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.

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。

乐山市茶树品种选育及特异种质资源研究现状

乐山市茶树种质资源丰富,已成功选育峨眉问春、川沐217、川茶2号、川茶3号、川茶10号、川沐28、马边绿1号、紫嫣、彝黄1号、川沐318等10个地方茶树品种。文章对这10个地方茶树品种授权情况进行了统计,并对物候期、新梢特点及主要化学成分含量进行了较全面的汇总比较,对其中的特异种质资源研究情况进行了简述,在此基础上分析了全市茶树育种工作存在的主要问题,提出下一步工作重点。

茶树CHS基因结构及编码区单核苷酸多态性分析

【目的】通过试验获得茶树CHS基因(CsCHS)gDNA序列,以进一步确定CsCHS基因结构;研究CsCHS编码区单核苷酸多态性,并结合茶树多酚含量进行关联分析,寻找基因中可能存在的与茶多酚含量存在显著或极显著相关关系的SNP位点。【方法】根据NCBI数据库中已有CsCHS序列设计特异性引物,再分别以基因组DNA和cDNA为模板进行PCR扩增,经克隆、测序获得CsCHS1、CsCHS2、CsCHS3三个基因的gDNA和cDNA全长序列,通过序列比对方法确定CsCHS结构。利用Compute pI/Mw、SOPMA等软件对所得序列进行生物信息学分析,预测和比较CsCHS1、CsCHS2和CsCHS3三者蛋白质结构。以茶多酚含量差异较大的57份茶树品种为材料,分别以57份材料的cDNA为模板,用特异性引物进行PCR扩增,然后利用PCR产物直接测序法筛查CsCHS编码区序列的单核苷酸多态性。结合CsCHS编码区序列单核苷酸多态性和57份材料多酚含量,利用软件TASSEL进行关联分析,筛选基因中可能与茶多酚含量存在显著或极显著相关关系的SNP位点。【结果】试验获得CsCHS1、CsCHS2和CsCHS3的cDNA序列长度分别为1 277、1 320和1 242 bp,各自均包含一个长度为1 170 bp的开放阅读框;CsCHS1、CsCHS2和CsCHS3的gDNA序列长度分别为1 600、1 330和1 607 bp。通过gDNA序列和cDAN序列比对,结合真核生物内含子GT-AG法则,确定CsCHS1、CsCHS3分别包含2个外显子和1个内含子,内含子大小分别为323和356 bp,CsCHS2可能没有内含子。根据CsCHS1、CsCHS2和CsCHS3 cDNA序列推导三者对应氨基酸序列,比较三条氨基酸序列发现三者氨基酸同源性较高,达到92.6%—95.4%,CHS蛋白亚家族中的特征性保守位点在这三条序列中都能找到,生物信息学分析结果显示CsCHS1、CsCHS2和CsCHS3三者蛋白质结构高度相似。CsCHS1编码区序列中共发现71个SNP位点,SNP出现频率为1SNP/16.48 bp,无Indel,基因核苷酸多样性(π)值为0.01088;CsCHS2编码区序列中共发现55个SNP位点,SNP出现频率分别为1SNP/21.27 bp,CsCHS2核苷酸多样性(π)值(0.00530)明显低于CsCHS1;因扩增CsCHS3 cDNA序列的PCR反应成功率低,未对该基因遗传多样性进行分析。通过关联分析分别从CsCHS1和CsCHS2中找到2个和4个与茶叶多酚含量相关的SNP位点。【结论】CsCHS1和CsCHS3属于保守型CHS基因,且CsCHS1、CsCHS2和CsCHS3蛋白质结构高度相似,推测三者可能在茶树的不同部位或不同生长阶段发挥类似作用;CsCHS1和CsCHS2活跃,二者编码区内可能存在突变热点区。

茶树多酚氧化酶基因的SNP分析

选取有代表性的40个茶树品种(系)为材料,先采用PCR-SSCP-银染检测技术,对供试材料的多酚氧化酶(PPO)基因进行SSCP分析,对PPO基因作初步基因分型;根据分型分析结果,对每一类型选择典型品种的PPO基因作进一步的DNA序列分析,用DNASTAR Program进行单核苷酸多态性(SNP)搜寻.结果表明,茶树PPO基因存在丰富的SNP,其类型包括:转换(AG,CT)、颠换(AT,GC,G→T,A→C)与杂合子(C/T,A/G,G/T,A/T,A/C,G/C).在发现的37个SNPs中,有13个SNPs发生在核酸内切酶酶切位点,有10个错义SNPs位点导致氨基酸的改变.在PPO基因的保守序列区,出现SNP的频率较低.

