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| Status |
Public on May 08, 2008 |
| Title |
Transcriptional profiling of Ascochyta blight resistance in Lentil |
| Platform organisms |
Cicer arietinum; Lathyrus sativus; Lens culinaris |
| Sample organism |
Lens culinaris subsp. culinaris |
| Experiment type |
Expression profiling by array
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| Summary |
Using a cDNA microarray themed on Ascochyta-Pulse interaction resistance response was studied in two lentil varieties, specifically in response to A. lentis inoculation in a highly resistant (ILL7537) and highly susceptible (ILL6002) lentil variety. The experiments were conducted in reference design, where samples from mock-inoculated controls acted as references against post-inoculation samples and the plants were grown using a uniform and standardized experimental system that minimized environmental effects. Robust and high quality data was obtained through the use of three biological replicates (including a dye-swap), the inclusion of negative controls, and stringent selection criteria for differentially expressed genes including a fold change cutoff determined by self-self hybridizations, Students t-test and FDR (Fasle Discovary Rate) multiple testing correction (P<0.05). Microarray observations were validated by quantitative real time RT-PCR using the RNA from one of the bioassay used in the original microarray experiment. Ninety genes were differentially expressed in ILL7537 and 95 genes were differentially expressed in ILL6002. The expression profiles of the two varieties showed substantial difference in type and time of genes that were expressed in response to A. lentis. The resistant variety showed early up-regulation of PR proteins and other defence related genes. The susceptible genotype showed mainly down-regulation of defence related genes. The microarray experiment, the first in lentil, conducted with a small number of genes themed on Asochyta and Pulse interactions was able to identify different components of the defence mechanism by comparing the transcriptional profiles of the susceptible and resistant genotypes. This study will thus form the basis of future experiments to elaborate and corroborate the genomics of lentils defence to A. lentis.
Keywords: time course, disease state analysis
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| Overall design |
Total RNA was extracted from pooled stem and leaf samples for each genotype at each time-point (including control samples) using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA). The quantity, quality and integrity of the total RNA samples were assessed by OD260/OD280 ratios and by gel electrophoresis. Fifty μg of total RNA from each sample was reverse transcribed using Superscript II (Invitrogen) with incorporation of amino-allyl dUTP (Sigma) into the cDNA for post labeling with Cy3- or Cy5-NHS esters (Amersham). For each time point after inoculation, hybridisations were performed with six technical replicates (corresponding to the six complete grids on each microarray slide) and three biological replicates and included a dye-swapping (i.e. reciprocal labeling of Cy3 and Cy5) to eliminate any dye bias. In total 180 images were analysed from 30 slides, resulting in 18 data points for each time-point of each genotype. An Affymetrix® 428™ array scanner (Santa Clara, CA) was used to scan hybridized microarray slides at 660 nm for Cy5 (red laser) and 532 nm for Cy3 (green laser) at 10 µm resolution and the image was captured with Affymetrix® Jaguar™ software (v. 2.0, Santa Clara, CA). Scanned image was analysed and quantified using Imagene™ 5 (BioDiscovery, Marina Del Rey, CA) software. Transformation of quantified spot data with GeneSight™ 3 (BioDiscovery, Marina Del Rey, CA) consisted of a local background correction (subtracting mean background intensity from mean signal intensity for each spot), omitting flagged spots (i.e. empty spots, negative spots (signal mean<background mean), poor spots and spots with mean signal intensity less than two times the local background), LOWESS normalisation of the entire population, creating a test/reference mean signal ratio, taking a shifted log (base 2), and combination of duplicated spot data. For identifying differentially expressed (DE) genes, expression ratios were filtered to eliminate genes whose mean fold change (FC) was less than 1.83-fold up or down at 95% confidence interval followed by Students t test and False Discovery Rate (FDR) multiple testing correction to retain only genes in which expression ratio changes were significant at P < 0.05. Datasets for ILL7537 and ILL6002 was built from mean expression ratios of a non-redundant list of genes differentially expressed at any time-point for any genotype at This dataset was then used to perform Figure of Merit (FOM) and k-means clustering using euclidean distance metrics with the MeV software.
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| Contributor(s) |
Mustafa BM, Coram TE, Pang E, Taylor P, Ford R |
| Citation |
B. M. Mustafa, T. E. Coram, E. C. K. Pang, P. W. J. Taylor and R. Ford. 2009. A cDNA microarray approach to decipher lentil (Lens culinaris) responses to Ascochyta lentis. Journal Australasian Plant Pathology Volume 38, Number 6, 617-631. DOI: 10.1071/AP09048 http://www.springerlink.com/content/514t6h36164r3262
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| Submission date |
May 07, 2008 |
| Last update date |
Aug 09, 2012 |
| Contact name |
barkat murtaja mustafa |
| E-mail(s) |
dipumustafa@hotmail.com, b.mustafa@pgrad.unimelb.edu.au
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| Phone |
+61-0433206060
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| Fax |
+61 3 83445037
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| URL |
http://www.landfood.unimelb.edu.au/research/biomarka/Profiles/pr-bmustafa.htm
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| Organization name |
University of melbourne
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| Department |
School of agriculture
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| Lab |
Biomarka
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| Street address |
Corner of Royal Parade & Tin Alley
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| City |
melbourne |
| State/province |
victoria |
| ZIP/Postal code |
3010 |
| Country |
Australia |
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| Platforms (1) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA106601 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE11374_RAW.tar |
35.9 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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