tissue samples grown under semi‐controlled conditions and infected with grey mould (Botrytis cinerea) or late blight (Phytophthora infestans). The target genes coding for U6 snRNA‐associated Sm‐like protein LSm7, calcineurin B‐like protein and V‐type proton ATPase were the most stable expressed of all the genes tested in three experimental repetitions. Evaluation of candidate reference genes with geNorm and NormFinder softwares yielded the lowest mean values for their respective primer sets LSM7, SlCBL1 and SlATPase, suggesting stable expression. However, SlATPase primer set revealed a comparably high intra‐group variation and was thus not considered further. In follow‐up experiments with P. infestans, the geNorm and NormFinder values of primer sets LSM7 and SlCBL1 were even lower, indicating the stability of their expression also under these conditions. Primer efficiency differed by ‐18 to +5 percentage points from values presented in the literature. Our findings show that a reference primer set which delivers the best results in one system may be outperformed by another under different experimental conditions, thus recommending a reassessment of both expression stability and qPCR efficiency whenever the biological or technical experimental set‐up is changed. On the basis of our results, we recommend the use of LSM7 and SlCBL1 as reference primer sets for gene expression studies on plant tissue derived from open or semi‐controlled conditions.

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“…2011; Müller et al . 2015), and the gene expression levels are given as the relative numbers of copies.…”

Section: Methodsmentioning

confidence: 99%

The pattern of 1‐aminocyclopropane‐1‐carboxylate oxidase induction in the tomato leaf petiole abscission zone is independent of expression of the ribonuclease‐LX‐encoding LeLX gene

et al. 2018

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The abscission of tomato leaves occurs in the petiole abscission zone, and its late stage includes two spatially divided processes: cell separation and programmed cell death (PCD). Both of these processes are regulated by ethylene. The last step in ethylene biosynthesis is conversion of 1‐aminocyclopropane‐1‐carboxylic acid to ethylene, which is catalysed by the enzyme 1‐aminocyclopropane‐1‐carboxylate oxidase (ACO); however, the location of ACO in the leaf petiole abscission zone is not known. The tomato gene LeLX encodes ribonuclease LX, which is a marker for PCD and is induced by ethylene during abscission, but its association with ACO has not been explored.In a tomato transgenic line 1‐7 with inhibited expression of LeLX showing delayed leaf abscission, the morphology and ultrastructure of the leaf petiole abscission zone was examined. In this zone of the cv.’VF36’ and of a transgenic line 1‐7, spatiotemporal differences in expression of LeACO1 and LeACO4 were analysed and ACO protein was detected immunohistochemically.In comparison to wild‐type plants, there were no obvious morphological and ultrastructural features in the abscission zone of plants of a transgenic line 1‐7 before and after abscission induction. LeACO1 expression was low before abscission induction, and increased 24 h after induction, although with no apparent spatial pattern. In contrast, LeACO4 was expressed before abscission induction, and its transcript level declined 24 h after induction on the distal side of the abscission zone fracture. In the LeLX‐inhibited transgenic line, there were no significant differences in LeACO1 and LeACO4 expression in the petiole abscission zone, in comparison to wild‐type plants. In addition, the ACO protein was immunolocalised to the vascular tissues that traverse the petiole abscission zone in plants of wild type and of a transgenic line 1‐7; and additionally in the plane of future abscission zone fracture of transgenic‐line plants.The results suggest temporal differential expression of the LeACO genes in tomato leaf petioles and vascular localisation of ACO1 protein. Additionally, the results indicate that expression of LeACO genes is not affected by suppression of the LeLX expression.

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Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses

Cited by 42 publications

References 75 publications

Evaluation of internal control genes for quantitative realtime PCR analyses for studying fruit development of dwarf tomato cultivar ‘Micro-Tom’

Evaluation of internal control genes for quantitative realtime PCR analyses for studying fruit development of dwarf tomato cultivar ‘Micro-Tom’

Expression of tomato reference genes using established primer sets: Stability across experimental set‐ups

The pattern of 1‐aminocyclopropane‐1‐carboxylate oxidase induction in the tomato leaf petiole abscission zone is independent of expression of the ribonuclease‐LX‐encoding LeLX gene

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