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.

Design and Research on Identification of Typical Tea Plant Diseases Using Small Sample Learning

Tea plants are susceptible to diseases during their growth.These diseases seriously affect the yield and quality of tea.The effective prevention and control of diseases requires accurate identification of diseases.With the development of artificial intelligence and computer vision,automatic recognition of plant diseases using image features has become feasible.As the support vector machine(SVM)is suitable for high dimension,high noise,and small sample learning,this paper uses the support vector machine learning method to realize the segmentation of disease spots of diseased tea plants.An improved Conditional Deep Convolutional Generation Adversarial Network with Gradient Penalty(C-DCGAN-GP)was used to expand the segmentation of tea plant spots.Finally,the Visual Geometry Group 16(VGG16)deep learning classification network was trained by the expanded tea lesion images to realize tea disease recognition.

Chlorophyllase is transcriptionally regulated by CsMYB308/CsDOF3 in young leaves of tea plant

Chlorophyll contributes to tea coloration, which is an important factor in tea quality. Chlorophyll metabolism is induced by light, but the transcriptional regulation responsible for light-induced chlorophyll metabolism is largely unknown in tea leaves. Here, we characterized a chlorophyllase1 gene CsCLH1 from young tea leaves and showed it is essential for chlorophyll metabolism, using transient overexpression and silencing in tea leaves and ectopic overexpression in Arabidopsis. CsCLH1 was significantly induced by high light. The DOF protein CsDOF3, an upstream direct regulator of CsCLH1, was also identified. Acting as a nuclear-localized transcriptional factor, CsDOF3 responded for light and repressed CsCLH1 transcription and increased chlorophyll content by directly binding to the AAAG cis-element in the CsCLH1 promoter. CsDOF3was able to physically interact with the R2R3-MYB transcription factor CsMYB308 and interfere with transcriptional activity of CsCLH1. In addition, CsMYB308 binds to the CsCLH1 promoter to enhance CsCLH1 expression and decrease chlorophyll content. CsMYB308 and CsDOF3 act as an antagonistic complex to regulate CsCLH1 transcription and chlorophyll in young leaves. Collectively, the study adds to the understanding of the transcriptional regulation of chlorophyll in tea leaves in response to light and provides a basis for improving the appearance of tea.

Core collection construction of tea plant germplasm in Anhui Province based on genetic diversity analysis using simple sequence repeat markers

The tea plant[Camellia sinensis(L.)O.Kuntze]is an industrial crop in China.The Anhui Province has a long history of tea cultivation and has a large resource of tea germplasm with abundant genetic diversity.To reduce the cost of conservation and utilization of germplasm resources,a core collection needs to be constructed.To this end,573 representative tea accessions were collected from six major tea-producing areas in Anhui Province.Based on 60 pairs of simple sequence repeat(SSR)markers,phylogenetic relationships,population structure and principal coordinate analysis(PCoA)were conducted.Phylogenetic analysis indicated that the 573 tea individuals clustered into five groups were related to geographical location and were consistent with the results of the PCoA.Finally,we constructed a core collection consisting of 115 tea individuals,accounting for 20%of the whole collection.The 115 core collections were considered to have a 90.9%retention rate for the observed number of alleles(Na),and Shannon’s information index(I)of the core and whole collections were highly consistent.Of these,39 individuals were preserved in the Huangshan area,accounting for 33.9%of the core collection,while only 10 individuals were reserved in the Jinzhai County,accounting for 8.9%of the core set.PCoA of the accessions in the tea plant core collection exhibited a pattern nearly identical to that of the accessions in the entire collection,further supporting the broad representation of the core germplasm in Anhui Province.The results demonstrated that the core collection could represent the genetic diversity of the original collection.Our present work is valuable for the high-efficiency conservation and utilization of tea plant germplasms in Anhui Province.

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.

