BMC Genomics 2009, 10:415 http://www.biomedcentral.com/1471-2164/10/415 Methods Plant material and in planta infection The chickpea (Cicer arietinum L.) seeds were sterilized with 70% ethanol and 0.1% HgCl2 followed by repetitive washing in autoclaved water. The seedlings were grown in sterile conditions in glass tubes containing MS basal medium solidified with 0.6% agar in an environmentally controlled growth room and maintained at 25 ± 2°C, 50 ± 5% relative humidity under 16 h photoperiod (270 mol m-2 s-1 light intensity). Fusarium oxysporum ciceri race 1 was grown in potato dextrose broth (PDB) at 28°C. The 3-week old seedlings were inoculated with Fusarium spore suspension at a concentration of 1 × 106/ml while the control plants were treated with water. Root and collar (root and shoot junction) tissue was sampled as experi- mental material, harvested at 6, 12, 24, 48 h and 5 days after inoculation and stored at -80°C after quick-freezing in liquid nitrogen. Construction of cDNA libraries Two suppression subtracted cDNA libraries were con- structed, one from a susceptible (JG-62) and the other from a resistant (WR-315) genotype using PCR select cDNA subtraction kit (Clontech, CA). Total RNA was iso- lated by GITC method as described earlier [107] from uninfected seedlings (used as driver) and Fusarium infected seedlings (used as tester) collected at different time points as mentioned above and subsequently com- bined. The poly (A)+ RNA from the pooled total RNA was purified by using Dyna beads oligo (dT) (Dynal biotech, USA). The cDNAs were cloned into pGEMT vector (Promega, Wisconsin) and transformed into E. coli cells (DH5Í). The individual clones were picked up and grown in deep well plates in 2× YT media containing ampicillin (75 g/ml) at 37°C for 16-18 hours under shaking at 250 rpm. All individual clones were stored in 96 well U bot- tom plates by mixing 220 l of grown culture and 30 l of 80% glycerol for long term storage. Sequencing, processing and assembly of ESTs into contigs Plasmid DNAs from both the subtracted cDNA libraries were isolated and purified by Perfectprep Plasmid 96 Vac Direct bind kit according to the manufacturer's instruc- tions (Eppendorf, Germany). The quality of the plasmid DNAs was analyzed by 0.8% agarose gel electrophoresis and the good quality plasmids having appropriate DNA concentration and free from any mix up were selected for sequencing. The individual plasmids were sequenced using the BigDye terminator cycle sequencing kit (Perkin Elmer, Applied Biosystems) with M13 forward and reverse primers for 5' and 3' single pass sequencing respectively, in ABI Prism 3700 sequencer (Applied biosystems, CA). Sequence base calls were made using Phred [108] with a quality cutoff of 15. Vector filtering was performed using the cross match program (P. Green, http://boze man.mbt.Washington.edu/phrap.docs/phrap.html) fol- lowed by trimming of low quality sequences. The sequences were individually inspected for chimeras, short reads, E. coli and mitochondrial sequences which were subsequently removed. The processed ESTs with 100 bases or longer were assembled into contigs by CAP3 pro- gramme using standard parameters [20]. The final assem- bly of contigs and singletons constituted the chickpea gene index. All EST sequences have been submitted to GenBank [GenBank: GR911733 to GR918004]. Blast analysis, annotation and comparison The EST contig and singleton sequences were annotated for homology using BLASTX and BLASTN algorithms against non redundant protein and nucleotide databases respectively. For BLASTX, an e-value cut-off of 10-15 was used and the sequences with e-value below this cutoff were then subjected to BLASTN analysis with e-value cut- off of 10-20. In addition, BLASTN was used to compare the chickpea sequences from this study to a database of leg- ume sequences. This database included sequences of Lotus japonicus (gene index release 3.0); Medicago trunculata (gene index release 8.0); Glycine max (gene index release 12.0) and Phaseolus vulgaris (gene index release 1.0) from The Institute for genomic Research (TIGR; Quackenbush et al, 2001) and Arachis hypogea, Cajanus cajan, Pisum sati- vum and Robinia pseudoacacia available from NCBI taxon- omy browser http://www.ncbi.nlm.nih.gov/Taxonomy/ taxonomyhome.html. The following criteria were used in stand-alone BLASTN comparison: (1) exact match bp = 11; (2) e-value  10-5 and (3) DNA identity  80% and 90%. Further, TBLASTX (with e-value cutoff of 10-15) was used for comparing chickpea sequences to GenBank's EST_others database for the identification of chickpea spe- cific sequences. Sequences were additionally functionally characterized in the context with the Gene Ontology (GO), Metacyc and COG. Cross species comparison of stress responsive transcriptome was carried out by search- ing each gene of unigene set against all classes of the stress responsive gene entries in the GO database http:// www.geneontology.org. Genes which had a match in more than one group of stress responsive genes were cate- gorized as ubiquitous and those which were earlier known to be involved in Fusarium stress as found from the fungal responsive group in the GO database were categorised as canonical. Remaining of the genes which did not show any match in any of the stress responsive gene groups in the GO database were classified as non canonical. Identification of gene families using single-linkage clustering In order to identify gene families, the chickpea contigs and singletons were combined into a single dataset. TBLASTX with e-value cutoff of 10-15 was used to compare the dataset against itself. Sequences with at least one Page 16 of 21 (page number not for citation purposes)