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.

Analysis of NPK in Camellia sinensis, Gliricidia sepium, and Musa acuminata Biomasses for Preparation of an Organic Fertilizers Formula for Young Tea Plants (Musa acuminata) and Studying of Their Nutrient Release Capacity in the Biodegradation Process

This study investigates the nitrogen (N), phosphorous (P), and potassium (K) contents in raw biomasses of Camellia sinensis, Gliricidia sepium, and Musa acuminata. Therein, the highest N and P content was seen in Camellia sinensis 116.80 ± 0.08 mg and 66.00 ± 0.14 mg respectively. The highest K content (106.80 ± 0.04 mg) was observed in Musa acuminata. Next, all three types of plant materials were allowed to decompose in water for 3 weeks, and a sample from each was analyzed for NPK after the 1st, 2nd, and 3rd week during decomposition. A significant increase in the release of N, P, and K by the Camellia sinensis to water (P Musa acuminate were not significantly changed (P > 0.05) over time. The ratio for N:P:K was calculated for raw biomass samples and decomposed samples to find the best fitting N:P:K ratio to apply to young tea plants as organic fertilizers. In addition to that, the microbial insight of these organic compounds was analyzed by observing how microbial population increased with decomposition by the enumeration of the total microbial count. A considerable increment in total microbial count was observed up to 3.28 × 106, 1.21 × 1010, 2.18 × 108, and 6.49 × 107 CFU/ml for Camellia sinensis, Gliricidia sepium, Musa accuminata (leaves), and Musa accuminata (trunk) respectively. The presence of phosphate solubilizing bacteria (PSB) and nitrogen solubilizing bacteria (NSB) throughout the decomposition period was confirmed by their growth on NBRIP and a modified nutrient medium that was specifically designed for the identification of ammonifiers respectively. Prepared fertilizer samples were applied to young tea plants that were grown in the Mawanella area in Sri Lanka (7°15'12.42"N 80°26'47.62"E) and according to the results, it is clear that fertilizer mixture 1 (N:P:K, 10:5:10, tea dust + Gliricidia + banana trunk) and fertilizer mixture 2 (N:P:K, 10:5:10, tea dust + Gliricidia + banana leaves) has the potential to increase the growth of young tea plants.

Identification of the lysine and histidine transporter family in Camellia sinensis and the characterizations in nitrogen utilization

In plants,the lysine and histidine transporter(LHT)family represent a class of proteins that mediate the uptake,translocation,and utilization of amino acids.The tea plant(Camellia sinensis)is a perennial evergreen with a relatively high level of amino acids.However,systematic identification and molecular characterization of the LHT gene family has rarely been reported in tea plants.In this study,22 CsLHTs were identified from the‘Shuchazao’genome and classified into two groups.The modeled three-dimensional structure and the conserved domains presented a high similarity among the LHTs proteins.Moreover,it was predicted that a few genes were conserved through the analysis of the physiochemical characters,structures and cis-elements in promoters.The expression patterns in tea plants revealed that CsLHT7 was mainly expressed in the roots,and CsLHT4 and CsLHT11 exhibited relatively high expression in both the roots and leaves.Moreover,the expression of all three genes could be induced by organic nitrogen.Additionally,heterogeneous expression of CsLHT4,CsLHT7 and CsLHT11 in Arabidopsis thaliana decreased the aerial parts biomass compared with that in WT plants while significantly increased the rosette biomass only for CsLHT11transgenic plants versus WT plants.Overall,our results provide fundamental information about CsLHTs and potential genes in N utilization for further analysis in tea plants.