茶树新品种‘紫娟’

茶树新品种‘紫娟’,新鲜嫩梢的芽、叶、茎均为紫色,花青素含量约为一般红芽茶的3倍;加工而成的紫娟绿茶色泽紫黑色,茶汤紫红色,味醇厚,香气特殊。经动物试验表明其降压效果优于云南大叶群体种茶。

15个茶树品种遗传多样性的RAPD分析

应用十聚体随机引物．对原产于福建、湖南、浙江、陕西、贵州、江西和湖北等省的15个无性系品种及其中2个品种的扦插繁殖后代的基因组DNA的遗传多样性进行RAPD分析，结果表明，20个随机引物在15个品种中共扩增出1050个位点．平均每个引物52．5个，每个品种70个位点。在得到的137条谱带中只有8条是所有品种共有的（单态的），129条是多态的，多态性程度达94．2％，证明了中国茶树品种资源在DNA分子水平上具有很高的遗传多样性。类平均法聚类结果表明，可将15个品种分成五个类群：A类群为江苦2号、蓝山苦茶、蓝标1号、北斗1号、福建水仙和政和大白菜等6个品种，B类群为早春早芽、乌牛早和黄叶早等3个品种，C类群为蒿坪茶、狗牯脑、大方贡茶和恩标等4个品种，汝城早芽、龙井43单独成为二个类群。从类群内多态度来看，类群B多态性最低，类群C次之，类群A最高。从类群间平均多态度和净遗传距离反映出类群A与B、B与C之间的差异程度较小且基本相似．而类群A与C之间的差异程度要大得多。

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。

茶树基因组DNA提纯与鉴定

采用在细胞核被裂解之前去除细胞质中的茶多酚和蛋白质，而后用ＳＤＳ裂解细胞核，异丙醇和乙醇沉淀基因组ＤＮＡ的方法，分别从不同茶树品种新梢、干梢及冰冻新梢中成功地提取和纯化基因组ＤＮＡ，并对其ＤＮＡ的得率和质量进行了鉴定。ＤＮＡ的得率在２０５～９６３ｎｇ／ｍｇ鲜重之间，所得到的ＤＮＡ样品片段均大于２１ｋｂ，适宜于进行限制性酶切和ＲＡＰＤ反应。实践证明，上述提取富含酚类物质植物基因组ＤＮＡ的方法，不仅迅速简单，而且经济有效。

云南古茶树(园)遗传多样性的ISSR分析

云南省保存有20多万亩古茶园，这些古茶树（阿萨姆变种Camellia sinensis vat．assamica）种质资源可能含有各种优良基因，对未来茶树良种选育有重要意义。本研究采用ISSR分子标记技术对云南省十个有代表性的古茶园遗传多样性进行分析，十个居群的多态位点百分率（P）为56．5％-90．9％；Nei’氏遗传距离（He）居群平均是0．281，阿萨姆变种水平内是0．461；Shannon多样性指数（HD）居群平均是0．418，阿萨姆变种水平内是0．653。而居群间遗传分化系数Gst=0.391，和Shannon多样性指数分析（36．0％）和AMOVA分析结果（39．7％）相一致，说明阿萨姆变种60．9％的遗传变异来自居群内的个体间，39．1％的遗传变异来自居群间。研究结果揭示阿萨姆变种居群遗传多样性高，居群间遗传变异存在中度的遗传分化，这可能是由于茶树种内高度异的特性和生境片段化所致。基于观察到的居群遗传信息，建议采取就地保护和迁地保护的保护策略。

茶树中植物激素研究进展

植物激素在植物生长发育中具有重要的调控作用。本文综述了植物激素对茶树生长发育和逆境反应的调控,及植物激素在茶树外植体繁殖中的应用,并概述了当前植物激素研究的方向和重点。针对茶树中激素研究现状并结合目前分子生物学的发展,提出茶树基因组测序完成后,植物激素对茶树茶芽萌发、抗逆等调控机理的研究将成为茶叶科学研究中一个新的热点。

不同品种茶树氮素效率差异研究

比较了6个茶树品种的氮素效率差异。结果表明,在4种施氮条件下,生物量增加值、新梢生长量、氮素吸收效率、氮素生理利用效率、氮素经济效率和总的氮素效率存在着显著的品种间差异。氮素效率的两个子性状—吸收效率和生理利用效率对其贡献大小不同,吸收效率是决定不同品种茶树氮素效率差异的主要因素。

茶树冷胁迫诱导抗寒基因CBF的克隆与表达分析

利用cDNA-AFLP技术进行了茶树低温胁迫处理的基因表达差异分析,获得低温诱导后特异表达的差异片段TDF11(transcript derived fragment,TDF)。经BLAST比对发现该片段与白菜、拟南芥、烟草的抗寒基因CBF(C-repeat binding factor)分别有94%、84%、81%的同源性。通过3’/5’RACE的方法获得该片段cDNA全长序列(GenBank,登录号EU857638)。所得序列全长981bp,其开放阅读框编码275个氨基酸。该基因与白菜、烟草、拟南芥中的CBF基因编码的氨基酸序列分别有74%、57%、57%的同源性。用RT-PCR的方法对其在低温处理过程中的表达进行分析,结果表明,茶树CBF基因在低温诱导4h开始表达,在8h左右表达量最高,然后开始下降,但仍维持在一个较高的水平。结果提示CBF基因可能在茶树抗寒分子机制的形成过程中发挥重要的作用。