Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant.

Tea is known for having a high catechin content, with the main component being (-)-epigallocatechin gallate (EGCG), which has significant bioactivities, including potential anti-cancer and anti-inflammatory activity. The poor intestinal stability and permeability of EGCG, however, undermine these health-improving benefits. O-methylated EGCG derivatives, found in a few tea cultivars in low levels, have attracted considerable interest due to their increased bioavailability. Here, we identify two O-methyltransferases from tea plant: CsFAOMT1 that has a specific O-methyltransferase activity on the 3''-position of EGCG to generate EGCG3''Me, and CsFAOMT2 that predominantly catalyzes the formation of EGCG4″Me. In different tea tissues and germplasms, the transcript levels of CsFAOMT1 and CsFAOMT2 are strongly correlated with the amounts of EGCG3''Me and EGCG4''Me, respectively. Furthermore, the crystal structures of CsFAOMT1 and CsFAOMT2 reveal the key residues necessary for 3''- and 4''-O-methylation. These findings may provide guidance for the future development of tea cultivars with high O-methylated catechin content.

Identifying Conserved Functional Gene Modules Underlying the Dynamic Regulation of Tea Plant Development and Secondary Metabolism.

Tea plants adjust development and metabolism by integrating environmental and endogenous signals in complex but poorly defined gene networks. Here, we present an integrative analysis framework for the identification of conserved modules controlling important agronomic traits using a comprehensive collection of RNA-seq datasets in Camellia plants including 189 samples. In total, 212 secondary metabolism-, 182 stress response-, and 182 tissue development-related coexpressed modules were revealed. Functional modules (e.g., drought response, theobromine biosynthesis, and new shoot development-related modules) and potential regulators that were highly conserved across diverse genetic backgrounds and/or environmental conditions were then identified by cross-experiment comparisons and consensus clustering. Moreover, we investigate the preservation of gene networks between Camellia sinensis and other Camellia species. This revealed that the coexpression patterns of several recently evolved modules related to secondary metabolism and environmental adaptation were rewired and showed higher connectivity in tea plants. These conserved modules are excellent candidates for modeling the core mechanism of tea plant development and secondary metabolism and should serve as a great resource for hypothesis generation and tea quality improvement.

Repressed Gene Expression of Photosynthetic Antenna Proteins Associated with Yellow Leaf Variation as Revealed by Bulked Segregant RNA-seq in Tea Plant Camellia sinensis.

The young leaves and shoots of albino tea cultivars are usually characterized as having a yellow or pale color, high amino acid, and low catechin. Increasing attention has been paid to albino tea cultivars in recent years because their tea generally shows high umami and reduced astringency. However, the genetic mechanism of yellow-leaf variation in albino tea cultivar has not been elucidated clearly. In this study, bulked segregant RNA-seq (BSR-seq) was performed on bulked yellow- and green-leaf hybrid progenies from a leaf color variation population. A total of 359 and 1134 differentially expressed genes (DEGs) were identified in the yellow and green hybrid bulked groups (Yf vs Gf) and parent plants (Yp vs Gp), respectively. The significantly smaller number of DEGs in Yf versus Gf than in Yp versus Gp indicated that individual differences could be reduced within the same hybrid progeny. Analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes revealed that the photosynthetic antenna protein was most significantly enriched in either the bulked groups or their parents. Interaction was found among light-harvesting chlorophyll a/b -binding proteins (LHC), heat shock proteins (HSPs), and enzymes involved in cuticle formation. Combined with the transcriptomic expression profile, results showed that the repressed genes encoding LHC were closely linked to aberrant chloroplast development in yellow-leaf tea plants. Furthermore, the photoprotection and light stress response possessed by genes involved in HSP protein interaction and cuticle formation were discussed. The expression profile of DEGs was verified via quantitative real-time PCR analysis of the bulked samples and other F1 individuals. In summary, using BSR-seq on a hybrid population eliminated certain disturbing effects of genetic background and individual discrepancy, thereby helping this study to intensively focus on the key genes controlling leaf color variation in yellow-leaf tea plants.

