Poncirus trifoliata CTV-challenged cDNA library - AGI2

Vector: Lambda Uni-ZAP XR, excised phagemid; Site_1: EcoRI; Site_2: XhoI; Plants were grown in the greenhouse at University of California, Riverside. The scion was a open-pollinated (very probably selfed) seedling of Poncirus trifoliata cv Pomeroy that was selected as homozygous for the Ctv resistance gene. The rootstock was sweet orange infected with citrus tristeza virus (CTV) isolate T514 over 1 year before sampling (CTV infects sweet orange, but not genotypes carrying the Ctv resistance gene). Shoots 10-30 cm long were harvested in October 2000, and the green phloem (bark) was removed and frozen quickly in dry ice. Total RNA was extracted using TRIZOL reagent (Gibco). Poly(A) RNA was purified, a cDNA library was made, and 0.5 million primary lambda cDNA clones were in vivo excised to give a population of pBluescript SK(-) phagemids. All steps to this point were performed in the ML Roose lab at the University of California, Riverside by X. Ye. Phagemids were plated, plasmid DNA purified, cDNA clones archived, and DNA sequences determined bi-directionally using an ABI3730 at the Arizona Genomics Institute, University of Arizona (Kim, Kudrna, Stum, Wissotski, Wing). Chromatogram files were downloaded to UC Riverside (Close), then processed at UC Riverside (Wanamaker) using the HarvEST pipeline (http://harvest.ucr.edu) to remove vector and cloning oligo sequences and various contaminants, and to trim to a high quality region. Sequences that retained a phred 17 region of at least 100 bases were deposited to GenBank.