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Tea plant is the most popular non-alcoholic beverage crop across the world. Tea was discovered more than 2000 years ago in China but is naturally distributed throughout the whole Asian Monsoon region (Banerjee, 1992). The main class of cultivated tea consist of Camellia sinensis (L.) O. Kuntze with small leaf, Camellia assamica with big leaf and Camellia assamica ssp. lasiocalyx (Planchon ex Watt) with the intermediate leaf size (Tapan, 2000). The 'China type' C. sinensis and 'Assam type' C. assamica originate around Southwest China, Myanmar and Northeast India (Assam) respectively, but the native range is obscured by a history of cultivation and introduction by man (Sealy, 1958). Tea is highly heterogeneous and all the above taxa freely interbreed resulting in a cline extending from extreme china types to those of Assam origin (Wight, 1959).
The tea plants have been cultivated widely in many Asian and African countries where they contribute significantly to the local economy (Freeman et al., 2004). The tea production can be categorised into six types, such as Green tea, Black tea, Oolong tea, White tea, Yellow tea and Dark tea according to their different processing procedures as shown in Figure 1 (Yao et al., 2008). Green tea, Black tea and Oolong tea are more popular in the world (Yao et al., 2008). China is currently the foremost producer, consumer and exporter of commercial tea, especially for Green and Oolong tea (Yao et al., 2008). On the other hand, India, Kenya and Sri Lanka are the largest producers and exporters of Black tea (Yao et al., 2008).
Figure 1. Major Tea Processing steps with corresponding types (Hilal and Engelhardt, 2007).
1.3 Tea Benefits
Tea is a popular beverage due to its stimulatory properties and physiological functions (Chen, 1999). It is reported to act against a number of abnormalities including artherosclerosis, radiation damage, antioxidative, anticancer, antiulceric, antiviral and germicidal (Chen, 1999).
Darjeeling Tea is the finest tea, because it's unique flavour and has traditionally been prized above all other black teas, especially in the United Kingdom and other countries (http://www.darjeelingtea.com/). It is cultivated, grown, processed and manufactured within the defined territory of hilly areas (namely: Sadar sub-division, Kalimpong sub-division and Kurseong subdivision) of Darjeeling district in the state of West Bengal in India (http://www.darjeelingtea.com/). The quality and reputation of Darjeeling Tea are essentially attributable to its geographical origin and processing. About 86 registered Tea Estates (gardens) located within the demarcated territory cultivate Camellia sinensis tea variety (http://www.darjeelingtea.com/).
Almost 40 million kg is sold as "Darjeeling Tea" (http://www.darjeelingnews.net/) when the actual production capacity of orthodox Darjeeling Tea varies from 9-10 million kg (http://www.darjeelingtea.com/). The low quantity with very high quality pushes up the demand to cause price escalation (http://www.darjeelingnews.net/). As a result, teas of non-Darjeeling origins blended with Darjeeling Tea are sold to the consumer through misuse of the word "Darjeeling" (http://www.darjeelingnews.net/). The steps involved in tea production (processing, wholesale buying, blending, shipping and repackaging allows for modification in origin (http://www.darjeelingnews.net/). This restricts the growth of other tea products such as Assam.
1.5 DNA Molecular Markers
DNA analysis can be used to monitor tea origin from producer to consumers. DNA molecular markers provide a good and informative approach to estimate the genetic diversity and genetic relationship of tea cultivars (Yao et al., 2008). Tea diversity has been studied with random amplified polymorphic DNA (RAPD) markers (Wachira 1995; Chen et al., 1998; Liyanage et al., 2001; Wachira et al., 2001; Kaundun & Park, 2002), amplified fragment length polymorphism (AFLP) markers (Wachira et al., 2001; Balasaravanan et al., 2003) and restriction fragment length polymorphism (RFLP) markers (Matsumoto et al., 2002, Kaundun and Matsumoto, 2003).
However, RAPDs and AFLP techniques generate dominant markers and in the case of RAPDs there are serious questions concerning reproducibility between laboratories (Freeman et al., 2004). The use of RFLP is limited by their slowness, the need for clone bank and requirement for large amounts of DNA (Tapan, 2002). The requirement of radioactive labelling and relatively better quantitative and qualitative DNA requirement also limits the utilization of AFLP in every laboratory (Tapan, 2002).
Mahipal Singh et al. (1999), demonstrated DNA extraction from processed tea but the DNA is unsuitable for RAPDs and AFLP because during processing the tea degrades much of the cells and their DNA, and only the degraded DNA can be isolated. As PCR technology finds increased use in genetic analysis, additional novel variations of this technique are emerging (Tapan, 2002).
