SAMN00176601
External Id
LIBEST_017751
External Id
University of California
INSDC center name
2005-08-17T00:00:00Z
INSDC first public
2023-09-28T21:06:09.044Z
INSDC last update
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
ERS16610537
SRA accession
Standard Sour Orange
cultivar
All plants were grown and inoculated at the Central California Tristeza Eradication Agency (CCTEA), under the direction of MaryLou Polek, with assistance by Cindy Wallen, who grew and inoculated the plants, and Tony Patino who collected tissue and ran the ELISA tests. All plants were Madame Vinous sweet orange (Citrus sinensis Osbeck) budded onto Standard Sour Orange rootstock (Citrus aurantium L.). They were inoculated onto the scion with bark patches infected with various strains of CTV. Two inoculation dates: 1/24/03, 7/14/04. Three collection dates: 8/19/04 (2 sets collected on this date), 10/21/04, and 12/9/04. Four plant 'days after inoculation' groups: 36, 99, 160, 573 days after inoculation. Number of plants collected for each set: 36-day (18 plants), 99-day (30 plants), 160-day (38 plants), and 573-day (18 plants). These included three virus-free control plants for each group, and 3 or more plants for each virus strain. Five quick-decline virus strains of CTV, identified by CCTEA isolate numbers, were used for the two sets of plants collected on 8/19/04: 107, 142, 96142, 96180, and 96208. Nine quick-decline virus strains of CTV were used for 10/21/04 and 12/9/04 collections: 107, 142, 96142, 96180, 96208, 160, 177, 146, 151. All of the 36-day plants failed to show positive ELISA tests of the scion before the sampling date, and continued to be negative in the rootstock when tested one and two months after the sampling date. The PCR test of the pooled RNA extracted from these plants was negative, so we had no convincing evidence that the virus was active at the bud union in these samples. Most of the 573 days-after-inoculation plants, which were growing outdoors, had tested ELISA positive for CTV at some time, but some of them gave negative tests shortly before sampling. However, they were showing decline symptoms, so all were judged to be infected. RNA was extracted from a pool of all the samples. This pool gave a strong RT-PCR positive result. ELISA tests indicated positive results for scion tissue from many of the plants of the 99-day and 160-day samples, but many were consistently negative. Some of the plants with negative ELISA scores did show some apparent symptoms of stunting, or stem pitting. RT-PCR was done by Claire Federici on some of the samples collected for the 99-day and 160-day samples, RNA from scion bark directly above bud-union sample piece. Those that were PCR tested had elevated ELISA scores, not quite positive, or they had symptoms that suggested they might be showing signs of CTV even though the ELISA titer did not indicate presence of the virus. Of the 27 99-day plants inoculated with CTV, 14 had positive ELISA tests. Of the 13 ELISA negative inoculated plants, 9 were tested by RTPCR and 2 of these gave a positive result, the others were all negative by both types of test. Of the 35 160-day plants inoculated with CTV, 6 had positive ELISA tests. Of the 29 ELISA negative inoculated plants, 14 were tested by RTPCR and all were negative. Which plants were used for the pools? All inoculated plants collected from the 36-day set and all inoculated plants from the 573-day set were used in two separate pools, regardless of ELISA scores. For the 99-day and 160-day plants, only ones with positive ELISA and/or RT-PCR results were used, and they were divided into four pools, with either high or low ELISA titer for each date. Samples were collected by Claire Federici, Mary Lou Polek, and Cindy Wallen. For each sample, a razor blade was used and the material collected in RNAlater (Ambion). The bud union with rootstock bark included the bark from just above the bud-union (this was the bottom cut of the scion bark piece) to about 1 cm below the bud union. That means this tissue included predominantly rootstock (C. aurantium) and some scion (C. sinensis) bark. For every tree there were two tubes of bud union/rootstock. One bud union/rootstock tube of each tree was to be pooled for the cDNA library production. The other was to be saved for gene expression studies. There was one departure from this plan; the pools for the 160-day plants were made from both tubes of rootstock/bud union bark from these plants since the tissue amount was small. Samples in RNALater were transported on ice to UC Riverside and then stored at -20( until extracted. CCTEA contact information: Dr. MaryLou Polek, Associate Plant Pathologist, Program Manager, cctea@lightspeed.net, Department of Food and Agriculture, Plant Health & Pest Prevention Services, Integrated Pest Control Branch, Central California Tristeza Eradic tion Agency, 22847 Road 140, Tulare, CA 93274. Jayati Mandal and Raymond Fenton (Close lab) purified RNA by a TRIzol method, purified poly(A) mRNA using PolyAT Tract mRNA Isolation Kit (Promega), produced a primary cDNA library using a lambda ZAP XR cDNA Synthesis Kit (Stratagene), then mass-excised 1 million pfu from the primary library to produce a phagemid population. The library was made from a mixture of equal quantities of RNA from each of the four sample types. Phagemids were plated, plasmid DNA purified, cDNA clones archived, and DNA sequences determined bi-directionally at the Arizona Genomics Institute (Kim, Kudrna, Collura, Wissotski, Byrne, Stum, Smart, Muller, Wing). Leftover plasmid DNA was re-sequenced on the 3' end using an ABI3730 at the UC Riverside Genomics Core Facility (Choi, Kingan). 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 (Wanamaker, Close, Roose). Sequences that survived all removal steps were submitted to GenBank. Clones from this library are archived at the Arizona Genomics Institute (http://www.genome.arizona.edu/orders/).
description
young trees
dev-stage
E. coli TJC121
lab-host
Citrus x aurantium
http://purl.obolibrary.org/obo/NCBITaxon_43166
organism
EcoRI
re_1
XhoI
re_2
bark below bud union, predominantly C. aurantium
tissue-type
Bark of Madame Vinous Sweet Orange Scion on Standard Sour Orange Rootstock, Citrus Tristeza Virus Challenged - UCRCA01-2
title
Phagemid pBluescript SK(-)
v_type
Lambda Uni-ZAP XR, excised phagemid
vector