Single-cell transcriptome atlas reveals developmental trajectories and a novel metabolic pathway of catechin esters in tea leaves.

The tea plant is an economically important woody beverage crop. The unique taste of tea is evoked by certain metabolites, especially catechin esters, whereas their precise formation mechanism in different cell types remains unclear. Here, a fast protoplast isolation method was established and the transcriptional profiles of 16 977 single cells from 1st and 3rd leaves were investigated. We first identified 79 marker genes based on six isolated tissues and constructed a transcriptome atlas, mapped developmental trajectories and further delineated the distribution of different cell types during leaf differentiation and genes associated with cell fate transformation. Interestingly, eight differently expressed genes were found to co-exist at four branch points. Genes involved in the biosynthesis of certain metabolites showed cell- and development-specific characteristics. An unexpected catechin ester glycosyltransferase was characterized for the first time in plants by a gene co-expression network in mesophyll cells. Thus, the first single-cell transcriptional landscape in woody crop leave was reported and a novel metabolism pathway of catechin esters in plants was discovered.

Menghai Huangye, a novel albino tea germplasm with high theanine content and a high catechin index.

Yunnan Province has a very wide diversity of tea germplasm resources. A variety of special tea germplasms with outstanding traits have been discovered, including tea germplasms with high anthocyanin content and low caffeine content. Albino tea cultivars generally have higher contents of theanine that contribute to the umami taste, and the quality of tea brewed from it is higher. The catechin index (CI), the ratio of dihydroxylated catechins (DIC) to trihydroxylated catechins (TRIC), is a crucial index of suitability for processing tea. In this study, the albino tea plant Menghai Huangye (MHHY) with yellow leaves was identified. Analysis of the biochemical components revealed that MHHY was enriched in theanine and the total catechins (TC) were lower than Yunkang 10 (YK10). In addition, the CI value of MHHY was extremely significantly higher than that of YK10. Metabolic profile of catechins and the related gene expression profile analysis found that the coordinated expression of the key branch genes F3'H and F3'5'Ha for the synthesis of DIC and TRIC in tea plant was closely related to the high CI and low TC of MHHY. Further analysis of the F3'H promoter showed that a 284-bp deletion mutation was present in the F3'H promoter of MHHY, containing the binding sites of the transcriptional repressor MYB4 involved in flavonoid metabolism, which might be an important reason for the up-regulated expression of F3'H in MHHY. Overall, this study provides a theoretical basis for understanding the characteristics of albino tea germplasm resources and efficiently utilizing high-CI tea germplasm resources.

Genome-Wide Identification and Expression Patterns of the C2H2-Zinc Finger Gene Family Related to Stress Responses and Catechins Accumulation in Camellia sinensis [L.] O. Kuntze.

The C2H2-zinc finger protein (C2H2-ZFP) is essential for the regulation of plant development and widely responsive to diverse stresses including drought, cold and salt stress, further affecting the late flavonoid accumulation in higher plants. Tea is known as a popular beverage worldwide and its quality is greatly dependent on the physiological status and growing environment of the tea plant. To date, the understanding of C2H2-ZFP gene family in Camellia sinensis [L.] O. Kuntze is not yet available. In the present study, 134 CsC2H2-ZFP genes were identified and randomly distributed on 15 chromosomes. The CsC2H2-ZFP gene family was classified into four clades and gene structures and motif compositions of CsC2H2-ZFPs were similar within the same clade. Segmental duplication and negative selection were the main forces driving the expansion of the CsC2H2-ZFP gene family. Expression patterns suggested that CsC2H2-ZFPs were responsive to different stresses including drought, salt, cold and methyl jasmonate (MeJA) treatment. Specially, several C2H2-ZFPs showed a significant correlation with the catechins content and responded to the MeJA treatment, which might contribute to the tea quality and specialized astringent taste. This study will lay the foundations for further research of C2H2-type zinc finger proteins on the stress responses and quality-related metabolites accumulation in C. sinensis.

Epigallocatechin-3-gallate activates the AMP-activated protein kinase signaling pathway to reduce lipid accumulation in canine hepatocytes.