基于金观音(Camellia sinensis)单体型基因组CsUGT等位基因的鉴定及表达

【目的】植物的尿苷二磷酸糖基转移酶(UGT)是催化次生代谢物糖基化的关键酶,UGT基因主要参与植物逆境胁迫的响应。对CsUGT等位基因进行鉴定与表达分析,为其在茶树抗性及乌龙茶加工中香气形成的作用提供理论参考。【方法】基于金观音(Camellia sinensis‘Jinguanyin’)单体型基因组,利用生物信息学方法分析,并通过实时荧光定量PCR技术分析CsUGT基因在低温(4℃)、干旱(10%PEG-6000溶液)和激素(100μmol·L^(−1)MeJA溶液)处理下0、3、6、12和24 h的表达,以及CsUGT基因在金观音乌龙茶加工过程中鲜叶、萎凋、一摇、二摇、三摇和杀青前的表达。【结果】生物信息学分析结果表明,从金观音单体型基因组中鉴定出了142对等位基因,多数定位于叶绿体中,并基于进化树将这些基因分为5个亚家族。CsUGT等位基因的结构较保守,相同基因的亲本等位基因的结构相似。转录组数据分析表明,发现多数CsUGT等位基因具有组织表达特性。实时荧光定量PCR检测发现,在MeJA处理下,CsUGT002、CsUGT047和CsUGT091的表达量在6 h达到峰值;在低温和干旱处理下,部分CsUGT基因的表达量分别在3或24 h时达到峰值。此外,在金观音乌龙茶加工过程中,CsUGT115的表达量显著上调,部分CsUGT基因在三摇时的表达量达到峰值。【结论】CsUGT蛋白多数为酸性和亲水性蛋白,并定位于叶绿体中;CsUGT等位基因结构较保守具有组织表达特性,部分CsUGT基因参与调控茶树的逆境胁迫及乌龙茶加工工艺中摇青时的香气合成。

Genome-Wide Identification of ABCC Gene Subfamily Members and Functional Analysis of CsABCC11 in Camellia sinensis

The ATP-binding cassette(ABC)transporter is a gene superfamily in plants.ATP-binding cassette subfamily C(ABCC)protein is a multidrug resistance-associated(MRP)transporter.They play various roles in plant growth,development,and secondary metabolite transport.However,there are few studies on ABCC transporters in tea plants.In this study,genome-wide association study(GWAS)analysis of epigallocatechin gallate(EGCG)content in 108 strains of Kingbird revealed that CsABCCs may be involved in EGCG transport.We identified 25 CsABCC genes at the genomic level of the tea plant,their phylogenetic tree,gene structure,targeted miRNA and other bioinformatics were analyzed.The expression patterns of CsABCCs in eight different tissues and abiotic stress indicate that they have potential roles in regulating the growth,development,and defense of tea plants.The correlation analysis revealed that the expression of the CsABCC11 gene was closely related to the EGCG content in tea buds of 108 strains of the Kingbird,and the subcellular localization experiments in tobacco showed that CsABCC11 protein was localized on the plasma membrane.The virus-induced gene silencing(VIGS)strategy in tea plants further verified that CsABCC11 was involved in EGCG accumulation.Our study laid a foundation for studying the biological function of CsABCC and provided a new candidate molecular marker gene for further EGCG-related variety breeding,which will be of great interest to breeders.

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.

Computational Identification of Conserved microRNAs and Their Targets in Tea (Camellia sinensis)

MicroRNAs (miRNAs) are a class of ~22 nucleotides long non coding RNA molecules which play an important role in gene regulation at the post transcriptional level. The conserved nature of miRNAs provides the basis of new miRNA identification through homology search. In an attempt to identify new conserved miRNAs in tea, previously known plant miRNAs were used for searching their homolog in a tea Expressed Sequence Tags and full length nucleotide sequence database. The sequences showing homolog no more than four mismatches were predicted for their fold back structures and passed through a series of filtration criteria, finally led us to identify 13 conserved miRNAs in tea belonging to 9 miRNA families. A total of 37 potential target genes in Arabidopsis were identified subsequently for 7 miRNA families based on their sequence complementarity which encode transcription factors (8%), enzymes (30%) and transporters (14%) as well as other proteins involved in physiological and metabolic processes (48%). Overall, our findings will accelerate the way for further researches of miRNAs and their functions in tea.

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.

