Natarajan et al. BMC Genomics 2010, 11:606 http://www.biomedcentral.com/1471-2164/11/606 emits less particulate matter than diesel upon combus- tion [3]. In fact, large scale jatropha cultivation will improve the environment by greening the area and transforming the wasteland to productive land by pre- venting soil erosion, causing accumulation of organic matter, increasing soil microbial activity, etc. The demand for biodiesel production is very huge that it cannot be met from wild grown plants. Increasing the jatropha production requires both to bring more area under cultivation and to enhance productivity. Though jatropha can grow and survive in wasteland with less water, nutrient, and virtually no pest and disease man- agement, productive growth and better yield under restrictive environmental conditions require the devel- opment of resilient genotypes. Genotypes with improved drought tolerance are preferred for plantations in mar- ginal lands. Large scale plantations may bring in new challenges which need to be addressed. It was reported that J. curcas planted in continuous stretches as a monocrop were devastated by flower and seed feeding insects Scutellera nobilis and Pempelia morosalis [4]. This indicates that plant breeding programs to develop pest and disease resistance are required when large scale cultivation of jatropha is planned. In addition, jatropha oil composition itself may have to be modified to make it the best feedstock for biodie- sel production. Oils with more of saturated fatty acids give higher cetane number, and oxidative stability which are desirable for combustion/ignition quality and shelf life of biodiesel, respectively. But jatropha oil contains less of saturated fatty acids (21.6%) and more of unsatu- rated fatty acids (78.4%) [5]. While viscosity of petro- diesel is 2.6 mm2/s, it is 4.8 mm2/s for biodiesel derived from jatropha oil. Viscosity affects atomization of the fuel upon injection into the combustion chamber, and thereby, increases the formation of engine deposits [6]. More the carbon number of saturated fatty acids higher will be the viscosity. Jatropha contains 84.5% 18-carbon fatty acids and only 14.9% 16-carbon fatty acids [5]. It is possible to make significant changes in the jatropha oil composition by genetic engineering of the metabolic pathway of oil biosynthesis. Therefore, there is a need and scope for genetic improvement of jatropha by using plant breeding and genetic engineering methods. Genetic intervention in jatropha requires understand- ing of the biosynthetic pathways, metabolic flux control points, cloning of the genes that code for the enzymes and proteins involved in the metabolic pathways and development of molecular markers. Molecular studies in jatropha are limited and only a few genes have been isolated from jatropha [7,8]. Currently, the dbEST of NCBI contains only 250 annotated Expressed sequence tags (ESTs) from jatropha. We have initiated a gene discovery project by large scale sequencing of ESTs. Page 2 of 14 ESTs are short, single pass sequence reads from 5’or 3’ end of randomly selected cDNA clones. Sequencing of ESTs has been successfully employed in several plants including tomato [9], citrus [10], castor [11], arabidop- sis [12], rice [13], water melon [14], radish [15] with the objective of gene discovery, metabolic pathway interpretation, gene cloning, molecular marker devel- opment, construction of genetic and physical map and comparative mapping and analysis. When sequencing of ESTs is employed for gene discovery purpose, nor- malization of the cDNA library will greatly increase the efficiency and economy of the process. Normalization reduces the frequency of abundant genes (hundreds of mRNA copies per cell) and enriches the library with rare genes (< 10 mRNA copies per cell) [16]. Since sequencing of ESTs is carried out with the ultimate objective of cloning the genes, it would be highly desir- able to combine the normalization with enrichment for full-length clones. This paper reports construction of a normalized and full-length enriched cDNA library from developing seeds of J. curcas and isolation of 7009 uni- genes by sequencing of 12,084 ESTs. Results and Discussion Construction of cDNA Library Construction of cDNA library and sequencing of ESTs helps in rapid gene discovery especially in non-model organisms where no prior sequencing data is available. Next generation sequencing technologies can circum- vent the need for constructing cDNA libraries and gen- erate extraordinarily huge amount of sequencing data to further speed up the gene discovery process. However, sequencing of cDNA clones has several advantages over the next generation sequencing methods such as higher average read length, virtually no assembly problem, abil- ity to isolate full length genes without going for RACE PCR and availability of physical clones for further char- acterization and applications. For the present study, a normalized and full-length enriched cDNA library was constructed from developing seeds of J. curcas. The normalization efficiency was monitored by using chloramphenicol reporter gene. Before normalization, the reporter gene was added to the cDNA population to a redundant rate of about 1% which was found to be reduced to less than 0.025% after normalization. This indicated 40 fold reduction in abun- dance due to normalization. This normalization will greatly help to enrich the library for rare genes. In addi- tion, it will increase the rate recovery of unigenes and reduce the cost of sequencing by avoiding redundant clones. In fact, the rate of recovery of unigenes in this study was about 58% (7009 unigenes out of 12,084 ESTs) which is much higher than 30 to 40% reported from non-normalized cDNA libraries [11,17,18].