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Identification and expression profiling of drought-regulated genes in mulberry (Morus sp.) by suppression subtractive hybridization of susceptible and tolerant cultivars

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Abstract

Mulberry, the backbone of sericulture industry, is a rainfed crop, and its biomass production is affected adversely under drought conditions. In this study, genes expressed differentially during drought stress response have been examined by PCR-Select subtractive hybridization. The sensitive and tolerant genotypes were identified based on physiological evaluation by determination of proline content, electrolyte leakage, and measurement of relative water content. In total, 1,920 clones were sequenced, representing 208 contigs and 151 singletons. The expressed sequence tags generated from this subtracted cDNA library comprises a broad repertoire of stress-responsive genes, which contribute to the process of drought tolerance in mulberry. Additionally, 23% of the cDNA library is represented by transcripts of unknown function. The expression of a select number of these drought-inducible genes was studied based on cDNA macroarray and Northern blot analyses. In order to unravel the crosstalk with other abiotic stresses, expression profile of Arabidopsis homologs of selected genes in response to a wide range of different stresses was studied using Genevestigator as a reference expression database. The results of this study show that subtractive hybridization coupled with validation steps for differential screening is an effective method for identification of stress/drought-induced genes in plants with limited sequence information available.

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Acknowledgments

This work was financially supported by grants received from the Department of Biotechnology (DBT), Government of India, New Delhi. VG acknowledges University Grants Commission (UGC) for the award of research fellowship.

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  1. Department of Plant Molecular Biology, University of Delhi South Campus, Dhaula Kuan, New Delhi, 110021, India

    Vibha Gulyani & Paramjit Khurana

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  1. Vibha Gulyani
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Correspondence to Paramjit Khurana.

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Communicated by J. Dean

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Supplementary Table 1

Contig assembly statistics of Morus sp. mature leaf cDNA library (DOC 29 kb)

Supplementary Table 2

List of differentially expressed genes from dehydration stress subtractive cDNA library of mulberry (XLS 268 kb)

Supplementary Table 3

List of conserved domains of mulberry genes with unknown functions in Arabidopsis (DOC 105 kb)

Supplementary Fig. 1

Occurrence of ESTs in contigs. Number above each bar represents frequency of contigs (PPT 86 kb)

Supplementary Fig. 2

Representative cDNA macroarray hybridization of water stress-inducible genes. 32P-labeled probes prepared from reverse-transcribed total RNA isolated from leaves of control K-2 genotype (a) and 2-h-stressed AR-12 genotype (b). Red arrows indicate clones were induced in the tolerant genotype AR-12, which were absent from sensitive genotype K-2 under control conditions (PPT 234 kb)

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Gulyani, V., Khurana, P. Identification and expression profiling of drought-regulated genes in mulberry (Morus sp.) by suppression subtractive hybridization of susceptible and tolerant cultivars. Tree Genetics & Genomes 7, 725–738 (2011). https://doi.org/10.1007/s11295-011-0369-3

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