Abstract
Understanding the plant-pathogen interactions is of utmost importance to design strategies for minimizing the economic deficits caused by pathogens in crops. With an aim to identify genes underlying resistance to downy mildew, a major disease responsible for productivity loss in pearl millet, transcriptome analysis was performed in downy mildew resistant and susceptible genotypes upon infection and control on 454 Roche NGS platform. A total of ~685 Mb data was obtained with 1 575 290 raw reads. The raw reads were pre-processed into high-quality (HQ) reads making to ~82% with an average of 427 bases. The assembly was optimized using four assemblers viz. Newbler, MIRA, CLC and Trinity, out of which MIRA with a total of 14.10 Mb and 90118 transcripts proved to be the best for assembling reads. Differential expression analysis depicted 1396 and 936 and 1000 and 1591 transcripts up and down regulated in resistant inoculated/resistant control and susceptible inoculated/susceptible control respectively with a common of 3644 transcripts. The pathways for secondary metabolism, specifically the phenylpropanoid pathway was up-regulated in resistant genotype. Transcripts up-regulated as a part of defense response included classes of R genes, PR proteins, HR induced proteins and plant hormonal signaling transduction proteins. The transcripts for skp1 protein, purothionin, V type proton ATPase were found to have the highest expression in resistant genotype. Ten transcripts, selected on the basis of their involvement in defense mechanism were validated with qRT-PCR and showed positive co-relation with transcriptome data. Transcriptome analysis evoked potentials of hypersensitive response and systemic acquired resistance as possible mechanism operating in defense mechanism in pearl millet against downy mildew infection.
Highlights
The 454 Roche sequencing has been extensively employed for transcriptome sequencing and is well-suitable for both model and non-model plants with better performance than conventional methods of gene expression (Chen et al, 2013)
To get a non-redundant data, duplicate reads were removed before assembling the reads into transcripts but were employed for back mapping while analyzing gene expression to decipher the level of transcript expression
We presented much attention to assembling the reads as it forms an essential view to determine how best to evaluate assemblies, in light of the variety of options available and absence of reference genome (O’Neil and Emrich, 2013)
Summary
India has seven million ha area under pearl millet with a production of 9.25 million tons (ICAR-AICPMIP Project co-ordinator review, 2015). It has wide adaptability and is looked at as one of the most significant crops in the scenario of food security and changing climate conditions. The crop productivity is severely constrained by several biotic stresses, major among them is downy mildew (DM) disease caused by the oomycete obligate pathogen, Sclerospora graminicola (Sacc.) Schroet. The oomycetes differ from fungi and includes economically important plant pathogens like downy mildews of poaceae, cucurbitaceae, vitaceae (Kamoun et al, 2015). The development of downy mildew disease is favored by high relative humidity (85–90%), moderate temperature (20– 30◦C) and characterized by leafy inflorescence, leaf chlorosis, and failure to set seeds (Thakur et al, 2008)
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