Tea Germplasm Research Articles

Genetic resources diversity of tea (Camellia sinensis (L.) Kuntze) in the southern region of the Caspian Sea

AbstractIn the fields of agricultural, food and medical research, the potential impacts of tea on human health are of special interest because tea (Camellia sinensis (L.) Kuntze) is continuously consumed by many people in the world. The evaluation of the diversity of genotypes found in tea germplasm can aid in the improvement of the breeding programme. In this study, the genetic diversity of 30 tea genotypes from two commercial sites of tea production in Iran was investigated by using morphological and 12 ISSR markers. Morphological analysis showed that the diversity between samples of tea was limited, and the narrow matching range was calculated. In cluster analysis at level 0.63, samples were divided into four groups. The application of 12 ISSR primers produced 91 polymorphic bands. PIC test showed a range of 0.41–0.48. Based on the ISSR data, the matching range was obtained in the range of 0.24–0.93. In cluster analysis, samples at level 0.58 were divided into five groups. According to the results, it can be understood that these series of traits and primers can very well recognize genetic differences. Using these markers, genetic diversity was observed among tea genotypes, but this diversity was not such as to be able to separate genotypes of various regions from each other. The results showed that the tea genotypes in Iran had high genetic diversity. As a consequence, the findings of our study will help the development of tea germplasm conservation strategies and their sustainable use in breeding programmes.

Population whole-genome resequencing reveals the phylogenetic relationships and population structure of four Hunan typical tea landraces

Hunan located at the evolutionary transitional zone edge of the Yunnan-Guizhou plateau, tea germplasms are abundant. At present, although morphological, enzymological and molecular markers have been used to study the genetic relationship of these tea germplasms. However, due to the complexity of the genetic background of tea germplasm resources, the evolution history and taxonomy of Hunan tea germplasm is still unclear. In this study, four Hunan tea landraces from the evolutionary transitional zone were collected, including 9 'Chengbu Dongcha' (CBDC), 8 'Jianghua Kucha' (JHKC), 11 'Rucheng Baimao' (RCBM) and 8 'Anhua Yuntai' (AHYT). We used whole-genome re-sequencing to identify genetic variation, phylogenetic placement, and population structure among the four tea landraces and then scanned the genome for regions related to population potential selective. Across the samples, we identified an average of 1.38 million (M) SNPs and 6.82 × 10³ INDELs. Based on the high-quality SNPs, we performed neighbor-joining phylogenetic tree, population structure, and principal component analysis, all of which supported that the 36 tea germplasms could been divided into two groups, CBDC and AHYT were clustered into a group, and RCBM and JHKC were clustered into another group. With CBDC, JHKC, and RCBM as control, 407, 380, and 147 selected genes were identified in AHYT, respectively, were in the top 5% of F_ST and Log₂^Ratioθπ scores. The identified genes are mainly involved in stress tolerance, biosynthesis of important secondary metabolites, adaptability, and yield. These results will be helpful in the classification of tea germplasms and investigating selection–related genes.

Integrated transcriptome and metabolome provide insights into flavonoid biosynthesis in 'P113', a new purple tea of <i>Camellia tachangensis</i>

Plants typically exhibit the purple phenomenon as a result of an increase in flavonoids and anthocyanins. A new tea germplasm 'P113' was recently selected from <italic>Camellia tachangensis</italic>, which is purple in tender shoots. In the present study, integrated transcriptome and metabolome were used to analyze the flavonoid metabolite components and the genes involved in flavonoid biosynthesis in 'P113'. A total of 86 flavonoid metabolites were identified, including 70 significantly differential metabolites (<italic>p</italic> &lt; 0.05) and they were enriched to the three metabolic pathways of ko00941, ko00942 and ko00944 through KEGG pathway analysis. A total of 136 flavonoid involved genes were obtained from transcriptomic study, of which 53 were significantly differentially expressed in developmental shoots. The correlation between metabolite profiling and transcriptome, transcriptome and protein interactions suggested that transcription factor MYB12 and glycosyltransferase UGT78D2 had a good facilitation on purple tender shoots. The metabolic pathways and potential genes that underlie the coloration of the shoots in 'P113' are clarified in this study. It also lays the groundwork for identifying potential genes involved in color variation and offers a theoretical framework for the creation and use of distinctive genetic resources and the breeding of new cultivars.

