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

Abstract

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.

Keywords: ABA, abscisic acid; ADF, actin depolymerizing factor; EST; ESTs, expressed sequence tags; Hsps, heat shock proteins; Novel miRNAs; PTGS, post-transcriptional gene silencing; REMs, remorins; SSA, snow pea pod senescence-associated; SUCEST, sugarcane EST database; Sugarcane; Suppression subtractive hybridization; TGS, transcriptional gene silencing; Waterlogging stress.

Fig. 1

(A) Size distribution of ESTs generated from water-logged leaf cDNAs (B) Reads assembled in contigs.

Fig. 2

Functional characterization of water-logged induced sugarcane leaf unigenes according to the best matched BLAST search results.

Fig. 3

Prediction of secondary structures for novel miRNA candidates under waterlogging condition of sugarcane. The putative miRNA sequences are highlighted in red and miRNA^∗ sequences are highlighted in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)