A GIS-based Study on Suitability Estimation of Tea Cultivation in Qimen County

On the basis of data from Soil Test and Formula Fertilization project in Qimen county,we employed Geographic Information System Software ArcGIS 9.2 and CLRMIS（Cultivated Land Resource Management Information System at County Scale） to establish region-specific estimation index system for tea cultivation in Qimen County by determining the estimation units,and carried out quantitative estimation analysis on tea cultivation there via comprehensive evaluation indices for natural elements of cultivated land.The results showed that the highly suitable,suitable,reluctantly suitable and unsuitable acreages are 5 665.8,8 327.1,8 415.9 and 11 979.9 hm2 respectively,accounting for 24.0%,35.3%,35.7% and 5.1% of the total acreage of cultivated land.These results would facilitate the production of high yield and quality tea in Qimen County.

Investigation of Pest in Xinyang Tea-Producing Area

In order to improve the development step of tea industry,the pest investigation was carried out in main Xinyang tea producing area from the year of 2005 to 2008,and 1 600 specimens were collected. The results showed that there were 70 species pests,containing 8 orders and 35 families. Among the pests,there were 11 species such as Boarmia obliqua Wehrli,Euproctis pseudoconspersa Strand,Dasychira barbarana Matsumura,Iragoides fasciata Morre,Clania minuscula Butler,Adoxophyes orana Fisher von Roslerstam,Hypomeces squamosus Fabricius,Sympiezomia citre Chao,Empoasca flavescens Fabricius,Toxoptera aurantii Boyer and Aleurocanthus spiniferus Quaintance,which were significantly harmful to the tea plant. Pest non-pollution control techniques were developed.

Preliminary Report on Application Effects of Slow-release Formula Fertilizer in Tea Plants

In order to accelerate the construction of green production mode for tea in Hubei Province, improve the yield of tea and use efficiency of fertilizers, and improve the quality of tea, application effects of special slow-release formula fertilizer in tea plants were investigated in Wuhan. The results showed that compared with conventional fertilization, application of tea plant-specific slow-release formula fertilizer improved the yield of tea by more than 30%. The amino acid content in tea was increased by 21.05% and 30.82%, respectively, and the income was increased 27 696 （33.88%） and 27 624 （33.79%） yuan/hm2, respectively.

Preliminary Study on the Application of Jiayuan Organic Fertilizer on Tea Plant

A trial was conducted by using Jiayuan organic fertilizer in Chibi to investigate its effect on tea plant and ascertain the possibility of high quality and high yield.The results indicated that such organic fertilizer could achieve the effects of promoting the growth of tea plant,increasing the weight of the plucked flesh leaves,improving tea yield,increasing the contents of main functional substances such as tea polyphenols and amino acids and improving tea quality.It also showed excellent effect in maintaining or improving soil environmental sanitation indicators and contained no plant growth hormones.

Transport of Nitrogen Assimilation in Xylem Vessels of Green Tea Plants Fed with NH_4-N and NO_3-N

An experiment was carried out to study the transport process of nitrogen （N） assimilation from tea roots by monitoring the dynamic composition of N compounds in xylem sap after 15^N-NO3 and 15^N-NH4 were fed to the root of tea plants （Camellia sinensis L.）. Results showed that the main amino acids were glutamine, theanine, axginine, asparic acid and glutamic acid, which accounted for 49%, 17%, 8%, 7%, and 4%, respectively, of the total amino acids in the xylem sap. After the tea plants were fed with 15^N-NO3 and 15^N-NH4 for 48 h, the amount of total amino acids in xylem sap significantly increased and those fed with 15^N-NH4 had higher increment than those with 15^N-NOa. Two hours after 15^N- NO3 and 15^N-NH4 were fed, 15N abundance in glutamine, asparagine, glutamic acid, alanine, and arginine were detected and increased quickly over time. This indicated that it took less than 2 h for NO3-N and NH4-N to be absorbed by tea roots, incorporated into the above amino acids and transported to the xylem sap. Rapid increase in 15^N-NO3 in the xylem sap of tea plants fed with 15^N-NO3 indicated that nitrate could be directly transported to the xylem sap. Glutamine, theanine, and alanine were the main amino acids transported in xylem sap of tea plants fed with both 15^N-NO3 and 15^N-NH4.

