Genetic analyses of ancient tea trees provide insights into the breeding history and dissemination of Chinese Assam tea(Camellia sinensis var.assamica)

Chinese Assam tea(Camellia sinensis var.assamica)is an important tea crop with a long history of cultivation in Yunnan,China.Despite its potential value as a genetic resource,its genetic diversity and domestication/breeding history remain unclear.To address this issue,we genotyped 469 ancient tea plant trees representing 26 C.sinensis var.assamica populations,plus two of its wild relatives(six and three populations of C.taliensis and C.crassicolumna,respectively)using 16 nuclear microsatellite loci.Results showed that Chinese Assam tea has a relatively high,but comparatively lower gene diversity(H_(S)=0.638)than the wild relative C.crassicolumna(H_S=0.658).Clustering in STRUCTURE indicated that Chinese Assam tea and its two wild relatives formed distinct genetic groups,with considerable interspecific introgression.The Chinese Assam tea accessions clustered into three gene pools,corresponding well with their geographic distribution.However,New Hybrids analysis indicated that 68.48%of ancient Chinese Assam tea plants from Xishuangbanna were genetic intermediates between the Puer and Lincang gene pools.In addition,10%of the ancient Chinese Assam tea individuals were found to be hybrids between Chinese Assam tea and C.taliensis.Our results suggest that Chinese Assam tea was domesticated separately in three gene pools(Puer,Lincang and Xishuangbanna)in the Mekong River valley and that the hybrids were subsequently selected during the domestication process.Although the domestication history of Chinese Assam tea in southwestern Yunnan remains complex,our results will help to identify valuable genetic resources that may be useful in future tea breeding programs.

Research advances in aluminum tolerance and accumulation in tea plant(Camellia sinensis)

Tea plant(Camellia sinensis)is widely planted on acidic soils where aluminum(Al)toxicity is considered as a primary factor limiting plant growth.Unlike most plant species,tea plant is Al tolerant and accumulates high levels of Al.Understanding the mechanisms underlying Al tolerance and accumulation in tea plant may contribute to the improvement of tea plant cultivation and development of Al-tolerant crops.In this review,we summarize recent advances in the uptake,transport and accumulation of Al in tea plant and the genetic and environmental factors that affect these processes.We further highlight recent studies of Al in tea plant based on omics approaches,including transcriptomics,proteomics,metabolomics,ionomics and microbiomics.We present perspectives for future research that will be helpful to decipher the mechanisms underlying Al tolerance and accumulation in tea plant.

Monitoring Thosea sinensis Walker in Tea Plantations Based on UAV Multi- Spectral Image

Thosea sinensis Walker(TSW)rapidly spreads and severely damages the tea plants.Therefore,finding a reliable operational method for identifying the TSW-damaged areas via remote sensing has been a focus of a research community.Such methods also enable us to calculate the precise application of pesticides and prevent the subsequent spread of the pests.In this work,based on the unmanned aerial vehicle(UAV)platform,five band images of multispectral red-edge camera were obtained and used for monitoring the TSW in tea plantations.By combining the minimum redundancy maximum relevance(mRMR)with the selected spectral features,a comprehensive spectral selection strategy was proposed.Then,based on the selected spectral features,three classic machine learning algorithms,including random forest(RF),support vector machine(SVM),and k-nearest neighbors(KNN)were used to construct the pest monitoring model and were evaluated and compared.The results showed that the strategy proposed in this work obtained ideal monitoring accuracy by only using the combination of a few optimized features(2 or 4).In order to differentiate the healthy and TSW-damaged areas(2-class model),the monitoring accuracies of all the three models were computed,which were above 96%.The RF model used the least number of features,including only SAVI and Bandred.In order to further discriminate the pest incidence levels(3-class model),the monitoring accuracies of all the three models were computed,which were above 80%,among which the RF algorithm based on SAVI,Band_(red),VARI__(green),and Band_(red_edge) features achieve the highest accuracy(OAA of 87%,and Kappa of 0.79).Considering the computational cost and model accuracy,this work recommends the RF model based on a few optimal feature combinations to monitor and distinguish the severity of TSW in tea plantations.According to the UAV remote sensing mapping results,the TSW infestation exhibited an aggregated distribution pattern.The spatial information of occurrence and severity can offer effective guidance for precise control of the pest.In addition,the relevant methods provide a reference for monitoring other leaf-eating pests,effectively improving the management level of plant protection in tea plantations,and guaranting the yield and quality of tea plantations.

