Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality

Abstract

Tea, one of the world's most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Keywords: catechins biosynthesis; comparative genomics; genome evolution; tea quality; theanine biosynthesis.

Fig. 1.

Landscape of the tea plant genome. (A) Global view. SNP markers in the genetic map (gray; a); simple sequence repeat density (orange; b); TE density (c; Copia, orange; Gypsy, blue); gene density (green; d); transcription factors (black; e); genes in syntenic blocks between tea and grape (each color for syntenic genes from each grape chromosome; f); and oriented paralogous genes (g). A 1-Mb sliding window was used to calculate the density of different elements. (B) Estimation of divergence time between CSS and CSA using orthologous gene pairs within collinear blocks. (C) DNA and protein sequence similarity of orthologous genes between CSS and CSA. The error bar indicates the maximum and minimum sequence similarity values of orthologous genes.

Fig. 2.

Evolution of the genes encoding subclade 1A of serine carboxypeptidase-like acyltransferases (SCPL1A) in tea and six other plant species. (A) Neighbor-joining phylogenetic tree of SCPL1A-encoding genes from seven plant species, including tea, kiwifruit, coffee, cacao, Arabidopsis, poplar, and grape. (B) Expression profiles of the 22 tea plant SCPL1A genes (column) in eight different tissues (rows): apical buds (AB), young leaves (YL), mature leaves (ML), old leaves (OL), young stems (ST), tender roots (RT), flowers (FL), and young fruits (FR). Gene expression level was evaluated using FPKM (fragments per kilobase per million reads mapped).

Fig. 3.

Evolution and expression of key genes involved in catechins biosynthesis. (A) Biosynthetic pathway of the principal catechins. CHS, CHI, F3H, F3′H, F3′5′H, DFR, ANS, LAR, ANR, and SCPL represent genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3′-hydroxylase, flavonoid 3′,5′-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, leucoanthocyanidin reductase, anthocyanidin reductase, and type 1A serine carboxypeptidase-like acyltransferases, respectively. (B) Expression profiles of key genes in different tissues of the tea plant in relation to their contents of different catechins. (B, Left) Expression levels of key genes associated with catechins biosynthesis in eight tea plant tissues: apical buds, young leaves, mature leaves, old leaves, young stems, flowers, young fruits, and tender roots. Expression data are plotted as log10 values. The horizontal axis of the boxplot (Right) shows statistics of catechins contents from different tissues, and the vertical axis exhibits different forms of catechins. “Cis” represents the contents of cis-flavan-3-ols, and “trans” represents the contents of trans-flavan-3-ols. The significant correlations of gene expression with the contents of ECG, EGCG, and cis-flavan-3-ols are indicated by black lines (Pearson’s correlation test, P < 0.05). The error bar represents the maximum and minimum catechins content in eight different tea plant tissues. (C) Transcriptional regulation of catechins biosynthetic genes. A coexpression network connecting structural genes in catechins biosynthesis with transcription factors represents the regulation of catechins biosynthetic genes. The color-filled hexagons represent the structural genes associated with catechins biosynthesis that was highly (green) or lowly (red) expressed in bud and leaf. Expression correlations between TFs (colored solid circles) and catechins-related genes (colored solid hexagons) are shown with colored lines (Pearson’s correlation test, P ≤ 1e-6).

Fig. 4.

Key genes involved in the theanine biosynthesis pathway. (A) The proposed pathway for theanine biosynthesis and expression of key genes upon precursor ethylamine feeding. TS, GS, GOGAT, GDH, and ADC represent genes encoding theanine synthetase, glutamine synthetase, glutamate synthetase, glutamate dehydrogenase, and arginine decarboxylase, respectively. Tea seedlings grown hydroponically were fed ethylamine chloride for different numbers of days before being sampled for amino acid profiling and transcriptome analyses. (B) Phylogenetic tree of tea TS and GS candidate genes and the available GS genes from prokaryotes, fungi, and plants. The tea TS candidate gene (CsTSI) shows high similarity to known GSI-type genes, and other GS candidate genes exhibit high homology with previously reported GSII-type genes in plants. (C) Assay of theanine synthesis activity of CsTSI in Arabidopsis seedlings. The candidate tea TS gene (CsTSI) that shows high similarity to known GSI-type genes was cloned into a binary vector and overexpressed in Arabidopsis driven by a 35S promoter. CsTSI-OE indicates CsTSI-overexpression lines, while WT represents wild type (control). Seedlings were fed with or without 10 mM EA chloride solution (with water as control) for 3 d. Theanine synthesized by the seedlings was extracted and measured. Data are expressed as means ± SD from at least three independent transgenic lines with replicate experiments. FW, fresh weight.