Chiang Mai J. Sci. 2010; 37(2) 293

Chiang Mai J. Sci. 2010; 37(2) : 293-303www.science.cmu.ac.th/journal-science/josci.htmlContributed Paper

Determination of Local Tobacco Cultivars Using ISSRMolecular MarkerJessada Denduangboripant*[a], Sornsuda Setaphan [b], Wilasinee Suwanprasart [c]and Somsak Panha [a][a] Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, 10330 Thailand.[b] Biotechnology Program, Faculty of Science, Chulalongkorn University, Bangkok, 10330 Thailand.[c] Research and Development Department, Thailand Tobacco Monopoly, Ministry of Finance,

Bangkok 10110, Thailand.*Author for correspondence; e-mail: jessada.d@chula.ac.th

Received: 19 October 2009Accepted: 11 December 2009

ABSTRACTIn many tobacco-growing countries, imported and local tobacco cultivars are subject

to different regulations in tariff collection. However, there is a major technical problem howto distinguish between the imported and local cultivars. No standard method has beenestablished to judge whether the plant has been grown for long time, enough to be legallycalled “local cultivar”. In this study, an Inter-Simple Sequence Repeat (ISSR) method wasintroduced to study genetic relationships between imported and local tobacco cultivars grownin Thailand. Of 20 screened-primers, five ISSR primers showed polymorphic PCR amplifiedproducts suitable for further phylogenetic analysis. Among them, two primers generated specificPCR bands that could separate local tobacco cultivars (Chorlare 1 and Chorlare 2) from otherimported cultivars. Genetic relationship trees revealed four pairs of tobacco cultivars havingbootstrap supporting-values higher than 50%. Among all 13 local tobacco cultivars collectedand examined, only the couple of Chorlare 1 and 2 was distantly separated from the importedcultivars, suggesting their long-history of being grown in Thailand. The other 11 local cultivarswere found to be morphologically and genetically similar to some imported cultivars andcould have originated from them. Therefore, the ISSR technique could be developed andimplemented as a simple, effective molecular marker to recognise early-introduced local tobaccocultivars.

Keywords: inter-simple sequence repeat, ISSR, local cultivar, molecular marker, Thailand,tobacco.

1. INTRODUCTIONTobacco (Nicotiana tabacum L.) is one of

important economic crops of the world, as araw material for the cigarette industry [1]. Thisplant originated in tropical America. Fromthere, it has rapidly spread throughout Europe,Africa, Asia and Australia [2]. Tobacco wasfirst introduced to Thailand in the 16th century

and has since been developed into many localcultivars in the country. Nowadays, mosttobacco plants are cultivated in the northernand northeastern parts of Thailand. Tobaccocultivars commercially grown in Thailand canbe separated into two major types: importedand local tobacco cultivars. Both groups are

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subject to different regulations in tariffcollection; the tariff for local cultivars beingmuch lower than for imported ones. However,there are major legal and technical problemsas to how to distinguish between the twogroups of tobacco cultivar. Only plants thathave been grown in Thailand for a long timecan be called “local” cultivars, even thoughthere is neither chemical nor physical standardmethod to verify this.

Recently, several molecular or geneticmarkers have been developed and increasinglyused as a modern technique for distinguishingthe genotypes of organisms. The techniqueprovides a powerful tool for DNA poly-morphism analysis, genetic and phylogeneticevaluation and genetic diversity assessment.A number of molecular marker techniques,such as Restriction Fragment Length Poly-morphism (RFLP) [3], Amplified FragmentLength Polymorphism (AFLP) [4], RandomAmplified Polymorphic DNA (RAPD) [5],Simple Sequence Repeat (SSR) [6] and Inter-Simple Sequence Repeat (ISSR) [7], have beendeveloped for crop plant genetic study.Each marker technique has its own advantagesand disadvantages. Common benefits ofmost markers include rapid analysis, highlyinformative results and independence fromenvironmental factors.