Quantitative Analysis of ATP Sulfurylase and Selenocysteine Methyltransferase Gene Expression in Different Organs of Tea Plant (<i>Camellia sinensis</i>)

Tea plant (Camellia sinensis) has unique biological features for the study of cellular and molecular mechanisms, an evergreen broad-leaved woody plant which can accumulate selenium in soil abundant of Selenium. Expression of the genes related to Selenium (Se) metabolism is an adaptation to the soil environment for a long period. The purpose of the present study was to explore if there exist differences of expression about these genes in tea plant between growing in Selenium-abundant and normal soil. A quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR) assay was done for quantification of ATP sulfurylase (APS) and selenocysteine methyltransferase (SMT) mRNA normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in tea plant. Young leaves, mature leaves and tender roots from tea plants growing in soil abundant of Selenium were respectively obtained from Shitai County, Anhui Province, and also the relevant materials of the selenium un-enriched tea plant planted at agricultural garden of Ahui Agriculture University were taken as control for real-time PCR analysis. The results showed that APS1, APS2 and SMT expression levels for either young or mature leaves in selenium-enriched tea plant were lower than that in ordinary (selenium un-enriched) tea plant. In contrast, the APS1, APS2 and SMT expression level of roots in selenium-enriched tea plant were all higher than that in ordinary tea plant. APS1 gene expression level of roots in selenium-enriched tea plant was about 1.6 times higher than that in the ordinary tea plant, APS2 gene expression level was about 4.8-fold higher than that in the ordinary tea plant, SMT gene expression level was about 3.3 times higher than that in the ordinary tea plant. Among various tissues of selenium-enriched tea plant, APS1 gene relative expression level of young leaves was similar to or slightly higher than mature leaves, and the one of roots was the lowest among them;APS2 gene relative expression level of young leaves was similar to or slightly higher than the roots, and the one of mature leaves was the lowest among them;SMT gene relative expression level of young leaves was similar to or slightly higher than mature leaves, and the one of roots was the highest among them. Our results suggest that there existed correlation between selenium and expression levels of these genes.

Genome-Wide Characterization of the Cellulose Synthase Gene Superfamily in Tea Plants(Camellia sinensis)

The cellulose synthase gene superfamily,including Cellulose synthase A(CesA)and cellulose synthase-like(Csl)gene families,is responsible for the synthesis of cellulose and hemicellulose,respectively.The CesA/Csl genes are vital for abiotic stress resistance and shoot tenderness regulation of tea plants(Camellia sinensis).However,the CesA/Csl gene family has not been extensively studied in tea plants.Here,we identified 53 CsCesA/Csl genes in tea plants.These genes were grouped into five subfamilies(CsCesA,CsCslB,CsCslD,CsCslE,CsCslG)based on the phylogenetic relationships with Arabidopsis and rice.The analysis of chromosome distribution,gene structure,protein domain and motif revealed that most genes in CsCesA,CsCslD and CsCslE subfamilies were conserved,whereas CsCslB and CsCslG subfamily members are highly diverged.The transcriptome analysis showed that most CsCesA/Csl genes displayed tissue-specific expression pattern.In addition,members of CsCslB4,CsCesA1/3/6,CsCslB3/4,CsCslD3,CsCslE1 and CsCslG2/3 subfamilies were up-regulated under drought and cold stresses,indicating their potential roles in regulating stress tolerance in tea plants.Furthermore,the expression levels of CsCslG2_6 and CsCslD3_5 in different tissues and cultivars,respectively,were positively correlated with the cellulose content that is negatively related with shoot tenderness.Thus,these two genes were speculated to be involved in the regulation of shoot tenderness in tea plants.Our findings may help elucidate the evolutionary relationships and expression patterns of the CsCesA/Csl genes in tea plants,and provide more candidate genes responsible for stress tolerance and tenderness regulation in tea plants for future functional research.

