Natarajan et al. BMC Genomics 2010, 11:606 http://www.biomedcentral.com/1471-2164/11/606 protocol (Biobasic Inc, Canada). Plasmid DNA was isolated from 12,810 cDNA clones. Sequencing of ESTs was per- formed using M13 reverse primer and BigDyeTM Termina- tor v3.1 Cycle Sequencing Kit in 3130xl Genetic Analyzer (Applied Biosystems, CA, USA). The raw sequence data was base called by Phred program [62] using DNA Sequen- cing Analysis Software Version 5.1 (Applied Biosystems, CA, USA). Only the bases having Phred value above 20 were considered for further analysis. The vector sequences and cDNA anchor sequences were removed using Codon Code Aligner (Codon Code Corporation, MA, USA). The clones having less than 100 bp good quality sequences were also removed from the analysis. Contigs assembly Contig assembly of ESTs was done using CAP3 program [63] and Codon Code Aligner (Codon Code Corporation, MA, USA). The default parameters set by CAP3 was used to assemble the ESTs in to contigs and singletons. The number of ESTs in the contigs was used to find the redundancy rate of the ESTs in the library. After the assembly result, the outputs were manually checked and redundant clones were removed. The total numbers of unique ESTs (unigenes) were calculated by adding all singletons and one EST from each contig. Annotation of Unigenes The unigenes were annotated using BLASTX and non- redundant database at NCBI [61]. All the sequences which showed significant similarity (e-value < e-10) were Page 11 of 14 assigned putative functions. Others were classified as unigenes with no significant similarity. Phylogenetic analysis Ten ESTs of J. curcas from the current study were selected and corresponding genes from five oilseed crops and Vitis vinifera were obtained from Genbank at NCBI. The Phylogenetic analysis was done using Clus- talW2 program [64]. Identification of full-length unigenes Full-length nature of the cDNAs at the 5’end was ana- lyzed from BLASTX output. A unigene was considered as potential full-length gene if (1) there was a bonafide 5’UTR, (2) the first amino acid in one of the positive reading frames of query sequence matches with the first amino acid in subject sequence from the database and (3) the reading frame covers the entire length of the query sequence. The unigenes that did not show signifi- cant similarity were analyzed for Open Reading Frames (ORF) using ORF Finder at NCBI [65]. The unigenes were considered as potential full-length gene if there was a bonafide 5’ UTR and the longest ORF covered almost the entire length of the sequence. Functional Classification of Unigenes The unigenes were annotated again using BLASTN and NT database of arabidopsis at The Arabidopsis Informa- tion Resource (TAIR) [66]. This resulted in locus identi- fier for each unigenes. These locus identifiers were S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Table 3 Primers and PCR product size of the genes used for semi-quantitative RT-PCR analysis Gene Name Forward primer Reverse primer Product size (bp) Carboxyl transferase of ACCase b subunit (CT-b) GAATGAGTTACTTCAGCTTCAC AATTCACCCTTCTTTCTTGTTGA 307 Biotin carboxyl carrier protein I of ACCase (BCCPI) GTCGGCTAATCTTAAAGCTATTC TGTTTATAGCTTCACTAGTGTAC 258 Biotin carboxyl carrier protein II of ACCase GATGCTCTCATTGCAATTCTC TAATGATAAATAACAATGAAGAAGG 278 (BCCPII) Malonyl-CoA:ACP transacylase (MCAT) ACTTCTCCTGTTCAATGGGAA TCTCTAGTACATGACGCTAATC 314 3-keto acyl ACP Reductase (KAR) GTTATCTCTCCCGAAAGTGTA CACAGTATCTGTCACCTTTTC 306 Beta-keto acyl ACP synthase I (KASI) GCATCTGGCTTGTCTCCAT GCAGAAGAACACAATTTTGATAC 402 Beta-keto acyl ACP synthase II (KASII) TGTTCCCAATTTGAAGAAGCAG AGGAACACCAAATCCAATCTTAT 307 Acyl carrier Protein (ACP) AAGATCAGTCTAGGAAATCCTTC GCATAAATTAGGAAATTTTAGAGTGT 180 Acyl ACP thioesterase (FATA) AATAATGTAGATTTCTTTATTTGTGT AGTAAACGTAAAACAATACAGTTGAT 285 ω-3-fatty acid Desaturase (ω-3-FAD) AAAGCTGCAAAATTTTTTATCTGCA CCCTCTCAAATCCAATCCAA 215 Acyl -CoA binding protein (ACBP) TGAACATTTCTATGCCGCTTG GAGAAATAAGGGTCACCATTATC 310 Long chain acyl-CoA synthetase (LCACS) ATCCATAGCATTTGGCTTTCAAA CGTGTAGAAGATGAATTGTATAAC 326 Acyl-CoA thioesterase (ACT) ACTCATCTTAGCTTTGTTATGTTC CCCATTTCAATCACCGTTTC 173 Glycerol-3-phosphate acyl transferase (GPAT) GGGTAACGTGTTGGGATTTG CACAGAATCCAAATAATTCTACATTT 200 Lysophosphatidic acid acyl transferase (LPAT) TGGTGTATGTTTGTGCTTGG CTGTACAAAATTGAATCAAGCTTTTT 238 Diacyl glycerol kinase (DGK) CGGCTATTCGGTTGGAAATAA GATTTTTGATACAACAAATTACCAGT 296 Triacyl glycerol lipase(TAGL) CTGGAGACAAGACGAGAATAA CTTCTATCATAATCAATTATTGTTCG 310 Actin CAAGTCATCACCATTGGAGCA GCCTCTTAATTTCGGCTTTAACA 590