The genetic marker chosen for this studyto solve the problem of tobacco cultivarclassification is the ISSR technique. The ISSRmarker is based on a PCR-amplification of100-3,000 basepair (bp) regions betweeninversely oriented SSRs (or microsatellites) [8].The ISSR marker is simple and has repro-ducibility. It requires small amounts of DNAand does not require information on DNAsequencing. ISSR primers are designed fromSSR motifs and can be undertaken for anyplant species containing a sufficient numberand distribution of SSR motifs in plantgenomes [9]. Therefore, the ISSR technique

has been widely used in many respects suchas the study of genetic diversity in barley [10],the analysis of polymorphism in cotton [11],the investigation of molecular variation andfingerprinting of Leucadendron cultivars [12]and the identification of olive cultivars [13].Since a genetic relationship analysis of tobacco(Nicotiana tabacum L.) cultivars in Thailandhas never been reported before, ISSR analysiswas introduced in this research to studyimported and local tobacco cultivars cultivatedin Thailand. We expected that this methodwould be able to give a clearer picture oftheir genetic relationship and might possiblybe developed into a standard classificationprocedure in the future.

2. MATERIALS AND METHODS2.1 Plant Materials and DNA Extraction

Fresh tobacco leaves from 53 importedcultivars were obtained from Maejo TobaccoExperiment Station of the Thailand TobaccoMonopoly in Chiang Mai province. Thirteenlocal cultivars were sampled from fiveprovinces of Thailand: Chiang Mai (North),Sukhothai (North), Nong Khai (Northwest),Nakhon Phanom (Northwest) and NakhonSi Thammarat (South). The names of all thetobacco cultivars used in this study are in Table1. Fresh leaf material from each cultivar wascut into small pieces and kept separately insilica-gel bags for DNA extraction. GenomicDNA extraction of all the studied tobaccowas performed using a DNeasy Plant MiniKit (QIAGEN, Germany) to produce rapidextraction and high quality extracted DNA.Genomic DNA was UV-visualised by 1%agarose gel electrophoresis followed bystaining with ethidium bromide.

2.2 ISSR AmplificationsTwenty ISSR-PCR primers (University of

British Columbia, Canada) were screened toamplify ISSR regions of the genomic DNA

Chiang Mai J. Sci. 2010; 37(2) 295

Cultivar Collecting localityRemark

groupCultivar name

(district, province)(Morphological

similarity)

Local Chorlare 1 and 2 Maejo Tobacco Experiment Station, -(13 cultivars) Chiang Mai

Nison Si Samrong, Sukhothai BurleyPetkhangsink Mueang, Sukhothai TurkishPu 001 and 002 Phon Phisai, Nong Khai Virginia:

coker-411White gold Tha Bo, Nong Khai Virginia: K-326Napanang That Phanom, Nakhon Phanom Virginia: Coker

187 hickE-bit That Phanom, Nakhon Phanom Burley: Ky-14Ya-glai Tha Sala, Nakhon Si Thammarat -Ya-chun Srichon, Nakhon Si Thammarat VirginiaYa Srichon, Nakhon Si Thammarat VirginiaLocal Nakhon Si Thammarat Chulabhorn, Nakhon Si Thammarat Burley & Virginia

Imported/ Bafra, Basma, Basma Maejo Tobacco Experiment Station, -Turkish xanthiyaka, Izmir, Samsun Chiang Mai

(9 cultivars) bafra, Samsun evkaf, Samsunmaden, Xanthiyaka, andZichan

Imported/ Coker-139, Coker-206, Maejo Tobacco Experiment Station, -Virginia Coker-254, Coker-319, Chiang Mai

(19 cultivars) Coker-347, Coker-371 gold,K-149, K-317, K-326, K-346,K-394, K-399, Speight G-52,Speight G-70, Speight G-140,Speight NC-82, NC-2326,NC 37 NF, and NC 89

