Abstract
BackgroundChickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers.ResultsA total of 20,162 (18,435 high quality) drought- and salinity- responsive ESTs were generated from ten different root tissue cDNA libraries of chickpea. Sequence editing, clustering and assembly analysis resulted in 6,404 unigenes (1,590 contigs and 4,814 singletons). Functional annotation of unigenes based on BLASTX analysis showed that 46.3% (2,965) had significant similarity (≤1E-05) to sequences in the non-redundant UniProt database. BLASTN analysis of unique sequences with ESTs of four legume species (Medicago, Lotus, soybean and groundnut) and three model plant species (rice, Arabidopsis and poplar) provided insights on conserved genes across legumes as well as novel transcripts for chickpea. Of 2,965 (46.3%) significant unigenes, only 2,071 (32.3%) unigenes could be functionally categorised according to Gene Ontology (GO) descriptions. A total of 2,029 sequences containing 3,728 simple sequence repeats (SSRs) were identified and 177 new EST-SSR markers were developed. Experimental validation of a set of 77 SSR markers on 24 genotypes revealed 230 alleles with an average of 4.6 alleles per marker and average polymorphism information content (PIC) value of 0.43. Besides SSR markers, 21,405 high confidence single nucleotide polymorphisms (SNPs) in 742 contigs (with ≥ 5 ESTs) were also identified. Recognition sites for restriction enzymes were identified for 7,884 SNPs in 240 contigs. Hierarchical clustering of 105 selected contigs provided clues about stress- responsive candidate genes and their expression profile showed predominance in specific stress-challenged libraries.ConclusionGenerated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding. Mapping of gene-based markers in chickpea will also add more anchoring points to align genomes of chickpea and other legume species.
Highlights
Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses
Root tissues from both drought and saline stressed plants were harvested for total RNA extraction and subsequent cDNA library construction
In case of chickpea, where crop production is adversely affected by drought and salinity, not much information is available on candidate genes or molecular markers associated with tolerance/resistance to these stresses
Summary
Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. The world's third most important food legume is grown in over 40 countries representing eight geographically diverse agro-climatic conditions. In addition to being a major source of protein for human food in semi-arid tropical regions, chickpea crop plays an important role in the maintenance of soil fertility, in the dry, rainfed areas [2,3]. Considering the small genome size, short seed-to-seed reproductive cycle of approximately three months and most importantly high economic importance as a food crop legume, chickpea is an interesting system for genomics research. Majority of the world's chickpea is grown in South Asia and India being the largest producer with an estimated annual production of 5.9 million tonnes (mt). Global annual production losses due to abiotic stresses alone are estimated to be around 3.7 mt, which amounts to 40-60% average loss
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