dc.contributor.author |
Coetzer, Nanette
|
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dc.contributor.author |
Gazendam, Inge
|
|
dc.contributor.author |
Oelofse, Dean
|
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dc.contributor.author |
Berger, David Kenneth
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dc.date.accessioned |
2011-01-11T06:46:34Z |
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dc.date.available |
2011-01-11T06:46:34Z |
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dc.date.issued |
2010-04 |
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dc.description.abstract |
BACKGROUD: Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (Vigna unguiculata (L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to
develop software tools to facilitate the process. RESULTS: Forward and reverse cDNA libraries enriched for cowpea drought response genes were screened on microarrays, and the R software package SSHscreen 2.0.1 was developed (i) to normalize the data effectively using
spike-in control spot normalization, and (ii) to select clones for sequencing based on the calculation of enrichment ratios with associated statistics. Enrichment ratio 3 values for each clone showed that 62% of the forward library and 34% of the reverse library clones were significantly differentially expressed by drought stress (adjusted p value < 0.05). Enrichment ratio 2 calculations showed that > 88% of the clones in both libraries were derived from rare transcripts in the original tester samples, thus supporting the notion that suppression subtractive hybridization enriches for rare transcripts. A set of 118 clones were chosen for sequencing, and drought-induced cowpea genes were identified, the most interesting encoding a late embryogenesis abundant Lea5 protein, a glutathione S-transferase, a thaumatin, a
universal stress protein, and a wound induced protein. A lipid transfer protein and several components of photosynthesis were down-regulated by the drought stress. Reverse transcriptase quantitative PCR confirmed the enrichment ratio values for the selected cowpea genes. SSHdb, a web-accessible database, was developed to manage
the clone sequences and combine the SSHscreen data with sequence annotations derived from BLAST and Blast2GO. The self-BLAST function within SSHdb grouped redundant clones together and illustrated that the SSHscreen plots are a useful tool for choosing anonymous clones for sequencing, since redundant clones cluster together on the enrichment ratio plots.
CONCLUSIONS: We developed the SSHscreen-SSHdb software pipeline, which greatly facilitates gene discovery using
suppression subtractive hybridization by improving the selection of clones for sequencing after screening the library
on a small number of microarrays. Annotation of the sequence information and collaboration was further enhanced
through a web-based SSHdb database, and we illustrated this through identification of drought responsive genes from cowpea, which can now be investigated in gene function studies. SSH is a popular and powerful gene discovery tool, and therefore this pipeline will have application for gene discovery in any biological system, particularly non-model organisms. SSHscreen 2.0.1 and a link to SSHdb are available from http://microarray.up.ac.za/SSHscreen. |
en |
dc.identifier.citation |
Coetzer, N, Gezendam, I, Oelofse, D & Berger, DK 2010, 'SSHscreen and SSHdb, generic software for microarray based gene discovery : application to the stress response in cowpea', Plant Methods, vol. 6, no. 10, pp. 1-20. [http://www.plantmethods.com/content/6/1/10] |
en |
dc.identifier.issn |
1746-4811 |
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dc.identifier.other |
10.1186/1746-4811-6-10 |
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dc.identifier.uri |
http://hdl.handle.net/2263/15613 |
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dc.language.iso |
en |
en_US |
dc.publisher |
BioMed Central |
en_US |
dc.rights |
© 2010 Coetzer et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
en |
dc.subject |
SSHscreen and SSHdb |
en |
dc.subject |
Generic software |
en |
dc.subject.lcsh |
DNA microarrays |
en |
dc.subject.lcsh |
Cowpea |
en |
dc.subject.lcsh |
Crops -- Effect of stress on |
en |
dc.title |
SSHscreen and SSHdb, generic software for microarray based gene discovery : application to the stress response in cowpea |
en |
dc.type |
Article |
en |