Imported/ Burley-21, Burley-64, Ky-9, Maejo Tobacco Experiment Station, -Burley Ky-10, Ky-15, Ky-17, Ky- Chiang Mai

(17 cultivars) 8959, Ky-907, Ky-908, MSKy-14 x L-8, TN-86, TN-90,TN-97, Va-182, Va-509,Va-528, and VS Burley-21 xKy-9

Imported/ MC Nair 135, Baisee, Blanket Maejo Tobacco Experiment Station, -unclassified A1, C.N.T., C.S.T., CDL 28, Chiang Mai

(8) Dimon 1, and E-bit

Table 1. Imported and local tobacco cultivars sampled from five provinces in Thailand andused in this study.

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of two local (Chorlare 1 and Chorlare 2) andnine imported tobacco cultivars (Turkish:Bafra, Basma xanthiyaka, and Samsun bafra;Burley: Ky-17, TN-86, and Va-182; Virginia:Coker-139, K-326, and Speight G-140). PCRreaction mixtures of 20 μl contained approxi-mately 20 ng of template DNA, 2.5 units ofDynazyme thermostable DNA polymerase(Finnzyme, Finland) and its optimised enzymebuffer (with 1.5 mM MgCl2), 0.5 μl of 2.5mM mixed dNTP, and 1 μl of each 15 μMISSR-primer. DNA amplifications werecarried out using a thermocycler (GeneAmpPCR system 9700, Applied Biosystem) asfollows: initial denaturation at 94oC for 5minutes; 45 cycles of denaturation at 94oCfor 1 minute, annealing at 42oC for 45 secondsand extension at 72oC for 2 minutes; and finalextension at 72oC for 5 minutes.

The PCR products were detected by gelelectrophoresis with 1.8% agarose gel at80 Volt. for 1 hour 10 minutes. Each PCRamplification reaction was repeated twice toensure reproducibility. Only primers thatproduced clearly polymorphic DNA bandswere selected for further ISSR analysis. Afterprimer screening, the PCR reactions of theselected ISSR primers were optimised usinghigher annealing temperature (45oC) and ahotstart-PCR method with HotstarTaq DNApolymerase and Q-solution (QIAGEN,Germany) for higher PCR specificity.

2.3 Genetic Relationship AnalysisISSR amplifications of the selected

primers for genetic relationship analysis wereperformed each time on 40 tobacco cultivars:all 13 local cultivars plus 27 representativesof the imported cultivars. These imported-cultivar representatives were chosen randomlyto have balanced numbers of cultivars fromeach group (eight Turkish, eight Virginia, eightBurley, and three unclassified cultivars). Onlyclear and reproducible amplified fragments

were scored for genetic relationship analysis.ISSR bands were coded numerically as “1”when present and “0” when absent. Data setsderived from the respective PCR bandingpatterns were used to generate a data matrixfor each reaction. Nei and Li’s coefficient [14]was employed to calculate pairwise bandsimilarities using PAUP* 4.0b10 software.Cluster analyses were performed to constructgenetic relationship tree diagrams using theUnweighted Pair Group Method with Arith-metic Mean (UPGMA) and the Neighbour-Joining (NJ) methods. Bootstrap analysis wasalso calculated based on 1,000 replicates toshow the degree of confidence of each branchon the trees. Only bootstrap values over 50%were considered significant and are mentionedin the diagrams.

3. RESULTSThe 20 ISSR primers preliminarily

screened for totally 11 tobacco samplesgenerated 128 PCR bands with sizesranging from 280 to 1,600 bp. The numberof polymorphic bands generated variedbetween one and 11 bands, with an averageof five bands per primer. Of them, fiveprimers (UBC-807, UBC-809, UBC-813,UBC-823, and UBC-836) were foundgenerating highly polymorphic and repro-ducible bands (Table 2). They were thenselected to investigate the relationshipbetween local and imported tobacco cultivars.Moreover, UBC-807 and UBC-809 primersalso gave distinctively bands specific forChorlare1 and Chorlare 2 local cultivars fromChiang Mai province. The cultivar-specificbands were approximately 680 bp and 780 bpin size (arrowed in Figures 1 and 2, respec-tively). The specificity of both Chorlare1-and-2 specific markers was confirmed after testedagainst all 53 imported cultivars (see anexample of the results in Figure 1).