1.5.1 Microsatellite or Simple Sequence Repeat (SSR)
Microsatellites or simple sequence repeats (SSR) have gained attention recently as an alternative means of characterizing complex eukaryotic genomes (Tapan, 2002). These are usually 2-5 bp long, short DNA sequence motif that occur at multiple sites (Wang et al., 1994) and reveal a high degree of allelic diversity which can be typed via PCR amplification of genomic segments flanked by inversely oriented (5' or 3' end), closely spaced microsatellite repeats (Schlotterer et al., 1991). The PCR products thus generated reveal multiple polymorphic products, which can be resolved on agarose gel electrophoresis (Tapan, 2002). Simple sequence repeat (SSR) markers have also been developed in tea plant (Kaundun and Matsumoto 2002; Freeman et al., 2004) and the SSR primers are available for diversity studies in tea plant. The CEQ 8000 Genetic Analysis System was used to analyse the samples along with SSR markers. CEQ is well suited for SSR analysis because of its ability to reproducibly size DNA fragments. Accurate genotyping of SSRs by fragment sizing relies on the constant relative migration of identical alleles, and prior knowledge of the spectrum of most or all possible apparent sizes that are derived from those relative migrations (Basu 2005).
1.6 The Present Project
In the present study, SSR markers were used (i) to study genetic relationship among Darjeeling tea from supermarkets own brands and other brands and (ii) to study genetic relationship among Darjeeling tea and mixed blends.
2.0 Materials and Methods
2.1 Tea Samples
The descriptions of 16 tea samples bought for the present study are given in Table 1. Camelia assamica was used as positive control.
Table 1. Tea Samples
Green Tea Single Estate
Supermarket own brands
Wild Needle Tea
Chinese Green Tea
Various High quality Tea
Camelia assamica (Assam)
2.2 DNA Extraction and Purification
Genomic DNA was extracted from dried tea leaf samples using a modified protocol by Dellaporta et al. 1983. Dried tea leaf (about 15mg) put into a 1.5ml eppendorf tube and 400Âµl of extraction buffer added (8Âµl mercaptoethanol and 10ml Extraction buffer Appendix ) were grind using a blue plastic pestle and votex briefly. The samples in the eppendorf tube were incubated at 65oC for 30 minutes with intermittent shaking and inverting after which were centrifuge at 13000 rpm for 10 minutes. Supernatant were decanted into new 1.5ml eppendorf tubes and centrifuged again at 13000 rpm for 5 minutes to remove leaf debris. The supernatant was decanted into new 1.5ml eppendorf tubes and an equal volume of cold isopropanol (200Âµl) was added to precipitate the DNA and then centrifuge at 13000 for 10 minutes. The supernatant was poured off and 200Âµl of 70% ethanol added to clean the DNA pellet and centrifuge at 13000 rpm for 5 minuntes. The DNA pellets were air-dried for 10 minutes and re-suspended in 50Âµl water.
The DNA extracted was purified using the Promega Wizard Clean-Up Kit (Appendix 1) according to manufacturer's instructions and the integrity and intactness of DNA were checked on 1% agarose gels.
2.3 PCR Amplification
Eight SSR primer pair labelled with the Beckman dye for detection and analysis on the CEQ 8000 Genetic Analysis System were used for PCR amplification of all the 16 tea samples and water (negative control). 0.2ml PCR reaction mastermix for each microsatellite primer pair was prepared. The chemical components and volume of the mastermix is shown in Table 2. An aliquot 17Âµl of the mastermix was pipette into 17 x 0.2ml PCR tubes (96 wells plate) and 3ul for each tea genomic DNA added (diluted to ~10ng/Âµl).
Table 2: Contents for 20Âµl reaction volume for 16 Tea samples. (Example Casmin10)
Volume for one sample (Âµl)
Mastermix Total volume (Âµl)
((x 17) + 5%)
10 x NEB PCR buffer inc. MgCl to 1.5 mM
20 ÂµM Camsin M10FGreen
20 ÂµM Camsin M10R
NEB Taq polymerase
Tea genomic DNA (~10ng/Âµl)
The contents were mixed gently and centrifuged briefly to collect the reaction. The following PCR cycles were performed:
One cycle consists of initial denaturation at 94oC for 3 minutes.
35 cycles of 45 seconds denaturing at 94oC, 1 minute annealing at 60 oC (temperature according to Table 3) and 1 minute extension at 72oC and then a final extension at 72oC for 7 minutes with a soak temperature at 4oC.
