2009 Annual Report 1a.Objectives (from AD-416) To characterize genetic expression profiles of drought stressed tall fescue containing novel endophytes strains and verify gene expression profiles of selected genes. 1b.Approach (from AD-416) Tall fescue, KY31, containing six novel endophyte strains, plus the endophyte-free and KY31 with wild-type endophyte will be evaluated under different drought stress regimes to characterize differences in gene expression profiles and correlate these with phenotypic response to the treatments. The KY31 lines differ in their response to drought stress and the goal is to compare gene expression levels and identify putative genes that are over- or under-expressed in response to the stress experiments that correlate with increased drought tolerance. ARS is developing an expression profiling tool for tall fescue for analysis of whole genome expression comparisons. This will be used to compare expression of the different KY31 lines containing the different endophyte strains to determine the effect of gene expression, using real time PCR techniques, in drought tolerance response. Three replications comparing non-stressed (beginning of the experiment) and three replications of stressed material for each of the lines will be evaluated. RNA will be isolated from pseudostems of each line for each treatment for analysis. 3.Progress Report Performance of this agreement is monitored by USDA-ARS, Forage-Animal Production Research Unit (FAPRU) scientists being intensely involved as collaborators in the agreement project, and meeting with the collaborator on an as needed basis to alter experimental design. Information exchange and planning is via face to face meetings, teleconferences or email on a regular basis. Tall [Schedonorus arundinaceus (Schreb.) Dumont. = Lolium arundinaceum (Schreb.)], has demonstrably greater adaptive advantages to environmental stresses when symbiotic with its fungal endophyte, Neotyphodium coenophialum (E+) compared to aposymbiotic tall fescue (E-). However the wild-type, common, endophyte (CE) found in the KY31 cultivar produces a number of ergot alkaloids that cause animal toxicosis resulting in significant economic losses for producers (approximately one billion dollars annually by some estimates). The presence of the endophyte provides the plant enhanced drought tolerance. However the basis for this remains obscure. A number of endophytes can produce beneficial alkaloids, such as lolines and peramines that are effective as insect deterrents, but do not produce ergot alkaloids. A number of tall fescue cultivars has been release that contain these non ergot alkaloid producing endophytes (also called novel endophytes, or NE). However, the effect on tall fescue growth and persistence by the presence of these NE’s is still not known, and anecdotal evidence has suggested that the symbiosis with some of the NE strains does not result in the robust persistence as is observed with the CE. Our objectives include isolating E+ and E- lines of Kentucky-31 tall fescue with endophyte UA (University of Arkansas) strains 4, 15, 16, 17, 18, 19, 20, 28 and 32, designated as seedlots KY4, KY15, KY16, KY17, KY18, KY19, KY20, KY28 AND KY32 and to isolate RNA from stressed experiments conducted at the University of Kentucky for transcriptome expression profiles by microarray and RT-PCR analysis To this end, we have generated a number of E+ and E- clone pairs for each of the seedlots above (KY4, 11; KY15, 6; KY16, 1; KY17, 7; KY18, 2; KY19, 4; KY20, 4; KY28, 7; KY32, 7). Additional putative clone pairs are presently being screened with a goal to obtain a minimum of 6 clone pairs for each seedlot (objective already met for KY4, KY15, KY17, KY28 and KY32). These then will be tested under drought stress treatments for phenotypic analysis of E+/E- comparisons and RNA isolation for gene expression analysis.