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Sample GSM250898 Query DataSets for GSM250898
Status Public on Nov 01, 2008
Title TcLr1_3dpi_inoculated_rep3
Sample type RNA
 
Source name Wheat flag leaves, 3 dpi, inoculated
Organism Triticum aestivum
Characteristics Genotype: Thatcher-Lr1, Growth stage: anthesis
Treatment protocol Tillers at anthesis (+/- two days) on each plant were tagged prior to inoculation. Flag leaves of each tiller were individually inoculated on the adaxial face either with Soltrol oil alone or with a suspension of P. triticina race 1 (BBB) spores in Soltrol oil (18 mg spores ml-1 oil). Inoculations employed two passes across the leaf surface with an atomizer nozzle. Spore densities applied to leaves were determined by inoculating 6 microscope slides during the course of the inoculations and then counting the number of spores in a defined area (2.67 mm2) on each slide under a microscope. Using this method, the average spore density on inoculated leaf surfaces was determined to be 592 spores cm-1. Spore germination was assessed by spraying water agar plates lightly with inoculum and then determining the percentage of spores that had germinated after 4 h. By this method, 75% of spores had germinated (germination tube evident), while a significant portion of the other spores were oblong in shape, indicating that they were in the early process of germinating. After allowing oil to dry off of leaf surfaces for 1.5 h, pots were placed in mist chambers (one rep per chamber) and subjected to 30 min initial misting. A 30 min mist/4 min no mist cycle was then employed for three h, followed by a mist cycle of 10 min on/10 min off for 5 h, and finally a mist regime of 6 min on/10 min off for 10 h 45 min. The temperature in the mist chamber during the mist period was 21ºC +/- 1ºC. Plant were then removed from the mist chambers and allowed to dry, and they were placed back into the growth chamber.
Growth protocol Seed of each genotype were stratified on wet filter paper for 4 days, and then planted three to a pot in 6 inch round plastic pots (Belden Plastics, Roseville, MN) filled with a potting mixture of field soil, compost, and sand. 20 grams of Nutricote 100 day slow release 13-13-13 pellets with micronutrients (SunGro, Vancouver, British Columbia, Canada) was placed into each pot. Pots were placed in a growth chamber (Conviron PGW36, Conviron, Winnipeg, Canada) initially set at 18°C day 16°C night, with 16 h day/8 h night light regime. The pots were laid out in a split plot with three reps. Genotypes were the main effect, and the subplots were treatments (mock-inoculated or pathogen-inoculated). For each rep, 6 pots (3 plants per pot) were planted per genotype. Pots were watered on an as-needed basis. After one month, the temperature was dropped to 15°C day/13°C night temperature to help ensure synchronized flowering, and subsequently the temperature was gradually stepped up to 20°C day/17°C night temperatures (16°C day/14°C night 9 days after setting to 15°C day/13°C night, 17°C/15°C two days later, 18°C/16°C two days later, and 20°C/17°C 9 days later). Pots were also watered thoroughly with a fertilizer solution (1.86 g L-1 Peters 20-20-20 soluble fertilizer) three weeks and 8 weeks after planting, and with a more dilute solution (0.62 g L-1) of the same soluble fertilizer 6 weeks after planting. Two months after sowing, plants were ready for inoculation.
Extracted molecule total RNA
Extraction protocol Trizol extraction of total RNA was performed according to the manufacturer's instructions, treated with RNase-free DNase, and further purified using RNeasy columns (Promega, Madison, WI) according to manufacturer's instructions.
Label biotin
Label protocol Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 μg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
 
Hybridization protocol Following fragmentation, 15 μg of cRNA were hybridized for 16 hr at 45ºC on GeneChip wheat Genome Array. GeneChips were washed and stained in the Affymetrix GeneChip Fluidics Station 450.
Scan protocol GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
Description TcLr1_3dpi_inoculated_rep3
Data processing Data analysis was conducted using the Expressionist software version Pro 3.1 (Genedata AG, CH-4016 Basel, Switzerland). The probe set values from all experiments were condensed together using the MAS 5.0 algorithm (Affymetrix, I. Statistical Algorithms Description Document. 2002.). The MAS 5.0 signal data were normalized to a median target intensity of 500 as follows: P. triticina- and mock-inoculated data were normalized together for each genotype at each timepoint for a total of six comparisons. The experiment was a randomized complete block design with three replicates for each genotype, and employed univariate analysis (t-tests) between mock- and P. triticina-inoculated plants within each genotype at each timepoint, for a total of six comparisons across the entire experiment.
 
Submission date Dec 17, 2007
Last update date Dec 17, 2007
Contact name David Garvin
E-mail(s) David.Garvin@ars.usda.gov
Organization name USDA
Department Plant Science Research Unit
Street address 411 Borlaug Hall, 1991 Upper Buford Circle
City St. Paul
State/province MN
ZIP/Postal code 55108
Country USA
 
Platform ID GPL3802
Series (1)
GSE9915 Transcript profiling of Lr1- and Lr34-mediated leaf rust resistance in wheat

Data table header descriptions
ID_REF
VALUE MAS5 signal

Data table
ID_REF VALUE
AFFX-BioB-5_at 2551.738334
AFFX-BioB-M_at 3097.022868
AFFX-BioC-5_at 5156.536246
AFFX-BioDn-5_at 7155.592174
AFFX-CreX-5_at 54868.98404
AFFX-CreX-3_at 66349.88937
AFFX-ThrX-5_at 1237.652451
AFFX-r2-Bs-thr-5_s_at 1392.426113
Ta.10110.2.S1_a_at 1744.204106
Ta.10110.2.S1_x_at 1971.557903
Ta.10110.1.S1_at 1197.243533
Ta.1022.1.S1_at 829.1893145
TaAffx.49630.1.S1_at 2098.353227
Ta.11112.1.S1_at 1028.58398
Ta.22524.1.S1_at 1824.554087
Ta.345.2.S1_x_at 4010.12022
Ta.6303.1.S1_at 146.9453584
Ta.7683.1.S1_at 36.4325831
Ta.18757.1.S1_at 160.367019
Ta.9991.1.A1_at 759.9209948

Total number of rows: 61290

Table truncated, full table size 1861 Kbytes.




Supplementary file Size Download File type/resource
GSM250898.CEL.gz 8.5 Mb (ftp)(http) CEL
Processed data included within Sample table
Raw data provided as supplementary file

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