Development of ESTs from chickpea roots and their use in diversity analysis of the Cicer genus

BACKGROUND: Chickpea is a major crop in many drier regions of the world where it is an important protein-rich food and an increasingly valuable traded commodity. The wild annual Cicer species are known to possess unique sources of resistance to pests and diseases, and tolerance to environmental stresses. However, there has been limited utilization of these wild species by chickpea breeding programs due to interspecific crossing barriers and deleterious linkage drag. Molecular genetic diversity analysis may help predict which accessions are most likely to produce fertile progeny when crossed with chickpea cultivars. While, trait-markers may provide an effective tool for breaking linkage drag. Although SSR markers are the assay of choice for marker-assisted selection of specific traits in conventional breeding populations, they may not provide reliable estimates of interspecific diversity, and may lose selective power in backcross programs based on interspecific introgressions. Thus, we have pursued the development of gene-based markers to resolve these problems and to provide candidate gene markers for QTL mapping of important agronomic traits. RESULTS: An EST library was constructed after subtractive suppressive hybridization (SSH) of root tissue from two very closely related chickpea genotypes (Cicer arietinum). A total of 106 EST-based markers were designed from 477 sequences with functional annotations and these were tested on C. arietinum. Forty-four EST markers were polymorphic when screened across nine Cicer species (including the cultigen). Parsimony and PCoA analysis of the resultant EST-marker dataset indicated that most accessions cluster in accordance with the previously defined classification of primary (C. arietinum, C. echinospermum and C. reticulatum), secondary (C. pinnatifidum, C. bijugum and C. judaicum), and tertiary (C. yamashitae, C. chrossanicum and C. cuneatum) gene-pools. A large proportion of EST alleles (45%) were only present in one or two of the accessions tested whilst the others were represented in up to twelve of the accessions tested. CONCLUSION: Gene-based markers have proven to be effective tools for diversity analysis in Cicer and EST diversity analysis may be useful in identifying promising candidates for interspecific hybridization programs. The EST markers generated in this study have detected high levels of polymorphism amongst both common and rare alleles. This suggests that they would be useful for allele-mining of germplasm collections for identification of candidate accessions in the search for new sources of resistance to pests / diseases, and tolerance to abiotic stresses

A legume genomics resource: The Chickpea Root Expressed Sequence Tag Database

Chickpea, a lesser-studied grain legume, is being investigated due to its taxonomic proximity with the model legume genome Medicago truncatula and its ability to endure and grow in relatively low soil water contents making it a model legume crop for the study of agronomic response to drought stress. Public databases currently contain very few sequences from chickpea associated with expression in root tissues. However, root traits are likely to be one of the most important components of drought tolerance in chickpea. Thus, we have generated a set of over 2800 chickpea expressed sequence tags (ESTs) from a library constructed after subtractive suppressive hybridization (SSH) of root tissue from two closely related chickpea genotypes possessing different sources of drought avoidance and tolerance (ICC4958 and Annigeri respectively). This database provides researchers in legume genomics with a major new resource for data mining associated with root traits and drought tolerance. This report describes the development and utilization of the database and provides the tools we have developed to facilitate the bioinformatics pipeline used for analysis of the ESTs in this database. We also discuss applications that have already been achieved using this resource

Laboratory Information Management Software for genotyping workflows: applications in high throughput crop genotyping

BACKGROUND: With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow. RESULTS: A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics. CONCLUSION: A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from

RESEARCH ARTICLE - A legume genomics resource: The Chickpea Root Expressed Sequence Tag Database

Chickpea, a lesser-studied grain legume, is being investigated due to its taxonomic proximity with the model legume genome Medicago truncatula   and its ability to endure and grow in relatively low soil water contents making it a model legume crop for the study of agronomic response to drought stress. Public databases currently contain very few sequences from chickpea associated with expression in root tissues. However, root traits are likely to be one of the most important components of drought tolerance in chickpea. Thus, we have generated a set of over 2800 chickpea expressed sequence tags (ESTs) from a library constructed after subtractive suppressive hybridization (SSH) of root tissue from two closely related chickpea genotypes possessing different sources of drought avoidance and tolerance (ICC4958 and Annigeri respectively). This database provides researchers in legume genomics with a major new resource for data mining associated with root traits and drought tolerance. This report describes the development and utilization of the database and provides the tools we have developed to facilitate the bioinformatics pipeline used for analysis of the ESTs in this database. We also discuss applications that have already been achieved using this resource

Chickpea molecular breeding: New tools and concepts

Chickpea is a cool season grain legume of exceptionally high nutritive value and most versatile food use. It is mostly grown under rain fed conditions in arid and semi-arid areas around the world. Despite growing demand and high yield potential, chickpea yield is unstable and productivity is stagnant at unacceptably low levels. Major yield increases could be achieved by development and use of cultivars that resist/tolerate abiotic and biotic stresses. In recent years the wide use of early maturing cultivars that escape drought stress led to significant increases in chickpea productivity. In the Mediterranean region, yield could be increased by shifting the sowing date from spring to winter. However, this is hampered by the sensitivity of the crop to low temperatures and the fungal pathogen Ascochyta rabiei. Drought, pod borer (Helicoverpa spp.) and the fungus Fusarium oxysporum additionally reduce harvests there and in other parts of the world. Tolerance to rising salinity will be a future advantage in many regions. Therefore, chickpea breeding focuses on increasing yield by pyramiding genes for resistance/tolerance to the fungi, to pod borer, salinity, cold and drought into elite germplasm. Progress in breeding necessitates a better understanding of the genetics underlying these traits. Marker-assisted selection (MAS) would allow a better targeting of the desired genes. Genetic mapping in chickpea, for a long time hampered by the little variability in chickpea’s genome, is today facilitated by highly polymorphic, co-dominant microsatellite-based markers. Their application for the genetic mapping of traits led to inter-laboratory comparable maps. This paper reviews the current situation of chickpea genome mapping, tagging of genes for ascochyta blight, fusarium wilt resistance and other traits, and requirements for MAS. Conventional breeding strategies to tolerate/avoid drought and chilling effects at flowering time, essential for changing from spring to winter sowing, are described. Recent approaches and future prospects for functional genomics of chickpea are discussed