Distribution of Aluminum and Fluoride in Tea Plant and Soil of Tea Garden in Central and Southwest China

The distribution of Al and F contents and the relationship between Al and F in tea plants and soils of 12 tea gardens in Central and Southwest China were investigated from October 31 to November 14, 2006. The results show that there were differences in pH, CEC, the contents of organic matter （OM）, Al and F in the different soils of the tea gardens. The Al content ranged from 1196 to 7976mg/kg for old leaf, 370 to 2681mg/kg for young leaf and 285 to 525mg/kg for stem, whereas the content of F ranged from 221 to 1504mg/kg for old leaf, 49 to 602mg/kg for young leaf and 13.5 to 77.5mg/kg for stem. The concentrations of labile Al varied obviously in the different soils, but the distribution law of labile Al content for the same garden was Alexchangeable≈AlFe.Mn oxide〉Alorganic〉mlwater.soluble. The contents of different labile F fractions varied slightly in the different soils and the different soil layers, though the exchangeable F content was lowest among the labile F in the soils. The concentrations of Al and F in tea plants increased with increasing amount of water-soluble Al or F, especially the amount of water-soluble fractions in the soil layer of 0-20cm. The correlation between Al content and F content in the tea leaf was more significant than that in the tea stem. Furthermore, the correlation between Al content and F content in whole tea plant was strongly significant （r=0.8763, p〈0.01, n=36）. There were evident tendency that Al concentration increased with the increase of F concentration in different soil layers. The correlation of water-soluble Al with water-soluble F in all soils was also strongly significant （r=0.7029, p〈0.01, n=34）. The results may provide a proof that Al and F are jointly taken up by tea plants to some extent in natural tea gardens.

Evaluation of the Resistance of Different Tea Cultivars to Tea Aphids by EPG Technique

In order to investigate the resistance of tea plant to tea aphid, the feeding behavior of tea aphids on six different tea cultivars was monitored by （EPG） technique. The result showed that the duration of El, E2 as well as （E I＋E2） which was feeding waveforrns of Toxoptera aurantii （Boyer） in tea phloem was significantly different by variance analysis （P〈0.05） and so did the duration of the probing of T. aurantii in all the tea tissues. And the six tea cultivars were classified into three groups with cluster analysis, based on the six major parameters which were durations of np, C, El, E2, F, and G waveforms, then the six major parameters of these three groups were detected by variance analysis, and the rank of six different tea cultivars＇ resistance to T. aurantii from strong to weak was Zhongcha 108, Sucha 1, Anjibaicha, Longjing 43, Xicha 5, and Sucha 120. As this result was in accord with the investigation into the tea field, we concluded that EPG technique was one of the most important means to examine tea plant resistance to T. aurantii and the durations of waveform El, E2 and （El＋E2） were important parameters for evaluating resistance of tea plants to tea aphids.

Preparation of the UPOV Guidelines for the Conduct of Tests for Distinctness, Uniformity and Stability–Tea Plant [Camellia sinensis (L.) O. Kuntze]

Distinctness, Uniformity and Stability （DUS） testing is the technical base of Plant Variety Protection （PVP） and the scientific basis for the approval of Plant Breeder＇s Rights （PBR）. DUS Test Guidelines are not only the technical manuals for the DUS testing authorities to conduct the testing, but also the technical standards for the competent authorities to examine the DUS of new varieties of plants. Tea plant, originated from Yunnan Province, China, is a very important woody cash species in the world. The International Union for the Protection of New Varieties of Plants （UPOV） DUS Test Guidelines for tea plant is the first Test Guidelines prepared by China for the UPOV. In this article, the subject, selection, and determination of characteristics, states of expression of characteristics and the selection of example varieties, assessment of the UPOV DUS Test Guidelines for tea plant were elucidated in detail. Finally, the proposal of PVP for tea plant in China was also proposed. The preparation of UPOV DUS Test Guidelines for tea plant will have important significance both for promoting the development of PVP and increasing the status of international PVP fields for China.

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.

