Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

TPIA2: an updated tea plant information archive for Camellia genomics.

The genus Camellia consists of about 200 species, which include many economically important species widely used for making tea, ornamental flowers and edible oil. Here, we present an updated tea plant information archive for Camellia genomics (TPIA2; http://tpia.teaplants.cn) by integrating more novel large-scale genomic, transcriptomic, metabolic and genetic variation datasets as well as a variety of useful tools. Specifically, TPIA2 hosts all currently available and well assembled 10 Camellia genomes and their comprehensive annotations from three major sections of Camellia. A collection of 15 million SNPs and 950 950 small indels from large-scale genome resequencing of 350 diverse tea accessions were newly incorporated, followed by the implementation of a novel 'Variation' module to facilitate data retrieval and analysis of the functionally annotated variome. Moreover, 116 Camellia transcriptomes were newly assembled and added, leading to a significant extension of expression profiles of Camellia genes to 13 developmental stages and eight abiotic/biotic treatments. An updated 'Expression' function has also been implemented to provide a comprehensive gene expression atlas for Camellia. Two novel analytic tools (e.g. Gene ID Convert and Population Genetic Analysis) were specifically designed to facilitate the data exchange and population genomics in Camellia. Collectively, TPIA2 provides diverse updated valuable genomic resources and powerful functions, and will continue to be an important gateway for functional genomics and population genetic studies in Camellia.

Analysis of Functional Single-Nucleotide Polymorphisms (SNPs) and Leaf Quality in Tea Collection under Nitrogen-Deficient Conditions.

This study discusses the genetic mutations that have a significant association with economically important traits that would benefit tea breeders. The purpose of this study was to analyze the leaf quality and SNPs in quality-related genes in the tea plant collection of 20 mutant genotypes growing without nitrogen fertilizers. Leaf N-content, catechins, L-theanine, and caffeine contents were analyzed in dry leaves via HPLC. Additionally, the photochemical yield, electron transport efficiency, and non-photochemical quenching were analyzed using PAM-fluorimetry. The next generation pooled amplicon-sequencing approach was used for SNPs-calling in 30 key genes related to N metabolism and leaf quality. The leaf N content varied significantly among genotypes (p ≤ 0.05) from 2.3 to 3.7% of dry mass. The caffeine content varied from 0.7 to 11.7 mg g-1, and the L-theanine content varied from 0.2 to 5.8 mg g-1 dry leaf mass. Significant positive correlations were detected between the nitrogen content and biochemical parameters such as theanine, caffeine, and most of the catechins. However, significant negative correlations were observed between the photosynthetic parameters (Y, ETR, Fv/Fm) and several biochemical compounds, including rutin, Quercetin-3-O-glucoside, Kaempferol-3-O-rutinoside, Kaempferol-3-O-glucoside, Theaflavin-3'-gallate, gallic acid. From our SNP-analysis, three SNPs in WRKY57 were detected in all genotypes with a low N content. Moreover, 29 SNPs with a high or moderate effect were specific for #316 (high N-content, high quality) or #507 (low N-content, low quality). The use of a linear regression model revealed 16 significant associations; theaflavin, L-theanine, and ECG were associated with several SNPs of the following genes: ANSa, DFRa, GDH2, 4CL, AlaAT1, MYB4, LHT1, F3'5'Hb, UFGTa. Among them, seven SNPs of moderate effect led to changes in the amino acid contents in the final proteins of the following genes: ANSa, GDH2, 4Cl, F3'5'Hb, UFGTa. These results will be useful for further evaluations of the important SNPs and will help to provide a better understanding of the mechanisms of nitrogen uptake efficiency in tree crops.

Evolutionary Landscape of Tea Circular RNAs and Its Contribution to Chilling Tolerance of Tea Plant.

Chilling stress threatens the yield and distribution pattern of global crops, including the tea plant (Camellia sinensis), one of the most important cash crops around the world. Circular RNA (circRNA) plays roles in regulating plant growth and biotic/abiotic stress responses. Understanding the evolutionary characteristics of circRNA and its feedbacks to chilling stress in the tea plant will help to elucidate the vital roles of circRNAs. In the current report, we systematically identified 2702 high-confidence circRNAs under chilling stress in the tea plant, and interestingly found that the generation of tea plant circRNAs was associated with the length of their flanking introns. Repetitive sequences annotation and DNA methylation analysis revealed that the longer flanking introns of circRNAs present more repetitive sequences and higher methylation levels, which suggested that repeat-elements-mediated DNA methylation might promote the circRNAs biogenesis in the tea plant. We further detected 250 differentially expressed circRNAs under chilling stress, which were functionally enriched in GO terms related to cold/stress responses. Constructing a circRNA-miRNA-mRNA interaction network discovered 139 differentially expressed circRNAs harboring potential miRNA binding sites, which further identified 14 circRNAs that might contribute to tea plant chilling responses. We further characterized a key circRNA, CSS-circFAB1, which was significantly induced under chilling stress. FISH and silencing experiments revealed that CSS-circFAB1 was potentially involved in chilling tolerance of the tea plant. Our study emphasizes the importance of circRNA and its preliminary role against low-temperature stress, providing new insights for tea plant cold tolerance breeding.