After the preliminary primer screening,

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Primer Sequence 5’ to 3’ Number ofname (T

m = melting temperature) PCR bands

Band size (bp)

UBC-807 AGA GAG AGA GAG AGA GT 520, 580, 680, 720, 800,(Tm = 45.25oC) 11 950, 1000, 1100, 1200, 1300

and 1400

UBC-809 AGA GAG AGA GAG AGA GG 6 280, 480, 500, 600, 780(Tm = 46.27oC) and 1500

UBC-813 CTC TCT CTC TCT CTC TT 8 350, 450, 580, 650, 850,(Tm = 44.15oC) 900,950 and 1200

UBC-823 TCT CTC TCT CTC TCT CC 400, 500, 550, 620, 700,(Tm = 46.13oC) 11 800, 900, 950, 1100, 1200

and 1500

UBC-836 AGA GAG AGA GAG AGA GYA 260, 280, 320, 380, 420,(Tm = 45.41-49.32oC; Y=C,T) 11 520, 700, 720, 800, 1200

and 1500

Table 2. PCR products obtained from ISSR-PCR amplification with five selected primerssuitable to investigate different bands between nine imported and two local tobacco cultivars(see cultivar names in the text).

we optimised the PCR reaction with a higherannealing temperature and hot-start techniqueto prevent the formation of misprimed non-specific PCR products. The genomic DNAof totally 40 cultivars was amplified with thefive selected primers (UBC-807, UBC-809,UBC-813, UBC-823, and UBC-836) and theoptimised PCR pattern results were sharperand brighter than before (see the examplein Figure 2). We found that most of the PCRpatterns of the imported tobacco cultivarsin the Virginia and Burley groups were verymuch alike while most of the Turkish cultivarswere found to be more closely related to eachother rather than to the Burley or Virginiacultivars.

Genetic relationship trees were con-structed using NJ and UPGMA methods.A phylogram based on NJ analysis revealedfour clusters of tobacco cultivars which hadbootstrap values higher than 50% as shownin Figure 3a. Group I consisted of both

Chorlare 1 and 2 cultivars distantly separatedfrom the others and considered as very closelyrelated with 100% bootstrap support. GroupII included Nison and Petkhangsink whichwere grouped with 58% bootstrap. Ya (Local

Kaset) and Local Nakhon Si Thammaratcultivars were paired as Group III with 65%bootstrap. NC 37 NF (Virginia) and Ky-10(Burley) were also grouped together with 63%bootstrap. Other paired branches with <50%bootstrap supporting values were not labeledsince they might not indicate any significantrelativeness between the cultivars.

A UPGMA dendrogram revealed thesame four groupings for some tobaccocultivars found in the NJ tree as shown inFigure 3b. Group I also consisted of Chorlare1 and Chorlare 2 separated from the otherswith a very high 99% bootstrap value. GroupII of Nison and Petkhangsink had 54%bootstrap while the other two local tobaccocultivars, Ya (Local Kaset) and Local

298 Chiang Mai J. Sci. 2010; 37(2)

Nakhon Si Thammarat, were clusteredtogether as Group III with 53% bootstrapsupport. Lastly, group IV consisting of NC37 NF (Virginia) and Ky-10 (Burley) wereseparated from the other tobaccos with a61% bootstrap. It was noted that four

cultivars of the Turkish group (Bafra, Izmir,Samsun maden, and Samsun evkaf) weregrouped together in both NJ and UPGMAtrees but with <50% bootstrap supportingvalues.