Baiyacha, a wild tea plant naturally occurring high contents of theacrine and 3″-methyl-epigallocatechin gallate from Fujian, China.

Baiyacha (BYC) is a kind of wild tea plant growing and utilizing in the remote mountain area of Fujian province, Southeastern China. However, scientific studies on this plant remain limited. Our results showed that BYC exhibits the typical morphological characteristics of Camellia gymnogyna Chang, a closely related species of C. sinensis (L.) O. Kuntze, which was not found in Fujian before. Chemical profiling revealed that parts of BYC plants are rich in purine alkaloids and catechins, especially featuring high levels of theacrine and 3″-methyl-epigallocatechin gallate (EGCG3″Me), chemical compounds with multiple biological activities that are rarely observed in regular tea plants. The contents of EGCG3″Me and theacrine in BYC both increased with the leaf maturity of tea shoots, whereas the caffeine content decreased significantly. The obtained results provide abundant information about the morphology and chemical compounds of BYC and may be used for tea production, breeding, and scientific research in the future.

Gene Coexpression Networks Reveal Key Drivers of Flavonoid Variation in Eleven Tea Cultivars (Camellia sinensis).

Following the recent completion of the draft genome sequence of the tea plant, high-throughput decoding of gene function, especially for those involved in complex secondary metabolic pathways, has become a major challenge. Here, we profiled the metabolome and transcriptome of 11 tea cultivars, and then illustrated a weighted gene coexpression network analysis (WGCNA)-based system biological strategy to interpret metabolomic flux, predict gene functions, and mine key regulators involved in the flavonoid biosynthesis pathway. We constructed a multilayered regulatory network, which integrated the gene coexpression relationship with the microRNA target and promoter cis-regulatory element information. This allowed us to reveal new uncharacterized TFs (e.g., MADSs, WRKYs, and SBPs) and microRNAs (including 17 conserved and 15 novel microRNAs) that are potentially implicated in different steps of the catechin biosynthesis. Furthermore, we applied metabolic-signature-based association method to capture additional key regulators involved in catechin pathway. This provides important clues for the functional characterization of five SCPL1A acyltransferase family members, which might be implicated in the production balance of anthocyanins, galloylated catechins, and proanthocyanins. Application of an "omics"-based system biology strategy should facilitate germplasm utilization and provide valuable resources for tea quality improvement.

Isolation and Characterization of CsWRKY7, a Subgroup IId WRKY Transcription Factor from Camellia sinensis, Linked to Development in Arabidopsis.

WRKY transcription factors (TFs) containing one or two WRKY domains are a class of plant TFs that respond to diverse abiotic stresses and are associated with developmental processes. However, little has been known about the function of WRKY gene in tea plant. In this study, a subgroup IId WRKY gene CsWRKY7 was isolated from Camellia sinensis, which displayed amino acid sequence homology with Arabidopsis AtWRKY7 and AtWRKY15. Subcellular localization prediction indicated that CsWRKY7 localized to nucleus. Cis-acting elements detected in the promotor region of CsWRKY7 are mainly involved in plant response to environmental stress and growth. Consistently, expression analysis showed that CsWRKY7 transcripts responded to NaCl, mannitol, PEG, and diverse hormones treatments. Additionally, CsWRKY7 exhibited a higher accumulation both in old leaves and roots compared to bud. Seed germination and root growth assay indicated that overexpressed CsWRKY7 in transgenic Arabidopsis was not sensitive to NaCl, mannitol, PEG, and low concentration of ABA treatments. CsWRKY7 overexpressing Arabidopsis showed a late-flowering phenotype under normal conditions compared to wild type. Furthermore, gene expression analysis showed that the transcription levels of the flowering time integrator gene FLOWERING LOCUS T (FT) and the floral meristem identity genes APETALA1 (AP1) and LEAFY (LFY) were lower in WRKY7-OE than in the WT. Taken together, these findings indicate that CsWRKY7 TF may participate in plant growth. This study provides a potential strategy to breed late-blooming tea cultivar.

Quantitative Trait Loci Mapping for Theobromine and Caffeine Contents in Tea Plant ( Camellia sinensis).

