Selection of reference genes for quantitative real-time PCR normalization in Ganoderma-infected oil palm (Elaeis guineensis) seedlings

Kwan, Yee Min; Meon, Sariah; Ho, Chai Ling; Wong, Mui Yun

doi:10.1007/s13313-016-0417-4

Cited by 12 publications

(8 citation statements)

References 27 publications

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“…Quantification of defense response-related gene expression is an important method for elucidating the molecular mechanisms of plant-pathogen and plant-environmental factor interactions [ 47 ]. Compared to Northern blotting, ribonuclease protection assay (RPA), semi-qPCR, molecular in situ hybridization, and cDNA microarray, the combination of qRT-PCR and data normalization using PCR reference genes is a more rapid, convenient, and reliable way of assessing gene transcriptional levels [ 46 , 48 ].…”

Section: Discussionmentioning

confidence: 99%

Identification and evaluation of PCR reference genes for host and pathogen in sugarcane-Sporisorium scitamineum interaction system

et al. 2018

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BackgroundSugarcane (Saccharum L. plant) is an important crop for sugar and bio-energy production around the world. Among sugarcane diseases, smut caused by Sporisorium scitamineum is one of the major fungal diseases causing severe losses to the sugarcane industry. The use of PCR reference genes is essential to the normalization of data on gene expression involving the sugarcane-S. scitamineum interaction system; however, no report that addresses criteria in selecting these reference genes has been published to date.ResultsIn this study, 10 sugarcane genes and eight S. scitamineum genes were selected as candidate PCR reference genes in the sugarcane-S. scitamineum interaction system. The stability and reliability of these 18 candidate genes were analyzed in smut-resistant (NCo376) and -susceptible (YC71–374) genotypes using the statistical algorithms geNorm, NormFinder, BestKeeper, and deltaCt method. Subsequently, the relative expression levels of the sugarcane chitinase I-3 gene and S. scitamineum chorismate mutase gene were determined to validate the applicability of these sugarcane and S. scitamineum PCR reference genes, respectively. We finally found that the acyl-CoA dehydrogenase gene (ACAD), serine/arginine repetitive matrix protein 1 gene (SARMp1), or their combination (ACAD + SARMp1) could be utilized as the most suitable reference genes for normalization of sugarcane gene expression in sugarcane bud tissues after S. scitamineum infection. Similarly, the inosine 5′-monophosphate dehydrogenase gene (S10), the SEC65-signal recognition particle subunit gene (S11), or their combination (S10 + S11) were suitable for normalization of S. scitamineum gene expression in sugarcane bud tissues.ConclusionsThe PCR reference genes ACAD, SARMp1, S10, and S11 may be employed in gene transcriptional studies involving the sugarcane-S. scitamineum interaction system.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4854-z) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Identification and evaluation of PCR reference genes for host and pathogen in sugarcane-Sporisorium scitamineum interaction system

et al. 2018

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“…Previously, actin has been studied extensively for its use as a reference gene for data normalisation in many species [15,[42][43][44] due to its conserved function in cytoskeleton assembly. Even though its expression can vary across different fungal (and plant) species, and experimental conditions, it has proven to be a stable gene to be used for gene expression analysis in N. ditissima.…”

Section: Plos Onementioning

confidence: 99%

Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

et al. 2020

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European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

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“…Gene expression of β-1,3-glucanase was investigated in this experiment while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene (Kwan et al, 2016). For each treatment, destructive sampling of two seedlings harvested at 0, 2, 4, 6 and 8 months after inoculation was done.…”

Section: Expression Of Defense-related Gene β-13-glucanasementioning

confidence: 99%

PYRACLOSTROBIN SUPPRESSED Ganoderma BASAL STEM ROT (BSR), PROMOTED PLANT GROWTH AND INDUCED EARLY EXPRESSION OF β-1,3-GLUCANASE IN OIL PALM (Elaeis guineensis)

Shamiera.Said¹

2019

JOPR

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Pyraclostrobin, a new group of fungicide, was evaluated on its efficacy to suppress G. boninense as well as its effect on oil palm physiology, growth promotion, and induction of defense-related gene, β-1,3-glucanase.Hexaconazole was also used as a positive control. In vitro, the best inhibitory result was achieved using pyraclostrobin at 0.75 µg a.i. ml -1 with percentage inhibition of radial growth (PIRG) of 80%, followed by pyraclostrobin at 0.50 µg a.i. ml -1 (PIRG 73%) and hexaconazole at 0.15 µg a.i. ml -1 (PIRG 73%). Probit analysis also indicated that emulsifiable concentrate (EC) values of pyraclostrobin towards G. boninense mycelial growth were 0.25 μg a.i. ml -1 for EC 50 and 0.58 μg a.i. ml -1 for EC 75 , which were further tested in vivo. In general, in vivo results indicated that the oil palm seedlings treated with pyraclostrobin at EC 75 had significantly lower basal stem rot (BSR) infection, higher plant growth, and positive effects on plant physiology compared to pyraclostrobin at EC 50 and were comparable to hexaconazole. Moreover, gene expression of β-1,3-glucanase in the seedlings treated with pyraclostrobin at EC 75 indicated the highest level at the early stage. These results suggest that pyraclostrobin at EC 75 was able to suppress BSR with high efficacy and significantly improved plant growth.

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Selection of reference genes for quantitative real-time PCR normalization in Ganoderma-infected oil palm (Elaeis guineensis) seedlings

Cited by 12 publications

References 27 publications

Identification and evaluation of PCR reference genes for host and pathogen in sugarcane-Sporisorium scitamineum interaction system

Identification and evaluation of PCR reference genes for host and pathogen in sugarcane-Sporisorium scitamineum interaction system

Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

PYRACLOSTROBIN SUPPRESSED Ganoderma BASAL STEM ROT (BSR), PROMOTED PLANT GROWTH AND INDUCED EARLY EXPRESSION OF β-1,3-GLUCANASE IN OIL PALM (Elaeis guineensis)

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