Epigallocatechin-3-gallate (EGCG) plays a crucial role in hepatic lipid metabolism. However, the underlying regulatory mechanism of hepatic lipid metabolism by EGCG in canine is unclear. Primary canine hepatocytes were treated with EGCG (0.01, 0.1, or 1 µM) and BML-275 (an AMP-activated protein kinase [AMPK] inhibitor) to study the effects of EGCG on the gene and protein expressions associated with AMPK signaling pathway. Data showed that treatment with EGCG had greater activation of AMPK, as well as greater expression levels and transcriptional activity of peroxisome proliferator activated receptor-α (PPARα) along with upregulated messenger RNA (mRNA) abundance and protein abundance of PPARα-target genes. EGCG decreased the expression levels and transcriptional activity of sterol regulatory element-binding protein 1c (SREBP-1c) along with downregulated mRNA abundance and protein abundance of SREBP-1c target genes. Of particular interest, exogenous BML-275 could reduce or eliminate the effects of EGCG on lipid metabolism in canine hepatocytes. Furthermore, the content of triglyceride was significantly decreased in the EGCG-treated groups. These results suggest that EGCG might be a potential agent in preventing high-fat diet-induced lipid accumulation in small animals.

Multiple-genotypes transcriptional analysis revealed candidates genes and nucleotide variants for improvement of quality characteristics in tea (Camellia sinensis (L.) O. Kuntze).

Tea quality is a polygenic trait that exhibits tremendous genetic variability due to accumulation of array of secondary metabolites. To elucidate global molecular insights controlling quality attributes, metabolite profiling and transcriptome sequencing of twelve diverse tea cultivars was performed in tea shoots harvested during quality season. RP-HPLC-DAD analysis of quality parameters revealed significant difference in catechins, theanine and caffeine contents. Transcriptome sequencing resulted into 50,107 non-redundant transcripts with functional annotations of 81.6% (40,847) of the transcripts. Interestingly, 2872 differentially expressed transcripts exhibited significant enrichment in 38 pathways (FDR ≤ 0.05) including secondary metabolism, amino acid and carbon metabolism. Thirty-eight key candidates reportedly involved in biosynthesis of fatty acid derived volatiles, volatile terpenes, glycoside hydrolysis and key quality related pathways (flavonoid, caffeine and theanine-biosynthesis) were highly expressed in catechins-rich tea cultivars. Furthermore, enrichment of candidates involved in flavonoid biosynthesis, transcriptional regulation, volatile terpene and biosynthesis of fatty acid derived volatile in Protein-Protein Interactome network revealed well-coordinated regulation of quality characteristics in tea. Additionally, ascertainment of 23,649 non-synonymous SNPs and validation of candidate SNPs present in quality related genes suggests their potential utility in genome-wide mapping and marker development for expediting breeding of elite compound-rich tea cultivars.

Transcriptomic analysis of tea plant (Camellia sinensis) revealed the co-expression network of 4111 paralogous genes and biosynthesis of quality-related key metabolites under multiple stresses.

The tea plant is an essential economic plant in many countries. However, its growing season renders them vulnerable to stresses. To understand the transcriptomic influences of these stresses on tea plants, we sequenced and analyzed the transcriptomes under drought, high-temperature, and pest. Paralogs were identified by comparing 14 evolutionarily close genomes. The differentially expressed paralog (DEPs) genes were analyzed regarding single or multiple stresses, and 1075 of the 4111 DEPs were commonly found in all the stresses. The co-expression network of the DEPs and TFs indicated that genes of catechin biosynthesis were associated with most transcription factors specific to each stress. The genes playing a significant role in the late response to drought and pest stress mainly functioned in the early response to high-temperature. This study revealed the relationship between stress and regulation of QRM synthesis and the role of QRMs in response to these (a)biotic stresses.

Integrative Transcriptomic and Metabolic Analyses Provide Insights into the Role of Trichomes in Tea Plant (Camellia Sinensis).