Table 3. Microsatellite Characterisation
Size Range (bp)
Annealing Temperature (oC)
Dye tag Colour
2.4 Agarose Gel Electrophoresis
2.4.1 Gel Preparation
The 2.5% agarose gel make up of 500 ml (250ml TBE in each flask) was prepared by melting 10g agarose (5g in each flask) in 0.5x TBE buffer (89 mM Tris-Hcl (pH 8.3), 89 mM boric acid, 5 mM EDTA) in a microwave. The content was cool for 10 to 15 minutes and 2.5Âµl Ethidium Bromide added to each flask and mixed gently by swirling the conical flask. The gel mixture was poured into a maxi-gel tray with 8 combs and allowed to set.
2.4.2 Gel Loading and Running
5Âµl DNA size marker (2-Log ladder) used for band sizing was loaded in the first and last well of each row followed by 4Âµl PCR product mixed with 2Âµl 6x loading buffer of 17 samples for each microsatellite marker. The PCR products were then separated on 2.5% agarose gel run at a constant 120 V for 45 minutes and detected by silver staining (Panaud et al., 1996) and gel images were recorded for analysis.
Analysis of SSR fragments
The Beckman Coulter CEQ 8000 Genetic Analysis System was used for the analysis of SSR fragments. The CEQ 8000 is a fully automated genetic analysis system. The system automatically fills the capillary array with a patented linear polyacrylamide (LPA) gel, denatures and loads the sample, applies the voltage and analyse the data base on the dye signal. Dye D1 (red) is reserved for use in the size standard ladder. The remaining three dyes used for primer labelling: D2 (Black), D3 (green), and D4 (Blue) with fluorescence emission of the dyes in the order: D4>D3>D2.
Sample Preparation and loading into CEQ
The amplified PCR products were separated by capillary electrophoresis using a CEQ 8000 genetic analysis system (Beckman Coulter Inc.). For each tea sample 2 microsatellites PCR products were mixed given 2 pools. An aliquot of total volume of 217Âµl was prepared by mixing 215Âµl of sample loading solution (SLS) and 2Âµl size standard 400 (SS4000) for each row of 8 wells of 96 wells plate. 25Âµl of aliquot was pipette into each of the 8 wells of one row. To this was added 3Âµl of pooled PCR products for each sample and layered with a drop of mineral oil to prevent evaporation of the reagents while running on the CEQ system. This was repeated for the all the pooled PCR products for the rest of the samples.
Data analysis was performed using the CEQ 8000 Fragment Analysis software version 7.0.55 according to manufacturer's recommendations (Beckman Coulter Inc). The SSR sizes peaks generate were scored on presence (1) or absence (0) for each sample base on the four SSR primer pairs in a binary mode. The data obtain was entered into excel and a dendrogram was constructed by the unweighted pair group method (UPMG) and Nei & Li's similarity coefficient was estimated using MVSP software. Multivariate Statistical Package (MSVP) ordination method makes no assumptions about the distribution of the variates or about their population genetics (Kovach, 2006). Euclidian distance was chosen in preference to other distance measures, as it does not class common absence of an allele as a shared characteristic (Kovach, 2006).
3.0 Results and Discussion
3.1 PCR-SSR Analysis
The DNA from all the 16 Tea samples used for PCR reaction was quantified on a 1% agarose gels and results are shown in Figure 2. Although clear bands were not observed, DNA was present for all the Tea samples.
A B C D E F G H I J K L M N O P Q R
Figure 2. Quantification of genomic DNA from 16 tea samples. (A), Whittard-Darjeeling (B), Sainsbury-Darjeeling (C), Tesco-Darjeeling (D), Twinnings-Darjeeling (E), Harrods-Darjeeling (F), Chinese Green Tea (G) Whittard-Earl Grey (H), Camelia assamica (Assam) (I) Whittard-Assam (J), Whittard-Oolong (K), Whittard-White Tea (L), Clipper-Green Tea (M), PG Tips (N), Typhoo (O), Tetley (P), Waitrose-Darjeeling (Q), Water (R).
Microsatellite markers have been used for characterisation in this study, they are known for their co dominance, multi-allelic nature, reproducibility, extensive genome coverage and ease of detection by polymerase chain reaction with unique primer pairs that flank the repeat motif (Sharma 2009). Among the 8 primers used for DNA amplification, only 4 primers (M9, M10, M12 and P18) produce stable SRR polymorphic fingerprints ranging in size from 1000bp to 3000bp base on 2-Log DNA ladder indication for 1% agarose gel as show in Figure 3 and 4.