Anti-inflammatory properties of oolong tea(Camellia sinensis)ethanol extract and epigallocatechin gallate in LPS-induced RAW 264.7 cells

Objective: To evaluate the anti-inflammatory activity of oolong tea ethanol extract(OTEE) and epigallocatechin gallate(EGCG) on lipopolysaccharide-induced murine macrophage cell line(RAW 264.7).Methods: A cytotoxic assay using MTS tetrazolium was conducted to find a nontoxic level of OTEE and EGCG toward RAW 264.7 cells. Interleukins(IL-6, IL-1 b), tumor necrosis factor-a(TNF-a), and cyclooxigenase-2(COX-2) levels were measured by ELISA, and nitric oxide(NO) levels measured by a nitrate/nitrite colorimetric assay to determine the inhibition activity of OTEE and EGCG.Results: Lipopolysaccharide induction increases NO, COX-2, IL-6, IL-1 b, and TNF-a levels compared with the untreated cell(negative control). The positive control,lipopolysaccharide-induced RAW 264.7 without treatments showed the highest level of all pro-inflammatory cytokines and modulators tested in this study. The positive control was used as standard to obtain OTEE and EGCG inhibition activity toward NO, COX-2,IL-6, IL-1 b, and TNF-a. OTEE had a higher inhibition activity toward NO, COX-2, IL-6,and IL-1 b than EGCG; the reverse was seen for TNF-a. However, both OTEE and EGCG suppressed production of NO, COX-2, IL-6, IL-1 b, and TNF-a.Conclusions: OTEE and EGCG have the potential for use as anti-inflammatory drugs,which is shown by their ability to reduce the production of NO, COX-2, IL-6, IL-1 b, and TNF-a in active macrophages.

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.

Differential metabolites and their transcriptional regulation in seven major tea cultivars (Camellia sinensis) in China

Various genetic and biochemical characteristics exist in tea plant cultivars,and they largely determine production suitability and tea quality.Here,we performed transcriptomic and metabolomic analyses of young shoots of seven tea cultivars and identified major regulatory transcription factors(TFs)for the characteristic metabolites in different cultivars based on weighted gene co-expression network analysis(WGCNA).Phenotypically,we found that‘Tieguanyin’(TGY)and‘Fujian Shuixian’(FJSX),which are suitable for oolong tea,had higher catechin contents.The metabolites of‘Jinxuan’(JX)were more prominent,especially the contents of phenolic acids,flavonoids,terpenes,and tannins,which were higher than those of the other six cultivars.Moreover,‘Fudingdabai’(FDDB),which is suitable for white tea,was rich in amino acids,linolenic acid,and saccharides.At the molecular level,hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase(HCT)(CsTGY12G0001876,and CsTGY06G0003042)led to the accumulation of chlorogenic acid in TGY.The main reason for the higher l-ascorbic acid content in FJSX was the high expression levels of L-galactono-1,4-lactone hydrogenase(GalLDH)(CsTGY13G0000389)and Myo-inositol oxygenase(MIOX)(CsTGY14G0001769,and CsTGY14G0001770),which were regulated by WRKY(CsTGY11G0001197).Furthermore,FDDB,‘Longjing 43’(LJ43),‘Shuchazao’(SCZ)and‘Baihaozao’(BHZ)had higher free fatty acid contents,among which MYB(CsTGY14G0002344)may be a hub gene for the regulation of palmitoleic acid accumulation.More importantly,we found that the shoots of TGY were green with purple,mainly due to the accumulation of anthocyanins and the downregulation of the Mg-protoporphyrin IX nonomethyl ester cyclase(MPEC)(CsTGY10G0001989)gene that affects chlorophyll synthesis.These results will provide a theoretical reference for tea cultivar breeding and suitability.

The high-resolution three-dimensional(3D)chromatin map of the tea plant(Camellia sinensis)

Dear Editor,Tea plant(Camellia sinensis(L.)O.Kuntze)is one of the world’s most important non-alcoholic beverages,with great economic,health,and cultural value.Recently,several genomes of C.sinensis var.assamica(CSA)(Yunkang 10),C.sinensis var.sinensis(CSS)(Shuchazao,Biyun,Longjing 43,Tieguanyin,Huangdan),and ancient tea plant(DASZ)have been deciphered[1-3].