Understanding the genetic basis of theobromine and caffeine accumulation in the tea plant is important due to their contribution to tea flavor. Quantitative trait loci (QTL) analyses were carried out to identify genetic variants associated with theobromine and caffeine contents and ratio using a pseudo-testcross population derived from an intervarietal cross between two varieties of Camellia sinensis. A total of 10 QTL controlling caffeine content (CAF), theobromine content (TBR), sum of caffeine and theobromine (SCT), and caffeine-to-theobromine ratio (CTR) were identified over four measurement years. The major QTL controlling CAF, qCAF1, was mapped onto LG01 and validated across years, explaining an average of 20.1% of the phenotypic variance. The other QTL were detected in 1 or 2 years, and of them there were four, two, and three for TBR, SCT, and CTR, respectively. The present results provide valuable information for further fine mapping and cloning functional genes and for genetic improvement in tea plant.

Hongyacha, a Naturally Caffeine-Free Tea Plant from Fujian, China.

Hongyacha (HYC) is a type of new wild tea plant discovered in Fujian Province, China. This tea is helpful to the healing or prevention of disease in its original growing area. However, research on this tea is limited. Our results showed that HYC displayed obvious differences in its morphological characteristics compared with Cocoa tea ( Camellia ptilophylla Chang), a famous caffeine-free tea plant in China. Theobromine and trans-catechins, but not caffeine and cis-catechins, were the dominant purine alkaloids and catechins detected in HYC. HYC might contain abundant gallocatechin-(4 → 8)-gallocatechin gallate, 1,3,4,6-tetra- O-galloyl-ß-d-glucopyranose, and (-)-gallocatechin-3,5-di- O-gallate, which were not detected in regular tea. We also found that the TCS1 of HYC was distinct, and the responding recombinant protein exhibited only theobromine synthase activity. The obtained results showed that HYC is a new kind of caffeine-free tea plant and may be used for scientific protection and efficient utilization in the future.

A Novel F3' 5' H Allele with 14 bp Deletion Is Associated with High Catechin Index Trait of Wild Tea Plants and Has Potential Use in Enhancing Tea Quality.

Catechins are important chemical components determining the quality of tea. The catechin index (CI, ratio of dihydroxylated catechin (DIC)/trihydroxylated catechin (TRIC)) in the green leaf has a major influence on the amounts of theaflavins in black tea. In this work, the major catechin profiles of wild tea plants originating from Guizhou Province with high CI trait were investigated. We identified a novel flavonoid 3',5' hydroxylase gene ( F3' 5' H) allele with a 14 bp deletion in the upstream regulation region and developed an insertion/deletion (InDel) marker accordingly. The 14 bp deletion in the novel  F3' 5' H allele was associated with low F3' 5' H mRNA expression, thereby resulting in low TRIC content and high CI value. The allelic variant in the novel F3' 5' H allele associated with high CI values and DIC contents was confirmed by the introgression lines derived from a distant cross population. The novel F3' 5' H allele in wild tea plants is a valuable gene resource, which could be applied to breeding improvement on tea quality.

Comprehensive Dissection of Metabolic Changes in Albino and Green Tea Cultivars.

Albino tea cultivars are special mutants of tea plants with white or yellow leaf color. In this study, three albino tea cultivars, including 'Anji Baicha', 'Huangjinya', and 'Baijiguan', and two green tea cultivars, 'Longjing 43' and 'Fuding Dabaicha', were applied to metabolite profiling by gas chromatography-mass spectrometry and ultraperformance liquid chromatography-mass spectrometry. Multivariate analyses revealed significantly different metabolite phenotypes in leaves among albino cultivars and green cultivars. The differential metabolite-related pathways included galactose metabolism, tryptophan metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. For the young leaves of albino cultivars, the sugar (sorbitol and erythrose) and amino acid (mainly proline, isoleucine, ornithine, aspartic acid, threonine, and valine) concentrations increased, whereas gallocatechin and epigallocatechin gallate concentrations decreased. These results reveal the divergence in metabolic profiling between tea plant cultivars with different leaf colors. With the development of leaves, the concentrations of flavonoids increased largely in the older leaves of albino cultivars.

