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United States Department of Agriculture

Agricultural Research Service

Title: Long Oligonucleotide Microarrays in Wheat: Evaluation of Hybridization Signal Amplification and An Oligonucleotide-Design Computer Script

Authors
item Skinner, Daniel
item Okubara, Patricia
item Hyun-Baek, Kwang - WASHINGTON STATE UNIV
item Call, Douglas - WASHINGTON STATE UNIV

Submitted to: Functional and Integrative Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 22, 2004
Publication Date: January 29, 2005
Citation: Skinner, D.Z., Okubara, P.A., Hyun-Baek, K., Call, D.R. 2005. Long oligonucleotide microarrays in wheat: evaluation of hybridization signal amplification and an oligonucleotide-design computer script. Functional and Integrative Genomics. Vol 5, Number 2, pgs 70-79.

Interpretive Summary: The expression level of many genes change in wheat plants exposed to stress factors. Microarrays are a tool used to simultaneously measure changes in expession levels of many genes. We developed the techniques necessary to use microarrays with wheat, and measured the responses of 40 genes to stress imposed by heat, cold, and root-colonizing bacteria. Several genes were identified that responded similarly to these stresses, indicating common pathways involved in response to multiple stresses. Knowledge of these genes provides targets for wheat breeders to use in improving the tolerance of wheat to many different stress factors.

Technical Abstract: A novel computer script was written in the PERL language to design equal-length long oligonucleotides from DNA sequences. The script allows the user to specify G+C content, melting temperature, self-complementarity, the maximum number of contiguous duplicate bases, whether to start with the first start codon and whether to report reverse-complements. Microarrays were fabricated with 95 oligonucleotides (60 mers) representing 41 genes. The microarray was interrogated with cDNA from roots and shoots of two near-isogenic lines and a commercial cultivar of Triticum aestivum L. (hexaploid wheat) challenged with cold temperature, hot temperature, or the biological control bacterium Pseudomonas fluorescens. Self-complementarity of the oligonucleotides was negatively correlated with signal intensity in 23 of 54 arrays (39%; P <0.01). Tyramide signal amplification was essential for signal generation and detection. Genes involved in signal transduction pathways responded similarly following exposure to cold, heat and P. fluorescens, suggesting intersection of the pathways involved in response to these disparate stress factors. We conclude that long oligonucleotide microarrays for interrogation with cDNA from hexaploid wheat should be constructed from oligonucleotides having minimal self complementarity that also meet user-specified requirements of length, G+C content and melting temperature, and that multiple oligonucleotides should be used to represent each gene.

Last Modified: 10/22/2014
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