Plant & Animal Genome IX Conference

Poster: Sequencing & EST
P01_17.html

THE USEFULNESS OF cDNA MACROARRAYS FOR THE IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN SUGARCANE

¹ Biotechnology Department, South African Sugar Association Experiment Station, Private Bag X02, Mount Edgecombe, 4300, South Africa

² Institute for Plant Biotechnology, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa

A sugarcane Expressed Sequence Tag (EST) database has been established which has led to the identification of genic sequences expressed in the leaf roll and maturing culm. Although this has provided some preliminary information about the types of genes being expressed in the two tissues, it is insufficient for the analysis of expression patterns of individual genes. This investigation aimed to assess the usefulness of cDNA macroarrays in a small-scale study to identify tissue-specific sugarcane expressed sequences. A random sample of 1000 bacterial clones containing partial cDNA sequences from a leaf roll and maturing culm cDNA library were arrayed onto nylon membranes in a 4X4 duplication pattern. The macroarrays were screened with total cDNA probes prepared from leaf, leaf roll, immature culm and maturing culm. Dideoxynucleotides were included during probe preparation according to a recently published method. This resulted in an improvement in the variations in hybridisation signal intensities between clones, which allowed for good discrimination between individual clones as "high", "medium" or "low" expressers. The signal intensity was consistent between duplicate samples. Expression results indicated that only 8.5% of clones exhibited variable expression patterns, with the remainder being constitutively expressed. Northern blot analysis of a 20% subset of variably expressed clones revealed no tissue-specific expression. Possible reasons for this are discussed. In addition, alternative approaches such as enrichment by subtraction for differentially expressed genes are suggested.