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.

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.

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.

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.

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.

Physiological Foundations of Sustainability <i>Camellia sinensis</i>(L.) O. Kuntze and <i>Corylus pontica</i>C. Koch. in the Conditions of Humid Subtropics of Russia

The study of the water status and activity of catalase in Chinese tea plants (Camellia sinensis (L.) O. Kuntze) and hazelnut (Corylus pontica C. Koch.). The indicators, which not only describe the physiological state of plants in the stressful period, but also allow it to identify more adaptive varieties within a given area was determined. Analysis of catalase activity data in Chinese tea and hazelnut revealed the presence of similar patterns: there is a change in enzymatic activity in response to hydrothermal factors. It is established that the stable varieties Camellia sinensis (L.) O. Kuntze and Corylus pontica C. Koch. characterized by a more active catalase, which is of particular importance during droughts. The water regime parameters variance scale for Camellia sinensis was developed, which allows differentiating varieties into groups of varying degrees of stability. It is shown that all methods that are used to estimate stability of plant species to extreme environmental conditions are based on several basic principles connected with peculiarities of adaptation mechanisms. The result was a selection of diagnostic indicators proposed for assessing adaptability: the water content of the leaf tissue, water-holding capacity of leaves, and concentration of cellular juice sprouts and activity of catalase.

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.

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.

Cloning and Expression of Two MYB Transcription Factors in Tea Plant(Camellia sinensis)

MYB transcription factors represent a family of genes that include the conserved MYB DNA-binding domain,and they are widely involved in the regulation of plant development and secondary metabolism.In this study,Part of sequences of two MYB transcription factors was determined through the cDNA microarray hybridization and selection of cDNA library derived from tender shoots.The full-length cDNAs of the genes were obtained with RT-PCR and RACE,and they were 1 132 bp and 1 020 bp,named as CsMYB1 and CsMYB2 (GenBank accession No.HQ660373 and HQ660374), and contained ORFs of 879 bp and 675 bp encoding 292 and 224 amino acids,respectively.Sequences analysis showed that the deduced protein molecular weight of the two genes were 32.9 ku and 25.4 ku, and the proteins contained two conserved MYB domains near the N-terminus and a conserved C1 motif near the R3 domains.The deduced amino acid sequence of CsMYB1 and CsMYB2 from tea plant showed high identity with that of other plants,for instance CsMYB1 shared 57%homology with MYB1 of Gossypium hirsutum and CsMYB2 shared 75% homology with MYBC2 of Vitis vinifera.The result of real time-PCR analysis showed the two genes were expressed constitutively in all tissues with different expression levels,e.g.the relative expression level of CsMYB2 in leaf was hundred times higher than that in root.Additionally,shading enhanced CsMYB1 expression,while the treatment did not alter the expression level of CsMYB2.

茶树新品系‘福茗2号’区域试验报告

【目的】选育早生、高产、高香茶树新品种,满足消费需求的多样化。【方法】以‘黄棪’为对照,在福建福安、松溪、福州3个区试点对‘福茗2号’新品系的种植成活率、物候期(一芽一叶、一芽二叶和一芽三叶期)、鲜叶产量、制茶品质、抗性等方面进行为期6年(2016—2021年)的茶树品种区域试验。【结果】‘福茗2号’新品系种植成活率高,达89%以上;物候期比对照早1~2 d;鲜叶产量比对照高5%左右;制乌龙茶品质比对照高出0.5~1.1分;耐寒、耐旱性强,茶炭疽病和茶小绿叶蝉抗性均表现为抗。【结论】‘福茗2号’是一个早生、高产、优质、高抗的乌龙茶新品种,适宜在福建福安、松溪、福州及相似乌龙茶地区种植和推广应用。

