Characterization of subtracted cDNA library for detecting expressed sequence tags (ESTs) specific to drought tolerance in black pepper (Piper nigrum L.)

Abstract

Despite many decades of research, drought continues to be a major challenge to agriculture. This is due to the unpredictability of its occurrence, duration and interaction with other biotic and abiotic stresses. So, it becomes necessary to identify the varieties which are tolerant to water stress conditions in the field and to evolve management strategies for conserving available water resources. Various genotypes of black pepper are reported to vary in their response to water stress and the variety ‘Kalluvally’ has been identified as a drought tolerant one among the cultivated genotypes.

Plants respond to stress through various physiological and biochemical adaptations which are governed by different genes and these genes are differentially expressed during drought conditions. Suppression subtractive hybridization (SSH) is one of the powerful techniques to identify genes that are differentially expressed during water stress conditions. A subtracted cDNA library was constructed in black pepper by SSH using mRNA from stress induced drought tolerant plant. Colony plates containing 280 independent clones were screened by colony hybridization with already characterized 20 clones as probes. DNA sequencing and in silico analysis of 35 novel sequences revealed good homology with known sequences which play important role during water stress conditions directly or indirectly. These genes include protein kinase, MYB transcription factor, Ribonuclease enzyme, peptidylprolyl isomerase, beta tubulin, NADH-ubiquinone oxidoreductase family protein, Eukaryotic peptide chain release factor, Glutamine synthetase, etc. Also these sequences had conserved domains for the above mentioned proteins. In addition to this, all the sequences possessed Open Reading Frames (ORFs) many had transmembrane helices and some were found to have signal peptide. The rest of the clones did not show any homology and therefore it was difficult to assign any reported role to these. The results thus obtained could be very well utilized for improving drought tolerance in other susceptible varieties of black pepper through molecular breeding