Development of SNP Markers for Original Analysis and Germplasm Identification in Camellia sinensis.

Tea plants are widely grown all over the world because they are an important economic crop. The purity and authenticity of tea varieties are frequent problems in the conservation and promotion of germplasm resources in recent years, which has brought considerable inconvenience and uncertainty to the selection of parental lines for breeding and the research and cultivation of superior varieties. However, the development of core SNP markers can quickly and accurately identify the germplasm, which plays an important role in germplasm identification and the genetic relationship analysis of tea plants. In this study, based on 179,970 SNP loci from the whole genome of the tea plant, all of 142 cultivars were clearly divided into three groups: Assam type (CSA), Chinese type (CSS), and transitional type. Most CSA cultivars are from Yunnan Province, which confirms that Yunnan Province is the primary center of CSA origin and domestication. Most CSS cultivars are distributed in east China; therefore, we deduced that east China (mainly Zhejiang and Fujian provinces) is most likely the area of origin and domestication of CSS. Moreover, 45 core markers were screened using strict criteria to 179,970 SNP loci, and we analyzed 117 well-Known tea cultivars in China with 45 core SNP markers. The results were as follows: (1) In total, 117 tea cultivars were distinguished by eight markers, which were selected to construct the DNA fingerprint, and the remaining markers were used as standby markers for germplasm identification. (2) Ten pairs of parent and offspring relationships were confirmed or identified, and among them, seven pairs were well-established pedigree relationships; the other three pairs were newly identified. In this study, the east of China (mainly Zhejiang and Fujian provinces) is most likely the area of origin and domestication of CSS. The 45 core SNP markers were developed, which provide a scientific basis at the molecular level to identify the superior tea germplasm, undertake genetic relationship analysis, and benefit subsequent breeding work.

Hyperspectral machine-learning model for screening tea germplasm resources with drought tolerance.

Drought tolerance and quality stability are important indicators to evaluate the stress tolerance of tea germplasm resources. The traditional screening method of drought resistant germplasm is mainly to evaluate by detecting physiological and biochemical indicators of tea plants under drought stresses. However, the methods are not only time consuming but also destructive. In this study, hyperspectral images of tea drought phenotypes were obtained and modeled with related physiological indicators. The results showed that: (1) the information contents of malondialdehyde, soluble sugar and total polyphenol were 0.21, 0.209 and 0.227 respectively, and the drought tolerance coefficient (DTC) index of each tea variety was between 0.069 and 0.81; (2) the comprehensive drought tolerance of different varieties were (from strong to weak): QN36, SCZ, ZC108, JX, JGY, XY10, QN1, MS9, QN38, and QN21; (3) by using SVM, RF and PLSR to model DTC (drought tolerance coefficient) data, the best prediction model was selected as MSC-2D-UVE-SVM (R2 = 0.77, RMSE = 0.073, MAPE = 0.16) for drought tolerance of tea germplasm resources, named Tea-DTC model. Therefore, the Tea-DTC model based on hyperspectral machine-learning technology can be used as a new screening method for evaluating tea germplasm resources with drought tolerance.

Variations in oolong tea key characteristic floral aroma compound contents among tea (Camellia sinensis) germplasms exposed to postharvest stress

Tea (Camellia sinensis) germplasm affects the tea aroma quality, especially in oolong tea. However, the mechanism underlying the diversity in aroma formation induced by postharvest stresses among different tea cultivars remains unclear. In this study, correlation analyses between sensory evaluation scores and aroma compound contents of oolong teas made from 12 different cultivars was carried out. The results confirmed the contribution of three characteristic aroma compounds [i.e., indole, jasmine lactone, and (E)-nerolidol] to the oolong tea floral aroma quality. However, only the indole was positively correlated with the jasmonic acid (JA) in the simulated withering (wounding stress due to plucking) and turnover (continuous wounding stress) stages. Furthermore, the expression levels of transcription factor CsMYC2c and structural genes for the three aroma compounds were positively correlated in the simulated withering stage. Transient transcriptional activation analyses in tobacco revealed that CsMYC2c regulated the promoter activity and expression of structural genes. However, yeast one-hybrid assay showed that CsMYC2c did not directly bind to the gene promoters. The information has expanded our understanding of the causes for the variations in aromas among diverse tea cultivars in response to postharvest stress. Furthermore, it may provide the basis for breeding and selection of high-aroma tea cultivars.

