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

Abstract

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.

Figure 1

A summary of the number of EST clones and respective gene contigs classified in various functional categories based on alignments with public databases. Putative identifications were assigned to 2130 of the 2858 sequences generated from a chickpea root subtractive hybridization library. Percentages indicate the proportion of unigenes from the total number of unigenes identified in whole dataset.

Figure 2

Polymorphic profiles of EST primers AGLC34, AGLC45, AGLC51 and AGLC52 screened on representatives of 8 wild species (IG69947, IG69960, IG69961, IG69974, IG69976, IG69986, IG69992, IG70029, ICC17116, ICC17121, ICC17122, ICC17126, ICC17141, ICC17148) and one cultivated genotype (ICC4958), separated by 6% non-denatured polyacrylamide gel electrophoresis and visualized by silver staining.

Figure 3

Principle Coordinate Analysis (PCoA) plot from diversity analysis of 44 EST markers screened across 14 accessions representing 8 wild Cicer species and one cultivated genotype (ICC4958).

Figure 4

Dendogram of 14 Cicer accessions (representing 8 wild species and one cultivated genotype, ICC4958) based on parsimony analysis of 103 EST alleles.