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Title: Microarray and Real-Time PCR Comparisons of Tall Fescue Gene Expression in Endophyte-Infected and Endophyte-Free Plants

item Dinkins, Randy

Submitted to: ASA-CSSA-SSSA Annual Meeting Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 6/18/2007
Publication Date: 5/11/2007
Citation: Dinkins, R.D., Barnes, A., Waters, W. 2007. Microarray and Real-Time PCR Comparisons of Tall Fescue Gene Expression in Endophyte-Infected and Endophyte-Free Plants. ASA-CSSA-SSSA Annual Meeting Abstracts. November 7, 2007, Session No. 274.

Interpretive Summary:

Technical Abstract: Many grasses have mutualistic symbioses with fungi of the family Clavicipitaceae. Tall fescue [Schedonorus arundinaceus (Schreb.) Dumont. = Festuca arundinacea (Schreb.)] can harbor the obligate endophyte, Neotyphodium coenophialum, that is asexually propagated and transmitted via host seeds. In an effort to dissect the host plant endophyte cross-talk, tall fescue global gene expression was analyzed using the Affymetrix Wheat Genome Array GeneChip® and Barley1 Genome Array GeneChip®. Total RNA was isolated from pseudostems of known endophyte-infected and endophyte-free plants and tested in triplicate. Analysis of the results was combined using four microarray analysis methods (MAS5, Plier, RMA and GCRMA) that were significantly (P<0.05) differentially expressed (greater than two-fold) over both the wheat and barley microarray chips. This combinatorial approach yielded 32 similar probe sets (genes) that were differentially expressed on both the wheat and barley microarray chips. Tall fescue ESTs were identified by BLASTn in GenBank for 20 of the probe sets. PCR and real-time PCR expression was evaluated on cDNA’s constructed using the Stratagene First Strand Synthesis kit of the original RNA samples evaluated by microarray, plus samples collected the following year, using PCR primers designed to the tall fescue EST’s using the IDT Primer Express software. Some primers gave results similar to that observed on the microarray suggesting that these genes are differentially expressed in response to the presence of the endophyte in tall fescue. In other cases no differences were observed between the endophyte-infected and endophyte-free or the results were contrary to what was previously observed. It is unknown whether the primers that did not give expected results are indicative of the particular gene or allele that was used for primer design compared to what was detected on the array, or if the incorrect tall fescue EST was used for PCR primer design due to the limited EST information presently in the databases for tall fescue.