Trichomes, which develop from epidermal cells, are regarded as one of the key features that are involved in the evaluation of tea quality and tea germplasm resources. The metabolites from trichomes have been well characterized in tea products. However, little is known regarding the metabolites in fresh tea trichomes and the molecular differences in trichomes and tea leaves per se. In this study, we developed a method to collect trichomes from tea plant tender shoots, and their main secondary metabolites, including catechins, caffeine, amino acids, and aroma compounds, were determined. We found that the majority of these compounds were significantly less abundant in trichomes than in tea leaves. RNA-Seq was used to investigate the differences in the molecular regulatory mechanism between trichomes and leaves to gain further insight into the differences in trichomes and tea leaves. In total, 52.96 Gb of clean data were generated, and 6560 differentially expressed genes (DEGs), including 4471 upregulated and 2089 downregulated genes, were identified in the trichomes vs. leaves comparison. Notably, the structural genes of the major metabolite biosynthesis pathways, transcription factors, and other key DEGs were identified and comparatively analyzed between trichomes and leaves, while trichome-specific genes were also identified. Our results provide new insights into the differences between tea trichomes and leaves at the metabolic and transcriptomic levels, and open up new doors to further recognize and re-evaluate the role of trichomes in tea quality formation and tea plant growth and development.

Preferential assimilation of NH4+ over NO3- in tea plant associated with genes involved in nitrogen transportation, utilization and catechins biosynthesis.

Physiological effects of ammonium (NH4+) and nitrate (NO3-) on tea have confirmed that tea plants prefer NH4+ as the dominant nitrogen (N) source. To investigate the possible explanations for this preference, studies of 15NH4+ and 15NO3- assimilation using hydroponically grown tea plants were conducted. During the time course of 15NH4+ and 15NO3- assimilation, the absorption of 15N from 15NH4+ was more rapid than that from 15NO3-, as there was a more efficient expression pattern of NH4+ transporters compared with that of NO3- transporters. 15NH4+-fed tea plants accumulated more 15N than 15NO3- fed plants, which was demonstrated by that genes related to primary N assimilation, like CsNR, CsNiR, CsGDH and CsGOGAT, were more affected by 15NH4+ than 15NO3-. Markedly higher NH4+ concentrations were observed in 15NH4+-fed tea roots in comparison with NO3- treatment, whereas tea plants maintained a balanced concentration of NH4+ in tea leaves under both these two N forms. This maintenance was achieved through the increased expression of genes involved in theanine biosynthesis and the inhibition of genes related to catechins derived from phenylpropanoid pathway. The current results suggest that efficient NH4+ transportation, assimilation, and reutilization enables tea plant as an ammonium preferring plant species.

Discerning between Two Tuscany (Italy) Ancient Apple cultivars, 'Rotella' and 'Casciana', through Polyphenolic Fingerprint and Molecular Markers.

Ancient apple cultivars usually have higher nutraceutical value than commercial ones, but in most cases their variability in pomological traits does not allow us to discriminate among them. Fruit of two Tuscany ancient apple cultivars, 'Casciana' and 'Rotella', picked from eight different orchards (four for each cultivar) were analyzed for their pomological traits, organoleptic qualities, polyphenolic profile and antiradical activity. The effectiveness of a polyphenol-based cluster analysis was compared to molecular markers (internal transcribed spacers, ITS1 and ITS2) to unequivocally discern the two apples. 'Casciana' and 'Rotella' fruit had a higher nutraceutical value than some commercial cultivars, in terms of phenolic abundance, profile and total antiradical activity. Although pedo-climatic conditions of different orchards influenced the phenolic profile of both apples, the polyphenolic discriminant analysis clearly separated the two cultivars, principally due to higher amounts of procyanidin B2, procyanidin B3 and p-coumaroylquinic acid in 'Casciana' than in 'Rotella' fruit. These three polyphenols can be used proficiently as biochemical markers for distinguishing the two apples when pomological traits cannot. Conversely, ITS1 and ITS2 polymorphism did not allow us to distinguish 'Casciana' from 'Rotella' fruit. Overall, the use of polyphenolic fingerprint might represent a valid tool to ensure the traceability of products with a high economic value.

Characterization of the transcriptional regulator CsbHLH62 that negatively regulates EGCG3"Me biosynthesis in Camellia sinensis.

Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) in tea (Camellia sinensis (L.) O. Kuntze) is a major source of O-methylated catechin and renowned for a wide range of health effects. However, the transcriptional regulation mechanisms of EGCG3"Me biosynthesis remain unclear. In the present work, the basic Helix-Loop-Helix (bHLH) transcription factor, designated as CsbHLH62, belonging to GBOF group of bHLH families, was isolated and characterized from Camellia sinensis. CsbHLH62 contains an Open Reading Frame of 1662 bp and encodes a polypeptide of 553 amino acids. Subcellular location and transcriptional activity analysis showed it as a nucleus protein and possessed transcriptional inhibition activity. Furthermore, the expression of CsbHLH62 was decreased during EGCG3"Me accumulation. More importantly, E-box motifs (5'-CANNTG-3') were found in the promoters of CCoAOMT, CsLAR, and CsDFR, and further transient expression assays showed that CsbHLH62 repressed the transcription of CCoAOMT, CsLAR, and CsDFR. Collectively, these results suggest that CsbHLH62 acts as a transcriptional repressor that might be negatively affecting the accumulation of EGCG3"Me. These findings provide novel insights into the regulatory mechanism of EGCG3"Me biosynthesis, which might help to breed high EGCG3"Me-content tea plants.

The acaricidal activity and mechanism of eugenol on Psoroptes cuniculi.

In this study, the acaricidal effect of eugenol was measured and its mechanism of action investigated. The results showed that eugenol possessed the effect of killing Psoroptes cuniculi, and could regulate the mRNA expression of glutathione S-transferase (GST), catechinic acid (Ca) and thioredoxin (Trx). PPAR, NF-kappa B, TNF, Rap 1 and Ras signaling pathways might be the main pathways that involved into the process of killing mites. These findings suggested that eugenol could be developed into a new kind of acaricide, and further expand current knowledge on the mechanisms of eugenol for killing Psoroptes cuniculi of eugenol.

Knockdown of P120 catenin aggravates endothelial injury under an impinging flow by inducing breakdown of adherens junctions.

At present, the mechanisms underlying intracranial aneurysm (IA) development remain unclear; however, hemodynamics is considered a crucial factor in the induction of IA. To elucidate the association between hemodynamics and endothelial cell (EC) functions, a modified T chamber system was designed to simulate the adjustable hemodynamic conditions of an artery bifurcation. Normal human umbilical vein ECs (HUVECs) and HUVECs with P120 catenin (P120ctn) knockdown were cultured on coverslips and placed in the chamber. A flow rate of 250 or 500 ml/min impinged on the cell layer. Subsequently, the expression levels of P120ctn and other proteins, and EC morphological alterations, were examined. In normal HUVECs, after 3 h under a flow rate of 500 ml/min, the expression levels of P120ctn, vascular endothelial (VE)­Cadherin, Kaiso and α­catenin were decreased, whereas matrix metalloproteinase­2 (MMP­2) was increased. In HUVECs with P120ctn knockdown, the period during which ECs adhered to the coverslip was reduced to 1 h under a flow rate of 500 ml/min. In addition, the expression levels of VE­Cadherin, Kaiso and α­catenin in ECs were decreased, whereas those of MMP­2 were increased after 1 h; more prominent alterations were detected under a 500 ml/min flow rate compared with a 250 ml/min flow rate. Adherens junctions (AJs) are critical to the maintenance of normal morphology and EC functioning in the vascular wall, and P120ctn is an important regulator of AJs. Loss of P120ctn may be induced by hemodynamic alterations. In response to changes in hemodynamic conditions, a loss of P120ctn may aggravate AJs between ECs, thus inducing inflammation in the vascular wall. Clinically, hemodynamic alterations may result in a loss of P120ctn and endothelial injury; therefore, P120ctn may have a critical role in inducing intracranial aneurysms.

Identification of key genes involved in catechin metabolism in tea seedlings based on transcriptomic and HPLC analysis.

Tea is a non-alcoholic beverage with many benefits to human health and thereby widely consumed in the world. It contains plenty of secondary metabolites and tea catechins are the characteristic compounds. To further elucidate the biosynthetic and regulatory mechanisms of catechins in tea, high performance liquid chromatography (HPLC) and transcriptome analysis were performed in tea seedlings of different growth stages. A combined method of differential expression and correlation analysis was then conducted. The results showed that the order of total catechin (TC) contents was leaves > stems > roots, irrespective of growth stages. For transcriptome analysis, a total of 355.81 million clean reads were generated and mapped to the referencing tea genome. Further real time PCR analysis of 18 selected genes confirmed RNA-Seq results. A total of 7 structural genes and 35 transcription factors (TFs) were identified to be significantly correlated with TC changes. Among them, three TFs homologous to ANL2, WRKY44 and AtMYB113 might play key roles in catechin regulation. The de novo transcriptome data of different organs in tea seedlings provided new insights into the biosynthetic and metabolic pathways of catechins.