Functional natural allelic variants of flavonoid 3',5'-hydroxylase gene governing catechin traits in tea plant and its relatives.

MAIN CONCLUSION: Functional allelic variants of the flavonoid 3',5'-hydroxylase (F3'5'H) gene provides new information of F3'5'H function of tea plant and its relatives. This insight may serve as the foundation upon which to advance molecular breeding in the tea plant. Catechins are the active components of tea that determine its quality and health attributes. This study established the first integrated genomic strategy for deciphering the genetic basis of catechin traits of tea plant. With the RNA-sequencing analysis of bulked segregants representing the tails of a F1 population segregated for total catechin content, we identified a flavonoid 3',5'-hydroxylase (F3'5'H) gene. F3'5'H had one copy in the genomic DNA of tea plant. Among 202 tea accessions, we identified 120 single nucleotide polymorphisms (SNPs) at F3'5'H locus. Seventeen significant marker-trait associations were identified by association mapping in multiple environments, which were involved in 10 SNP markers, and the traits including the ratio of di/tri-hydroxylated catechins and catechin contents. The associated individual and combination of SNPs explained 4.5-25.2 and 53.0-63.0% phenotypic variations, respectively. In the F1 population (validation population), the catechin trait variation percentages explained by F3'5'H diplotype were 6.9-74.3%. The genotype effects of ten functional SNPs in the F1 population were all consistent with the association population. Furthermore, the function of SNP-711/-655 within F3'5'H was validated by gene expression analysis. Altogether, our work indicated functional SNP allelic variants within F3'5'H governing the ratio of di/tri-hydroxylated catechins and catechin contents. The strong catechin-associated SNPs identified in this study can be used for future marker-assisted selection to improve tea quality.

Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in 'Anji Baicha' (Camellia sinensis).

BACKGROUND: The new shoots of the albino tea cultivar 'Anji Baicha' are yellow or white at low temperatures and turn green as the environmental temperatures increase during the early spring. 'Anji Baicha' metabolite profiles exhibit considerable variability over three color and developmental stages, especially regarding the carotenoid, chlorophyll, and theanine concentrations. Previous studies focused on physiological characteristics, gene expression differences, and variations in metabolite abundances in albino tea plant leaves at specific growth stages. However, the molecular mechanisms regulating metabolite biosynthesis in various color and developmental stages in albino tea leaves have not been fully characterized. RESULTS: We used RNA-sequencing to analyze 'Anji Baicha' leaves at the yellow-green, albescent, and re-greening stages. The leaf transcriptomes differed considerably among the three stages. Functional classifications based on Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed unigenes were mainly related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and carbon fixation in photosynthetic organisms. Chemical analyses revealed higher ß-carotene and theanine levels, but lower chlorophyll a levels, in the albescent stage than in the green stage. Furthermore, unigenes involved in carotenoid, chlorophyll, and theanine biosyntheses were identified, and the expression patterns of the differentially expressed unigenes in these biosynthesis pathways were characterized. Through co-expression analyses, we identified the key genes in these pathways. These genes may be responsible for the metabolite biosynthesis differences among the different leaf color and developmental stages of 'Anji Baicha' tea plants. CONCLUSIONS: Our study presents the results of transcriptomic and biochemical analyses of 'Anji Baicha' tea plants at various stages. The distinct transcriptome profiles for each color and developmental stage enabled us to identify changes to biosynthesis pathways and revealed the contributions of such variations to the albino phenotype of tea plants. Furthermore, comparisons of the transcriptomes and related metabolites helped clarify the molecular regulatory mechanisms underlying the secondary metabolic pathways in different stages.

Association mapping of caffeine content with TCS1 in tea plant and its related species.

