Subtractive hybridization-mediated analysis of genes and in silico prediction of associated microRNAs under waterlogged conditions in sugarcane (Saccharum spp.)

• Waterlogging adversely affects sugarcane productivity and quality.
• A subtractive cDNA library was prepared from sugarcane leaf tissue and sequenced to generate ESTs.
• EST sequences were used to identify transcripts induced by waterlogging.
• The sequenced clones were classified by predicted functions and stress-related genes formed the largest class.
• EST sequences were also used to identify putative novel microRNAs and their targets.

Sugarcane is an important tropical cash crop meeting 75% of world sugar demand and it is fast becoming an energy crop for the production of bio-fuel ethanol. A considerable area under sugarcane is prone to waterlogging which adversely affects both cane productivity and quality. In an effort to elucidate the genes underlying plant responses to waterlogging, a subtractive cDNA library was prepared from leaf tissue. cDNA clones were sequenced and annotated for their putative functions. Major groups of ESTs were related to stress (15%), catalytic activity (13%), cell growth (10%) and transport related proteins (6%). A few stress-related genes were identified, including senescence-associated protein, dehydration-responsive family protein, and heat shock cognate 70 kDa protein. A bioinformatics search was carried out to discover novel microRNAs (miRNAs) that can be regulated in sugarcane plants subjected to waterlogging stress. Taking advantage of the presence of miRNA precursors in the related sorghum genome, seven candidate mature miRNAs were identified in sugarcane. The application of subtraction technology allowed the identification of differentially expressed sequences and novel miRNAs in sugarcane under waterlogging stress. The comparative global transcript profiling in sugarcane plants undertaken in this study suggests that proteins associated with stress response, signal transduction, metabolic activity and ion transport play important role in conferring waterlogging tolerance in sugarcane.