Figure 1. Cultivar-specific ISSR marker using primer UBC-807 for two Chorlare local cultivarsand compared with 53 imported cultivars. (M = 1.5 kb + 100 bp DNA marker, lanes C1 andC2 = local cultivars: Chorlare 1 and Chorlare 2, No. 1-9 = Turkish cultivars: Bafra, Basma,Basma xanthiyaka, Izmir, Samsun bafra, Samsun evkaf, Samxun maden, Xanthiyaka, and Zichan,No. 10-28 = Virginia cultivars: Coker-139, Coker-206, Coker-254, Coker-319, Coker-347,Coker-371 gold, K-149, K-317, K-326, K-346, K-394, K-399, Speight G-52, Speight G-70,Speight G-140, Speight NC-82, NC-2326, NC 37 NF, and NC 89, No. 29-45 = Burleycultivars: Burley-21, Burley-64, Ky-9, Ky-10, Ky-15, Ky-17, Ky-8959, Ky-907,Ky-908, MS Ky-14 x L-8, TN-86, TN-90, TN-97, Va-182, Va-509, Va-528,and VS Burley-21 x Ky-9, No. 46-53: Unclassified cultivars: MC Nair 135, Baisee, Blanket A1,C.N.T, C.S.T, CDL 28, Dimon 1, and E-bit).

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Figure 2. PCR patterns of totally 40 local and imported tobacco samples after ISSR-PCRamplification with UBC-809 primer. (lanes 1-13 = Local cultivars: Chorlare 1, Chorlare 2,Nison, Petkhangsink, Pu 001, Pu 002, White gold, Napanang, E-bit, Ya-glai, Ya (LocalxKaset),Local Nakhon Si Thammarat, and Ya-chun; lanes 14-21 = Turkish cultivars: Bafra, Basma,Izmir, Samsun bafra, Samsun maden, Xanthiyaka, and Zichan; lanes 22-29 = Virginia cultivars:Coker-139, Coker-347, Coker-371 gold, K-394, K-399, Speight G-70, NC 37 NF, and NC89; lanes 30-37 = Burley cultivars: Ky-9, Ky-10, Ky-15, TN-86, TN-90, TN-97, Va-509, andVa-528; lanes 38-40 = Unclassified cultivars: Blanket A1, Dimon 1, and E-bit).

4. DISCUSSIONISSR-PCR technique is an arbitrarily

amplified dominant (AAD) marker, whichanalyses the dominant inheritant of speciesbased on PCR technique with arbitraryprimers similar to RAPD (Ramdon AmplifiedFragment DNA), another popular molecularmarker [8]. However, ISSR uses a longer(15-30 bp) single-simple sequence repeat (SSR)primer which allows for a higher annealingtemperature and results in a greater repro-ducibility of amplified bands. In most geneticdiversity analyses, selection of suitable primersis a very important step for the PCR reaction.Needed properties for suitable ISSR primersare that then be able to give highly polymorphicPCR products, high reproducibility and are

more informative. One successful example ofusing ISSR-PCR to study genetic variationis the work of Pharmawati et al. [12]. Theyselected two from 64 ISSR primers toconstruct a DNA fingerprinting of threeLeucadendron cultivars and concluded thatISSR profiling was a powerful method foridentification and molecular classification.

Although tobacco is one of the mostimportant plants in agriculture, there are onlya few previous studies of genetic diversity andthe relationships between tobaccos usingmolecular markers, especially ISSR technique.Yang et al. [15] screened ISSR primers toamplify tobacco genomic DNA and tenprimers were found giving reproduciblyamplified products. Comparing their results

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to ours, five ISSR primers (UBC-807, UBC-809, UBC-813, UBC-823, and UBC-836)were selected from 20 screened primers.The selected primers generated highlyreproducible bands and were further used toinvestigate the genetic relationship betweenlocal and imported tobacco cultivars. Thesefive primers gave higher numbers of poly-morphic bands than the other 15 primerswhich did not produce any band or couldproduce only some faint bands. This led toour optimisation experiment where theannealing temperature was raised and some

chemicals (HotstarTaq polymerase and Q-solution) were added to ensure a highspecificity of reaction.