Caffeine is the most abundant purine alkaloid in majority of tea plant and its related species. This purine alkaloid contributes to the important flavor and health attributes of tea. Tea caffeine synthase 1 (TCS1, EC 2.1.1.159/2.1.1.160) gene plays a crucial role in caffeine biosynthesis. The objective of this study was to investigate the genetic relationship between the TCS1 and caffeine content of tea plant and its related species using association mapping. We identified 87 single-nucleotide polymorphisms (SNPs, π = 0.00447) by resequencing the TCS1 locus of 44 tea accessions. Linkage disequilibrium (LD) analysis showed that LD did not extend over the entire gene (r(2) < 0.1, within 1000 bp). Two cleaved amplified polymorphism sequence (CAPS) markers were developed from sequence variations (SNP4318 and SNP6252). By association mapping, we identified SNP4318 associated with caffeine content in four environments, explaining 4.0%-7.7% of the phenotypic variance. We also validated the significant marker-trait associations in site-directed mutagenesis experiments. Examination of allelic variation and linkage disequilibrium by a candidate-gene-based approach can help to decipher the genetic basis of caffeine biosynthesis. Moreover, the SNP marker identified in this study can potentially be applied for future marker-assisted selection to improve tea quality.

Natural allelic variations of TCS1 play a crucial role in caffeine biosynthesis of tea plant and its related species.

Tea caffeine synthase 1 (TCS1) is an enzyme that catalyzes the methylation of N-3 and N-1 and considered to be the most critical enzyme in the caffeine biosynthetic pathway of tea plant. This study shows that TCS1 has six types of allelic variations, namely, TCS1a, TCS1b, TCS1c, TCS1d, TCS1e, and TCS1f, with a 252 bp insertion/deletion mutation in the 5'-untranslated region. Among tea plant and its related species, TCS1a is the predominant allele, and TCS1b-f are the rare alleles that mainly appear in few wild germplasms. The full-length cDNA sequences of three new alleles, namely, TCS1d, TCS1e, and TCS1f, were isolated from specific germplasms, and all of recombinant proteins have higher caffeine synthase (CS, EC 2.1.1.160) activity than theobromine synthase (TS, EC 2.1.1.159). Amino acid residue 269 is responsible for the difference in TCS activity and substrate recognition, which was demonstrated by using site-directed mutagenesis experiments. Furthermore, natural variations in TCS1 change the transcription levels. There are two molecular mechanisms controlling the caffeine biosynthesis in low-caffeine-accumulating tea germplasms, i.e., TCS1 allele with low transcription level or its encoded protein with only TS activity. Allelic variations of TCS1 play a crucial role in caffeine biosynthesis. Taken together, our work provides valuable foundation for a comprehensive understanding of the mechanism of caffeine biosynthesis in section Thea plants and useful guidance for effective breeding.

Transcriptomic Analysis of Tea Plant Responding to Drought Stress and Recovery.

Tea plant (Camellia sinensis) is an economically important beverage crop. Drought stress (DS) seriously limits the growth and development of tea plant, thus affecting crop yield and quality. To elucidate the molecular mechanisms of tea plant responding to DS, we performed transcriptomic analysis of tea plant during the three stages [control (CK) and during DS, and recovery (RC) after DS] using RNA sequencing (RNA-Seq). Totally 378.08 million high-quality trimmed reads were obtained and assembled into 59,674 unigenes, which were extensively annotated. There were 5,955 differentially expressed genes (DEGs) among the three stages. Among them, 3,948 and 1,673 DEGs were up-regulated under DS and RC, respectively. RNA-Seq data were further confirmed by qRT-PCR analysis. Genes involved in abscisic acid (ABA), ethylene, and jasmonic acid biosynthesis and signaling were generally up-regulated under DS and down-regulated during RC. Tea plant potentially used an exchange pathway for biosynthesis of indole-3-acetic acid (IAA) and salicylic acid under DS. IAA signaling was possibly decreased under DS but increased after RC. Genes encoding enzymes involved in cytokinin synthesis were up-regulated under DS, but down-regulated during RC. It seemed probable that cytokinin signaling was slightly enhanced under DS. In total, 762 and 950 protein kinases belonging to 26 families were differentially expressed during DS and RC, respectively. Overall, 547 and 604 transcription factor (TF) genes belonging to 58 families were induced in the DS vs. CK and RC vs. DS libraries, respectively. Most members of the 12 TF families were up-regulated under DS. Under DS, genes related to starch synthesis were down-regulated, while those related to starch decomposition were up-regulated. Mannitol, trehalose and sucrose synthesis-related genes were up-regulated under DS. Proline was probably mainly biosynthesized from glutamate under DS and RC. The mechanism by which ABA regulated stomatal movement under DS and RC was partly clarified. These results document the global and novel responses of tea plant during DS and RC. These data will serve as a valuable resource for drought-tolerance research and will be useful for breeding drought-resistant tea cultivars.

