Affymetrix GeneChip Barley Genome [Barley1]

Stage-Specific Suppression of Basal Defense Discriminates Barley Plants Containing Fast- and Delayed-Acting Mla Powdery Mildew Resistance Alleles

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A large-scale time course expression profiling of wild type (Mla12/Rar1/Rom1) and mutants (mla12-M66, M82 (rar1-1), M100 (rar1-2) and rom1) of barley cultivar Sultan 5 was conducted to understand the molecular mechanisms of delayed powdery mildew resistance. Barley plants were inoculated with powdery mildew pathogen isolate 5874. First leaves of inoculated and non-inoculated plants were harvested at six time points after pathogen inoculation. The experiment was laid out in split-split-plot design with 180 experimental units (3 replications x 2 treatments (inoculated and non-inoculated) x 5 genotypes x 6 time points).

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Transcription profiling of wild type and mutants of Sultan V barley plants

A large-scale time course expression profiling of wild type (Mla12/Rar1/Rom1) and mutants (mla12-M66, M82 (rar1-1), M100 (rar1-2) and rom1) of barley cultivar Sultan 5 was conducted to understand the molecular mechanisms of delayed powdery mildew resistance. Barley plants were inoculated with powdery mildew pathogen isolate 5874. First leaves of inoculated and non-inoculated plants were harvested at six time points after pathogen inoculation. The experiment was laid out in split-split-plot design with 180 experimental units (3 replications x 2 treatments (inoculated and non-inoculated) x 5 genotypes x 6 time points).


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4_450

Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-TABM-228

Seedlings were grown in the greenhouse for 7 days. Seven-day old seedlings were inoculated with powdery mildew isolates and then transferred to the growth chamber.

P-TABM-229

Inoculation was performed by tipping the flats at 45o angle and dusting the plants with a high density of fresh conidiospores. This procedure was repeated from the opposite angle to ensure complete coverage.

P-TABM-230

TRIzol-like RNA isolation and RNeasy clean up protocols TRIzol-like Reagent: Final Concentration Phenol, pH. 4.3 380 ml 38% Guanidine thiocyanate 118.6 g 1.0 M Ammonium thiocyanate 76.12 g 1.0 M 3M Sodium Acetate, pH 5.0 33.4 0.1 M Glycerol 50 ml H2O to 1 liter Total 1 liter **This reagent works better than commercial TRIzol and is significantly cheaper if you are preparing lots of RNA samples** 0.8 M Sodium citrate/1.2 M NaCl Isopropanol Chloroform 75% ETOH DEPC-H2O Work in the hood. Wear safety glasses when working with TRIzol and chloroform. Preheat the Trizol to 60oC for at least 1 hr. 1. Grind 1-gram tissue with a mortar and pestle in liquid nitrogen. 2. Add 10 ml TRIzol-like reagent (pre-heated to 60oC) to tissue in a falcon tube. Swirl to mix. 3. Incubate samples at 60oC for 5 minutes. 4. Centrifuge at 9500 x rpm at 4°C for 10 minutes. Transfer supernatant to 50 ml polypropylene copolymer tube (Nalgene Round Non-lipped Centrifuge tube #3110-0500) centrifuge tube that has been dipped in chloroform to rinse out any residual RNases. 5. Add 2 ml of chloroform (Fisher #C298-4). Vortex the sample until color shade is uniform, at least 5 seconds, and incubate at room temperature for 5 minutes. 6. Centrifuge @ 9000 x rpm for 15 minutes at 4°C. 7. Collect the upper aqueous layer and transfer to a new 50 ml pp (SS34) centrifuge tube dipped in chloroform. 8. Add 0.5 volume of isopropanol & Na-citrate/NaCl solution per 1 ml of aqueous solution. Mix gently. Incubate at room temperature for 20 minutes. 9. Centrifuge at 9000 rpm for 10 minutes at 4°C. 10. Wash the clear RNA pellet with 10 ml of cold 75% ethanol. Swirl & centrifuge at 9000 rpm for 5 minutes at 4°C. 11. Discard supernatant & air-dry the pellet for 10 minutes, inverted on a kimwipe. Dissolve pellet in 300 ul of AccuGENE Molecular Biology water (Cambrex #51200). Resuspend by pipeting up & down a few times. 12. Add 2 ul Recombinant RNasin Ribonuclease Inhibitor (Promega #N2515). Incubate at 55-60°C for 10 minutes to resuspend. 13. Transfer to 1.5 ml microcentrifuge tube and rinse the 50 ml pp (SS34) centrifuge tube with 100 ul DEPC-treated H2O. Check concentration and run a gel of 2 ul of RNA samples (see qualitative/quantitative analysis). At this stage we have very clean RNA at a 260/280 of ~1.9 –> 2.1, however for consistent labeling for GeneChip hybridizations, proceed to step #15. RNeasy Clean-up (Midi Kit) Mercaptoethanol must be added to Buffer RLT (10ul/1ml RLT). 15. Adjust sample to 0.5 ml volume with RNase-free water and add 2.0 ml Buffer RLT. Mix thoroughly. 16. Add 1.4 ml (96-100%) ethanol to the diluted RNA. Mix thoroughly. 17. Apply sample to a Midi column placed in a 15 ml centrifuge tube. Maximum loading volume is 4.0 ml. Close tube gently and centrifuge for 5 min at 4750 rpm. Discard the flow-through. 18. Add 2.5 ml Buffer RPE to the RNA easy column. Close the centrifuge tube gently, incubate for 3 min and centrifuge for 2 min at 4750 rpm. Discard the flow-through. 19. Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, incubate for 3 min and centrifuge for 5 min at 4750 rpm to dry the RNeasy silica-gel membrane. Remove flow-through and centrifuge again for another 3 min. 20. Transfer the RNA easy column to a new collection tube. Pipet 250 ml volume of RNase-free water directly onto the RNeasy silica-membrane. Close the tube gently. Let it stand for 1 min and then centrifuge for 4 min at 4750 rpm. Do a double elution for better recovery (Put the eluate back to the column and centrifuge again. We usually get a 50% recovery with double elution). Qualitative/Quantitative Analysis 1. To determine concentration, dilute 2 ul RNA to 78 ul H20. Measure the absorbancy 260/280 and calculate concentration. 2. Prepare and run a 1% gel (2 ul of RNA, 7 ul H2O and 1 ul of tracking dye). - use 1X TBE-agarose - run gel at 80 volts for 1.5 hrs

Transcription profiling of wild type and mutants of Sultan V barley plants

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