Accumulation and distribution of arsenic and cadmium by tea plants

It is important to research the rules about accumulation and distribution of arsenic and cadmium by tea plants,which will give us some scientific ideas about how to control the contents of arsenic and cadmium in tea.In this study,by field investigation and pot trial,we found that mobility of arsenic and cadmium in tea plants was low.Most arsenic and cadmium absorbed were fixed in feeding roots and only small amount was transported to the above-ground parts.Distribution of arsenic and cadmium,based on their concentrations of unit dry matter,in tea plants grown on un-contaminated soil was in the order:feeding roots>stems≈main roots>old leaves>young leaves.When tea plants were grown on polluted soils simulated by adding salts of these two metals,feeding roots possibly acted as a buffer and defense,and arsenic and cadmium were transported less to the aboveground parts.The concentration of cadmium in soil significantly and negatively correlated with chlorophyll content,photosynthetic rate,transpiration rate and biomass production of tea plants.

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.

Population Structure Analysis and Genome-Wide Association Study of Tea(Camellia sinensis(L.)Kuntze)Germplasm in Qiannan,China,Based on SLAF-Seq Technology

Duyun Maojian tea is a famous tea in China.In this study,the specific-locus amplified fragment(SLAF)sequencing method was used to analyze the population structure and conduct a genome-wide association study(GWAS)of 2 leaf traits of 123 tea plants in Qiannan,China.A total of 462,019 SLAF tags and 11,362,041 single-nucleotide polymorphism(SNP)loci were obtained.The results of phylogenetic tree analysis,cluster analysis,and principal component analysis showed that 123 tea germplasms were clustered into three groups,and the heterozygosity rates for Groups I,II,and II were 0.206,0.224,and 0.34,respectively.Generally,tea germplasms in a production area are clustered in a group,indicating that tea germplasms in different production areas have certain genetic diversity.The traditional Duyun Maojian tea core production areas,TS and DC-SJ,are clustered into Group I and Group II respectively,while the ZY production area is relatively independent in Group III.Furthermore,based on GWAS analysis,11 candidate genes related to leaf apex and 7 candidate genes related to leaf shape were obtained.This study clarified the genetic relationship among eight Duyun Maojian tea production areas and obtained candidate genes related to leaf apex and leaf shape development.The results showed that population structure and candidate genes are an effective basis for the breeding of Duyun Maojian tea germplasm.

Detection and Discrimination of Tea Plant Stresses Based on Hyperspectral Imaging Technique at a Canopy Level

Tea plant stresses threaten the quality of tea seriously.The technology corresponding to the fast detection and differentiation of stresses is of great significance for plant protection in tea plantation.In recent years,hyperspectral imaging technology has shown great potential in detecting and differentiating plant diseases,pests and some other stresses at the leaf level.However,the lack of studies at canopy level hampers the detection of tea plant stresses at a larger scale.In this study,based on the canopy-level hyperspectral imaging data,the methods for identifying and differentiating the three commonly occurred tea stresses(i.e.,the tea leafhopper,anthrax and sun burn)were studied.To account for the complexity of the canopy scenario,a stepwise detecting strategy was proposed that includes the process of background removal,identification of damaged areas and discrimination of stresses.Firstly,combining the successive projection algorithm(SPA)spectral analysis and K-means cluster analysis,the background and overexposed non-plant regions were removed from the image.Then,a rigorous sensitivity analysis and optimization were performed on various forms of spectral features,which yielded optimal features for detecting damaged areas(i.e.,YSV,Area,GI,CARI and NBNDVI)and optimal features for stresses discrimination(i.e.,MCARI,CI,LCI,RARS,TCI and VOG).Based on this information,the models for identifying damaged areas and those models for discriminating different stresses were established using K-nearest neighbor(KNN),Random Forest(RF)and Fisher discriminant analysis.The identification model achieved an accuracy over 95%,and the discrimination model achieved an accuracy over 93%for all stresses.The results suggested the feasibility of stress detection and differentiation using canopy-level hyperspectral imaging techniques,and indicated the potential for its extension over large areas.

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.

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.