Genetic Diversity of Tea Plant (Camellia sinensis (L.) Kuntze) Germplasm Resources in Wuyi Mountain of China Based on Single Nucleotide Polymorphism (SNP) Markers

Wuyi Mountain in Southeast China is the origin of black tea and oolong tea. It is also considered the ‘treasure trove of tea cultivars’ because of its rich tea germplasm resources. In the present study, the population structure and genetic diversity of 137 tea germplasms from Wuyi Mountain and its adjacent areas were analyzed by SNPs. The information index (I), observed heterozygosity (Ho), expected heterozygosity (He) and fixation index (F) polymorphisms of the selected SNPs were high, stable and reliable. Ho had an average of 0.389, while He had an average of 0.324, indicating that Wuyi Mountain tea germplasms had rich genetic diversity. The AMOVA results showed that genetic variation came mainly from intrapopulation variation, accounting for 66% of the total variation. The differences in the Fst and Nei values of tea germplasm between Wuyi Mountain and its adjacent areas are similar to the geographical differences. Multiple analyses based on high-quality SNPs found that the landraces of tea plants on Wuyi Mountain had different genetic backgrounds from the wild-type landraces and the landraces of Wuyi Mountain tea plants underwent population differentiation. This study provides a basis for the effective protection and utilization of tea germplasms on Wuyi Mountain and lays a foundation for identifying potential parents to optimize tea cultivation.

Metabolomic Profiling in Combination with Data Association Analysis Provide Insights about Potential Metabolic Regulation Networks among Non-Volatile and Volatile Metabolites in Camellia sinensis cv Baijiguan.

The non-volatile and volatile metabolites in tea confer the taste and odor characteristics of tea fusion, as well as shape the chemical base for tea quality. To date, it remains largely elusive whether there are metabolic crosstalks among non-volatile metabolites and volatile metabolites in the tea tree. Here, we generated an F1 half-sib population by using an albino cultivar of Camellia sinensis cv Baijiguan as the maternal parent, and then we quantified the non-volatile metabolites and volatile metabolites from individual half-sibs. We found that the EGC and EGCG contents of the albino half-sibs were significantly lower than those of the green half-sibs, while no significant differences were observed in total amino acids, caffeine, and other catechin types between these two groups. The phenylpropanoid pathway and the MEP pathway are the dominant routes for volatile synthesis in fresh tea leaves, followed by the MVA pathway and the fatty acid-derivative pathway. The total volatile contents derived from individual pathways showed large variations among half-sibs, there were no significant differences between the albino half-sibs and the green half-sibs. We performed a comprehensive correlation analysis, including correlations among non-volatile metabolites, between volatile synthesis pathways and non-volatile metabolites, and among the volatiles derived from same synthesis pathway, and we identified several significant positive or negative correlations. Our data suggest that the synthesis of non-volatile and volatile metabolites is potentially connected through shared intermediates; feedback inhibition, activation, or competition for common intermediates among branched pathways may co-exist; and cross-pathway activation or inhibition, as well as metabolome channeling, were also implicated. These multiple metabolic regulation modes could provide metabolic plasticity to direct carbon flux and lead to diverse metabolome among Baijiguan half-sibs. This study provides an essential knowledge base for rational tea germplasm improvements.

Physiological and biochemical evaluation of high anthocyanin pigmented tea (Camellia sinensis L. O. Kuntze) germplasm for purple tea production.

