Parent Washington Navel Orange Thrip-Challenged Flavedo cDNA Library UCRCS07

Overview
Library NameParent Washington Navel Orange Thrip-Challenged Flavedo cDNA Library UCRCS07
Unique NameParent Washington Navel Orange Thrip-Challenged Flavedo cDNA Library UCRCS07
OrganismCitrus sinensis (Sweet orange)
Typecdna_library
Vector: Lambda Uni-ZAP XR, excised phagemid; Site_1: EcoRI; Site_2: XhoI; Trees were grown in the field at University of California, Riverside using standard horticultural practices. Young fruits were placed in a cage with thrips (Scirtothrips citri). Infestations were conducted by Watkins (Morse lab) and flavedo collected by Federici (Roose lab) from May-June 2003. The thrips were collected from naturally infested Rhus plants by sucking into a tube. A flexible hose was attached to a tube that extended into a covered vial, and another tube stuck out of the vial at a right angle. The bent tube was held above the thrips, sucking on the flexible tube created a vacuum, pulling the thrips into the vial. Thrips were knocked off the Rhus plant onto a manila folder, then only second instars were captured. This was done repeatedly until enough were obtained. Approximately 7-10 thrips were caged on each fruit within a plastic vial made of a 8 cm long by 5 cm diameter tube that had a very fine mesh organdy fabric glued to the bottom. The plastic cap was slit from the edge to the center so it could be slipped over the stem of the fruit. It was put in place, the thrips were knocked into the vial and it was fastened onto the cap then all gaps were closed with masking tape. The thrips naturally move up to the fruit. The cages were left in place for two days, then removed. The fruit were checked to be sure the thrips had stayed on, and then brought to the lab to cut off the flavedo using a razor blade. Only the flavedo from the stem 1/3 to 1/2 of the fruit was used. For controls an equal number of comparable sized fruit were caged without thrips, and the peel collected from them in the same manner. Tissues were frozen in liquid nitrogen, 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 an Oligotex mRNA Kit (Qiagen), produced a primary cDNA library using a lambda ZAP XR cDNA Synthesis Kit (Stratagene), then mass-excised 0.77 million pfu from the primary library to produce a phagemid population. The library was made from equal portions of RNA from each of the two treatments. Phagemids were plated, plasmid DNA purified, cDNA clones archived, and DNA sequences determined bi-directionally using an ABI3730 at DNA Landmarks (Landry, Hubert, Laforest, Landry, Ligonde). Chromatogram files were downloaded by FTP by Close, then processed 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 assembled, then chimeras were removed following manual inspection of assemblies (Close, Roose, Federici, Wanamaker, Lyon, Ye, Jang, Collin, Kacar, Ikeda, Quinitio). Sequences that survived all removal steps were submitted to GenBank.
Features
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Feature NameUnique NameType
CX043371CX043371EST
CX043372CX043372EST
CX043373CX043373EST
CX043374CX043374EST
CX043375CX043375EST
CX043376CX043376EST
CX043377CX043377EST
CX043378CX043378EST
CX043379CX043379EST
CX043380CX043380EST
CX043381CX043381EST
CX043382CX043382EST
CX043383CX043383EST
CX043384CX043384EST
CX043385CX043385EST
CX043386CX043386EST
CX043387CX043387EST
CX043388CX043388EST
CX043389CX043389EST
CX043390CX043390EST
CX043391CX043391EST
CX043392CX043392EST
CX043393CX043393EST
CX043394CX043394EST
CX043395CX043395EST

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Properties
Property NameValue
Epicarptissue type
Genbank library cultivarParent Washington Navel
Genbank library dev stage11 year old trees
Genbank library noteVector: Lambda Uni-ZAP XR, excised phagemid; Site_1: EcoRI; Site_2: XhoI; Trees were grown in the field at University of California, Riverside using standard horticultural practices. Young fruits were placed in a cage with thrips (Scirtothrips citri). Infestations were conducted by Watkins (Morse lab) and flavedo collected by Federici (Roose lab) from May-June 2003. The thrips were collected from naturally infested Rhus plants by sucking into a tube. A flexible hose was attached to a tube that extended into a covered vial, and another tube stuck out of the vial at a right angle. The bent tube was held above the thrips, sucking on the flexible tube created a vacuum, pulling the thrips into the vial. Thrips were knocked off the Rhus plant onto a manila folder, then only second instars were captured. This was done repeatedly until enough were obtained. Approximately 7-10 thrips were caged on each fruit within a plastic vial made of a 8 cm long by 5 cm diameter tube that had a very fine mesh organdy fabric glued to the bottom. The plastic cap was slit from the edge to the center so it could be slipped over the stem of the fruit. It was put in place, the thrips were knocked into the vial and it was fastened onto the cap then all gaps were closed with masking tape. The thrips naturally move up to the fruit. The cages were left in place for two days, then removed. The fruit were checked to be sure the thrips had stayed on, and then brought to the lab to cut off the flavedo using a razor blade. Only the flavedo from the stem 1/3 to 1/2 of the fruit was used. For controls an equal number of comparable sized fruit were caged without thrips, and the peel collected from them in the same manner. Tissues were frozen in liquid nitrogen, 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 an Oligotex mRNA Kit (Qiagen), produced a primary cDNA library using a lambda ZAP XR cDNA Synthesis Kit (Stratagene), then mass-excised 0.77 million pfu from the primary library to produce a phagemid population. The library was made from equal portions of RNA from each of the two treatments. Phagemids were plated, plasmid DNA purified, cDNA clones archived, and DNA sequences determined bi-directionally using an ABI3730 at DNA Landmarks (Landry, Hubert, Laforest, Landry, Ligonde). Chromatogram files were downloaded by FTP by Close, then processed 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 assembled, then chimeras were removed following manual inspection of assemblies (Close, Roose, Federici, Wanamaker, Lyon, Ye, Jang, Collin, Kacar, Ikeda, Quinitio). Sequences that survived all removal steps were submitted to GenBank.
Genbank library tissue typeFlavedo