乌龙茶种质资源种群遗传多样性AFLP评价

以银染法 AFLP 分子标记技术用 5 对引物组合对来自福建武夷山市、安溪县、台湾省和广东潮安县 45份乌龙茶品种资源进行遗传多样性分析。结果表明,5 对引物扩增出 208 条多态性条带,多态性为 92.03%;最大遗传距离为 0.481,最小遗传距离为 0.124,种质资源间遗传多样性估值较高,达 0.311。按照地理分布分组分析表明,种群内遗传多样性以武夷山最高,其次为安溪乌龙茶种质资源,以台湾的乌龙茶种质资源遗传多样性最小;种群间遗传相似性,以武夷山与安溪种群间最高,达 0.9505,以台湾和潮安县类型间的相似性最低,相似性系数为 0.77。构建的种间和种群间进化树表明,可将 45 份乌龙茶品种划分为二大类型,福建类型和广东潮安类型。结合种群间相似系数,提出乌龙茶种质资源与其加工工艺的演化路径是一致的,也是由武夷山向安溪再向台湾传播。

凤凰单丛古茶树资源的遗传多样性AFLP分析

采用AFLP技术，选用分辨能力强、多态性高的5对引物组合对34个凤凰单丛古茶树资源进行遗传多样性分析。结果表明，5对引物共扩增出438条带，平均每对引物扩增87．6条带，多态性条带348条，多态性频率为79．3％。34个风凰单丛古茶资源之间遗传距离的变异范围在0.13-0.49之间，平均为0.33。其中大庵宋茶与棕蓑挟之间的遗传距离最低，为0.13，字茅黄栀香与福南蜜兰、白叶单丛与通天香之间的遗传距离最大，均为0.49。构建的聚类图表明，34个凤凰单丛古茶树资源的遗传关系和它们的生物学特性和香味类型没有必然的联系。

茶树炭疽菌Colletotrichum fructicola的鉴定及系统发育分析

从福建地区不同品种茶树上分离获得ZHG、WSX、WHG、FTG、ZTG、AHD、WRG、SLH等8株能侵染茶树的病原菌,采用形态学和rDNA-ITS序列分析相结合的方法对其进行鉴定。结果发现,SLH菌株为胶孢炭疽菌Colletotrichum gloeosporioides,其余7个菌株均为Colletotrichum fructicola。C.fructicola为能侵染茶树的新记录种炭疽菌,将其ITS序列与寄主分别为油茶、茉莉和番石榴等木本植物的9株炭疽菌的ITS序列进行聚类分析,结果显示,来自茶树的7个C.fructicola菌株种内存在碱基突变或缺失,且与寄主为茶树、油茶、茉莉等植物的胶孢炭疽菌C.gloeosporioides遗传距离较近。

利用cDNA-AFLP技术研究茶树花蕾发育基因差异表达片段

利用cDNA-AFLP技术,对茶树花蕾发育期的基因表达进行了分析。结果表明,茶树花蕾在发育早期和晚期基因的表达中有明显差异,共显示1110条带,早期发育花蕾和晚期发育花蕾显示的特异条带分别为35条和87条;选取特异表达的晚期发育花蕾重复性较好的15个TDFs(Transcript Derived Fragment)在GenBank进行序列分析,结果显示,9个分别与氧化还原酶、染色体13、14-3-3蛋白、Amyrel基因、钙网蛋白、ATP硫化酶、接触反应/铁离子结合蛋白及花粉特异蛋白的序列有相似性;2个与假拟蛋白相似,4个在GenBank数据库中未找到相似序列。

广东茶树种质资源AFLP分析

本研究采用AFLP技术，选用多态性高分辨能力强的5对引物对25个广东茶树群体品种和5个对照品种进行选择性扩增，获得了较清晰的DNA指纹图谱。5对引物共扩增出365条带，平均每对引物扩增73条带，多态性条带338条，占总带数的92．6％。扩增片段大小在50-600bp之间。采用DPS2000软件进行聚类，30份供试材料以清凉山茶和清远笔架茶遗传距离最近（0．12752），桂北大叶种和凤凰水仙之间的遗传距离最远（0．5），本实验结果表明广东地方茶树群体品种遗传多样性比较丰富。