Preparation of high molecular weight （HMW） genomic DNA from tea plant （Camellia sinensis） for BAC library construction

A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.

Study on the Transferability of Camellia sinensis EST-SSRs in Theaceae Plant

[Objective] The research aimed to study the transferability of Camellia sinensis EST-SSRs in Theaceae plant.[Method] Seven pairs of EST-SSRs primers which derived from Camellia sinensis EST sequence were used to amplify the nineteen materials of Theaceae plant.[Result] Five pairs in the seven pairs of EST-SSRs primers could effectively amplify the nineteen tested varieties,and the transferability rate was 71.43%.The amplification rate of Camellia retiacalate FengShanCha,Camellia japouica CaiXia and Camellia retiacalate JuBan was the highest.The amplification rate of Camellia synaptica Sealy and Adinandra sagonica var.wallichiana（oc）Ming was the lowest.Moreover,four pairs in the five pairs of primers which could effectively amplify showed the rich polymorphism whose difference was obvious in the tested materials.[Conclusion] SSR primers which were developed from Camellia sinensis genome had the higher transferability in the different genus and species of Theaceae plant,could be used in the comparative genome research and analysis mark research of Theaceae plant.

Comparative Effect of Ca, K, Mn and B on Post-Drought Stress Recovery in Tea [<i>Camellia sinensis</i>(L.) O <i>Kuntze</i>]

Tea (Camellia sinensis) is one of the most important economic crops. Being perennial in nature, tea plant often experiences natural drought, which affects its growth and productivity. The present investigation was undertaken to understand the mechanism of post-drought stress recovery on rehydration and the effect of nutrients in the recovery process of the selected clones of Camellia sinensis L. (TV-1, TV-20, TV-29 & TV-30). The results demonstrated that decrease in relative water content (RWC), dry mass of leaf and antioxidants like-ascorbate and glutathione in all the tested clones, as a result of imposed water stress, which caused damage was not permanent. Increase in phenolic content with decrease in O2-, H2O2 and lipid peroxidation was indication of the recovery of stress induced oxidative damage following the post stress rehydration. Further, the post drought recovery was enhanced by foliar spray of K, Ca, Mn & B. Differential activities of enzymes like SOD, CAT, POX, GR and PPO in response to foliar spray of nutrients in rehydrated plant improved the recovery process. The present study reveals that the tested nutrients (K, Ca, Mn & B) showed some positive response in influencing growth and antioxidative responses during post drought recovery process, where K and Ca showed comparatively better effect in improving post drought recovery potential in tea plant.

Evaluation on Suitability of Camellia sinensis Planting Based on GIS

Camellia sinensis is an important commercial crop in China. Suitability evaluation of tea tree planting, which is an embodiment of agricultural planting based on Geographic Information System（GIS）, includes overlay analysis, hierarchical analysis,artificial intellegence, multivariable linear regression and fuzzy evaluation. Via a series of functions of GIS such as data query, retrieve and management, we can be informed of current situation and problems in tea plant development, find out areas which are appropriate or inappropriate for Camellia sinensis planting and figure out corresponding planting schemes and policies. Science and technology are the basic solution to modernization of Camellia sinensis planting. It is necessary to set up decision information and plantation management systems in agriculture on account of GIS, which are important channels to regionalization of Camellia sinensis planting suitability.

In vitro anti-hyaluronidase activity of Sri Lankan low grown orthodox orange pekoe grade black tea(Camellia sinensis L.)

