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Pest Management in Horticultural El·osystems.Vol. 12. No. I pp 29-36 (2006)

MOLECULAR ANALYSIS OF Lellci/lodes orbollalis Guen.POPULATIONS WITHIN TAMIL NADU USING

LEPIDOPTERAN SPECIFIC RANDOM PRIMERS

S. GEETHARAJALAKSHMI, S. SUBRAMANIAN,P. S. SHANMUGASUNDARAM and S. MOHANKUMAR'

Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology,Tamil Nadu Agricultural University, Coimbatore - 3, Tamil Nadll, India

'Corresponding Author

Key Words: Genetic diversity, Leucinodes orbonalis. molecular markers, RAPD

ABSTRACT: Genomic DNA often different populations belonging to different eggplantgrowing regions were analyzed for RAPD profiles, for understanding the intraspecificvariation among them. Twenty-five "lepidopteran specific random primers" were used inthis analysis, which generated a lotal of279 markers revealing an average of 10-12 markerspel' primer in each popliialio~. The primers generated polymorphic markers (249),monomorphic markers (35) wilh a percentage of polymorphism (87.6). The per cent ofpolymorphism ranged from 46.15-100 for different primers. The results are discussed inrelation to the genetic relation~hjp among the ten populations.

1

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I

INTRODUCTION

Interpretation of genetic variation ll1

geographical populations can be an importantaspect to study the pest populations andmanagement. The nature of genetic variation,magnitude and distribution is necessary forunderstanding the behaviour, response toselection pressure, structure and dynamics ofdifferent populations and management. Within anecosystem the extent of genetic variationbetween geographical populations depends onseveral factors, including gene flow betweenpopulations, host range and time sinceseparation. Polymerase chain reaction (PCR)based Random Amplified Polymorphic DNA(RAPD) approach has been a handy and

29

convenient technique for investigations ofgenetic variation and genome mapping. Beingsimple nnd non-radioactive, the technique is quitesensitive and used to detect genetic variation inmany organisms. Eggplam (Solanum melongell11L.) is a major vegetable of worldwide significancegrown predominantly in South and South EastAsian countries (F.A.O, 1999). The eggplantshoot and fruit borer, Lel/ciJIodes orbonalisGuenee (Pyralidae: Lepidoptera) is the mostdestructive pest causing an average annual lossof 54-66 pel' cent (Singh and Kalda, 1997). Thisnecessitates higher levels of pesticideapplication, which is deleterious to human healthand environment. Altemative control strategiesare needed but, to be effective, they require asound understanding of ecology of the insect.

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Knowledge of insect population structure,coupled with an understanding of themechanisms that drive genetic changes in theinsect population, is essential for formulatinglong-term insect management strategies.Molecular marker technology is a powerful toolin analyzing the phylogeny, evolution andpopulation dynamics in insects (Symondson andLiddell, 1996; Smith and Wayne, 1996). 11,e fieldof molecular biology has greatly expanded andreconstruction of phylogenies from moleculardata has become a technology for getting newlevel of resolution for the study of insectecological systems and taxonomy and to identifyand track the insect populations. In the presentstudy, RAPD analysis was used 10 measure Ihegenetic variation in populations of Leucinodesorbollalis at various spatial scales in Tamil Nadu(South India). Ten populations along a 450kmtransect spanning the high, moderate andlow pesticide intensive eggplant ecosystemswere analyzed.

MATERIALS AND METHODS

Genomic DNA of ten different populationsbelonging to different eggplant growing regionsnamely Periyanaickenpalayam, Coimbatore,Alanlhur, Thondamuthur (low intensity), Erode,Harnr, Trichy (moderale intensity), Kalakkurichi,Tirnvannamalai, Tirnnelveli (high intensity) wereanalyzed for RAPD profiles, for understandingthe genetic variation among them. Twentyfive"lepidopteran specific random primers" wereused in Ihis analysis (NAPS Unit, The Universityof British Columbia, Vancouver, Canada). Theseprimer set was compiled by B. Crespi et al., whichis a compilation for insect systematics andpopulation biology, Ihe primers designed mostlywork for the Lepidopteran order insects as L.orbonalis comes under order lepidoptera theseprimers were used for this srudy (Table I), whichgenerated a total of 279 markers revealing anaverage of 10-12 markers per primer in eachpopulation. The plimers generated polymorphic