Finding promising purple tea germplasm that would target new tea products for diversification and value addition boost the tea industry’s economic growth. Accordingly, 10 tea germplasm viz. TRA St. 817, TRA St. 293, TRA St. 400, TRA 177/3, TRA 376/2, TRA 376/3, TRA 427/7, TRA P7, TRA P8, and TV1 were evaluated in terms of gas exchange parameters, multiplication performance, and biochemical markers such as chlorophyll, carotenoids, and anthocyanin content, which are related to the purple tea quality. The investigated gas exchange and biochemical parameters revealed significant differences. Germplasm TRA St.817 was physiologically more efficient (24.7 μmol m–2 s–1), followed by TRA St. 293, exhibiting the highest net photosynthesis, water use efficiency (19.02 μmol mmol–1), carboxylation efficiency (0.73), chlorophyll fluorescence or photochemical efficiency of PSII (0.754) and mesophyll efficiency (ci/gs ratio: 2.54). Net photosynthesis was positively correlated with water use efficiency, carboxylation efficiency, mesophyll efficiency, and photochemical efficiency of PSII (r = 0.965**, 0.937**, 0.857**, 0.867**; P = 0.05), respectively, but negatively correlated with the transpiration ratio (r = −0.878**; P = 0.05) based on Pearson correlation analysis. The total anthocyanin content (4764.19 μg.g–1 fresh leaf weight) and carotenoid content (3.825 mg.g–1 fresh leaf weight) were highest in the TRA St.817 germplasm, followed by germplasm TRA St. 293 (2926.18 μg.g–1 FW). In contrast, total chlorophyll content was significantly low (1.779 mg.g–1 fresh weight), which is very suitable for manufacturing purple tea. The highest carotenoid concentration in TRA St. 817 was 3.825 mg.g–1 FW, followed by TRA P8 (3.475 mg.g–1 FW), favoring the formation of more volatile flavor constituents. The promising germplasm, TRA St 817, has a multiplication success rate of 91.4% through cleft grafting. The outcome reveals that TRA St.817 is a promising germplasm that can be used to make speciality teas, i.e., purple tea.

Natural variation of main biochemical components, morphological and yield traits among a panel of 87 tea [Camellia sinensis (L.) O. Kuntze] cultivars

Many attentions have been previously focused to identify the multiple biochemical components related to tea quality and health benefits, however, the natural variation of biochemical components present in tea germplasm has not been adequately evaluated. In this study, the main biochemical components, leaf morphological and yield characteristics were evaluated for four rounds of tea leaves in a panel of 87 elite tea cultivars suitable for black, green, or oolong tea. Significant variations were observed among the tea cultivars, as well as seasonal differences in the levels of the free amino acid (FAA), caffeine (CAF), tea polyphenols (TP), water extract (WE) and TP to FAA ratio (TP/FAA). Results showed that the average levels of FAA showed a seasonal change, with the highest level of 4.0% in the 1st spring tea in the cultivars suitable for green tea and the lowest of 3.2% in summer tea in the cultivars suitable for black tea. The average CAF content was highest 3.2% in the cultivars suitable for oolong tea in the 1st spring and the lowest 2.5% in the cultivars suitable for green tea in summer. Limited seasonal and varietal variations were noticed in the average levels of WE among the three categories of tea. In addition, significant natural variation of the morphological characteristics, bud length varying from 2.5 cm to 8.7 cm, bud density from 190.3 buds · m-2 to 1 730.3 buds · m-2, mature leaves biomass from 128.4 kg · hm-2 to 2 888.4 kg · hm-2, and yield component traits of 100 buds (one bud with two leaves) dry weight from 3.7 g to 37.7 g, tea yield/round from 444.6 kg · hm-2 to 905.3 kg · hm-2, were observed. The aim of our evaluation was not only to identify the advantages of seasonal and clonal variations but also to provide a new viewpoint for their further application. Representative accessions were selected from the germplasm to promote the establishment of an inherent biochemical constituent expressing the quality of black, green, and oolong tea. The findings might be utilized to establish early selection criteria to enhance the tea breeding and production program.

Physicochemical analysis of Ugandan tea (Camellia sinensis) germplasm reveals potential commercial green and black tea varieties

ABSTRACT Tea (Camellia sinensis L.) is an important beverage consumed worldwide. In Uganda, it is the second-largest and highly prioritized export crop after coffee and provides the much-needed jobs to more than 800,000 people. Despite its importance in the country, the crop has received very limited research attention for its improvement and optimal utilization. This study was aimed at characterizing existing tea germplasm in Uganda to inform future breeding initiatives for market-preferred tea varieties. Fifty-eight advanced tea clones were randomly selected from the tea germplasm conserved at Uganda’s Tea Research Center in Rwebitaba and analyzed in the laboratory for eight physicochemical descriptors. Hierarchical cluster analysis performed on the 58 clones using Cluster-R package revealed four main clusters, indicating the existence of variation for physicochemical parameters within tested germplasm. This variation can be exploited to select among and/or improve the studied germplasm genetically for quality. High fermentation rate, which is one of the key indicators for good-quality black teas, revealed 15 fast-fermenter tea clones. Clone “UTR12/12” was identified as the most rapid fermenter, fully fermenting within 30 min, which implies a good clone for black tea. The clone also had the highest polyphenol content (26.7%), higher than the high-quality clone “UTR6/8” (control). Other tea clones whose polyphenol content was within the range of the control black tea clone were: “UTR144/10” (20.9%) and ‘UTR144/17, (20.66%). The identified promising black tea clones can be advanced to multi-location trials for further evaluation and selection for eventual variety release as commercial black-tea clones.

