Comparative analysis of expressed sequence tags (ESTs) between drought-tolerant and -susceptible genotypes of chickpea under terminal drought stress
TL;DR: This study compares not only genes that are up- and down-regulated in a drought-tolerant genotype under terminal drought stress and a drought susceptible genotype but also between the bulks of the selected RILs exhibiting extreme phenotypes to provide a better insight into the selection of candidate genes associated with drought tolerance.
Abstract: Chickpea (Cicer arietinum L.) is an important grain-legume crop that is mainly grown in rainfed areas, where terminal drought is a major constraint to its productivity. We generated expressed sequence tags (ESTs) by suppression subtraction hybridization (SSH) to identify differentially expressed genes in drought-tolerant and -susceptible genotypes in chickpea. EST libraries were generated by SSH from root and shoot tissues of IC4958 (drought tolerant) and ICC 1882 (drought resistant) exposed to terminal drought conditions by the dry down method. SSH libraries were also constructed by using 2 sets of bulks prepared from the RNA of root tissues from selected recombinant inbred lines (RILs) (10 each) for the extreme high and low root biomass phenotype. A total of 3062 unigenes (638 contigs and 2424 singletons), 51.4% of which were novel in chickpea, were derived by cluster assembly and sequence alignment of 5949 ESTs. Only 2185 (71%) unigenes showed significant BLASTX similarity (<1E-06) in the NCBI non-redundant (nr) database. Gene ontology functional classification terms (BLASTX results and GO term), were retrieved for 2006 (92.0%) sequences, and 656 sequences were further annotated with 812 Enzyme Commission (EC) codes and were mapped to 108 different KEGG pathways. In addition, expression status of 830 unigenes in response to terminal drought stress was evaluated using macro-array (dot blots). The expression of few selected genes was validated by northern blotting and quantitative real-time PCR assay. Our study compares not only genes that are up- and down-regulated in a drought-tolerant genotype under terminal drought stress and a drought susceptible genotype but also between the bulks of the selected RILs exhibiting extreme phenotypes. More than 50% of the genes identified have been shown to be associated with drought stress in chickpea for the first time. This study not only serves as resource for marker discovery, but can provide a better insight into the selection of candidate genes (both up- and downregulated) associated with drought tolerance. These results can be used to identify suitable targets for manipulating the drought-tolerance trait in chickpea.
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TL;DR: This review provides basic principles and a broad set of references useful for the management of phenotyping practices for the study and genetic dissection of drought tolerance and, ultimately, for the release of drought-tolerant cultivars.
Abstract: Improving crops yield under water-limited conditions is the most daunting challenge faced by breeders. To this end, accurate, relevant phenotyping plays an increasingly pivotal role for the selection of drought-resilient genotypes and, more in general, for a meaningful dissection of the quantitative genetic landscape that underscores the adaptive response of crops to drought. A major and universally recognized obstacle to a more effective translation of the results produced by drought-related studies into improved cultivars is the difficulty in properly phenotyping in a high-throughput fashion in order to identify the quantitative trait loci that govern yield and related traits across different water regimes. This review provides basic principles and a broad set of references useful for the management of phenotyping practices for the study and genetic dissection of drought tolerance and, ultimately, for the release of drought-tolerant cultivars.
448 citations
Cites background from "Comparative analysis of expressed s..."
...…(Johnson et al., 2000), maize (Tuberosa et al., 2003, 2007b, 2011b; Hammer et al., 2009; Landi et al., 2010; Hund et al., 2011), barley (Forster et al., 2005), wheat (Manschadi et al., 2006, 2010; Wasson et al., 2012), and especially, in rainfed rice (Nguyen et al., 1997; Price and Tomos,…...
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...…four major QTLs that control stay-green and GY have been identified (Harris et al., 2007) and near isogenic lines (NILs) for these QTLs have been derived, providing an opportunity for a detailed analysis of stay-green physiology and positional cloning of the underlying genes (Vadez et al., 2011)....
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...…et al., 2009), a peculiarity that allows plants to adapt better to the chemical and physical properties of the soil, particularly under drought conditions 1http://www.csiro.au/files/files/p2ki.pdf www.frontiersin.org September 2012 | Volume 3 | Article 347 | 3 (Bacon et al., 2002; Yu et al., 2007)....
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...Therefore, delaying leaf senescence maintains transpiration and increases cumulative photosynthesis over the crop life cycle (Borrell et al., 2001; Jiang et al., 2004; Vadez et al., 2011)....
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TL;DR: The most recent advances in plant physiology for precision phenotyping of drought response are discussed, a vital step before implementing the genetic and molecular-physiological strategies to unravel the complex multilayered drought tolerance mechanism and further exploration using molecular breeding approaches for crop improvement.
Abstract: Drought is one of the most serious production constraint for world agriculture and is projected to worsen with anticipated climate change. Inter-disciplinary scientists have been trying to understand and dissect the mechanisms of plant tolerance to drought stress using a variety of approaches; however, success has been limited. Modern genomics and genetic approaches coupled with advances in precise phenotyping and breeding methodologies are expected to more effectively unravel the genes and metabolic pathways that confer drought tolerance in crops. This article discusses the most recent advances in plant physiology for precision phenotyping of drought response, a vital step before implementing the genetic and molecular-physiological strategies to unravel the complex multilayered drought tolerance mechanism and further exploration using molecular breeding approaches for crop improvement. Emphasis has been given to molecular dissection of drought tolerance by QTL or gene discovery through linkage and association mapping, QTL cloning, candidate gene identification, transcriptomics and functional genomics. Molecular breeding approaches such as marker-assisted backcrossing, marker-assisted recurrent selection and genome-wide selection have been suggested to be integrated in crop improvement strategies to develop drought-tolerant cultivars that will enhance food security in the context of a changing and more variable climate.
