1a.Objectives (from AD-416):
To characterize and compare metabolite profiles in tall fescue endophyte-infected and endophyte-free clone pairs.
1b.Approach (from AD-416):
Tall fescue, KY31, containing the wild-type fungal endophyte (Neotyphodium coenophialum) strain has been used to generate endophyte-free lines, constituting of a genetically identical plant background with and without the endophyte. These clone pairs provide a tool to characterize and compare gene expression, metabolite profiles and phenotypic responses, in an identical genetic background, thus allows comparisons based solely on the presence or absence of the endophyte.
The clone pairs have been used initially in a project to determine the variation found in the endophyte that infects Kentucky-31 (KY31) tall fescue. The cloned pairs as well as seed that these clone pairs originated from were included in this initial project. At least 2 copies of each line is maintained in the Noble greenhouses and they are regularly (every 6 months) screened for endophyte infection using a Polymerase Chain Reaction (PCR) based screen. In all the screens, the E- and E+ lines have always remained true to form. However, some plants died following the initial transport of the plants, likely due to poor care, thus Noble now maintains 11 E+/E- clone pairs. Each line has been tested for the presence of two simple sequence repeat (SSR) markers (B10 and B11) that show variation within and across Neotyphodium (N) coenophialum strains. Original seed stock was also analyzed and indicated that a mix of endophytes was present in the line. Similarly, variation was observed across the clone pairs. Prior to metabolite analysis, a series of Ribonucleic acid (RNA)-seq experiments will be done on three related endophytes, N. coenophialum, N. sp. FaTG-2 and N. sp. FaTG-3, (representing five different isolates) compatible with tall fescue. Samples representing two tissue types (pseudostem and leaf blade) from two different host backgrounds (summer active and summer dormant tall fescue) will be evaluated. These samples include endophyte-infected and endophyte-free clonal material (clones 27 and 52). A number of libraries (including biological replications) have been generated with unique tags and pooled into eight libraries/HiSeq flowcell. These samples have been processed for sequencing and we anticipate RNA-seq data will be available by the end of July 2012. Once received, all data will be initially mapped to the Epichloae festucae gene models and then through a bioinformatics pipeline established by the Scientific Computing Core Facility (Noble Foundation). Once collated, data will be made accessible as an interactive web server for the N. coenophialum gene expression atlas.