Tea works is the most popular non-alcoholic drink harvest across the universe Tapan, 2002. Tea was discovered more than 2000 old ages ago in China but is of course distributed throughout the whole Asiatic Monsoon part ( Banerjee, 1992 ) . The chief category of cultivated tea consist of Camellia sinensis ( L. ) O. Kuntze with little foliage, Camellia assamica with large foliage and Camellia assamica ssp. lasiocalyx ( Planchon ex Watt ) with the intermediate foliage size ( Kaundun, 2000 ) . The ‘China type ‘ C. sinensis and ‘Assam type ‘ C. assamica originate around Southwest China, Myanmar and Northeast India ( Assam ) , but the native scope is obscured by a history of cultivation and debut by adult male ( Sealy, 1958 ) . Tea is extremely heterogenous and all the above taxa freely interbreed ensuing in a Cline widening from utmost China types to those of Assam beginning ( Wight, 1959 ) .
The tea workss have been cultivated widely in many Asiatic and African states where they contribute significantly to the local economic system ( 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 harmonizing to their different processing processs ( Yao et al. , 2008 ) . Green tea, Black tea and Oolong tea are more popular in the universe ( Yao et al. , 2008 ) . China is presently the foremost manufacturer, consumer and exporter of commercial tea, particularly for Green and Oolong tea ( Yao et al. , 2008 ) . On the other manus, India, Kenya and Sri Lanka are the largest manufacturers and exporters of Black tea ( Yao et al. , 2008 ) .
Tea is a popular drink due to its stimulatory belongingss and physiological maps ( Chen, 1999 ) . It is reported to move against a figure of abnormalcies including artherosclerosis, radiation harm, antioxidative, anticancer, antiulceric, antiviral and bactericidal ( Chen, 1999 ) .
Darjeeling Tea is the finest tea, because its alone spirit and has traditionally been prized above all other black teas, particularly in the United Kingdom and other states ( hypertext transfer protocol: //www.darjeelingtea.com/ ) . It is cultivated, grown, processed and manufactured within the defined district of hilly countries ( viz. : Sadar sub-division, Kalimpong sub-division and Kurseong subdivision ) of Darjeeling territory in the province of West Bengal in India ( hypertext transfer protocol: //www.darjeelingtea.com/ ) . The quality and repute of Darjeeling Tea are basically attributable to its geographical beginning and processing. About 86 registered Tea Estates ( gardens ) located within the demarcated district cultivate Camellia sinensis tea assortment ( hypertext transfer protocol: //www.darjeelingtea.com/ ) .
About 40 million kilogram is sold as “ Darjeeling Tea ” ( hypertext transfer protocol: //www.darjeelingnews.net/ ) when the existent production capacity of Orthodox Darjeeling Tea varies from 9-10 million kilogram ( hypertext transfer protocol: //www.darjeelingtea.com/ ) . The low measure with really high quality pushes up the demand to do monetary value escalation ( hypertext transfer protocol: //www.darjeelingnews.net/ ) . As a consequence, teas of non-Darjeeling beginnings blended with Darjeeling Tea are sold to the consumer through abuse of the word “ Darjeeling ” ( hypertext transfer protocol: //www.darjeelingnews.net/ ) . The stairss involved in tea production ( processing, sweeping purchasing, blending, transportation and repackaging allows for alteration in beginning ( hypertext transfer protocol: //www.darjeelingnews.net/ ) . This restricts the growing of other tea merchandises such as Assam.
1.4 DNA Molecular Markers
Deoxyribonucleic acid analysis can be used to supervise tea beginning from manufacturer to consumers. DNA molecular markers provide a good and enlightening attack to gauge the familial diverseness and familial relationship of tea cultivars ( Yao et al. , 2008 ) . Tea diverseness has been studied with random amplified polymorphous DNA ( RAPD ) markers ( Wachira 1995 ; Chen et al. , 1998 ; Liyanage et al. , 2001 ; Wachira et al. , 2001 ; Kaundun & A ; Park, 2002 ) , amplified fragment length polymorphism ( AFLP ) markers ( Wachira et al. , 2001 ; Balasaravanan et al. , 2003 ) and limitation fragment length polymorphism ( RFLP ) markers ( Matsumoto et al. , 2002, Kaundun and Matsumoto, 2003 ) .
However, RAPDs and AFLP techniques generate dominant markers and in the instance of RAPDs there are serious inquiries refering duplicability between research labs ( Freeman et al. , 2004 ) . The usage of RFLP is limited by their awkwardness, the demand for ringer bank and demand for big sums of DNA ( Tapan, 2002 ) . The demand of radioactive labelling and comparatively better quantitative and qualitative DNA demand besides limits the use of AFLP in every research lab ( Tapan, 2002 ) .