Transcriptomic analysis identifies differentially expressed genes in purple tender shoots and green mature leaves of Zijuan tea

Purple shoots of tea germplasm Zijuan (Camellia sinensis var. asssamica) have high anthocyanin content and gradually turn green with continued growth and development. To uncover the mechanism of this colour change, we performed transcriptome analysis to identify differentially expressed genes (DEGs). A total of 18 328 DEGs were identified in purple tender shoots of Zijuan (ZJP) compared with mature green leaves of Zijuan (ZJG). These included 85 DEGs associated with phenylpropanoid biosynthesis, 30 DEGs associated with flavonoid biosynthesis, and 3 DEGs involved in flavone and flavonol biosynthesis. We detected higher expression in ZJP than in ZJG of genes encoding the following key enzymes regulating anthocyanin biosynthesis and their corresponding metabolites: chalcone synthase (CHS), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), adenosine triphosphate (ATP)-binding cassette (ABC) transporters, bifunctional 3-dehydroquinate dehydratase (DHQ), chorismate mutase (CM), cinnamoyl-CoA reductase 1 (CCR), and cinnamyl alcohol dehydrogenase (CAD). In addition, upregulated expression of carbohydrate metabolism, glycolysis pathway, sucrose metabolism, and pyruvate metabolism suggested that glycolysis and upregulation of the sucrose synthase (SUS) gene may provide more intermediates as substrates for promoting anthocyanin accumulation in ZJP. Moreover, DEGs involved in anthocyanin biosynthesis, including phenylalanine ammonialyase (PAL), cinnamate acid 4-hydroxylase (C4H), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), ANR, ANS, CHS, and flavonoid 3',5'-hydroxylase (F3'5'H) were significantly (P &amp;lt; 0.01) correlated with the contents of anthocyanin components such as cyanidin (Cy), delphinidin (Dp), peonidin (Pn), petunidin (Pt), and malvidin (Mv). The above results suggested that these upregulated DEGs may contribute to anthocyanin accumulation in ZJP and may play important roles in the colour changes in Zijuan tea. This research provided a foundation for clarifying the mechanisms underlying colour changes in Zijuan tea.

Complete chloroplast genome of Ilex dabieshanensis: Genome structure, comparative analyses with three traditional Ilex tea species, and its phylogenetic relationships within the family Aquifoliaceae.

Ilex dabieshanensis K. Yao & M. B. Deng is not only a highly valued tree species for landscaping, it is also a good material for making kuding tea due to its anti-inflammatory and lipid-lowering medicinal properties. Utilizing next-generation and long-read sequencing technologies, we assembled the whole chloroplast genome of I. dabieshanensis. The genome was 157,218 bp in length, exhibiting a typical quadripartite structure with a large single copy (LSC: 86,607 bp), a small single copy (SSC: 18,427 bp) and a pair of inverted repeat regions (IRA and IRB: each of 26,092 bp). A total of 121 predicted genes were encoded, including 113 distinctive (79 protein-coding genes, 30 tRNAs, and 4 rRNAs) and 8 duplicated (8 protein-coding genes) located in the IR regions. Overall, 132 SSRs and 43 long repeats were detected and could be used as potential molecular markers. Comparative analyses of four traditional Ilex tea species (I. dabieshanensis, I. paraguariensis, I. latifolia and I. cornuta) revealed seven divergent regions: matK-rps16, trnS-psbZ, trnT-trnL, atpB-rbcL, petB-petD, rpl14-rpl16, and rpl32-trnL. These variations might be applicable for distinguishing different species within the genus Ilex. Phylogenetic reconstruction strongly suggested that I. dabieshanensis formed a sister clade to I. cornuta and also showed a close relationship to I. latifolia. The generated chloroplast genome information in our study is significant for Ilex tea germplasm identification, phylogeny and genetic improvement.