Objective:To access the anti-hyaluronidase activity of Sri Lankan low grown orthodox orange pekoe（OP） grade black tea with a view to develop an anti-aging skin formulation.Methods:Five concentrations（0.125,0.250,0.500,1.000 and 2.000 mg/mL） of black tea brew（BTB） were made using a freeze dried sample of Sri Lankan low grown orthodox OP grade black tea which was prepared according to international organization for standardization specification.Epigallocatechin gallate（EGCG） was used as the reference agent（concentrations tested:0.012,0.025,0.050,0.100 and 0.200 mg/mL）.Anti-hyaluronidase activity of BTB and EGCG in vitro were ascertained spectrometrically using hyaluronic acid（from rooster comb）and bovine testicular hyaluronidase.Results:The results revealed that BTB had moderate[IC50=（1.09±0.12） mg/mL]and dose dependent（r2=0.94） anti-hyaluronidase activity.EGCG also exhibited dose dependent（r2=0.93,P【0.05） anti-hyaluronidase activity which was superior[IC50=（0.09±0.00） mg/mL]to BTB.Conclusions:Sri Lankan low grown orthodox OP gradc black tca has promising antihyaluronidase activity in vitro and has the potential to be used as an anti-aging cosmaceutical.In addition,it may prove useful as a beverage in the management of allergy,some joint diseases and cnvenomation.

Identification of miRNAs and target genes regulating catechin biosynthesis in tea (Camellia sinensis)

Micro RNAs（miRNAs） are endogenous non-protein-coding small RNAs that play crucial and versatile regulatory roles in plants. Using a computational identification method, we identified 55 conserved miRNAs in tea（Camellia sinensis） by aligning miRNA sequences of different plant species with the transcriptome library of tea strain 1005. We then used quantitative real-time PCR（qRT-PCR） to analyze the expression of 31 identified miRNAs in tea leaves of different ages, thereby verifying the existence of these miRNAs and confirming the reliability of the computational identification method. We predicted which miRNAs were involved in catechin synthesis using ps RNAtarget Software based on conserved miRNAs and catechin synthesis pathway-related genes. Then, we used RNA ligase-mediated rapid amplification of c DNA ends（RLM-RACE） to obtain seven miRNAs cleaving eight catechin synthesis pathway-related genes including chalcone synthase（CHS）, chalcone isomerase（CHI）, dihydroflavonol 4-reductase（DFR）, anthocyanidin reductase（ANR）, leucoanthocyanidin reductase（LAR）, and flavanone 3-hydroxylase（F3H）. An expression analysis of miRNAs and target genes revealed that miR529d and miR156 g-3 p were negatively correlated with their targets CHI and F3H, respectively. The expression of other miRNAs was not significantly related to their target genes in the catechin synthesis pathway. The RLM-RACE results suggest that catechin synthesis is regulated by miRNAs that can cleave genes involved in catechin synthesis.

Induced Polyploidy as a Tool for Increasing Tea(Camellia sinensis L.) Production

Tea （Camellia sinensis L.） represents different ploidy levels. In the present paper, we reviewed the recent data on the diploid, aneuploid and polyploid formed their origin and chemically induced polyploidy and predicted it role in teagenetic improvement for better yield. Different polidy levels had different effects on tea physiology. Tetraploid and triploid had more vigour and hardness due to increased size of cells, while triploid could have even more vigours due to increased size of cells and sterilities. Chemically induced polyploidy had been found an important tool for improving plant physiology and production, therefore, induced polyploids should be produced to overcome the problem of low yield and limited rainfall in tea growing areas.

Peroxidase Inhibition and Antioxidant Activity of Bulk-Marketed Black Tea (<i>Camellia sinensis</i>L.) from the Democratic Republic of the Congo