The leaf extracts of Camellia sinensis (green tea) ameliorate sodium fluoride-induced oxidative stress and testicular dysfunction in rats

Objective:To investigate the effect of Camellia(C.)sinensis in mitigating oxidative damage and reproductive toxicity in testis induced by sodium fluoride in a rat model.Methods:Twenty-four adult male Wister rats were divided into 4 groups,with 6 rats in each group.Group 1 orally received distilled water(1 mL/100 g body weight)daily and served as the control group,while group 2 received drinking water with 100 ppm sodium fluoride per day for 21 consecutive days,group 3 was administered with only C.sinensis extract by gavage at a dose of 100 mg/kg body weight and group 4 received drinking water with 100 ppm sodium fluoride and 100 mg/kg body weight C.sinensis leaf extract per day for 21 consecutive days.At the end of the treatment,the rats were sacrificed under light ether anesthesia.The gonado-somatic index,sperm count and motility,serum level of luteinizing hormone and testosterone were assayed.Lipid peroxidation[malondialdehyde(MDA)level],nitric oxide(NO)production,and activities of antioxidant enzymes-superoxide dismutase(SOD),catalase,and reduced glutathione level(GSH)were also analysed.Results:Sodium fluoride treatment significantly decreased gonado-somatic index,sperm count and motility as well as the serum level of luteinizing hormone and testosterone(P<0.05).The histological examination of testes revealed atrophy and degenerative changes in several seminiferous tubules,along with enhanced interstitial space and a reduced number of Leydig cells.There was a highly significant increase in NO and MDA production(P<0.05),while SOD,catalase activities and GSH level decreased significantly(P<0.05).However,C.sinensis significantly restored testicular weight,sperm parameter,hormonal level(P<0.05),and also reversed MDA and NO generation and antioxidant enzymes activities in the testicular tissue(P<0.05).Conclusions:C.sinensis may have an ameliorative role against sodium fluoride-induced oxidative damage in the testis probably because of its antioxidant property.

Functional mechanism on stem cells by tea(Camellia sinensis)bioactive compounds

Camellia sinensis(tea),one of the most popular commercial crops,is commonly applied in all parts of the world.The main active ingredients of tea include polyphenols,alkaloids,polysaccharides,amino acids,aroma and volatile constitutes,all of which are potentially responsible for the activities of tea.Stem cells(SCs)are the immature and undifferentiated cells by a varying capacity for proliferation,self-renewal and the capability to differentiate into one or more different derivatives with specialized function or maintain their stem cell phenotype.Herein,a thorough review is conducted of the functional mechanism on SCs by tea bioactive compounds.

Influence of Location of Production, Season and Genotype on Caffeine and Flavan-3-ols in Young Green Tea （Camellia sinensis） Leaves in Kenya

Although tea is grown in agro-ecological regions widely varying in environmental factors affecting yield and quality, planters usually import genetic materials across the different growing regions assuming genotypes with good quality attributes in one location maintain their status in all regions. However, tea quality has not been replicated in new production regions. Black tea quality is influenced by the green leaf quality precursors including caffeine, total polyphenols and individual flavan-3-ols which are precursors of theaflavins and thearubigins, key plain black tea quality parameters. Factors influencing levels of the precursors compounds have not been quantified for popular Kenyan tea cultivars. The influence of geographical production location and seasons on levels and ratios of the quality precursors for 10 black tea popular clones grown in three locations in Kenya under uniform agronomic inputs were monitored. Caffeine varied significantly （P _〈 0.05） with clones and location but not with season. The flavan-3-ols and their ratios all varied （P 〈 0.05） with clones and site but not with season, with significant interactions （P 〈 0.05） between locations and clones, and locations and seasons. These results explain the observed variations in plain black tea quality due to clones and location of production. Thus, farmers in different locations are unlikely to produce black tea of same quality. It is therefore necessary to evaluate new tea genotypes in intended growing areas to establish cultivars suitable for producing high quality plain black teas in the locations. Seasonal variations in plain black tea in Kenya are unlikely to vary significantly due to flavan-3-ols.

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.

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.