Filippis et al. [16] commented upon theimportance of doing a reproducibility test.He advised that genetic markers usually havelimitations mainly because reproducibilityfrom sample to sample is difficult. Fromour optimisation experiment, the resultsshowed that all distinctively major ISSR bandswere still reproduced and confirmed that theISSR primers selected from the screeningexperiment could specifically combine to

(a) (b)

Figure 3. Genetic relationship trees from whole ISSR data among 40 tobacco samples basedon Nei and Li’s similarity coefficient using (a) neighbour-joining (NJ) and (b) UPGMA methods.Numbers along branches are bootstrap-supporting values (%) generated after 1,000 replications.

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SSR-regions within the tobacco genome.This finding agrees well with the worksof Bahulikar et al. [9]. They tested thereproducibility of the ISSR-PCR amplificationin Nicotiana attenuate and assumed that the ISSRmethod could produce reproducible bands.Moreover, optimisation approaches improvedthe PCR results as they could increase thespecificity of the reactions. These preventedthe formation of misprimed non-specificPCR products or primer-dimers, reducednon-specific amplification products andincreased amplified yields of the PCRproducts [12, 17].

After the ISSR-PCR amplifications wereperformed with five selected primers toanalyse genetic relationships among 27imported and 13 local tobacco cultivars,most primers were found to give an adequatenumber of amplified DNA fragments enoughto reconstruct genetic relationship trees.The PCR patterns of two from three majorgroups of the imported tobacco cultivars,Burley and Virginia, were similar to each otherand suggested that they were genetically closelyrelated to each other. These results were thesame as the ISSR study of Yang et al. [15] inwhich they suggested that 24 flue-curedtobacco cultivars (only Burley and Virginiacultivar groups) were closely related and hadlow genetic diversity. These findings wereexpected by some experts such as Dr. RamseyLewis (Crop Science Department, NorthCarolina State University). As a foreignscientific advisor to Thailand Tobacco Mono-poly, he suggested that an identification ofgenetic polymorphism between tobaccocultivars with simple molecular markers likeISSR would be problematic. Instead, herecommended using a very high polymorphicAFLP technique (personal communication).

AFLP marker has been used to analysegenetic polymorphism among differentNicotiana species and cultivars since 2001

[18, 19]. Recent AFLP studies research ongenetic diversity among tobacco cultivars inChina and India revealed narrow geneticdiversity among cultivars [20, 21]. Moreover,Zhang et al. [22] assessed AFLP and RAPDmarkers for genetic diversity among tobaccocultivars and confirmed that AFLP generatedlarger number of bands than RAPD, whichcannot indicate any clear pattern of relationshipamong tobaccos. Although the latest RAPDanalysis of Sarala and Rao [23] could distinguishBurley cultivars from Virginia cultivars, wewould rather introduce AFLP marker techniqueto our future work and hope it could give aclearer picture of genetic differences betweenlocal and imported tobacco cultivars grownin Thailand.

From our results, the ISSR-PCR patternsof most local tobacco cultivars examined,except the Chorlare 1 and Chorlare 2 cultivars,were similar to those of some importedcultivars. Chorlare 1 and Chorlare 2 showedsome cultivar-specific bands and their PCR-pattern differences from the other localcultivars suggested that some local tobaccosin Thailand (such as Chorlare cultivars) reallyhave distant genetic characteristics from otherlocal and imported cultivars. Likewise, geneticrelationship trees based on NJ and UPGMAmethods also supported the distinctiveness ofboth Chorlare cultivars since they had 100%bootstrap supporting-values higher along thebranch. Therefore, we proposed that theChorlare cultivars presumably had a long-history of growth in Thailand and they couldbe legally pronounced as “true” local cultivarsof the country.

