Natarajan et al. BMC Genomics 2010, 11:606 http://www.biomedcentral.com/1471-2164/11/606 (191) did not have significant similarity with any genes available in the non-redundant database at NCBI. This is significantly lower than 8 - 24% of such genes reported in other plants [55,12,56]. These genes are very important as they may be specific to jatropha. Full-length unigenes Almost all of the unigenes will be full-length at the 3’ end because first strand cDNA synthesis was carried out using oliog-dT(15) primer which initiates synthesis from poly (A) tail of the mRNAs. Therefore, full-length nat- ure of the cDNAs at the 5’ end was analyzed using the BLASTX results. This could be done for 6818 of the 7009 unigenes which showed significant similarity with genes in the non-redundant database at NCBI. It was found that 91.4% of the unigenes (6233) potentially code for full-length genes. This is significantly higher than 60-75% full-length genes reported before [55,10,57]. The remaining 191 unigenes that could not be analyzed by BLASTX for lack of similarity with genes in the data- base at NCBI were analyzed by predicting open reading frames (ORFs). In 14 unigenes, 5’UTR and single longest ORF covering almost the entire lengths of the sequences were identified (Figure 4). These unigenes could also be considered as potential full-length unigenes. In 18 uni- genes, 5’UTRs were present but the ORFs terminated prematurely. We could not predict ORFs in the remain- ing 159 unigenes. These results show that the library is highly enriched with full length genes that it can be highly useful for gene discovery purpose. Functional Classification Functional coverage of the unigenes was identified by comparing the functional distribution of the genes from fully sequenced A. thaliana genome. Unigenes were searched against A. thaliana genome for functional annotation and locus identifiers using BLASTN at TAIR. The Gene Ontology annotations were assigned for each unigene based on the locus identifiers using GO annotation and categorization tool at TAIR. The 7009 unigenes from J. curcas were classified under three broad functional categories using GO slim terms. Distri- bution of J. curcas unigenes and A. thaliana genome under these three broad functional categories is shown in Figure 5. This classification provides information on percentage of J. curcas unigenes involved in the signal transduction, anabolism, catabolism, reproduction and so on. The results showed that the unigenes cover all the GO slim terms in Arabidopsis. The unigenes were first classified according their func- tion in 16 different cellular compartments and anatomi- cal structures such as endoplasmic reticulum, plastid, mitochondria, nucleus, cell wall, golgi apparatus etc. which may have unique genes or specific expression Page 7 of 14 profiles. The proportion of genes identified under each class was comparable with A. thaliana genome except those unigenes which were classified under ‘unknown cellular components’ (Figure 5A). Majority of the uni- genes were grouped under ‘other intra-cellular compo- nents’, ‘unknown cellular components’, ‘other cytoplasmic components’ and ‘chloroplast’, which accounted for about 55% of the unigenes. Though, non- green tissue was used for the cDNA library construction, about 12% of the unigenes belonged to chloroplast cellu- lar component. The unigenes were then classified according to their involvement in 14 different biological processes such as protein metabolism, developmental processes, response to stress, transcription, signal transduction etc. These processes are very important for a cell to live and repro- duce. Hence, the genes that cover the biological pro- cesses are of great importance for functional study. It was found that the larger part of the J. curcas unigenes were grouped under ‘other cellular process’, ‘other metabolic process’ and ‘unknown biological processes’ accounting for 23.4%, 20.6% and 13.3%, respectively (Figure 5B). The unigenes were finally classified under 15 different molecular functions which mainly correspond to the activities performed by the gene products from indivi- dual gene or group of genes such as transferase activity, hydrolase activity, kinase activity, receptor binding, receptor activity etc. Greater part of the unigenes was classified under ‘unknown molecular function’, ‘other enzyme activity’, ‘other binding’, ‘transferase activity’ which accounted for 49.3% of the unigenes (Figure 5C). Validation of ESTs In order to validate the expression of ESTs, a set of 17 ESTs for representing oil biosynthesis genes were selected and their expression was studied in roots, mature leaves, flowers and developing seeds of J. curcas. These ESTs were first sequenced from the 3’ ends and primers specific to 3’UTR were designed to increase their specificity to the respective transcripts. Gene expression was studied by using semi-quantitative RT-PCR and actin was used as an internal control (Figure 6). The results confirmed that the transcripts representing all the selected ESTs are actively expressed in J. curcas. All the transcripts were found to be expressed in all the tis- sues without significant variation in the level of expres- sion, except ACP gene which showed significantly higher expression in flower. O’Hara et al. [58] have also reported that in flowers, the ACP gene is expressed at higher level than the other genes involved in fatty acid biosynthesis. Conclusions Normalized and full-length enriched cDNA library was constructed and 12,084 clones were sequenced from