Sugarcane ESTs differentially expressed in immature and maturing internodal tissue

Two subtracted cDNA libraries were constructed by reciprocal subtractive hybridisation between sugarcane immature (low sucrose-accumulating) and maturing (high sucrose-accumulating) internodal tissue. The subtracted libraries contained high, moderate and low abundance transcripts. To isolate cDNAs differentially expressed during culm maturation. 400 random clones (200 from each library) were systematically arrayed onto nylon filters and screened with total cDNA probes prepared from immature and maturing culm poly (A)(+) RNA. Results indicated that 36 and 30% of the total number of cDNAs analysed were preferentially expressed in the immature and maturing culm, respectively. Northern analysis of selected clones confirmed culm developmental stage-specific and -preferential expression for most of the clones tested. Expressed Sequence Tags (ESTs) generated by partial sequence analysis for all 132 differentially expressed clones indicated 95 unique transcripts. Partial sequence information could assign putative identities to 66% of the differentially expressed ESTs. The majority of ESTs with a putative identity were homologous to genes associated with cell wall metabolism. carbohydrate metabolism. stress responses and regulation, where the specific ESTs identified in the immature and maturing culm were distinct from each other. No developmentally regulated ESTs directly associated with sucrose metabolism were detected. This suggests that growth and maturation of the sugarcane culm is associated with the expression of genes for a variety of processes. This study demonstrates that a combination of cDNA subtraction with macroarray screening is an effective strategy to identify and analyse candidate developmentally regulated genes in sugarcane. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.