|Barnes, Adam -|
|Waters, Whitney -|
Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 7, 2010
Publication Date: September 15, 2010
Citation: Dinkins, R.D., Barnes, A., Waters, W. 2010. Microarray analysis of Endophyte-infected and Endophyte-free tall fescue. Journal of Plant Physiology. 167:1197-1203. Interpretive Summary: The goal for this work is to study gene expression differences in tall fescue [Lolium arundinaceum (Schreb.) = Schedonorus arundinaceus (Schreb.) Dumont.] due to the presence of the symbiotic endophyte (Neotyphodium coenophialum). Whole genome expression profiling is a good method to begin dissection of the genetic interaction, however there are presently no whole genome expression profiling tools available for tall fescue. In this work we sought to determine whether the Affymetrix Wheat Genome Array GeneChip® or Barley1 Genome Array GeneChip® would be a good platform to screen for tall fescue whole genome expression. Pseudostems of tall fescue, KY31, with and without endophyte, were collected in triplicate, total RNA isolated, and used to probe the Affymetrix arrays. The results demonstrated that overall only 14-15% and 17-18% of the probe sets on the wheat and barley chips, respectively, were called present. Tall fescue ESTs were identified for a number of the probe sets that were called positive and PCR experiments were done to verify the expression profile observed in the microarray experiments. The PCR experiment demonstrated that some primers gave results similar to that observed on the arrays, although in other cases no differences were observed between the endophyte-positive and endophyte-negative plants as suggested by the arrays, or the results were contrary to what was observed on the arrays. Our results suggest that while some differentially expressed genes were identified in tall fescue using the wheat and barley microarray chips, they appear to have limited utility for tall fescue whole genome expression analysis.
Technical Abstract: Many grasses have mutualistic symbioses with fungi of the family Clavicipitaceae. Tall fescue [Lolium arundinaceum (Schreb.) = Schedonorus arundinaceus (Schreb.) Dumont.] can harbor the obligate endophyte, Neotyphodium coenophialum that is asexually propagated and transmitted via host seeds. Total RNA was isolated from pseudostems of known endophyte-infected and endophyte-free plants and tested in triplicate on the Affymetrix Wheat Genome Array GeneChip® and Barley1 Genome Array GeneChip®. Overall only 14-15% and 17-18% of the probe sets were called present on the wheat and barley chips, respectively. In order to identify genes that were specifically differentially expressed between the E+ and E- tall fescue, a combination of both barley and wheat target sequences that were differentially expressed (greater than two-fold) by four microarray analysis methods. This combinatorial approach yielded 32 probe sets (genes) that were similar on both chips. Tall fescue ESTs were identified for 20 of the probe sets. PCR primers were designed to the fescue EST’s and tested to verify the expression profile observed in the microarray experiments. Some primers gave results similar to the expected, although in other cases no differences were observed between the E+ and E- plants, or the results were contrary to what was expected. Our results suggest that while some differentially expressed genes were identified by this method, these chips appear to have limited utility for tall fescue whole genome expression analysis.