Comparative transcriptomic analysis unveils the deep phylogeny and secondary metabolite evolution of 116 Camellia plants.

Camellia plants include more than 200 species of great diversity and immense economic, ornamental, and cultural values. We sequenced the transcriptomes of 116 Camellia plants from almost all sections of the genus Camellia. We constructed a pan-transcriptome of Camellia plants with 89 394 gene families and then resolved the phylogeny of genus Camellia based on 405 high-quality low-copy core genes. Most of the inferred relationships are well supported by multiple nuclear gene trees and morphological traits. We provide strong evidence that Camellia plants shared a recent whole genome duplication event, followed by large expansions of transcription factor families associated with stress resistance and secondary metabolism. Secondary metabolites, particularly those associated with tea quality such as catechins and caffeine, were preferentially heavily accumulated in the Camellia plants from section Thea. We thoroughly examined the expression patterns of hundreds of genes associated with tea quality, and found that some of them exhibited significantly high expression and correlations with secondary metabolite accumulations in Thea species. We also released a web-accessible database for efficient retrieval of Camellia transcriptomes. The reported transcriptome sequences and obtained novel findings will facilitate the efficient conservation and utilization of Camellia germplasm towards a breeding program for cultivated tea, camellia, and oil-tea plants.

Diverse roles of MYB transcription factors in regulating secondary metabolite biosynthesis, shoot development, and stress responses in tea plants (Camellia sinensis).

Tea (Camellia sinensis) is concocted from tea plant shoot tips that produce catechins, caffeine, theanine, and terpenoids, which collectively determine the rich flavors and health benefits of the infusion. However, little is known about the integrated regulation of shoot tip development and characteristic secondary metabolite biosynthesis in tea plants. Here, we demonstrate that MYB transcription factors (TFs) play key and yet diverse roles in regulating leaf and stem development, secondary metabolite biosynthesis, and environmental stress responses in tea plants. By integrating transcriptomic and metabolic profiling data in different tissues at a series of developmental stages or under various stress conditions, alongside biochemical and genetic analyses, we predicted the MYB TFs involved in regulating shoot development (CsMYB2, 98, 107, and 221), epidermal cell initiation (CsMYB184, 41, 139, and 219), stomatal initiation (CsMYB113 and 153), and the biosynthesis of flavonoids (including catechins, anthocyanins, and flavonols; CsMYB8 and 99), caffeine (CsMYB85 and 86), theanine (CsMYB9 and 49), carotenoids (CsMYB110), mono-/sesquiterpenoid volatiles (CsMYB68, 147, 148, and 193), lignin (CsMYB164 and 192), and indolic compounds (CsMYB139, 162, and 198), as well as the MYB TFs that are likely involved in hormone signaling-mediated environmental stress and defense responses. We characterized the functions of some key MYBs in regulating flavonoid and carotenoid biosynthesis for tea quality and flavor. This study provides a cross-family analysis of MYBs in tea alongside new insights into the coordinated regulation of tea plant shoot development and secondary metabolism, paving the way towards understanding of tea quality trait formation and genetic improvement of quality tea plants.

CsMYB1 integrates the regulation of trichome development and catechins biosynthesis in tea plant domestication.

Tea trichomes synthesize numerous specialized metabolites to protect plants from environmental stresses and contribute to tea flavours, but little is known about the regulation of trichome development. Here, we showed that CsMYB1 is involved in the regulation of trichome formation and galloylated cis-catechins biosynthesis in tea plants. The variations in CsMYB1 expression levels are closely correlated with trichome indexes and galloylated cis-catechins contents in tea plant populations. Genome resequencing showed that CsMYB1 may be selected in modern tea cultivars, since a 192-bp insertion in CsMYB1 promoter was found exclusively in modern tea cultivars but not in the glabrous wild tea Camellia taliensis. Several enhancers in the 192-bp insertion increased CsMYB1 transcription in modern tea cultivars that coincided with their higher galloylated cis-catechins contents and trichome indexes. Biochemical analyses and transgenic data showed that CsMYB1 interacted with CsGL3 and CsWD40 and formed a MYB-bHLH-WD40 (MBW) transcriptional complex to activate the trichome regulator genes CsGL2 and CsCPC, and the galloylated cis-catechins biosynthesis genes anthocyanidin reductase and serine carboxypeptidase-like 1A. CsMYB1 integratively regulated trichome formation and galloylated cis-catechins biosynthesis. Results suggest that CsMYB1, trichome and galloylated cis-catechins are coincidently selected during tea domestication by harsh environments for improved adaption and by breeders for better tea flavours.

Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants.

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.

HAK/KUP/KT family potassium transporter genes are involved in potassium deficiency and stress responses in tea plants (Camellia sinensis L.): expression and functional analysis.

BACKGROUND: Tea plant is one of the most important non-alcoholic beverage crops worldwide. While potassium (K+) is an essential macronutrient and greatly affects the growth and development of plants, the molecular mechanism underlying K+ uptake and transport in tea plant root, especially under limited-K+ conditions, is still poorly understood. In plants, HAK/KUP/KT family members play a crucial role in K+ acquisition and translocation, growth and development, and response to stresses. Nevertheless, the biological functions of these genes in tea plant are still in mystery, especially their roles in K+ uptake and stress responses. RESULTS: In this study, a total of 21 non-redundant HAK/KUP/KT genes (designated as CsHAKs) were identified in tea plant. Phylogenetic and structural analysis classified the CsHAKs into four clusters (I, II, III, IV), containing 4, 8, 4 and 5 genes, respectively. Three major categories of cis-acting elements were found in the promoter regions of CsHAKs. Tissue-specific expression analysis indicated extremely low expression levels in various tissues of cluster I CsHAKs with the exception of a high root expression of CsHAK4 and CsHAK5, a constitutive expression of clusters II and III CsHAKs, and a moderate cluster IV CsHAKs expression. Remarkably, the transcript levels of CsHAKs in roots were significantly induced or suppressed after exposure to K+ deficiency, salt and drought stresses, and phytohormones treatments. Also notably, CsHAK7 was highly expressed in all tissues and was further induced under various stress conditions. Therefore, functional characterization of CsHAK7 was performed, and the results demostrated that CsHAK7 locates on plasma membrane and plays a key role in K+ transport in yeast. Taken together, the results provide promising candidate CsHAKs for further functional studies and contribute to the molecular breeding for new tea plants varieties with highly efficient utilization of K+. CONCLUSION: This study demonstrated the first genome-wide analysis of CsHAK family genes of tea plant and provides a foundation for understanding the classification and functions of the CsHAKs in tea plants.

Comparative analysis of phenolic compound metabolism among tea plants in the section Thea of the genus Camellia.

Wild tea plants, which are classified into different species in the section Thea of the genus Camellia, are widely distributed in southern China. Tea produced from these plants has a unique flavor, which is different from that of tea produced from tea cultivars. In this study, we performed a comparative analysis of morphology, phylogenetic relationships, and phenolic compound metabolism between two wild tea plants (Gujing and Siqiu) and a tea cultivar (Shuchazao). Siqiu and Gujing tea plants had similar morphological traits and could be phylogenetically classified into a same cluster, which was entirely separate from the cluster containing widely cultivated cultivars such as Camellia sinensis cv. Shuchazao. Combined metabolomic and transcriptome analyses revealed that UGT84a22 was highly expressed in Gujing leaves compared with Shuchazao and Siqiu leaves, which may lead to the high accumulation of galloylquinic acid in Gujing leaves. A 14-bp deletion spanning the -765-(-7 5 1) range in the F3'5'H promoter potentially led to low F3'5'H expression levels in Siqiu and Gujing tea plants, which severely disrupted the accumulation of trihydroxy flavonoids in Gujing and Siqiu tea leaves. The high astringency intensity in Gujing tea could be due to the high accumulation of proanthocyanidins and galloylquinic acid. The results of the present study may improve our understanding of the metabolic characteristics of each evolutionary group of species or varieties in the section Thea of the genus Camellia.

The Reference Genome of Tea Plant and Resequencing of 81 Diverse Accessions Provide Insights into Its Genome Evolution and Adaptation.

