Digital Gene Expression Profiling to Explore Differentially Expressed Genes Associated with Terenoid Biosynthesis during Fruit Development in _Litsea cubeba_  
[PERSON]  
1 State Key Laboratory of Forest Genetics and Tree Breeding, Chinese Academy of Forestry, No.1 [PERSON], Beijing 10086, China; [EMAIL_ADDRESS] (M.G. Citral belongs to the terpenoids. Terpenoids, represented mainly by isoprene (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), are the largest category of plant secondary metabolites. They play important roles in plant development, the interactions of plants with the environment, and reproduction through attraction of pollinators and seed disseminators [3]. The biosynthetic pathway of plant terpenoids have been well studied [4; 5] and a large number of enzymes are involved this pathway. In the past several years, there has been a significant progress in the identification and characterization of genes encoding the enzymes involved in terpenoid biosynthesis in many plant species, especially in the early steps of biosynthesis. For example, the first plant acetoacetyl-CoA thiolase (AACT) was cloned from radish (_Raphanus sativus_) [6]. 3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) was identified in _Arabidopsis_[7], _Brassica juncea_[8] and _Heeva brasiliensis_[9]. Three genes encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) have been identified in snapdragon (_Antitrhinum majus_) [10]. A cDNA encoding a protein with homology to 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) have been identified in tomato [11]. However, there are few studies about the enzymes involved in terpenoid biosynthesis in _L. cubeba._ [PERSON] reported the isolation and functional characterization of three monoterpene synthase (mono-TPS) genes in _L. cubeba_[12]. cDNA encoding DXR was isolated in _L. cubeba_, but functional characterization of the corresponding enzymes was not studied [13]. At the same time, there is still no integrated model describing differentially expressed genes (DEGs) involved in terpenoid biosynthesis during the fruit development of _L. cubeba_. Significant improvements in oil accumulation must be accompanied by changes in the expression of the terpenoids biosynthetic genes during fruit development. Identification of these genes and their regulatory pathways would provide not only new genetic information to understand the development of _L. cubeba_ fruit, but also to control gene expression to alter oil accumulation. In recent years, the rapid development of next-generation sequencing (NGS) technologies has offered new cost-efficient high-throughput approaches for global measurements of gene expression of model or non-model species in recent years [14; 15; 16]. Digital gene expression (DGE), a tag-based transcriptome sequencing method, can be applied to detect new genes and changes in gene expression with an unbiased view of the transcriptome, greater precision, simpler preprocessing, and consistent results compared to qPCR [17; 18]. Furthermore, this technology is useful for estimating the overall gene expression levels at different developmental stages [19; 20]. An RNA sequencing project for _L. cubeba_ has been carried out using the Illumina platform [2] aiming to promote the further gene expression studies of _L. cubeba_. In this study, we employed DGE tag profiling using the Illumina NGS platform to analyze the gene expression profiles of five different developmental stages of fruit (60, 75, 90, 105 and 135 days after flowering; DAF) and to identify differentially expressed genes. DGE generated over eight million reads per sample, which produced expression data for approximately 19354 genes. Twenty six candidate genes were selected using pathway analysis and characterized as those responsible for terpenoid biosynthesis. Our results provide a comprehensive understanding of DGE patterns and expression levels during _L.