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.

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.

Effects of Sinochem Microbial Organic Fertilizer on Tea Plants

[Objectives]This study was conducted to explore the application effects of Sinochem microbial organic fertilizer on tea plants.[Methods]Through the experiment and demonstration of Sinochem microbial organic fertilizer,its effects on the yield and quality of tea and the physical and chemical quality of soil were explored.[Results]Compared with the conventional organic fertilizer cake fertilizer,the fertilizer could increase the weight of freshly picked bud leaves,increase the yield of tea,and thus promote the growth of tea plants.It increased the contents of chlorophyll components,amino acids and tea polyphenols,the main functional substances of tea,which was conducive to improving the internal quality of tea.It improved the physical and chemical properties of soil,and increased the contents of organic matter,alkali-hydrolyzable nitrogen and available phosphorus in tea garden soil.It also had a good effect in improving soil environmental health indexes and physical and chemical properties,and did not contain heavy metals and plant growth hormones.[Conclusions]This study provides a theoretical basis for the popularization and application of Sinochem microbial organic fertilizer.

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.

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.

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.

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.

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.

New Standardized Cultivation Techniques for Tea Seed Production

The tea from tea plants,a kind of traditional leaf plant,is deeply loved by people,but the production of tea fruit and its important functional value have been seriously underestimated for a long time.To this end,the oil plant function and comprehensive utilization value of tea oil from tea fruit were introduced,and a set of new standardized cultivation technique of tea plants that could fully exert the potential of tea seed yield was put forward.The technique could increase the tea seed yield per mu from less than 50kg in traditional tea gardens to more than 200 kg,which broke through the production mode of single leaf picking in traditional tea gardens,gave play to the natural reproductive growth advantages of tea plants,achieved"leaf-seed dual harvest",and promoted the improvement of tea industry quality and efficiency,thereby creating a new path to achieve the"leaf-fruit dual use"of tea plants.

Review of the application of in-situ sensing techniques to address the tea growth characteristics from leaf to field

The tea plant is a valuable and evergreen crop that is extensively cultivated in China and many other countries.Currently,there is growing research interest in this plant.For the tea industry,it is crucial to develop rapid and non-invasive methods to evaluate tea plants in their natural environment.This article provides a comprehensive overview of non-invasive sensing techniques used for in-situ detection of tea plants.The topics covered include leaf,canopy,and field-level assessments,as well as statistical analysis techniques and characteristics specific to the research.Non-invasive testing technology is primarily used for monitoring and predicting tea pests and diseases,monitoring quality,and nutrients,determining tenderness and grade,identifying tea plant varieties,automatically detecting,and identifying tea buds,monitoring tea plant growth,and extracting tea garden areas through remote sensing.It also helps to evaluate planting suitability,assess disasters,and estimate yields.Additionally,the article examines the challenges and prospects of emerging techniques aimed at resolving the in-situ detection problem for tea plants.It can assist researchers and producers in comprehensively understanding the tea environment,quality characteristics,and growth process,thereby enhancing tea production quality,and fostering tea industry development.

Heterologous Expression of Camellia sinensis Late Embryogenesis Abundant Protein Gene 1(CsLEA1)Confers Cold Stress Tolerance in Escherichia coli and Yeast

Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily protein,was successfully cloned froma tea plant[Camellia sinensis(L.)O.Kuntze].Bioinformatics analysis and prokaryotic expression assays showed that CsLEA1 is a typical hydrophilic protein with a molecular weight of approximately 10.4 kD.Expression analyses revealed that the transcription of CsLEA1 in C.sinensis leaves was significantly induced by cold stress.In addition,the heterologous expression of CsLEA1 increased the tolerance of Escherichia coli and yeast to cold stress,which might be closely related to the low molecular weight and high hydrophilicity of the CsLEA1.Taken together,our results suggest that CsLEA1 might have an important function in the tolerance of C.sinensis to cold stress,thus providing a potential application in molecular breeding to enhance the cold stress tolerance of tea plants.

Optimal flight parameters of unmanned helicopter for tea plantation frost protection

To determine proper flight parameters of an unmanned helicopter for tea plantation frost protection,field experiments were conducted to study the impact of flight height,speed and interval on airflow disturbance and temperature rise around tea canopies based on the analysis and simulation of frost protection with a certain helicopter.The relationship between temperature rise after flight and the above flight parameters was established through a regression orthogonal experiment,based on which the optimal combination of flight parameters was obtained through the single-factor golden section method.The results showed that wind speed around tea canopies decreased with the increase of flight height when flight speed was constant.There was a multivariate linear relationship between temperature rise and flight parameters,and the sequence of flight parameters’influence on frost protection effect was flight interval,flight height,flight speed.The optimal combination of flight parameters were flight height of 4.0 m,flight speed of 6.0 m/s and flight interval of 20 min.After the flight with the above parameters air temperature around tea canopies increased 1.6℃ when background thermal inversion strength was 3.8℃.

The Reference Genome of Tea Plant and Resequencing of 81 Diverse Accessions Provide Insights into Its Genome Evolution and Adaptation

Tea plant is an important economic crop,which is used to produce the world's oldest and most widely consumed tea beverages.Here,we present a high-quality reference genome assembly of the tea plant(Camellia sinensis var.sinensis)consisting of 15 pseudo-chromosomes.LTR retrotransposons(LTR-RTs)account for 70.38%of the genome,and we present evidence that LTR-RTS play critical roles in genome size expansion and the transcriptional diversification of tea plant genes through preferential insertion in promoter regions and introns.Genes,particularly those coding for terpene biosynthesis pro-teins,associated with tea aroma and stress resistance were significantly amplified through recent tandem duplications and exist as gene clusters in tea plant genome.Phylogenetic analysis of the sequences of 81 tea plant accessions with diverse origins revealed three well-differentiated tea plant populations,support-ing the proposition for the southwest origin of the Chinese cultivated tea plant and its later spread to western Asia through introduction.Domestication and modern breeding left significant signatures on hundreds of genes in the tea plant genome,particularly those associated with tea quality and stress resis-tance.The genomic sequences of the reported reference and resequenced tea plant accessions provide valuable resources for future functional genomics study and molecular breeding of improved cul-tivars of tea plants.