On the other hand, all other 11 localcultivars were found to be placed closely toimported tobacco cultivars on the trees. Thegenetic relationship results from the PCRanalyses agreed well with the previoussuggestions of officers of the tobacco regionalstations that they were morphologically

302 Chiang Mai J. Sci. 2010; 37(2)

similar to some imported cultivars. Theofficers proposed that these local cultivarswere descended from some importedtobacco cultivars have industrially promotedand given by the Thailand Tobacco Monopolyto farmers since 30 years ago. Thus, if therewere any healthy, high yield and disease-resistant plants left, the farmers might havesecretly kept tobacco seeds for their owngrowing in the next cultivating season. Shouldthis hypothesis be true, these tobaccos couldnot be called “true” local cultivars, althoughthey have their own Thai names and have beenrecognised as local cultivars.

5. CONCLUSIONSIn conclusion, although the ISSR-PCR

markers developed in this experiment couldnot clearly indicate the genetic relationshipsbetween imported and local tobacco cultivarsgrown in Thailand, some of them should beused as effective markers to help distinguishlong-cultivated local cultivars from recentlycultivated ones. The results from this researchthen shed more light on the basic knowledgeof tobacco germplasm development andbreeding in Thailand. Moreover, the cultivar-specific markers found in this study (forexample, the 680-bp band for both Chorlarecultivars from the UBC-807 primer) couldbe further converted to SCAR (SequenceCharacterised Amplified Region) markers forDNA fingerprinting purpose. Last but notleast, we are currently in the process ofexamining some more advanced molecularmarker techniques, such as AFLP (AmplifiedFragment Length Polymorphism) which isa highly sensitive method for detectingpolymorphisms in DNA, to give a betterresolution of the genetic relationships betweenimported and local tobacco cultivars.

ACKNOWLEDGEMENTSSpecial thanks to Mr. Chiwin Kunalai,

Mr. Decha Sutrarashoun and officers of theTobacco Product Analysis Subdivision,Thailand Tobacco Monopoly for their greathelp in organising the expeditions to collecttobacco samples. We thank the GraduateSchool of Chulalongkorn University and theThailand Tobacco Monopoly for financialsupport for laboratory equipments andchemicals. We also thank Assistant ProfessorSimon Wright, Chulalongkorn University, forhis kind help on English grammar correction.The first author was supported by fundingfrom Thailand Research Fund (under theresearch grant no. MRG-4680001).

REFERENCES

[1] Arslan B. and Okumus A., Genetic andGeographic Polymorphism of CultivatedTobacco (Nicatiana tabacum) in Turkey,Genetika, 2006; 42: 667-671.

[2] Albert F.H., Hill’s Economic Botany, TataMcGraw-Hill Publishing CompanyLimited, India, 1996.

[3] Botstein D., White R.L., Skolnick M. andDavid R., Construction of a GeneticLinkage Map in Man Using RestrictionFragment Length Polymorphisms, Am.J. Hum. Genet., 1980; 32: 314-331.

[4] Vos P., Hogers R., Bleeker M., Van delee T., Hormes M., Fritjer A., Pot J.,Peleman J., Kuiper M. and Zabeau M.,AFLP: a New Technique for DNAFingerprinting, Nucl. Acids Res., 1995; 23:4407-4414.

[5] Williams J.G.K., Kubelic A.R., Livak K.J.,Rafalski J.A. and Tingey S.V., DNAPolymorphisms Amplified by ArbitraryPrimers are Useful as Genetic Markers,Nucl. Acids Res., 1990; 18: 6531-6535.

[6] Morgante M. and Olivieri A.M., PCR-amplified Microsatellites as Markers inPlant Genetics, Plant J., 1993; 3: 175-182.

[7] Zietkiewicz E., Rafalsik A. and Labuda

Chiang Mai J. Sci. 2010; 37(2) 303

S., Genome Fingerprinting by SimpleSequence Repeat (SSR)-anchored Poly-merase Chain Reaction Amplification,Genomes, 1994; 20:176-183.

