De Novo Characterization of Leaf Transcriptome Using 454 Sequencing and Development of EST-SSR Markers in Tea (Camellia sinensis)

Abstract

Although tea leaves are rich in secondary metabolites, not enough transcriptomic information is available to aid understanding of the molecular mechanisms underlying plant growth, development, and secondary metabolite production. In this study, a total of 437,908 reads were generated from the tea leaf transcriptome using 454 sequencing. De novo assembly yielded 25,637 unigenes, 22,872 of which were annotated by BLAST searches against public databases. Most of these unigenes mapped to carbohydrate metabolism, energy metabolism and secondary metabolite biosynthetic pathways. Some abundant transcripts related to photomorphogenesis and development in plants, including ubiquitin/26S proteasome, lipid transfer protein, PPR-containing protein, small GTPase, expansin, transport inhibitor response 1 and thioredoxin, were identified in the transcriptome. Most of the genes encoding the main enzymes involved in flavonoid, caffeine and theanine biosynthesis were also found, and six MYB and two bHLH genes known to regulate flavonoid synthesis were identified. ABC transporter and glutathione S-transferase, generally responsible for secondary metabolite transport, and CYP450, broadly involved in oxidation steps in secondary metabolism, were also present in a large number of unigenes. Additionally, 3,767 EST-SSRs were identified as potential molecular markers in our unigenes. A total of 100 PCR primer pairs used in initial screening tests among 20 tea genotypes successfully identified 36 polymorphic loci. Overall, the tea leaf transcriptome sequences generated in this study reveal novel gene expression profiles and offer important clues for further study of the molecular mechanism of tea leaf growth, development and secondary metabolite synthesis. The thousands of EST-SSR markers identified will facilitate marker-assisted selection in tea breeding.