Singh et Al. ( 1999 ) , demonstrated DNA extraction from processed tea but the DNA is unsuitable for RAPDs and AFLP because during treating the tea degrades much of the cells and their Deoxyribonucleic acid, and merely the debauched Deoxyribonucleic acid can be isolated. As PCR engineering discoveries increased usage in familial analysis, extra novel fluctuations of this technique are emerging ( Tapan, 2002 ) .
1.4.1 Microsatellite or Simple Sequence Repeat ( SSR )
Microsatellites or simple sequence repetitions ( SSR ) have gained attending late as an alternate agencies of qualifying complex eucaryotic genomes ( Tapan, 2002 ) . These are normally 2-5 bp long, short DNA sequence motive that occur at multiple sites ( Wang et al. , 1994 ) and uncover a high grade of allelomorphic diverseness which can be typed via PCR elaboration of genomic sections flanked by reciprocally oriented ( 5 ‘ or 3 ‘ terminal ) , closely separated microsatellite repetitions ( Schlotterer et al. , 1991 ) . The PCR merchandises therefore generated reveal multiple polymorphous merchandises, which can be resolved on agarose gel cataphoresis ( Tapan, 2002 ) . Simple sequence repetition ( SSR ) markers have besides been developed in tea works ( Kaundun and Matsumoto 2002 ; Freeman et al. , 2004 ) and the SSR primers are available for diverseness surveies in tea works. The CEQ 8000 Genetic Analysis System was used to analyze the samples along with SSR markers. CEQ is good suited for SSR analysis because of its ability to reproducibly size Deoxyribonucleic acid fragments. Accurate genotyping of SSRs by fragment sizing relies on the changeless comparative migration of indistinguishable allelomorphs, and prior cognition of the spectrum of most or all possible evident sizes that are derived from those comparative migrations.
1.5 The Present Project
In the present survey, SSR markers were used ( I ) to analyze familial relationship among Darjeeling tea from supermarkets ain trade names and other trade names and ( two ) to analyze familial relationship among Darjeeling tea and assorted blends.
2.0 Materials and Methods
2.1 Tea Samples
The descriptions of 16 tea samples bought for the present survey are given in Table 1. Camelia assamica was used as positive control.
Table 1. Tea Samples
Charles digby harrods
Green Tea Single Estate
Supermarket ain trade names
Wild Needle Tea
Chinese Green Tea
Assorted High quality Tea
Camelia assamica ( Assam )
2.2 DNA Extraction and Purification
Genomic DNA was extracted from dried tea foliage samples utilizing a modified protocol by Dellaporta et Al. 1983. Dried tea foliage ( about 15mg ) put into a 1.5ml eppendorf tubing and 400Aµl of extraction buffer added ( 8Aµl mercaptoethanol and 10ml Extraction buffer Appendix ) were grind utilizing a bluish plastic stamp and votex briefly. The samples in the eppendorf tubing were incubated at 65oC for 30 proceedingss with intermittent shaking and inverting after which were extractors at 13000 revolutions per minute for 10 proceedingss. Supernatant were decanted into new 1.5ml eppendorf tubings and centrifuged once more at 13000 revolutions per minute for 5 proceedingss to take leaf dust. The supernatant was decanted into new 1.5ml eppendorf tubings and an equal volume of cold isopropyl alcohol ( 200Aµl ) was added to precipitate the Deoxyribonucleic acid and so centrifugate at 13000 for 10 proceedingss. The supernatant was poured away and 200Aµl of 70 % ethyl alcohol added to clean the DNA pellet and extractor at 13000 revolutions per minute for 5 minuntes. The Deoxyribonucleic acid pellets were air-dried for 10 proceedingss and re-suspended in 50Aµl H2O.
The Deoxyribonucleic acid extracted was purified utilizing the Promega Wizard Clean-Up Kit ( Appendix 1 ) harmonizing to maker ‘s instructions and the unity and intactness of DNA were checked on 1 % agarose gels.
2.3 PCR Amplification
Eight SSR primer brace labelled with the Beckman dye for sensing and analysis on the CEQ 8000 Genetic Analysis System were used for PCR elaboration of all the 16 tea samples and H2O ( negative control ) . 0.2ml PCR reaction mastermix for each microsatellite primer brace was prepared. The chemical constituents and volume of the mastermix is shown in Table 2. An aliquot 17Aµl of the mastermix was pipette into 17 ten 0.2ml PCR tubings ( 96 Wellss home base ) and 3ul for each tea genomic DNA added ( diluted to ~10ng/Aµl ) .