[8] Bussell J.D., Waycot M. and Chappill J.A.,Arbitrarily Amplified DNA Markers asCharacters for Phylogenetic Inference,Perspect. Plant Ecol. Evol. Syst., 2005; 7:3-26.

[9] Buhulikar R.A., Stanculescu D., PrestonC.A. and Baldwin I.T., ISSR and AFLPAnalyses of the Temporal and SpatialPopulation Structure of the Post-fireAnnual Nicotiana attenuate in SW Utah,BMC Ecology, 2004; 4: 1-13.

[10] Brantestem A.K., Bothmer R.V., DaytegC., Rashal I., Tuvesson S. and Weibull J.,Inter Simple Sequence Repeat Analysis ofGenetic Diversity and Relationship inCultivated Burley of Nordic and BalticOrigin, Hereditas, 2004; 141: 186-192.

[11] Liu B. and Wendel J.F., IntersimpleSequence Repeat (ISSR) Polymorphismsas a Genetic Marker System in Cotton,Mol. Eco. Notes, 2001; 1: 205-208.

[12] Pharmawati M., Yan G. and FinneganP.M., Molecular Variation and Finger-printing of Leucadendron Cultivars(Proteaceae) by ISSR Marker, Ann. Bot.,2005; 95: 1163-1170.

[13] Terzopoulos P.J., Kolano B., Bebeli P.J.,Kaltsikes J. and Metzidkis I., Indentifi-cation of Olea europaea L. Cultivars usingInter-simple Sequence Repeat Markers,Scientia Horticulturae, 2005; 105: 45-51.

[14] Nei M. and Li W.H., Mathematical Modelfor Studying Genetic Variation in Termsof Restriction Endonucleases, Proc. Natl.Acad. Sci. USA., 1979; 76: 5269-5273.

[15] Yang B.C., Xiao B.G., Chen X.J. and ShiC.H., Genetic Diversity of Flue-curedTobacco Varieties Band on ISSR markers,Hereditas (Beijing), 2005; 27: 753-758.

[16] Filippis L.D., Hoffmann E. and HamppR., Identification of Somatic Hybrids ofTobacco Generated by Electrofusionand Culture of Protoplasts using RAPD-PCR, Plant Sci., 1996; 121: 39-46.

[17] Soltis D.E., Soltis P.S. and Doyle J.J.,Molecular Systematics of Plants II: DNASequencing, 3rd Edn., Kluwer AcademicPublisher, USA, 2000.

[18] Ren N. and Timko M.P., AFLP Analysisof Genetic Polymorphism and Evolu-tionary Relationships among Cultivatedand Wild Nicotiana species. Genome, 2001;44: 559-571.

[19] Rossi L., Bindler G., Pijnenburgh H., IsaacP.G., Giraud-Henry I., Mahe M., OrvainC. and Gadani F., Potential of MolecularMarker Analysis for Variety Identificationin Processed Tobacco. Plant Var. Seeds,2001; 14: 89-101.

[20] Zhang H.Y., Liu X.Z., Li T.S. and YangY.M., Genetic Diversity among Flue-cured Tobacco (Nicotiana tabacum L.)Revealed by Amplified Fragment LengthPolymorphism. Bot. Studies, 2006; 47:223-229.

[21] Siva Raju K., Madhav M.S., Sharma R.K.,Murthy T.G.K. and Mohapatra T.,Genetic Polymorphism of IndianTobacco Types as Revealed by AmplifiedFragment Length Polymorphism. Curr.Sci., 2008; 94: 633-639.

[22] Zhang H.Y., Liu X.Z., He C.S. and YangY.M., Genetic Diversity among Flue-cured Tobacco based on RAPD andAFLP Markers. Braz. Arch. Biol. Techn.,2008; 51: 1097-1101.

[23] Sarala K. and Rao R.V.S., GeneticDiversity in Indian FCV and BurleyTobacco Cultivars. J. Genet., 2008; 87:159-163.