Differential Metabolic Profiles during the Albescent Stages of 'Anji Baicha' (Camellia sinensis).

'Anji Baicha' is an albino tea cultivar with white shoots at low air temperature and green shoots at high air temperature in early spring. The metabolite contents in the shoots dynamically vary with the color changes and with shoot development. To investigate the metabolomic variation during the albescent and re-greening stages, gas chromatography-mass spectrometry combined with multivariate analysis were applied to analyze the metabolite profiles in the different color stages during the development of 'Anji Baicha' leaves. The metabolite profiles of three albescent stages, including the yellow-green stage, the early albescent stage, and the late albescent stage, as well as the re-greening stage were distinguished using principal component analysis, revealing that the distinct developmental stages were likely responsible for the observed metabolic differences. Furthermore, a group classification and pairwise discrimination was revealed among the three albescent stages and re-greening stage by partial least squares discriminant analysis. A total of 65 differential metabolites were identified with a variable influence on projection greater than 1. The main differential metabolic pathways of the albescent stages compared with the re-greening stage included carbon fixation in photosynthetic organisms and the phenylpropanoid and flavonoid biosynthesis pathways. Compared with the re-greening stage, the carbohydrate and amino acid metabolic pathways were disturbed during the albescent stages. During the albescent stages, the sugar (fructofuranose), sugar derivative (glucose-1-phosphate) and epicatechin concentrations decreased, whereas the amino acid (mainly glycine, serine, tryptophan, citrulline, glutamine, proline, and valine) concentrations increased. These results reveal the changes in metabolic profiling that occur during the color changes associated with the development of the albino tea plant leaves.

Global transcriptome and gene regulation network for secondary metabolite biosynthesis of tea plant (Camellia sinensis).

BACKGROUND: Major secondary metabolites, including flavonoids, caffeine, and theanine, are important components of tea products and are closely related to the taste, flavor, and health benefits of tea. Secondary metabolite biosynthesis in Camellia sinensis is differentially regulated in different tissues during growth and development. Until now, little was known about the expression patterns of genes involved in secondary metabolic pathways or their regulatory mechanisms. This study aimed to generate expression profiles for C. sinensis tissues and to build a gene regulation model of the secondary metabolic pathways. RESULTS: RNA sequencing was performed on 13 different tissue samples from various organs and developmental stages of tea plants, including buds and leaves of different ages, stems, flowers, seeds, and roots. A total of 43.7 Gbp of raw sequencing data were generated, from which 347,827 unigenes were assembled and annotated. There were 46,693, 8446, 3814, 10,206, and 4948 unigenes specifically expressed in the buds and leaves, stems, flowers, seeds, and roots, respectively. In total, 1719 unigenes were identified as being involved in the secondary metabolic pathways in C. sinensis, and the expression patterns of the genes involved in flavonoid, caffeine, and theanine biosynthesis were characterized, revealing the dynamic nature of their regulation during plant growth and development. The possible transcription factor regulation network for the biosynthesis of flavonoid, caffeine, and theanine was built, encompassing 339 transcription factors from 35 families, namely bHLH, MYB, and NAC, among others. Remarkably, not only did the data reveal the possible critical check points in the flavonoid, caffeine, and theanine biosynthesis pathways, but also implicated the key transcription factors and related mechanisms in the regulation of secondary metabolite biosynthesis. CONCLUSIONS: Our study generated gene expression profiles for different tissues at different developmental stages in tea plants. The gene network responsible for the regulation of the secondary metabolic pathways was analyzed. Our work elucidated the possible cross talk in gene regulation between the secondary metabolite biosynthetic pathways in C. sinensis. The results increase our understanding of how secondary metabolic pathways are regulated during plant development and growth cycles, and help pave the way for genetic selection and engineering for germplasm improvement.