Table 2: Contentss for 20Aµl reaction volume for 16 Tea samples. ( Example Casmin10 )
Volume for one sample ( Aµl )
Mastermix Total volume ( Aµl )
( ( x 17 ) + 5 % )
10 ten NEB PCR buffer inc. MgCl to 1.5 millimeters
20 AµMiˆ Camsin M10FGreen
20 AµM iˆ Camsin M10R
Tea genomic DNA ( ~10ng/Aµl )
The contents were assorted gently and centrifuged briefly to roll up the reaction. The undermentioned PCR rhythms were performed:
One rhythm consists of initial denaturation at 94oC for 3 proceedingss.
35 rhythms of 45 seconds denaturing at 94oC, 1 minute tempering at 60 oC ( temperature harmonizing to table 3 ) and 1 minute extension at 72oC and so a concluding extension at 72oC for 7 proceedingss with a soak temperature at 4oC.
Table 3. Microsatellite Word picture
Size Range ( bp )
Annealing Temperature ( oC )
Dye tag Colour
2.4 Agarose Gel Electrophoresis
2.4.1 Gel Preparation
The 2 % agarose gel make up of 500 milliliter ( 250ml TBE in each flask ) was prepared by runing 10g agarose ( 5g in each flask ) in 0.5x TBE buffer ( 89 mM Tris-Hcl ( pH 8.3 ) , 89 millimeter boracic acid, 5 millimeter EDTA ) in a microwave. The content was cool for 10 to 15 proceedingss and 2.5Aµl Ethidium Bromide added to each flask and assorted gently by twirling the conelike flask. The gel mixture was poured into a maxi-gel tray with 8 combs and allowed to put.
2.4.2 Gel Loading and Running
5Aµl DNA size marker ( 2-Log ladder ) used for set size was loaded in the first and last well of each row followed by 4Aµl PCR merchandise mixed with 2Aµl 6x lading buffer of 17 samples for each microsatellite marker. The PCR merchandises were so separated on 1 % agarose gel tally at a changeless 120 V for 45 proceedingss and detected by Ag 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 to the full automated familial analysis system. The system automatically fills the capillary array with a patented additive polyacrylamide ( LPA ) gel, denatures and loads the sample, applies the electromotive force and analyze the informations base on the dye signal. Dye D1 ( ruddy ) is reserved for usage in the size standard ladder. The staying three dyes used for primer labelling: D2 ( Black ) , D3 ( green ) , and D4 ( Blue ) with fluorescence emanation of the dyes in the order: D4 & gt ; D3 & gt ; D2.
Sample Preparation and lading into CEQ
The amplified PCR merchandises were separated by capillary cataphoresis utilizing a CEQ 8000 familial analysis system ( Beckman Coulter Inc. ) . For each tea sample 2 microsatellites PCR merchandises were assorted given 2 pools. An aliquot of entire volume of 217Aµl was prepared by blending 215Aµl of sample lading solution ( SLS ) and 2Aµl size criterion 400 ( SS4000 ) for each row of 8 Wellss of 96 Wellss home base. 25Aµl of aliquot was pipette into each of the 8 Wellss of one row. To this was added 3Aµl of pooled PCR merchandises for each sample and layered with a bead of mineral oil to forestall vaporization of the reagents while running on the CEQ system. This was repeated for the all the pooled PCR merchandises for the remainder of the samples.
Data analysis was performed utilizing the CEQ 8000 Fragment Analysis package version 7.0.55 harmonizing to maker ‘s recommendations ( Beckman Coulter Inc ) . The SSR sizes extremums generate were scored on presence ( 1 ) or absence ( 0 ) for each sample base on the four SSR primer braces in a binary manner. The information obtain was entered into excel and a dendrogram was constructed by the unweighted brace group method ( UPMG ) and Nei & A ; Li ‘s similarity coefficient was estimated utilizing MVSP package. Multivariate Statistical Package ( MSVP ) ( Kovach, 2006 ) this ordination method makes no premises about the distribution of the random variables or about their population genetic sciences. Euclidian distance was chosen in penchant to other distance steps, as it does non category common absence of an allelomorph as a shared feature.
3.0 RESULTS AND DISCUSSION
A B C D E F G H I J K L M N O P Q R3.1 DNA Quantification
Figure ( .. ) Quantification of genomic Deoxyribonucleic acid 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 ) .