Transcriptomic analyses of cacao cell suspensions in light and dark provide target genes for controlled flavonoid production.

Catechins, including catechin (C) and epicatechin (E), are the main type of flavonoids in cacao seeds. They play important roles in plant defense and have been associated with human health benefits. Although flavonoid biosynthesis has been extensively studied using in vitro and in vivo models, the regulatory mechanisms controlling their accumulation under light/dark conditions remain poorly understood. To identify differences in flavonoid biosynthesis (particularly catechins) under different light treatments, we used cacao cell suspensions exposed to white-blue light and darkness during 14 days. RNA-Seq was applied to evaluate differential gene expression. Our results indicate that light can effectively regulate flavonoid profiles, inducing a faster accumulation of phenolic compounds and shifting E/C ratios, in particular as a response to switching from white to blue light. The results demonstrated that HY5, MYB12, ANR and LAR were differentially regulated under light/dark conditions and could be targeted by overexpression aiming to improve catechin synthesis in cell cultures. In conclusion, our RNA-Seq analysis of cacao cells cultured under different light conditions provides a platform to dissect key aspects into the genetic regulatory network of flavonoids. These light-responsive candidate genes can be used further to modulate the flavonoid production in in vitro systems with value-added characteristics.

Molecular Evidence for Catechin Synthesis and Accumulation in Tea Buds (Camellia sinensis).

Early spring buds of the Camellia sinensis variety Shuchazao were separated into two parts, including the shoot tip (ST) and non-expanded young leaves (YL), in which the synthesis and accumulation of catechins in the two parts were assessed by high-performance liquid chromatography (HPLC), p-dimethylaminocinnamaldehyde (DMACA) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and in situ hybridization. HPLC showed that (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) amounts in YL were increased significantly by 74.0 and 71.8%, respectively. The results of DMACA staining indicated that catechins in buds accumulated mainly in mesophyll cells and the bud shaft of YL. Meanwhile, qRT-PCR demonstrated that the relative expression levels of genes related to flavonoid metabolism, including CsPAL1, CsC4H1, CsC4H2, CsCHS2, CsF3'5'H1, CsDFR1, CsDFR2, and CsANR1, were significantly higher in YL than in the ST. In situ hybridization revealed that CsDFR1, CsDFR2, CsLAR, and CsANR1 were expressed in leaf primordia and YL but not in the apical meristem. These findings highlight the synthesis and accumulation patterns of catechins in different parts of the ST in C. sinensis, providing a theoretical basis for the assessment of synthesis, accumulation, and transfer patterns of catechins in tea plants.

Effects of (-)-epicatechin on frontal cortex DAPC and dysbindin of the mdx mice.

INTRODUCTION: Multiple components of the dystrophin-associated protein complex (DAPC) are expressed in numerous tissues including the brain. Members of the DAPC and dysbindin are abnormally expressed in the brain of Duchenne Muscular Dystrophy (DMD) patients, which has been associated with cognitive impairments. However, little is known about the expression pattern of individual members of the DAPC in animal models of DMD and their relationship with dysbindin. METHODS: Ten mdx mice were randomly allocated into a control and intervention group [(-)-epicatechin (Epi) 1mg/kg/day for four weeks] and results compared to a wild-type mice. After sacrifice, brain pre-frontal cortices were collected for Western blotting and immunoprecipitation assays, and sagittal sections processed for immunohistochemistry. RESULTS: Epi promotes a partial recovery of DAPC members [α1-Syntrophin, sarcoglycans (SG), dystrophin 71 (Dp71)], dysbindin, and utrophin protein levels. Epi also appears to restore the association of DAPC between dysbindin, and utrophin with Dp71 and ε-SG. Co-immunostaining evidence increased protein levels of dysbindin, dystrophin, and ε-SG and their colocalization. CONCLUSIONS: Altogether, results suggest that Epi is capable of restoring pre-frontal cortex DAPC and dysbindin levels of mdx mice towards that of healthy brains. The functional implications of such studies warrant further investigation.

Transcriptional profiling of catechins biosynthesis genes during tea plant leaf development.