Large-Scale SNP Discovery and Genotyping for Constructing a High-Density Genetic Map of Tea Plant Using Specific-Locus Amplified Fragment Sequencing (SLAF-seq).

Genetic maps are important tools in plant genomics and breeding. The present study reports the large-scale discovery of single nucleotide polymorphisms (SNPs) for genetic map construction in tea plant. We developed a total of 6,042 valid SNP markers using specific-locus amplified fragment sequencing (SLAF-seq), and subsequently mapped them into the previous framework map. The final map contained 6,448 molecular markers, distributing on fifteen linkage groups corresponding to the number of tea plant chromosomes. The total map length was 3,965 cM, with an average inter-locus distance of 1.0 cM. This map is the first SNP-based reference map of tea plant, as well as the most saturated one developed to date. The SNP markers and map resources generated in this study provide a wealth of genetic information that can serve as a foundation for downstream genetic analyses, such as the fine mapping of quantitative trait loci (QTL), map-based cloning, marker-assisted selection, and anchoring of scaffolds to facilitate the process of whole genome sequencing projects for tea plant.

Determination of catechin content in representative Chinese tea germplasms.

To understand tea germplasms better and to use them effectively for production and breeding, the catechin content of 403 accessions of representative tea germplasms collected from various locations in China were studied using HPLC. The catechin content of these tea germplasms varied from 56.6 to 231.9 mg/g and averaged 154.5 ± 18.1 mg/g. One germplasm with low total catechin (TC) content (<60 mg/g) and three with high TC (>200 mg/g) contents were found. Averages of the TC content of the three varieties of Camellia sinensis (L.) O. Kuntze, namely, sinensis, assamica, and pubilimba, were 152.9 ± 16.2 mg/g, 162.8 ± 22.3 mg/g, and 165.1 ± 21.3 mg/g, respectively. The TC content of the sinensis variety was significantly lower (P < 0.05) than that of the other two varieties. The assamica variety had the highest levels of (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC), whereas the pubilimba variety had the highest levels of (-)-epigallocatechin gallate (EGCG), (+)-gallocatechin (GC), (+)-catechin (C), and (-)-gallocatechin gallate (GCG). Factor analysis indicated that GC, C, GCG, catechin index, and ECG greatly influenced the classification. The TC content of germplasms collected from the various provinces showed significant differences (P < 0.05). Tea germplasms of the southern provinces had higher degrees of variation in TC.

Construction of a SSR-based genetic map and identification of QTLs for catechins content in tea plant (Camellia sinensis).

Catechins are the most important bioactive compounds in tea, and have been demonstrated to possess a wide variety of pharmacological activities. To characterize quantitative trait loci (QTLs) for catechins content in the tender shoots of tea plant, we constructed a moderately saturated genetic map using 406 simple sequence repeat (SSR) markers, based on a pseudo-testcross population of 183 individuals derived from an intraspecific cross of two Camellia sinensis varieties with diverse catechins composition. The map consisted of fifteen linkage groups (LGs), corresponding to the haploid chromosome number of tea plant (2n = 2x = 30). The total map length was 1,143.5 cM, with an average locus spacing of 2.9 cM. A total of 25 QTLs associated with catechins content were identified over two measurement years. Of these, nine stable QTLs were validated across years, and clustered into four main chromosome regions on LG03, LG11, LG12 and LG15. The population variability explained by each QTL was predominantly at moderate-to-high levels and ranged from 2.4% to 71.0%, with an average of 17.7%. The total number of QTL for each trait varied from four to eight, while the total population variability explained by all QTLs for a trait ranged between 38.4% and 79.7%. This is the first report on the identification of QTL for catechins content in tea plant. The results of this study provide a foundation for further cloning and functional characterization of catechin QTLs for utilization in improvement of tea plant.