Profiling of Few of the Tea Accessions on Physiological Growth and Stress Tolerance

Tea [Camellia sinensis(L) O Kuntz] is one of the oldest, most widely consumed and least expensive natural beverages, known to the world over for its heritage brew with various flavor and antioxidant properties. The productivity of tea in world as well as in India has been raised by developing high yielding clones and seed stocks but the genetic base of the existing diversity of the crop is rapidly narrowing down. In present scenario, apart from yield and quality, physiological parameters and abiotic stress tolerance are also raising up to be the most important environmental indicator impacting on sustainability and productivity of tea. In the published research work, an effort was made to characterize and estimate the variation of 20 tea germplasm named as THT1 to THT20, growing in the Experimental Garden for Plantation Crops, AAU, Jorhat on the basis of growth parameters and abiotic stress tolerance capability of tea.&#x0D; In the experiment all the germplasm showed highly significant difference amongst them regarding growth parameters, pigment content and on abiotic stress tolerance. THT 1, THT 10, THT 3 contained higher total chlorophyll and THT 4, THT 10, THT 20 showed high carotene content. In case of growth parameters THT 11, THT 7, THT5 showed higher plucking point density whereas THT 11, THT 17, THT 7 showed good abiotic stress tolerance with high value of proline content, relative turgidity, water saturation deficit and epicuticular wax content.&#x0D; When clustering was done based on abiotic stress tolerance, pigment and growth parameters respectively, THT11 and THT 17 are found to form a discriminated cluster in the dendogram. The parameters taken especially for abiotic stress tolerance study may be subjected to environmental factors for which more elaborated field trial may be required for these clones to assess their yield and quality parameters.

Nontargeted and targeted metabolomics analysis provides novel insight into nonvolatile metabolites in Jianghua Kucha tea germplasm (Camellia sinensis var. Assamica cv. Jianghua).

Jianghua Kucha (JHKC) is a special tea germplasm with high bitterness growing in China; however, the chemical characteristics of JHKC are not completely understood. In this study, 61 differential metabolites were identified between 11 wild JHKC individuals and 3 control cultivars of Fudingdabai, Yunkang 10, and Zhuyeqi using comprehensive nontargeted and targeted metabolomics approach. The JHKC accessions mainly possessed significantly higher levels of purine alkaloids of theacrine (12.06±5.23mg/g) and 1,3,7-trimethyluric acid, non-epi-form flavanols (catechin, gallocatechin, catechin gallate, and gallocatechin gallate), and methylated flavanols of epigallocatechin-3-O-(3″-O-methyl)-gallate (4.79±1.45mg/g) and epicatechin-3-O-(3″-O-methyl)-gallate (1.02±0.34mg/g), as well as significantly lower levels of flavonol glycosides, which indicated that caffeine metabolism, flavonoid biosynthesis, and flavonol and flavone biosynthesis are mostly differential metabolic pathways. Our study demonstrated that JHKC germplasm is a promising resource for breeding novel tea cultivars with high contents of theacrine, non-epi-form flavanols, and methylated flavanols.

Genetic Diversity and Genome Size Variability in the Russian Genebank Collection of Tea Plant [Camellia sinensis (L). O. Kuntze

The tea collection of the FRC SSC RAS (Sochi, Maykop in Russia) represents one of the northernmost germplasm comprising a number of locally derived cultivars and ɣ-irradiation mutants. The latter are often characterized by larger genome size, which may lead to better adaptation to biotic and abiotic stress. Such genotypes may be a valuable genetic resource for better adaptability to extreme environmental conditions, which could enable tea cultivation outside global growing regions. Microsatellite markers are often the best choice for genetic diversity analysis in genebank collections. However, their use in polyploid species is questionable because simple sequence repeat (SSR) allele dosage cannot be readily determined. Therefore, the efficiency of SSR and start codon targeted (SCoT) markers was investigated using 43 selected cultivars from the Russian genebank collection derived from mutant breeding and clonal selection. Previously, the increase in genome size was confirmed in 18 mutants within this collection. Despite the presence of polyploid tea genotypes, our study revealed higher efficiency of SSR markers than SCoT markers. Subsequent SSR analysis of the 106 genotypes in the Russian genebank collection revealed three distinct genetic clusters after STRUCTURE analysis. Greater genetic variation was observed within genetic clusters than between clusters, indicating low genetic variation between collections. Nevertheless, the northernmost tea collection exhibited a greater genetic distance from the other two clusters than they did from each other. Close genetic relationships were found between many cultivars with particularly large leaves and mutant forms. Pearson’s correlation analysis revealed a significant, moderate correlation between genome size and leaf area size. Our study shows that microsatellite fingerprinting is useful to estimate the genetic diversity and genetic background of tea germplasm in Russia despite polyploid tea accessions. Thus, the results of our study contribute to the development of future tea germplasm conservation strategies and modern tea breeding programs.

