Washington Navel Orange Stored Fruit Pulp cDNA Library

Vector: Lambda Uni-ZAP XR, excised phagemid; Site_1: EcoRI; Site_2: XhoI; Fruits were collected January-March 2003 (Federici, Roose lab; Focht, Sievert & Robinson, Arpaia lab). Four samples related to storage conditions were produced: 1) fresh-picked in Mentone (Arnott Brothers Enterprises, Mentone, CA), 2) after 21 days storage at 5C at Kearney then transported to UC Riverside on ice, 3) after 5 additional days storage at 11C at Kearney, sampled immediately, 4) fruit grown in southern CA were obtained from Redlands Foothill Packing House after commercial packing, X-ray irradiated at 300 Gy by Surebeam, then stored 1 day at ambient temperature. Pulp tissue (juice vesicles) were collected. Tissues were snap frozen and then stored at -80C until further processing. Fenton (Close lab) purified RNA by the phenol method described in J. Japanese Soc. Hort. Sci. 1996. 64 (4): 809-814, purified poly(A) mRNA using a PolyATtrack mRNA Isolation System IV (Promega), produced a primary cDNA library using a lambda ZAP XR cDNA Synthesis Kit (Stratagene), then mass-excised one million pfu from the primary library to produce a phagemid population. 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, Yost, Wing). Chromatogram files were downloaded by FTP to UC Riverside (by Close), then processed at UC Riverside (by Wanamaker, Close lab) 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.