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ARS Home » Southeast Area » Mississippi State, Mississippi » Crop Science Research Laboratory » Corn Host Plant Resistance Research » Research » Publications at this Location » Publication #366635

Research Project: Enhanced Resistance of Maize to Aspergillus flavus Infection, Aflatoxin Accumulation, and Insect Damage

Location: Corn Host Plant Resistance Research

Title: Reference gene selection for RT-qPCR analysis in maize kernels inoculated with Aspergillus flavus

item OLIVERIA, DAFNE - Mississippi State University
item TANG, JULIET - Forest Products Laboratory
item Warburton, Marilyn

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/25/2021
Publication Date: 5/28/2021
Citation: Oliveria, D.A., Tang, J.D., Warburton, M.L. 2021. Reference gene selection for RT-qPCR analysis in maize kernels inoculated with Aspergillus flavus. Toxins. 13(6):386.

Interpretive Summary: The study of which genes are responsible for important traits in crop plants can be studied by seeing which are turned on in response to a stimulus. For example, disease resistance genes are often turned on when the plant senses the disease organisms have begun the infection process. In order to study genes that may be responsible for resistance to the fungus Aspergillus flavus, we will look at both fungal resistant and fungal susceptible corn plants that have been inoculated with fungal spores and compared them to resistant and susceptible plants that have not been. Genes that are turned on (or “up-regulated”, or show “increased expression”) are good candidates for breeding more resistant corn plants. However, to determine if genes are more up-regulated in resistant plants in response to infection, we needed to identify genes to use as controls. These would be genes that all plants need for normal function and growth, and are expected to be turned on at approximately the same level in all plants, under all conditions. This allows us to see what differences in gene expression is not statistically significant, in order to record only those genes whose up-regulation is greater than these non-significant differences. We identified three genes to use as controls in our future studies of candidate gene expression differences and we will proceed with those studies now.

Technical Abstract: Resistance against infection by the fungus Aspergillus flavus L. in commercial maize (Zea mays) is the target of many studies. Quantification of gene expression patterns via RT-qPCR is a straightforward method to reveal expression profiles of genes of interest in a study, and has been used to find genes that change expression following infection by A. flavus. However, there is no current information on validated reference genes suitable for use as controls in the study of gene expression changes involved in the interaction of A. flavus and maize. The selection of stable reference genes is a crucial step to ensure reliable RT-qPCR data interpretation. Thus, in this study, six candidate reference genes (ACT1, ß-Tub2, eIF4A2, TATA, EFIa, and GAPDH) were evaluated and ranked according to their expression stability. The genes were amplified from first-strand cDNA samples synthesized from kernels of two susceptible and two resistant maize lines that were either inoculated with A. flavus or water, or not inoculated. To calculate and rank the stability of the genes, three software packages were used: geNorm, NormFinder, and BestKeeper. The analysis revealed that the most stable genes to normalize expression levels from maize kernels responding to A. flavus inoculation and wounding are ACT1, EFIa, and eIF4A2. This work delivers a set of reliable reference genes that can be used in future studies of expression profiles of maize kernels during an outbreak of A. flavus or in response to wounding.