The Diversity and Classification of Tea Germplasm Based on Morpho-Agronomic Characteristics

The information on diversity needs to be disclosed as a basis for consideration in the use of genetic resources. The objective of this study is to reveal the diversity and the classification of tea germplasm based on morpho-agronomic characteristics. The experiment was carried out from June to December 2018 in Gunung Putri Experimental Garden, Cianjur, West Java, Indonesia. The materials of the study used were 13 first generation tea clones. The research used was the observation method.s. The level of kinship among the clones was calculated using cluster analysis with the PBSTAT program. The results of observations showed that there was a diversity of leaf margin characteristics, leaf shape, mature leaf color, and immature leaf color. The high diversity was seen in pecco+2 weight, pecco+3 weight, and petiole length. The highest weight of pecco+3 was found in clone Cin 1. The coefficient of dissimilarity of 0.34 was divided into 2 main groups. Group I consisted of the clone Cin 1 and the clone Cin 28. The second group consisted of 11 tea clones. The clone Cin 1 and the clone Cin 28 and the clone Mal 17 are the tea clones with the smallest similarity values.

A novel TcS allele conferring the high-theacrine and low-caffeine traits and having potential use in tea plant breeding.

Theacrine (1,3,7,9-tetramethyluric acid) is a natural product with remarkable pharmacological activities such as antidepressant, sedative and hypnotic activities, while caffeine (1,3,7-trimethylxanthine) has certain side effects to special populations. Hence, breeding tea plants with high theacrine and low caffeine will increase tea health benefits and promote consumption. In this study, we construct an F1 population by crossing 'Zhongcha 302' (theacrine-free) and a tea germplasm 'Ruyuan Kucha' (RY, theacrine-rich) to identify the causal gene for accumulating theacrine. The results showed that the content of theacrine was highly negatively correlated with caffeine (R2 > 0.9). Bulked segregant RNA sequencing analysis, molecular markers and gene expression analysis indicated that the theacrine synthase (TcS) gene was the candidate gene. The TcS was located in the nucleus and cytoplasm, and the theacrine can be detected in stably genetic transformed tobacco by feeding the substrate 1,3,7-trimethyluric acid. Moreover, an in vitro enzyme activity experiment revealed that the 241st amino acid residue was the key residue. Besides, we amplified the promoter region in several tea accessions with varied theacrine levels, and found a 234-bp deletion and a 271-bp insertion in RY. Both GUS histochemical analysis and dual-luciferase assay showed that TcS promoter activity in RY was relatively high. Lastly, we developed a molecular marker that is co-segregate with high-theacrine individuals in RY's offspring. These results demonstrate that the novel TcS allele in RY results in the high-theacrine and low-caffeine traits and the developed functional marker will facilitate the breeding of characteristic tea plants.

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.

Genetic diversity, population structure and core collection analysis of Hunan tea plant germplasm through genotyping-by-sequencing

Tea is considered to be a well-known and widely consumed beverage and Hunan province is rich in tea plant germplasm. In order to better conserve and utilize Hunan tea plant resources, 110 tea accessions from seven geographical origins were used to assess genetic diversity of Hunan tea plant germplasm through genotyping by sequencing (GBS) technology. As a result, a total of 311,044 high-quality single nucleotide polymorphism (SNP) markers were obtained. Population structure, phylogenetic relationships and principal component analysis (PCA) divided the entire accessions into three groups. The genetic diversity and population differentiation analysis showed that the mean observed heterozygosity (Ho) ranged from 0.16 to 0.24, while the mean polymorphic information content (PIC) ranged from 0.14 to 0.17, and mean minor allele frequency (MAF) ranged from 0.11 to 0.14. Analysis of molecular variance (AMOVA) indicated that 81.38% of the total variance was derived from within populations, which suggested a rich genetic diversity in Hunan tea germplasms. Furthermore, a core tea germplasm set was developed, which was comprised of 22 tea plant accessions and maintained the whole genetic diversity of the entire collection. This work should be valuable for conservation and utilization of tea germplasm in Hunan.