The widely known leaves of Camellia sinensis (L.) derive the most consumed beverage globally. All over the world, like in the Democratic Republic of the Congo (DR Congo), they are marketed in the form of packaged and in bulk black tea. The aim of this study was to report the microscopic botanical features, the chromatographic fingerprints, the antioxidant activities, and the peroxidase inhibition of bulk-marketed black teas consumed in DR Congo. The microscopic analysis of powders from unpackaged black tea marketed in DR Congo allowed the identification of specific microscopical features, which are characteristic to C. sinensis (L.) such as numerous asterosclerites, numerous fragments of unicellular trichomes;fragments of abaxial epidermis consisting of cells with slightly wavy walls, numerous anomocytic stomata, and the detection of biological contaminants, e.g. the dust mites. TLC and HPLC analysis indicated that flavonoids (hyperoside, rutin) and phenolic acids (chlorogenic acid, gallic acid) were major phenolic compounds, along with other secondary metabolites. Aqueous extracts from all bulk-marketed black teas exhibited good antioxidant activities at the concentrations range of 1 - 20 μg·mL-1 using in vitro ABTS and DPPH assays. These extracts showed equally inhibitory effects on myeloperoxidase (MPO) and horseradish peroxidase (HRP) activities at the concentration ranges of 1 - 20 μg·mL-1 and 20 - 100 μg·mL-1 respectively. Bulk-marketed black teas consumed in DR Congo possess antioxidant and anti-inflammatory properties comparable to packaged Ceylon tea largely marketed. The observed bioactivities support the health benefits of the consumed black tea by Congolese people, despite the storage conditions to be improved in order to preserve its nutritional and therapeutic assets.

Screening of Adoptive Elite Tea (Camellia sinensis) Clones

The research screening of adoptive elite tea clones was conducted at NTRI, Mansehra during 2011-2012. Nine clones 101Aa, 105aa, 108aa, 561aa, ll7aa, 219ab, 470bb and 180bd were evaluated for seedling performance. Randomized complete block design was used with three replications. Data was recorded on various morphological characters after 8 months. The results showed that high survival percentage, shoot length, number of roots plant-1, number of leaves plant1 and root length were observed in clone 105aa. While the highest fresh weight and dry weight of leaves were observed in clones 117aa and 105aa. The clone 105aa was drought resistant, high survival percentage and root growth. On the basis of the results, clone 105aa was recommended for cultivation through cuttings in the hilly areas of Pakistan where unequal rainfall distribution was a major hitch.

Effect of Enriched Cattle Manure on Soil Nutrient Status, Nitrogen Uptake and Yield of Tea （Camellia sinensis）

Inorganic fertilizer NPK （nitrogen, phosphorus and potassium） （S） 25：5：5：5 is generally recommended for optimum yield and quality of tea （Camellia sinensis）. Non-judicious use of this inorganic fertilizer however acidifies the soils and pollutes the environment. Integrated soil fertility management （ISFM） which involves the combined use of organic and inorganic fertilizer is recommended for improved crop yield and soil health. An experiment was carried out to determine the effect of enriching cattle manure with different ratios of inorganic fertilizers （OM： NPKS at ratios 1：2 and 1：4）, and rates on soil nutrient status, nitrogen uptake and yield of tea in the east of Rift Valley, Kenya. Enriching manures and organic manure up to a rate of 150 kg N/ha increased the level of P mature leaf. A higher N and K level in the mature leaf was observed when NPKS was applied at higher rates. In the soil, fertilizer rate up to 150 kg N/ha showed higher pH and K where organic manure and enriched manures were applied while NPKS treatment showed higher P content throughout the soil depths. Enriching organic manures with inorganic fertilizers increased yield significantly.

Functional Components of Tea(Camellia Sinensis)Germplasms Collected in Korea

This study was carried out to obtain useful information and to search quality related components for breeding of tea by analysis of Korean local tea leaves at Sulloc-Cha R&D center in Jeju Island,Korea.It was also conducted to measure the major agronomic characteristics of 300 local germplasms.These results allowed us to select some superior lines as genetic resources for green-tea breeding and research genotypes.

Analysis and Evaluation of Biochemical Components in Bitter Tea Plant Germplasms

Bitter tea is a special kind of tea germplasm in China.The major biochemical components of 24 bitter teas and other 8 Camellia sinensis var.sinensis and 8 C.sinensis var.assamica tea germplasms,which were stored in the China National Germplasm Hangzhou Tea Repository(CNGHTR),were analyzed and evaluated.The results showed that no significant differences of major biochemical components affecting the tea quality were found between bitter tea and common tea.According to the processing suitability index,bitter tea was suitable for the manufacturing of black tea;while according to evolutionary indices such as the composition and content of catechin,bitter tea was similar to C.sinensis var.assamica belonging to the relatively primitive type in evolution.The results of cluster analysis indicated that bitter tea was clustered with C.sinensis var.assamica,so it could be considered to belong to C.sinensis var.assamica.