markers (249), monomorphic markers (35) with apercentage of polymorphism (87.6). 11,e per cenlof polymorphism ranged from 46.!5-1 00 fordifferent primers. The results arc discussed inrelation to the genetic relationship among the tenpopulations. The borer affected fruits werecollected from ten different places. The larvaewere reare-d in the same fruits and after the adultemergence, five female adults were collected. fromwhich DNA was isolated and nsed for RAPD­PCR analysis. Five female adults (due to scxualdimorphism there will be different bandingpattems for male and female adults narurally, inorder to prevent this only female ildults weretaken in this study) were ground with 15m! ofCTAB (Cetyl Trimethyl Ammonium Bromide)(CTAB 4% buffer, 100mM Tris-HCI (pH 8.0),1.4mM sodium chloride, 20mM EDTA, 0.5Mglucose, 0.1 % 2-Mercaptoethanol added just priorto lise} and suspended with the same buffer. Thesuspension was incubated at 65°C for 2 hours andthen 15ml of Chlorofoml: isoamylaleohol (24: I)was added. The suspension was centrifuged at4000rpm for 30 minutes at 4°C. The supernatant

J was transferred to a fresh tube and DNA wasprecipitated by adding 0.7 volume of ice-coldisopropanol. The DNA pellet was dissolved in

• 50~1 TE (lOmM Tris lmM EDTA) and purifiedfrom RNA contaminants following standardprocedures (Sambrook el al., 1989). The genomicDNA from ten different populations weresubjected to polymerase chain reaction (peR)with 25 lepidopteran specific random primers(Table 1). Amplification of genomic DNA wascarried out in 10 ~l reaction mixture containing40 -50 ng DNA as template. DNA amplificationwas performed in a DNA thermal cycler (modelPTC-IOO MJ Research. Inc., USA) programmed forI cycle of initial denaturation at 940 C for 4minfollowed by 44 cycles of 94°C for I min(denaturation), 3rC to 63"C according to theprimer used for I min (annealing temperature)(The annealing temperature ofprimers differs fromeach other based on their base composit ion. Thesame annealing temperature of a primer should

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Table 1. List of RAPD primers used in the study and their sequences

Primer Name Sequence (5'-3') Annealin\!-code temperanlr~ (ee)

in 1 M3 CACAT(CT) AACATT GTC GT(GC)AT(CT)GG 51

in 2 Rc44.9 CIT GAT GAAATC (CT)CTGTGCC 53

in 4 rcM5l-l CAT(AG)TTGTC(GT)CCGTGCCA(GT)CC 53

in 8 rcM53-2 GCAATG TG(AG) GCIGTGTGGCA 55

ill 9 M52.7 CGTCAAGGA(AGXG1)TGCGTCGTGG 57

in 10 rcM4 ACAGC(ACG)AC(GT)GT(cnTG(CT)CTCAT(AG)TC 49

in 12 EFA923 ACGTTCITCACGTTGAA(AG)CCAA 55

in 15 19.5Df GG(AGCT)GA(cnGA(cnATIGC(CGT)TGG ATG 47

in 19 22.5drc GAACCA(AG)TT(AG)AC(AG)TG(AG)AAGATC 45

in 21 G6PDG209R TT(cnTG(AGCJ}ACCAT(cnTC(CT)TT(AGCT)C J7

in 22 LEPWGI GA(A G)TG(CT)AA(AG)TG(CT)CA(CT)GG(CT)ATG TCTGG 55

ill 27 CK7-3' CAGGTGCTCGTTCCACATGAA 59

ill 28 ARK7-'3 GTGCCAAGGTIGGT(GT)qGGCA 57

ill 29 ACTI5' GCTGTTTTCCCGTCCATTGT 59

in 35 EF2-3' ATGTGAGCAGTGTGGCAATCCAA 63

in 36 Tubl-5' CAGGCTGGTCAATGTCC(cnAA(CT)CA 59

ill 37 Tub2i-3' CC(AG)TG(CT)TCATCTTA(GT)AT(CT)ACCATGGA 59

ill 38 Tub3-5' ATACGGTCTGGGD\CTC(cnTC(AGCT)CG 61

ill 39 Tub4-3' GCCGAGCTGCA(AG)GA(cnATGATCAA 61

in 40 Cal-I GTGTCCTTCATTTT(AGcnC(GT)TGCCATCAT 59

ill 41 Ca12 TAATGATCCTTCCGCAGGTTCA 63

ill 43 18S-S22 AG(cnTCCATGTAGGCATTGTTGA 59

ill45 A1669 AGCGTATGGC(AC)TC(AG)AAGAACTG 55

ill 49 S1I24 CAGTTCIT(CT)GA(GT)GCCATACocr 55

in 50 AlJ03 CAG TTCTT(cn GA(G1)GCCATA cocr 55

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Table 2. Total number of RAPD amplified products generated by the selectedpopulations of L. orbo/lalis

s. No. Selected regions (Tamil Nadu) Number of bands (Fig. I)