Tea plant is an important economic crop, which is used to produce the world's oldest and most widely consumed tea beverages. Here, we present a high-quality reference genome assembly of the tea plant (Camellia sinensis var. sinensis) consisting of 15 pseudo-chromosomes. LTR retrotransposons (LTR-RTs) account for 70.38% of the genome, and we present evidence that LTR-RTs play critical roles in genome size expansion and the transcriptional diversification of tea plant genes through preferential insertion in promoter regions and introns. Genes, particularly those coding for terpene biosynthesis proteins, associated with tea aroma and stress resistance were significantly amplified through recent tandem duplications and exist as gene clusters in tea plant genome. Phylogenetic analysis of the sequences of 81 tea plant accessions with diverse origins revealed three well-differentiated tea plant populations, supporting the proposition for the southwest origin of the Chinese cultivated tea plant and its later spread to western Asia through introduction. Domestication and modern breeding left significant signatures on hundreds of genes in the tea plant genome, particularly those associated with tea quality and stress resistance. The genomic sequences of the reported reference and resequenced tea plant accessions provide valuable resources for future functional genomics study and molecular breeding of improved cultivars of tea plants.

Transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root (Camellia sinensis L.).

Free amino acids, including theanine, glutamine and glutamate, contribute greatly to the pleasant taste and multiple health benefits of tea. Amino acids in tea plants are mainly synthesized in roots and transported to new shoots, which are significantly affected by nitrogen (N) level and forms. However, the regulatory amino acid metabolism genes have not been systemically identified in tea plants. Here, we investigated the dynamic changes of free amino acid contents in response to N deficiency and forms in tea plant roots, and systemically identified the genes associated amino acid contents in individual metabolism pathways. Our results showed that glutamate-derived amino acids are the most dynamic in response to various forms of N and N deficiency. We then performed transcriptomic analyses of roots treated with N deficiency and various forms of N, and differentially expressed amino acid metabolic genes in each pathway were identified. The analyses on expression patterns and transcriptional responses of metabolic genes to N treatments provided novel insights for the molecular basis of high accumulation of theanine in tea plant root. These analyses also identified potential regulatory genes in dynamic amino acid metabolism in tea plant root. Furthermore, our findings indicated that the dynamic expression levels of CsGDH, CsAlaDC, CsAspAT, CsSDH, CsPAL, CsSHMT were highly correlated with changes of amino acid contents in their corresponding pathways. Herein, this study provides comprehensive insights into transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root.

Optimized sequencing depth and de novo assembler for deeply reconstructing the transcriptome of the tea plant, an economically important plant species.

BACKGROUND: Tea is the oldest and among the world's most popular non-alcoholic beverages, which has important economic, health and cultural values. Tea is commonly produced from the leaves of tea plants (Camellia sinensis), which belong to the genus Camellia of family Theaceae. In the last decade, many studies have generated the transcriptomes of tea plants at different developmental stages or under abiotic and/or biotic stresses to investigate the genetic basis of secondary metabolites that determine tea quality. However, these results exhibited large differences, particularly in the total number of reconstructed transcripts and the quality of the assembled transcriptomes. These differences largely result from limited knowledge regarding the optimized sequencing depth and assembler for transcriptome assembly of structurally complex plant species genomes. RESULTS: We employed different amounts of RNA-sequencing data, ranging from 4 to 84 Gb, to assemble the tea plant transcriptome using five well-known and representative transcript assemblers. Although the total number of assembled transcripts increased with increasing sequencing data, the proportion of unassembled transcripts became saturated as revealed by plant BUSCO datasets. Among the five representative assemblers, the Bridger package shows the best performance in both assembly completeness and accuracy as evaluated by the BUSCO datasets and genome alignment. In addition, we showed that Bridger and BinPacker harbored the shortest runtimes followed by SOAPdenovo and Trans-ABySS. CONCLUSIONS: The present study compares the performance of five representative transcript assemblers and investigates the key factors that affect the assembly quality of the transcriptome of the tea plants. This study will be of significance in helping the tea research community obtain better sequencing and assembly of tea plant transcriptomes under conditions of interest and may thus help to answer major biological questions currently facing the tea industry.

The tea plant reference genome and improved gene annotation using long-read and paired-end sequencing data.

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.

Tea Plant Information Archive: a comprehensive genomics and bioinformatics platform for tea plant.

Tea is the world's widely consumed nonalcohol beverage with essential economic and health benefits. Confronted with the increasing large-scale omics-data set particularly the genome sequence released in tea plant, the construction of a comprehensive knowledgebase is urgently needed to facilitate the utilization of these data sets towards molecular breeding. We hereby present the first integrative and specially designed web-accessible database, Tea Plant Information Archive (TPIA; http://tpia.teaplant.org). The current release of TPIA employs the comprehensively annotated tea plant genome as framework and incorporates with abundant well-organized transcriptomes, gene expressions (across species, tissues and stresses), orthologs and characteristic metabolites determining tea quality. It also hosts massive transcription factors, polymorphic simple sequence repeats, single nucleotide polymorphisms, correlations, manually curated functional genes and globally collected germplasm information. A variety of versatile analytic tools (e.g. JBrowse, blast, enrichment analysis, etc.) are established helping users to perform further comparative, evolutionary and functional analysis. We show a case application of TPIA that provides novel and interesting insights into the phytochemical content variation of section Thea of genus Camellia under a well-resolved phylogenetic framework. The constructed knowledgebase of tea plant will serve as a central gateway for global tea community to better understand the tea plant biology that largely benefits the whole tea industry.