用cDNA-AFLP技术研究茶树种子在低温贮藏过程中差异基因的表达

以茶树无性系龙井43种子为材料,利用cDNA-AFLP差异显示方法,分析它在低温贮藏后的基因表达,为顽拗性植物种子的中长期保存提供理论依据。从64对引物筛选出的差异片段中克隆出10个低温差异表达片段,有7个在GenBank数据库中与小分子量热激蛋白、MYB类转录因子、衰老相关蛋白、F-box家族蛋白、蛋白激酶、磷酸二酯酶等基因有较高同源性。

茶树多酚氧化酶基因的PCR-RFLP多态性分析

采用PCR-RFLP技术,检测茶树多酚氧化酶(PPO)基因中4个限制性核酸内切酶酶切位点在不同品种中的多态性,以研究其与品种适制性及遗传背景的关系。遗传参数分析表明:4个位点均处于Hardy-Weinberg平衡状态(P>0.05),HpaⅡ酶切位点(引物L7/L8扩增区段)呈现高度多态,BsuRⅠ酶切位点呈现中度多态,这2个酶切位点在不同品种中的基因型分布与品种的遗传背景有直接的关联,可作为遗传标记应用于茶树品种遗传亲源关系分析与亲子鉴定。HpaⅡ酶切位点在引物L7/L8扩增区段存在丰富的多态性,且与品种适制性具有明显关联,适制红茶的品种多为AA基因型,此位点能被HpaⅡ内切酶完全酶切;适制绿茶与乌龙茶的品种多为BB型或AB型,此位点不能被酶切或不能完全被酶切,该HpaⅡ酶切位点可作为茶树品种适制性早期鉴定的遗传标记。对引物L7/L8扩增区域的HpaⅡ酶切位点在祁门4号×潮安大乌叶F1代群体进行分型检测的结果表明,该位点在F1代的分离符合孟德尔遗传规律(x2=0.23)。

茶树α-tubulin基因的原核表达及其多克隆抗体的制备

根据茶树α-微管蛋白(α-tubulin)的mRNA全长序列(GenBank登录号DQ340766)设计引物,以RT-PCR方法扩增茶树α-tubulin基因,将扩增产物克隆到原核表达载体pET-32a(+)上,并转化至大肠杆菌BL21trxB(DE3)中,经异丙基-β-D硫代半乳糖苷(IPTG)诱导后以包涵体形式表达。以纯化后的α-tubulin融合蛋白制备兔多克隆抗体,用Westernblot方法检测抗体特异性。结果显示α-tubulin蛋白以包涵体形式大量表达,以融合蛋白制备的抗体对茶树体内的α-tubulin具有良好的特异性。

基于SSR标记的古茶树(Camellia sinensis)遗传关系分析

本研究利用30个SSR标记,结合已有的128个茶树品种指纹,对福建省园岭林场的4株古茶树和1株铁观音进行了遗传关系分析。结果表明,30个SSR标记共产生了124个等位片段,每个标记的等位片段数1~9个;多态性信息量较高,平均为0.507。遗传聚类分析表明,铁观音品种‘Tieguanyin2’与Fujian组的‘Tieguanyin’、‘Jinmudan’、‘Mingke1’和‘Zimudan’等遗传距离较近,属于铁观音品种。4株古茶树(YL01,YL02,YL03和YL04)间遗传距离较近,属于Fujian组,推测为八仙茶古茶树品种。本研究的5株茶树与已知的128个茶树品种间没有直接的亲子关系。YL02、YL03和YL04聚类较近,不匹配位点数较少,可能来源于共同的亲本。DNA分子指纹检测表明,8对核心SSR标记可对所有参试茶树进行有效的鉴别,这为古茶树的品种鉴定和产权保护提供了有效的分子证据。