I Periyanaickenpalayam 1382 Coimbatore 1083 Alanthur 125

4 Thondamulhur 1455 Erode 1436 Harur 146

7 Trichy 1168 Kalakkurichi 143

9 Tiruvannamalai 131

10 Tirunelveli 170

Table 3. Number of markers and per cent of polymorphism developed by the primers

Primers Total number of Number of Polymorphismmarkers generated polymorphic markers (%)

ill 1 14 • 12 85.71ill 2 14 12 85.71in 4 13 12 92.30in 8 8 •• 5 62.15in 9 9 7 6428in 10 13 12 92.30in 12 8 8 100ill 15 7 6 85.71in 19 11 10 90.90ill 21 12 10 83.33ill 22 11 11 100.00ill 27 12 12 100.00in 28 13 13 100.00ill 29 8 7 87.50in 35 13 6 46.15ill 36 II 10 81.81in 37 10 10 100.00in 38 15 11 73.33in 39 13 13 100.00ill 40 10 8 80.00in 41 13 12 92.30in 43 10 10 100.00ill 45 14 10 71.42in 49 7 7 100.00in 50 10 10 100.00

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be used for amplification for all the experimentsamples. However, the annealing temperature canbe varied with different primers). 72° C for 2min(extension), followed by final extension at 72"Cfor 5min. Amplification products were analyzedby electrophoresis on 1.5% agarose gel (w/v) inIx TBE and photographed. One kilobase DNAladder (MBI Fermentas, USA) was used as themolecular size marker. The RAPD bands werescored for the presence labsence ignoring theintensity of the bands (Fig. I). Reproducibility ofmarkers is the main limitation in RAPD techniques,hence only the bands which are prominentlyscorable and reproducible has been used in thisstudy. Percentage of polymorphism wascalculated as the proportion of the polymorphicmarkers to the total number ofmarkers. Similarityco-efficient was calculated and clustering wasdone following unweighted pair group with

arithmetic mean average (UPGMA) using theNTSYS pc-2.0 software statistical package(Loxdale and Hollander, 1989).

RESULTS

The dendrogram construcled by UPGMA(Fig.2) method resulted into two major groups Aand B in the populations. The group A comprisedof 9 populations while the distinct second groupis subdivided to A 1 to A2 at a similaritycoefficient of0.43, where A2 comprised only onepopulation from Alanthur. The A 1 groupcomprising of 8 populations divide into twodistinct groups A1a and Alb; Alb comprising ofTrichy, Tiruvannamalai, Tirunelveli and A 1aThondamuthur, Harur, Kallakurichi, Erode. Withinthe populations Harur and Kallakurichi showedthe highest similarity of 77 %.

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TIRUNELVEU

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KALLAKURICHI

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ALANTHUR

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0.41 0.50 0.59 0.77

Fig. 2. Dendrogram based on Jaccard's similarity index showing relationship among different~.

populations of L. orbonalis (The dendrogram was constructed according to the unweightedpair-group method with arithmetic mean).

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DISCUSSION

In this study the number of polymorphicmarkers was higher indicating the diversityamong the populations (Lynch and Milligan,1994; Williams et aI., 1990). The mean similarityindex was worked out for 10 geographicpopulations of L. orbonalis, which shows thatthe populations are closely related. Similar resultswere recorded in other pests like screwonn flies(Barum et al., 1995) and Mediterranean fruit fly(Haymer et al., 1997; InfanleMalachias et al.,1999; Boulding, 1998). Genetic similarities amonggeographical populations from the data werewithin the similarity coefficients ranging from 0.41to 0.77. Host mediated variation has also beenreported (John et al., 2002). The population fromCoimbatore was collected from Tamil NaduAgricultural University orchard where the insectexposure to pesticides is very low as comparedto intensive pesticide sprayed fields of Alanthur.The maximum similarity index value was observedbetween Harur and Kallakurichi populations,these places are far away from each othe!' Butthey show the maximum similarity among them.