Metabolomics and Transcriptomics Analyses Reveal Nitrogen Influences on the Accumulation of Flavonoids and Amino Acids in Young Shoots of Tea Plant ( Camellia sinensis L.) Associated with Tea Flavor.

Tea-specialized metabolites contribute to rich flavors and healthy function of tea. Their accumulation patterns and underlying regulatory mechanism are significantly different under different nitrogen (N) conditions during adaptation stage. Here, we find that flavonoids associated with tea flavor are dominated by different metabolic and transcriptional responses among the four N conditions (N-deficiency, nitrate, ammonia, and nitric oxide). Nitrogen-deficiency tea plants accumulate diverse flavonoids, corresponding with higher expression of hub genes including F3H, FNS, UFGT, bHLH35, and bHLH36. Compared with N-deficiency, N-supply tea plants significantly increase proline, glutamine, and theanine, which are also associated with tea flavor, especially under NH4+-supply. As NH4+-tolerant species, tea plant exploits the adaptive strategy by substantial accumulation of amino acids including theanine to adapt excess NH4+, which attributes to, at least in part, efficient N transport and assimilation, and active protein degradation. A distinct divergence of N reallocation in young shoots of tea plant under different N sources contributes to diverse tea flavor.

Gene co-expression network analysis reveals coordinated regulation of three characteristic secondary biosynthetic pathways in tea plant (Camellia sinensis).

BACKGROUND: The leaves of tea plants (Camellia sinensis) are used to produce tea, which is one of the most popular beverages consumed worldwide. The nutritional value and health benefits of tea are mainly related to three abundant characteristic metabolites; catechins, theanine and caffeine. Weighted gene co-expression network analysis (WGCNA) is a powerful system for investigating correlations between genes, identifying modules among highly correlated genes, and relating modules to phenotypic traits based on gene expression profiling. Currently, relatively little is known about the regulatory mechanisms and correlations between these three secondary metabolic pathways at the omics level in tea. RESULTS: In this study, levels of the three secondary metabolites in ten different tissues of tea plants were determined, 87,319 high-quality unigenes were assembled, and 55,607 differentially expressed genes (DEGs) were identified by pairwise comparison. The resultant co-expression network included 35 co-expression modules, of which 20 modules were significantly associated with the biosynthesis of catechins, theanine and caffeine. Furthermore, we identified several hub genes related to these three metabolic pathways, and analysed their regulatory relationships using RNA-Seq data. The results showed that these hub genes are regulated by genes involved in all three metabolic pathways, and they regulate the biosynthesis of all three metabolites. It is notable that light was identified as an important regulator for the biosynthesis of catechins. CONCLUSION: Our integrated omics-level WGCNA analysis provides novel insights into the potential regulatory mechanisms of catechins, theanine and caffeine metabolism, and the identified hub genes provide an important reference for further research on the molecular biology of tea plants.

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

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.

The Tea Tree Genome Provides Insights into Tea Flavor and Independent Evolution of Caffeine Biosynthesis.

Tea is the world's oldest and most popular caffeine-containing beverage with immense economic, medicinal, and cultural importance. Here, we present the first high-quality nucleotide sequence of the repeat-rich (80.9%), 3.02-Gb genome of the cultivated tea tree Camellia sinensis. We show that an extraordinarily large genome size of tea tree is resulted from the slow, steady, and long-term amplification of a few LTR retrotransposon families. In addition to a recent whole-genome duplication event, lineage-specific expansions of genes associated with flavonoid metabolic biosynthesis were discovered, which enhance catechin production, terpene enzyme activation, and stress tolerance, important features for tea flavor and adaptation. We demonstrate an independent and rapid evolution of the tea caffeine synthesis pathway relative to cacao and coffee. A comparative study among 25 Camellia species revealed that higher expression levels of most flavonoid- and caffeine- but not theanine-related genes contribute to the increased production of catechins and caffeine and thus enhance tea-processing suitability and tea quality. These novel findings pave the way for further metabolomic and functional genomic refinement of characteristic biosynthesis pathways and will help develop a more diversified set of tea flavors that would eventually satisfy and attract more tea drinkers worldwide.