3.2 PCR-SSR Analysis
Microsatellite markers have been used for word picture in this survey, they are known for their carbon monoxide laterality, multi-allelic nature, duplicability, extended genome coverage and easiness of sensing by polymerase concatenation reaction with alone primer braces that flank the repetition motive ( Sharma 2009 ) . Among the 8 primers used for DNA elaboration, merely 4 primers ( M9, M10, M12 and P18 ) produce stable SRR polymorphous fingerprints runing in size from 1000bp to 3000bp base on 2-Log DNA ladder indicant for 1 % agarose gel as show in Figure ( … . ) , ( … ) and ( … ) .
Figure ( .. ) Amplification of genomic Deoxyribonucleic acid from 16 tea samples with primer M12 and P18. Tea samples are label in the order as 2-log DNA ladder ( Appendix 2 )
Figure ( .. ) Amplification of genomic Deoxyribonucleic acid from 16 tea samples with primer M9and 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 really weak sets as show in Figure ( … . ) for primer P16 whilst the last two primers ( P7and P19 ) failed to magnify DNA for all the 16 tea samples as show in Figure ( … ) for primer P19. Hence these primers ( P3, P16, P7 and P19 ) were excluded from capillary cataphoresis. The failure of these primers to magnify the Deoxyribonucleic acid could be attributed to inefficiencies during lading or wrong annealing temperature. The annealing temperature is a cardinal factor on the quality of SSR fingerprints and it had to be determined for each primer ( Yao et al. 2008 ) . In this survey, the optimum annealing temperature of the SSR primers ranged from 55oC to 60oC as show in table 3.
Figure ( .. ) Faint Band/ absence of sets observed with P19.
3.3 SSR-CEQ Fragment Analysis
Due to little fragment size of the Deoxyribonucleic acid and big pore size of the agarose gel, clear sets may non been observed therefore capillary cataphoresis was done for the four primers ( M9, M10, M12 and P18 ) and CEQ 8000 Fragment Analysis package was used to analyze the SSR fragment sizes. SSR fragment sizes were automatically calculated to two denary topographic points by the CEQ 8000 Genetic Analysis System. However whole figure sizes for extremums naming were used and these are reported in Table ( … . ) .
Dye Tag Colour
Size ( bp )
Size ( bp )
Size ( bp )
Size ( bp )
Size ( bp )
Size ( bp )
Size ( bp )
Size ( bp )
Table ( … ) Primers used CQE: Dye label coloring material and Size
Clear extremums for allelomorphs we non observed for primer P18 in 5 tea samples although clear stria form for this primer was observed when cataphoresis was carried out on its PCR merchandises. This show as losing informations ( changeless migration speed ) in Figure ( … ) and may be due to the presence of a bubble in the capillary home base Wellss during pipetting therefore forestalling the Deoxyribonucleic acid from being picked up when current is passed through. Thus the CEQ 8000 fragment size for primer P18 have been excluded from the informations used for bunch analysis.
Figure ( .. ) Missing Data with Primer M18
Figure ( aˆ¦ ) Peaks observed with Primer M10 ( Pool 1 )
Figure: Extremum OF P9
3.4 Cluster Analysis
3.4.1 Familial Variation among all Tea Trade names
A dendrogram generated utilizing unweighted brace group method and step of similarity estimated utilizing Nei & A ; Li ‘s similarity coefficient is shown in Figure. Similarity among the tea samples range from 0 % similarity coefficient between Whittard-Assam and the other 15 tea samples to a upper limit of 83 % similarity coefficient between Tetley and PG tips blend tea samples. Three blend trade names made up of PG tips, Tetley, Typhoo and Clipper Green tea a high quality trade name separate into one group at 70 % similarity coefficient. Tesco and Twinnings Darjeeling tea samples has 80 % similarity coefficient.
Figure ( aˆ¦ ) Dendogram demoing similarity between all the Tea Brands used in the analysis ( generated utilizing MVSP )
3.4.2 Familial Variation among Darjeeling Tea Brands
Nei and Li ‘s similary coefficient was used to asses the diverseness amongt the Darjeeling tea sold under different trade name names. Two chief bunchs were observed at 34 % similarity with Sainsbury in one group and the five trade names in the other. 48 % similarity observed among the five trade names out of which four are sold as high quality trade names. Maximum familial similarity of 80 % seen between Tesco and Twinnings Darjeeling assortments. The familial relationship between the Darjeeling tea samples was portrayed diagrammatically in the signifier of a dendogram in Figure
Figure ( aˆ¦ ) Dendogram demoing Similarity between all the Darjeeling types used in the analysis ( generated utilizing MVSP )
The bunch obtained of the assorted blends is consistent with predicted associations. Typically those trade names being sold under Darjeeling assortment should constellate together, nevertheless in this survey they did non constellate together. As mentioned above a 48 % similarity coefficient.