MAIN CONCLUSION: A total of 299,113 unigenes were generated and 15,817 DEGs were identified. We identified candidate genes associated with the regulation of catechins biosynthesis during leaf development in tea plant. The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most economically significant crops worldwide because of its positive effects on human health. The health benefits of tea are mainly attributed to catechins, which are the predominant polyphenols that accumulate in tea. Catechins are products of the phenylpropanoid and flavonoid biosynthetic pathways. Although catechins were identified in tea leaves long ago, the molecular mechanisms regulating catechins biosynthesis remain unclear. To identify candidate genes involved in catechins biosynthesis, we analyzed the transcriptomes of tea leaves during five different leaf stages of development using RNA-seq. Approximately 809 million high-quality reads were obtained, trimmed, and assembled into 299,113 unigenes with an average length of 565 bp. A total of 15,817 unigenes were differentially expressed during the different stages of leaf development. These differentially expressed genes were enriched in a variety of processes such as the regulation of the cell cycle, starch and sucrose metabolism, photosynthesis, phenylpropanoid biosynthesis, phenylalanine metabolism, and flavonoid biosynthesis. Based on their annotations, 51 of these differentially expressed unigenes are involved in phenylpropanoid and flavonoid biosynthesis. Furthermore, transcription factors such as MYB, bHLH and MADS, which may involve in the regulation of catechins biosynthesis, were identified through co-expression analysis of transcription factors and structural genes. Real-time PCR analysis of candidate genes indicated a good correlation with the transcriptome data. These findings increase our understanding of the molecular mechanisms regulating catechins biosynthesis in the tea plant.

Fine-tuning of the flavonoid and monolignol pathways during apple early fruit development.

MAIN CONCLUSION: A coordinated regulation of different branches of the flavonoid pathway was highlighted that may contribute to elucidate the role of this important class of compounds during the early stages of apple fruit development. Apple (Malus × domestica Borkh.) is an economically important fruit appreciated for its organoleptic characteristics and its benefits for human health. The first stages after fruit set represent a very important and still poorly characterized developmental process. To enable the profiling of genes involved in apple early fruit development, we combined the suppression subtractive hybridization (SSH) protocol to next-generation sequencing. We identified and characterized genes induced and repressed during fruit development in the apple cultivar 'Golden Delicious'. Our results showed an opposite regulation of genes coding for enzymes belonging to flavonoid and monolignol pathways, with a strong induction of the former and a simultaneous repression of the latter. Two isoforms of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, key enzymes located at the branching point between flavonoid and monolignol pathways, showed opposite expression patterns during the period in analysis, suggesting a possible regulation mechanism. A targeted metabolomic analysis supported the SSH results and revealed an accumulation of the monomers catechin and epicatechin as well as several forms of procyanidin oligomers in apple fruitlets starting early after anthesis, together with a decreased production of other classes of flavonoids such as some flavonols and the dihydrochalcone phlorizin. Moreover, gene expression and metabolites accumulation of 'Golden Delicious' were compared to a wild apple genotype of Manchurian crabapple (Malus mandshurica (Maxim.) Kom.). Significant differences in both gene expression and metabolites accumulation were found between the two genotypes.

Development of CAPS markers to identify Indian tea (Camellia sinensis) clones with high catechin content.

Tea leaves are rich in plant secondary phenolics, especially flavonoids. Catechins are considered to be the most valuable flavonoids, and the catechin content in tea is an important trait for determining its quality. We have developed cleaved amplified polymorphic sequence (CAPS)-based markers for evaluating total catechin content that target two important secondary metabolite pathway genes, PAL (phenylalanine ammonia-lyase) and CHS (chalcone synthase). Catechin content levels in the tea samples tested ranged from 9 to 33 mg/mg. The CAPS technique identified clones with the homozygous profile PRc1, which has relatively lower catechin content than clones with the heterozygous profile PRc2. A significant difference (t = 16.85) in the level of catechin content was also detected between heterozygotes and homozygotes in the tea seed stock TS379. We found a polynomial relationship between the marker developed for CHS2 and catechin content in these tea samples with R2 = 0.9788. Moreover, PAL has less of a relationship with catechin content. Therefore, we recommend tea clones with heterozygous CAPS profiles for the gene CHS2+RcaI for the further improvement in these clones.