Figure 3. Amplification of genomic DNA from 16 tea samples with primer M12 and P18. Tea samples are label in the order as 2-log DNA ladder (Appendix 2)
Figure 4. Amplification of genomic DNA from 16 tea samples with primer M9 and M10. Tea samples are label in the order as 2-log DNA ladder (Appendix 2)
Among the other 4 primers, P3 and P16 produce unstable or very weak bands whilst the last two primers (P7 and P19) failed to amplify DNA for all the 16 tea samples as show in Figure 5 for primer P19. Hence these primers (P3, P16, P7 and P19) were excluded from capillary electrophoresis. The failure of these primers to amplify the DNA could be attributed to inefficiencies during loading or incorrect annealing temperature. The annealing temperature is a key factor on the quality of SSR fingerprints and it had to be determined for each primer (Yao et al. 2008). In this study, the optimal annealing temperature of the SSR primers ranged from 55oC to 60oC as show in Table 3.
Figure 5. Faint Band/ absence of bands observed with primer P19.
3.2 SSR-CEQ Fragment Analysis
Due to small fragment size of the DNA and large pore size of the agarose gel, clear bands may not been observed hence capillary electrophoresis was done for the four primers (M9, M10, M12 and P18) and CEQ 8000 Fragment Analysis software was used to analyse the SSR fragment sizes. SSR fragment sizes were automatically calculated to two decimal places by the CEQ 8000 Genetic Analysis System. However whole number sizes for peaks calling were used and these are reported in Table 4.
Dye Tag Colour
Table 4. SSR Primers Sizes Scored and Dye tag colour
Clear peaks for alleles we not observed for primer P18 in 5 tea samples although clear banding pattern for this primer was observed when electrophoresis was carried out on its PCR products. This show as missing data (constant migration velocity) in Figure 6 and may be due to the presence of a bubble in the wells of capillary plate during pipetting thus preventing the DNA from being picked up when current is passed through. Thus the CEQ 8000 fragment size for primer P18 have been excluded from the data used for cluster analysis. Peaks were however observed for the other primers as show in Figure 7 (primer P18) and Figure 8 (primer M9).
Figure 6. Electropherogram for a sample considered as missing data with Primer P18
Figure 7. Electropherograms showing peak sizes for Primer M10 to for Tea Samples
Figure 8. Electropherogram showing peak sizes for Primer M9 to for Tea Samples
3.3 Cluster Analysis
3.3.1 Genetic Variation among all Tea Brands
A dendrogram generated using unweighted pair group method and measure of similarity estimated using Nei & Li's similarity coefficient is shown in Figure 9. Similarity among the tea samples range from 0% similarity coefficient between Whittard-Assam and the other fifteen tea samples to a maximum of 83% similarity coefficient between Tetley and PG tips blend tea samples. Three blend brands made up of PG tips, Tetley, Typhoo and Clipper Green tea a high quality brand separate into one group at 70% similarity coefficient. Earl Grey blend (black tea blend with bergamot oil) is however different from the other three tea blends (49% similarity coefficient). Tesco and Twinnings Darjeeling tea samples has 80% similarity coefficient.
Figure 9. UPGMA dendrogram showing Nei & Li's similarity coefficient among all the Tea Brands using SSR makers. (Appendix 3)
3.3.2 Genetic Variation among Darjeeling Tea Brands
Nei and Li's similary coefficient was used to assess the diversity among the Darjeeling tea sold under different brand names. The genetic relationship between the Darjeeling tea samples was portrayed graphically in the form of a dendogram in Figure 10. At a minimum similarity coefficient of 14% Dajeeling tea separate into one cluster whilst Chinese green tea and Camilia assamica (Assam) are in the second cluster. Sainsbury Darjeeling tea is different from the other Darjeeling types (37% similarity coefficient). A maximum similarity coefficient of 80% was observed between Tesco Darjeeling and Twinnings Darjeeling.
Figure 10. UPGMA dendrogram showing Nei & Li's similarity coefficient among all the Darjeeling types using SSR markers. (Appendix 3)
3.4 Principle Coordinate Analysis
Figure 11: Scatter plot of Principal Coordinates case scores (Euclidean) (Appendix 4).
Principal coordinate analysis for the first two axis give 48% total variation as shown in Figure 11 with Axis 1 representing 28% variation and axis 2 representing 20% variation with case score (Appendix 4). Axis 1 is strongly positively correlated to all the four blends used in the analysis. It's weakly positively correlated to Clipper Green Tea, Twinning Darjeeling and Harrods Darjeeling. Sainsbury Darjeeling and Whittard Darjeeling are weakly negatively correlated to axis 1.
