Natarajan et al. BMC Genomics 2010, 11:606 http://www.biomedcentral.com/1471-2164/11/606 The cDNA library can be more efficiently used for gene discovery if normalization is combined with enrichment for full-length genes. This was done by removing cDNAs smaller than 0.5 kb. Removal of smal- ler fragments will also increase the cloning efficiency of longer cDNAs. The cDNA library constructed for this study was estimated to contain about 1 × 106 clones. Ninety six clones were randomly selected to test for the enrichment of full-length genes in these clones. The insert size ranged between 0.8 kb and 3.2 kb with an estimated average insert size of 2.1 kb (Figure 1). BLASTX analysis of the sequences revealed that 94% of the clones could potentially encode for full-length genes. These results indicated that this cDNA library could be reliably used for the gene discovery project in jatropha. Sequencing of Expressed Sequence Tags (ESTs) From the normalized and full-length enriched cDNA library, 13,220 clones were patched for plasmid DNA isolation. Clones which did not grow in the selection medium or did not yield sufficient quantity or quality of plasmid DNA were discarded. Plasmid DNA suitable for sequencing was isolated from 12,810 clones. These clones were sequenced using M13 reverse primer (CAG- GAAACAGCTATGAC) which directly reads the cDNAs from the 5’ end. Nucleotide bases having Phred value less than 20 were discarded. Vector backbone and addi- tional sequences that were added during cDNA Figure 1 Construction of cDNA library from J. curcas. (A) Fruits of J. curcas in different sizes and green in colour (A) and the seeds were removed for total RNA isolation (B). Average insert size of the cDNA library was determined by colony PCR analysis of randomly selected clones(C & D). 1.0 kb ladder DNA size markers (lane M) were used for size determination. Page 3 of 14 synthesis were removed. After the trimming exercise 144 empty clones (resulted in 0 bases after trimming) and 12,084 high quality ESTs were obtained. The read length of the high quality ESTs ranged between 105 bp and 874 bp with an average length of 576 bp which is comparable with other reports [19,20,18]. Contig Assembly of ESTs Contig assembly of the 12, 084 ESTs was done to remove the redundant ESTs so that the unique ESTs (unigenes) can be annotated. The summary of the contig assembly is given in Table 1. It showed 2258 contigs and 4751 singletons. The contig size ranged between 2 and 23 (Figure 2) and there were 7333 ESTs in the 2258 contigs indicating the presence of 5075 redundant ESTs. The 2258 contigs were manually checked and the long- est EST from each contig was selected as unigene. These representative ESTs from contigs and the 4751 singletons together resulted in 7009 unigenes from the 12,084 ESTs assembled. Sequencing of ESTs from developing seeds often showed the abundance of genes coding for seed sto- rage proteins [11,21,22]. On the contrary, the current study the showed the abundance of genes related to stress response, disease resistance and plant develop- ment (Table 2). The largest contig contained 23 ESTs coding for phosphoethanolamine N-methyltransferase which is essential for phosphatidylcholine biosynthesis. It is reported that 60.5% phospholipid in jatropha seed is composed of phosphatidylcholine [23]. Phosphatidyl- choline is hydrolyzed into phosphatidic acid and cho- line. Phosphatidic acid acts as second messenger in stress signaling, and choline is a precursor for glycine betaine synthesis. Glycine betaine is a compatible solute and its accumulation is widely reported to con- fer salt and oxidative stress [24,25]. Other contigs con- tained ESTs coding for indole-3 acetic acid amido synthetase, ccr4 associated factor, ethylene responsive transcription factor and calcineurin B which are also involved in biotic and abiotic stress responses in plants [26-29]. The contigs also represented ESTs coding for protein disulfide isomerase, copine and sucrose synthase which are involved in seed development [30,31] and seed size [32]. Table 1 Summary of Contig assembly Description Total number of ESTs assembled Number of contigs Number of ESTs in contigs No. of ETS as singletons Number of unique ESTs (unigenes) 12084 2258 7333 4751 7009 - - 60.6 39.3 58.0 Number Percentage