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Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...
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SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in

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Summary

This study developed new software (SSHscreen-SSHdb) to improve gene discovery in cowpea using suppression subtractive hybridization (SSH). The tools enhance clone selection and data management, identifying key drought-responsive genes.

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Area of Science:

  • Plant Molecular Biology
  • Bioinformatics
  • Genomics

Background:

  • Suppression subtractive hybridization (SSH) is crucial for gene discovery in non-model organisms like cowpea.
  • Existing SSH methods face challenges in library screening and data management.
  • Development of specialized software tools is needed to streamline the SSH process.

Purpose of the Study:

  • To develop and validate software tools for efficient gene discovery using SSH in cowpea.
  • To enrich for and identify drought-responsive genes in a drought-tolerant cowpea line.
  • To improve the selection and management of candidate gene clones.

Main Methods:

  • Construction of forward and reverse cDNA libraries enriched for drought response genes.
  • Screening of libraries using microarrays and analysis with the R package SSHscreen 2.0.1.
  • Development of a web-accessible database (SSHdb) for managing sequence data and annotations.

Main Results:

  • SSHscreen 2.0.1 effectively normalized microarray data and selected clones based on enrichment ratios.
  • Significant differential expression (p < 0.05) was observed in 62% of forward and 34% of reverse library clones.
  • SSHdb facilitated clone management and annotation, leading to the identification of drought-induced genes (e.g., Lea5, glutathione S-transferase) and down-regulated genes (e.g., lipid transfer protein, photosynthesis components).

Conclusions:

  • The SSHscreen-SSHdb pipeline significantly enhances gene discovery efficiency using SSH, particularly for non-model organisms.
  • The software improves clone selection for sequencing and facilitates data annotation and collaboration.
  • This approach is broadly applicable for identifying functional genes in various biological systems under specific stress conditions.