The cluster obtained of the various blends is consistent with predicted associations. Typically those brands being sold under Darjeeling variety should cluster together, however in this study they did not cluster together. The tea samples used in the study proved to be tedious to work with and intact DNA fragments could not be extracted due industrial processing of the tea leaves , this also meant that a high amount of impurities were present. This study demonstrated that SSRs offer a suitable detection of genetic variability and molecular study of tea genotypes. Several features of the protocol contributed to this including the PCR cycles, melting and annealing temperatures. The SSR technique proved to be fast and sufficiently reliable (Tapan 2002). Though the number of primers used in the final analysis was small, hence it is not fully representative of the total diversity present in the tea types used
4.0 Conclusions and Recommendations
Results of present investigation reveal variability in genetic nature of the tea samples used in the current study. However, this conclusion is base on the results generated from three SSR primers; hence by considering more primers in future may reveal the actual genetic diversity among the tea samples. To estimate genetic relationship among tea genotype it is important to screen primers and select those that produced repeatable and stable banding pattern (Tapan, 2002)
Further studies (Biochemical) can be carried out complement molecular biology techniques (Magoma 2000). Tea contains a wide range of phenolic compounds including flavanols, flavandiols, flavonoids, and phenolic acids; these compounds may account for up to 30% of the dry weight of the tea leaves according to the literature (Hilal and Engelhardt, 2007). Studies can beconducted on the main quality components of tea such as tea polyphenols, catechins and amino acid content (http://www.nbtea.co.uk/tea-manufacture). The type of antioxidants (polyphenol or flavenoids) differs - green tea has simple flavenoids called catechins, while black tea has more complex flavenoids called the aflavins and the arubigins (http://www.nbtea.co.uk/tea-manufacture).
The main advantage of this method of diversity detection is that it is robust, cheap and has high throughput. Also it can be developed as one of the markers of tea quality and can be used to monitor the type of tea coming from the supermarkets and other processing companies (Magoma 2000). In addition, certification by the tea tasters to have the distinctive features of Darjeeling tea such as aroma, colour, liquor, flavour and taste may be considered for further studies.
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Darjeeling Tea Facts http://www.darjeelingnews.net/
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6.1 APPENDIX 1: Wizard DNA Clean Up System
Purification without a Vacuum Manifold (Using 3 ml Luer - Lok Syringes)
Before You Begin
Thoroughly mix the wizard DNA Clean-Up Resin before removing an aliquot. If crystals or aggregates are present, dissolve by warming the resin to 37ËšC for 10 minutes. The resin itself is insoluble. Cool to 25-30ËšC before use.
The sample volume must be between 50-500Âµl .If the sample volume is less than 50Âµl, bring the volume at least 50Âµl with sterile water. Pre-warm water for elution at 65-70ËšC.
Binding of DNA
Use one WizardÂ® Minicolumn for each sample. Remove and set aside the plunger from the 3ml disposable syringe. Attach the syringe barrel to the Leur-Lok Â® extension of each Minicolumn.
Add 1ml of Wizard DNA clean up resin to a 1.5ml micro centrifuge tube. Add the sample to the Clean -up Resin and mix by inversion.
Pipette the WizardÂ®DNA Clean -Up Resin containing the bound DNA into the Syringe Barrel. Insert the syringe plunger slowly and gently push the slurry into the Minicolumn with the syringe plunger.
Detach the syringe from the Minicolumn and remove the plunger from the syringe. Reattach the syringe Barrel to the Minicolumn. Pipette 2ml of 80% isopropanol into the syringe. Reinsert the plunger and push the solution through the Minicolumn.
Remove the Syringe Barrel and transfer the Minicolumn to a 1.5 ml micro-centrifuge tube. Centrifuge the Minicolumn at maximum speed in a micro-centrifuge for 2 minutes to dry the resin.
Transfer the Minicolumn to a new micro centrifuge tube .Apply 50Âµl of pre-warmed (65-70ËšC) water or TE buffer to the Minicolumn and wait for 1 minute. Centrifuge the Minicolumn fro 20 seconds at maximum speed to elute the bound DNA.
Remove and discard the Minicolumn. The purified DNA may be stored in the micro centrifuge tube at 4ËšC or -20ËšC.
6.2 APPENDIX 2:
2-Log DNA ladder
6.3 APPENDIX 3:
CLUSTER ANALYSIS Darjeeling Types
Analysing 19 variables x 9 cases
Nei &Li's Coefficient
Harrods D organic