SignificanceA high-quality genome assembly of Camellia sinensis var. sinensis facilitates genomic, transcriptomic, and metabolomic analyses of the quality traits that make tea one of the world’s most-consumed beverages. The specific gene family members critical for biosynthesis of key tea metabolites, monomeric galloylated catechins and theanine, are indicated and found to have evolved specifically for these functions in the tea plant lineage. Two whole-genome duplications, critical to gene family evolution for these two metabolites, are identified and dated, but are shown to account for less amplification than subsequent paralogous duplications. These studies lay the foundation for future research to understand and utilize the genes that determine tea quality and its diversity within tea germplasm.

BackgroundTea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes.ResultsUsing high-throughput Illumina RNA-seq, the transcriptome from poly (A)+ RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR).ConclusionsAn extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis.

Theanine is the most abundant non-protein amino acid in Camellia sinensis, but it is not known how a tea plant accumulates such high levels of theanine. The endophyte isolated from in vitro grown plantlets of C. sinensis cultivars was identified as Luteibacter spp., showing strong biocatalytic activity for converting both glutamine and ethylamine to theanine. Theanine was secreted outside of the bacteria. The endophyte isolated from in vitro plantlets of Camellia oleifera cultivar was identified as Bacillus safensis and did not convert glutamine and ethylamine to theanine. Enzymatic assays in vitro indicated that γ-glutamyltranspeptidases rCsEGGTs from the endophyte Luteibacter strains converted glutamine and ethylamine to theanine at higher rates than rCsGGTs from C. sinensis. This is the first report on theanine biosynthesis by an endophyte from C. sinensis, which provides a new pathway to explore the mechanism of theanine biosynthesis in C. sinensis and the interactions between an endophyte and tea plants.

Tea is a globally consumed non-alcohol beverage with great economic importance. However, lack of the reference genome has largely hampered the utilization of precious tea plant genetic resources towards breeding. To address this issue, we previously generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology, which produced a total of 2,124 Gb short and 125 Gb long read data, respectively. A hybrid strategy was employed to assemble the tea genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more comprehensive omics datasets with improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be utilized to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, thus help to breed new varieties with high yield and better quality in the future.

No abstract