3.5 Principle Coordinate Analysis
Figure ( aˆ¦ ) : Scatter secret plan of PCO instance tonss ( Euclidean ) ( Appendix 4 ) .
Axis 1 represents 28 % fluctuation ; axis 2 represents 20 % fluctuation ( Appendix 4 ) . Axis 1 is strongly positively correlated to all the four blends used in the analysis. Its hebdomadal positively correlated to Clipper Green Tea, Twining Darjeeling and Harrods Darjeeling. Sainsbury Darjeeling and Whittard Darjeeling are hebdomadal negatively correlated to axis 1.
4 Decisions and Recommendations ( WE NEED TO ADD PROBLEMS FACED )
Consequences of present probe reveal variableness in familial nature of the tea samples used in the current survey. It besides indicated the false claim on quality indicated by assorted high quality trade names. However, this decision is base on the consequences generated from three SSR primers ; hence by sing more primers in future may uncover the existent familial diverseness among the tea samples. To gauge familial relationship among tea genotype it is of import to screen primers and select those that produced quotable and stable stria form ( Tapan, 2002 )
Further surveies ( Biochemical ) can be carried out complement molecular biological science techniques ( Magoma 2000 ) . The chief quality constituents of tea of assorted supermarket trade names, 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 theaflavins and thearubigins ( hypertext transfer protocol: //www.nbtea.co.uk/tea-manufacture ) . Besides enfranchisement by the tea taste testers to hold the typical characteristics of Darjeeling tea such as olfactory property, colour, spirits, spirit and gustatory sensation may be considered for farther surveies.
The chief advantage of this method of diverseness sensing is that it is robust, inexpensive and has high throughput. Besides it can be developed as one of the markers of tea quality and can be used to supervise the type of tea coming from the supermarkets and other processing companies.
5.1 APPENDIX 1
Wizard DNA Clean Up System
Purification without a Vacuum Manifold ( Using 3 milliliter Luer – Lok Syringes )
Before You Begin
Thoroughly mix the ace DNA Clean-Up Resin before taking an aliquot. If crystals or sums are present, fade out by warming the rosin to 37EsC for 10 proceedingss. The rosin itself is indissoluble. Cool to 25-30EsC before usage.
The sample volume must be between 50-500Aµl.If the sample volume is less than 50Aµl, conveying the volume at least 50Aµl with unfertile H2O. Pre-warm H2O for elution at 65-70EsC.
Binding of DNA
Use one WizardA® Minicolumn for each sample. Remove and set aside the speculator from the 3ml disposable syringe. Attach the syringe barrel to the Leur-Lok A® extension of each Minicolumn.
Add 1ml of Wizard DNA clean up rosin to a 1.5ml micro extractor tubing. Add the sample to the Clean -up Resin and mix by inversion.
Pipette the WizardA®DNA Clean -Up Resin incorporating the edge DNA into the Syringe Barrel. Insert the syringe speculator easy and gently force the slurry into the Minicolumn with the syringe speculator.
Detach the syringe from the Minicolumn and take the speculator from the syringe. Reattach the syringe Barrel to the Minicolumn. Pipette 2ml of 80 % isopropyl alcohol into the syringe. Reinsert the speculator and force the solution through the Minicolumn.
Remove the Syringe Barrel and reassign the Minicolumn to a 1.5 milliliter micro-centrifuge tubing. Centrifuge the Minicolumn at maximal velocity in a micro-centrifuge for 2 proceedingss to dry the rosin.
Transfer the Minicolumn to a new micro extractor tubing.Apply 50Aµl of pre-warmed ( 65-70EsC ) H2O or TE buffer to the Minicolumn and delay for 1 minute. Centrifuge the Minicolumn fro 20 seconds at maximal velocity to elute the edge DNA.
Remove and fling the Minicolumn. The purified Deoxyribonucleic acid may be stored in the micro extractor tubing at 4EsC or -20EsC.
2-Log DNA ladder
Analysis begun: Sunday, February 07, 2010 7:11:43 Autopsy
Analyzing 19 variables x 7 instances
Nei & A ; Li ‘s Coefficient
Harrods D organic
ALL TEA BRANDS
Analysis begun: Friday, February 05, 2010 8:25:56 Autopsy
Analyzing 19 variables x 16 instances
Nei & A ; Li ‘s Coefficient
Charles digby harrods Darjeeling