茶(Camellia sinensis)蜂花粉及其蜂粮的营养成分研究

测定中国茶 (Camellia sinensis)的新鲜蜂花粉、5 d、1 0 d、1 5 d茶蜂粮中水解氨基酸的含量 ,茶蜂粮中平均含量为2 3 .3 6% ,比新鲜茶蜂花粉高 0 .2 6% ;新鲜茶蜂花粉和混合 (5～ 1 0 d)茶蜂粮样品中游离氨基酸含量分别为 1 0 3 7.46mg/1 0 0 g和 80 1 .75 mg/1 0 0 g。混合 (5～ 1 0 d)茶蜂粮和新鲜茶蜂花粉样品中维生素 A、B1 、B2 、C、D、E的含量 (mg/1 0 0 g)分别是0 .1 9、0 .1 5、1 .1 5、0 .3 1、0 .0 1 5 6、1 0 .2和 0 .79、0 .0 9、2 .74、1 .2 0、0 .0 1 5 8、6.6。新鲜茶蜂花粉和混合茶蜂粮样品中软脂酸、硬脂酸、亚油酸、亚麻酸的含量分别为 1 .3 %、0 .2 %、0 .1 6%、2 .5 %和 1 .1 %、1 .0 %、0 .3 %、1 .0 %。1 d茶蜂粮中的过氧化氢酶、淀粉酶的活力比新鲜茶蜂花粉高 ,而过氧化物歧化酶的活力则比新鲜茶蜂花粉略低 ;随着酿制时间的延长 ,茶蜂粮中过氧化氢酶、过氧化物歧化酶的活力下降 。

茶(Camellia sinensis)花粉及其蜂粮中花粉粒的显微结构

采用光学显微镜和扫描电镜观察茶蜂花粉及其不同酿制时间的蜂粮中花粉粒的外部形态和萌发沟,未发现花粉壁和花粉粒结构被破坏的迹象.通过体外模拟消化试验,采用光学显微镜和透射电镜观察,发现经模拟消化的蜂粮中花粉粒内含物外吐的数量比蜂花粉多,而且随着酿制时间的延长,其内含物外吐的花粉粒所占的比例增加.结果提示,同种粉源的蜂粮比蜂花粉更易于被消化.

Genetic Diversity Analysis of Wild Tea Plants in Yunnan Province Using EST-SSR Markers

In this study, 27 pairs of EST-SSR primers were employed to analyze the genetic diversity and genetic relationship of 100 wild tea plant germplasm re- sources and 22 cultivars, according to the results, a total of 88 polymorphic bands were amplified with 27 pairs of primers; the variation of effective alleles accounted for 69.01% ; a total of 183 genotypes were detected, with a variation range of 4 -11 ; averagely 6.78 genotypes were amplified with each primer pair; Shannon index （I） of 27 primer pairs ranged from 0.32 to I. 35, with an average of 0.88 ; the observed heterozygosity （0.52） was basically consistent with the expected het- erozygosity （0.52） ; the average polymorphism heterozygosity was 0.48, which was very close to 0.50 ; the average Nei＇s index was 0.51, which was higher than 0. 50 ; the average polymorphism information content （PIC） was 0.52, which was higher than 0.50, indicating high genetic diversity among wild tea germplasm resources in Yuunan Province. According to the clustering results, based on geographical origins and genetic backgrounds, 122 materials were clustered into 14 categories. Dendrogram based on Nei＇s genetic distance revealed complex genetic relationships among wild tea germplasm resources in Yunnan Province. This study provided certain reference for subsequent preservation, development and research of wild tea germplasm resources in China.