418 citations
Cites methods from "Comparative analysis of expressed s..."
...This study was further extended by the National Research Centre on Plant Biotechnology (NRCPB) in India and 5,494 highquality drought-responsive ESTs were isolated using suppression subtraction hybridization (SSH) of droughtchallenged root and shoot tissues (Deokar et al. 2011)....
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International Crops Research Institute for the Semi-Arid Tropics1, University of Florida2, Hokkaido University3, Leibniz Association4, Indian Institute of Pulses Research5, Indian Agricultural Research Institute6, Central Arid Zone Research Institute7, University of Agricultural Sciences, Bangalore8
TL;DR: Nine QTL clusters containing QTLs for several drought tolerance traits have been identified that can be targeted for molecular breeding and one cluster present on CaLG04 has a high potential to enhance drought tolerance in chickpea improvement.
Abstract: Key message
Analysis of phenotypic data for 20 drought tolerance traits in 1–7 seasons at 1–5 locations together with genetic mapping data for two mapping populations provided 9 QTL clusters of which one present on CaLG04 has a high potential to enhance drought tolerance in chickpea improvement.
293 citations
Cites background from "Comparative analysis of expressed s..."
...Towards understanding complexity of drought tolerance in chickpea, a few expression and functional genomics (Varshney et al. 2009; Deokar et al. 2011) and physiological (Zaman-Allah et al. 2011) studies were conducted in recent past; however, the genetics and molecular mechanisms for drought…...
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TL;DR: The impact of terminal drought on leaf development and senescence, light harvesting and carbon fixation, and grain development and grain composition is discussed and the mechanisms of resistance, management options, and innovative breeding and functional genomics strategies to improve resistance to terminal drought in grain legumes are discussed.
Abstract: Water scarcity is a major constraint limiting grain legume production particularly in the arid and semi-arid tropics. Different climate models have predicted changes in rainfall distribution and frequent drought spells for the future. Although drought impedes the productivity of grain legumes at all growth stages, its occurrence during reproductive and grain development stages (terminal drought) is more critical and usually results in significant loss in grain yield. However, the extent of yield loss depends on the duration and intensity of the stress. A reduction in the rate of net photosynthesis, and poor grain set and grain development are the principal reasons for terminal drought-induced loss in grain yield. Insight into the impact and resistance mechanism of terminal drought is required for effective crop improvement programmes aiming to improve resistance to terminal drought in grain legumes. In this article, the impact of terminal drought on leaf development and senescence, light harvesting and carbon fixation, and grain development and grain composition is discussed. The mechanisms of resistance, management options, and innovative breeding and functional genomics strategies to improve resistance to terminal drought in grain legumes are also discussed.
277 citations
276 citations
References
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15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: The 2-Delta Delta C(T) method as mentioned in this paper was proposed to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and it has been shown to be useful in the analysis of realtime, quantitative PCR data.
139,407 citations
TL;DR: A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original.
Abstract: The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.
70,111 citations
TL;DR: Blast2GO (B2G), a research tool designed with the main purpose of enabling Gene Ontology (GO) based data mining on sequence data for which no GO annotation is yet available, is presented.
Abstract: Summary: We present here Blast2GO (B2G), a research tool designed with the main purpose of enabling Gene Ontology (GO) based data mining on sequence data for which no GO annotation is yet available. B2G joints in one application GO annotation based on similarity searches with statistical analysis and highlighted visualization on directed acyclic graphs. This tool offers a suitable platform for functional genomics research in non-model species. B2G is an intuitive and interactive desktop application that allows monitoring and comprehension of the whole annotation and analysis process.
Availability: Blast2GO is freely available via Java Web Start at http://www.blast2go.de
Supplementary material:http://www.blast2go.de -> Evaluation
Contact:[email protected]; [email protected]
10,092 citations
"Comparative analysis of expressed s..." refers methods in this paper
...3) [15], following the standard procedure of BLASTX for unigenes dataset (parameters: nr database, high scoring segment pair (HSP) cutoff length 33, report 20 hits, maximum E-value 1....
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TL;DR: KEGG PATHWAY is now supplemented with a new global map of metabolic pathways, which is essentially a combined map of about 120 existing pathway maps, and the KEGG resource is being expanded to suit the needs for practical applications.
Abstract: KEGG (http://www.genome.jp/kegg/) is a database of biological systems that integrates genomic, chemical and systemic functional information. KEGG provides a reference knowledge base for linking genomes to life through the process of PATHWAY mapping, which is to map, for example, a genomic or transcriptomic content of genes to KEGG reference pathways to infer systemic behaviors of the cell or the organism. In addition, KEGG provides a reference knowledge base for linking genomes to the environment, such as for the analysis of drug-target relationships, through the process of BRITE mapping. KEGG BRITE is an ontology database representing functional hierarchies of various biological objects, including molecules, cells, organisms, diseases and drugs, as well as relationships among them. KEGG PATHWAY is now supplemented with a new global map of metabolic pathways, which is essentially a combined map of about 120 existing pathway maps. In addition, smaller pathway modules are defined and stored in KEGG MODULE that also contains other functional units and complexes. The KEGG resource is being expanded to suit the needs for practical applications. KEGG DRUG contains all approved drugs in the US and Japan, and KEGG DISEASE is a new database linking disease genes, pathways, drugs and diagnostic markers.
5,352 citations
"Comparative analysis of expressed s..." refers background in this paper
...Several hormone pathways, such as of abscisic acid, ethylene, salicylic acid, and jasmonic acid, are involved in one or more environmental stresses, including drought stress and other abiotic stresses processes [38-42]....
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