Populations from similar eggplant growingareas need not always express genetic similaritybecause an adaptational/cropping patternvariation is continuously taking place and henceconcluding that RAPD markers are useful tool indetecting polymorphism among the populations.GenetIc differences of the insect population wereevaluated at different eggplant growing regionsand sites within a region, which will help inunderstanding the insect resistance genes. Insectmorphological features have been extensivelyused previously to describe the insectpopulations. However, these morphologicalfeatures are highly influenced by both geneticand environment factors and makes themwueliable. Recent development in biotechnology,especially in the molecular markers, help to studythe variation among the insect population at theDNA level which is not affected by theenvironment.

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As like other insects, in this study the,variations among the populations are distinct inthe DNA level, (Lu and Rank, 1996; Roehrdanzand Johnson, 1988; Roehrdanz, 1989, Dowdy andMcGaughey, 1996; Haymer, 1994), the populationsexamined in this study has shown 41 to 77%similarity among the populations al DNA level.This shows that numerous genetic, environmental,and ecological factors are involved in thepopulations and molecular markers can be usedto study the variation among the populations.Selection could result from differences in theeggplant cultivars planted locations or otheraspects of the environment and agriculturalpractices. The first study of genetic variationamong the natural populations of L. orbonalishas shown that lepidopteran insect specificRAPD markers are useful tools to identify geneticvariability and therefore cnn be concluded that awide range of variation exists among theL. orbonalis populations within Tamil Nadu. Thepresent study undertaken forms a basis for furtherresearch on this pest.

REFERENCES

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Boulding, E.G., 1998. Molecular evidence againstphylogenetically distinct host races of thepea aphid (Acrythosiphon pisum).Genome,42: 769-775.

Dowdy, A. K., and McGaughey, W. H. 1996.Using Random amplified polymorphic DNAto differentiate strains of the Indian mealmoth (Lepidoptera: pyralidae).Environmental Entomology, 25: 396-400.

F.A.O.1999. http://www.fao.org/ Annual report,FAO, Rome.

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Haymer, D. S. 1994. Random amplifiedpolymorphic DNA and microsatellites. Whatare they and can they tell us anything wedon't know? Annuals of EntomologicalSociety ofAmerica, 87: 717-722.

Haymer, D. S., Mei, H., and Mcinnis, D.O., 1997.Genetic· marker analysis of spatial andtemporal relationships among existingpopulations and new infestations of theMediterranean fruit fly ( Ceratitis capitata).Heredity, 79: 302-309.

JnfanteMalachias, M. E., Yotoko, K. S ., and deAzeredoEspin, A. M. L., 1999. Randomamplified polymotphic DNA ofsereworm flypopulation (Diptera: Calliphoridae) fromSoutheastern Brazil and Northern Argentina.Genome. 42: 772-779.

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John, D.N., Steplien, B.H., and Frederick, R. W.2002. Evalwiol/, 56: 1475-1488.

Loxdale, H. D., and Hollander, D. 1989.Electrophoresis studies on agricuhuralpests. Systematics Association SpecialVolume No 39, Clarendon Press; Oxford.

Lu, R., and Rank, G H. 1996. Use of RAPDanalysis to estimate population geneticparameters in the alfalfa leaf cutting bee,Megachile rowndata. Gel/ome, 39: 655-663.

Lynch, M., and Milligan, B. G 1994. Estimationof relatedness by DNA fingetprinting.Molecular Ecology, 3: 91-99.

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Roehrdanz, R. L. 1989.lntraspecific geneticvariability in mitochondrial DNA of screwworm fly, Cochliomyia hominivorax.Biochemical Gel/etics, 27: 55 1-559.

Roehrdanz, R. L., and Johnson, D.A.1988.Mitochondrial DNA variation amonggeographical populations of the screw womlfly, Cochliomyia homillivorllx. Jortmal ofMedical Entomology. 25: 136-141.

Sambrook,1. E., Fritsh, F., and Maniatis, T 1989Molecular c1aning- A labora/my manual.Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York.

Singh, T.H and Kalda, TS. 1997.Sources ofresistance to shoot and fruit borer ineggplant (Solallum melollgella L.) PKV.Res.J.,21: 126-128.

Smith, T B and Wayne, R.K.1996. Moleculargenetic approaclJe!i in conservation, OxfordUniversity Press, Oxford.

.$ymondson, W.O.C and Liddell, J. E. 1996. Theecology of agricultural pests: biochemicalapproaches. Chapman & Hall, London

• (1996)

Williams, J. G K., KubeJik, A. R., Livak, K. J.,Rafalski,J. A., and Tingey, S.V., 1990. DNApolymorphisms arnpilified by arbitraryprimers are useful as genetic markers.Nucleic Acids Research. 18: 6531-6535.