Detection of genetic polymorphism in the …nopr.niscair.res.in/bitstream/123456789/23158/1/IJEB 43(6) 548-551.pdfDetection of genetic polymorphism in the populations of brinjal shoot

Indian Journal of Experimental B iology Vol. 43, June 2005, pp. 548-55 1

Detection of genetic polymorphism i n the populations of brinjal shoot and fruit borer, Leucinodes orbonalis (Guenee)

K A M Karth ikeyan, I Vijayakumar, P Murali , P Suresh & S Janmthanan*

Thiagarajar Centre for Entomological Science, Department of Zoology, Thiagarajar College (Autonomous), Madurai 625 009, India

Received 20 November 2004

In the present study six different populations of L. orbonalis were collected and subjected to analysis of genetic variability in terms of carboxyl esterase isozyme pattern and DNA polymorphism using RAPD-PCR. Pattern of carboxylesterase revealed a simi lar isozyme cluster in the populations namely, sivaganga (population-3), d indigal (population-4), virudhunagar (population-5) and coimbatore (population-6). Similarly, the populations of L. (lrbollaiis recorded 3 distinct randomly amplified polymorphic DNA markers in all populations grouped above. This pattern of genetic variabi lity in the populations was also supported by the analysis of the similarity i ndices and UPGMA dendrogram.

Keywords: Carboxylesterase, LeLicillodes orbollalis, RAPD-PCR

Leucinodes orbonalis (Guenee) is the most injurious and a ubiquitous pest of brinjaJ, Solanum melongena L. , which is found to feed on the shoot and frui ts and occur throughout the year with varying degrees of infestation intensity. The pest is known to cause about 1 6 and 70 per cent damage to shoots and fruits, respectively ' . The larva confines i ts feeding activi ties on shoot in the early stages of crop and later on, on frui ts, which become unfit for human consumption. Varied control measures including the chemicals and other non-chemical approaches applied against the control of L. orbonalis have modified and resulted in heterogeneity among the populations. In response to the stresses, the populations of L. orbonalis have changed to adapt the i l l-effects by changing their eco­behavioural pattern, feeding physiology and reproduction, in addition to the changes in their molecular machineries2 . Hence, i t is of practical importance to precisely trace the genetic variabi l i ty in the expanding geographical distribution of the pest. Genetic characterization of populations of i nsect pests plays a pivotal role in determining management strategies of the insect pese.

Use of isozymes as biochemical marker to study the insect populations began in the l ate 1 950s and continues to be widely appl ied4. Within the last few years, techniques using the 'polymerase chain reaction

*Correspondent author Phone: 9 1 -0452-23 1 1 875 Fax: 9 1 -0452- 23 1 2 375 E-mai l : janarthanans@rediffmai l.com

(PCR) to amplify specifi c genomic regions have become w idespread5, 6. Wil liams et al.7 developed the technique that uses a single . decamer primer to amplify arbitrary regions of a genome. They demonstrated that RAPD-PCR process reveals polymorphisms in the genomes of a wide variety of i nsect species. In this paper, we describe carboxylesterase and RAPD-PCR patterns to i l l ustrate their use as molecular markers in six different popLiations of L. orbonalis.

Materials and Methods

Populat ions of L. orbonalis were collected from si>-. different localities (population- I : Madurai . population-2: Theni , population-3 : Sivaganga. population-4: Dindigal , population-5: V irudhunagar. population-6: Coimbatore) of southern parts of Tami l Nadu, India. The fifth instar l arvae were used for both carboxylesterase and RAPD-PCR analysis . The larval tissues were di ssected out and used for the enzyme extraction and gel electrophoresis by non-denatured polyacrylamide gel as described by Georghiou and Pasteur8. DNA was also isolated from the tissues using genomic DNA purification kit supplied by M BI Fermentas, USA. DNA (20 ng) was dissolved i n 20111 of PCR reaction buffer containing 10 mM Tris-HCI (PH 8.8) ; l .5 mM, MgCh; 50 mM, KCl ; O. l %, Triton X- IOO; 0.2 mM, dNTPs; 5 pM of all the primers and 0.5 U of DNA polymerase. Ten primers (OPI- I I to OPI-20) obtained from Operon Technologies (USA) were used for RAPD-PCR studies. PCR wa�

KARTHIKEY AN et ai.: DETECTION OF GENETIC POLYMORPHISM IN L. ORBONALIS 549

conducted according to the methods of Will iams et

al. 7 : initial heat step (94°C for 4 min.), 40 cycles of denaturation (94°C for 45 sec.), annealing (37°C for 45 sec.) and extension (72°C for 90 sec.) and a final extension step (72°C for 7 min.) . Amplification was performed using a programmable Thermal Cycler PTC- I 50 (M] Research, USA). The products of PCR and DNA size markers [A DNA digested with EcoRI and HindII I (MBI Fermentas, USA)] were loaded onto a 1 .7% tris-borate-EDT A agarose gel and run for 4 hr at 50 V. The gels were stained with ethidium bromide and photographed. Each lane of enzyme and RAPD profiles were subjected to gel documentation system (Vi lbert-Lourmat, France) . The dendrogram analysis was carried out using Bioprofil 1 0 software.

Results

The pattern of carboxylesterase isozyme and RAPD profile of L. orbonalis in this study showed characteristics of genetic variability of each population. Figure l a shows native PAGE analysis of carboxylesterase isozymes of tissue samples of L. orbonalis. The isozyme pattern showed one slow moving esterase fraction at Rm 0.46 and three fast moving esterase fractions at Rm 0.64, 0.68 and 0.8 1 . The slow moving esterase fraction was observed in all the populations except the population-2 (Theni). The fast moving esterase fractions, Rm 0.68 and Rm 0.8 1 , were found i n all the populations with differential staining activity. The fraction electrophoresed at Rm 0.64 was recorded to be unique to population- l (Madurai). The similarity i ndex analysis

' of all the

carboxylesterase fractions among various populations resulted in the generation of 1 00% similarity among the population-4 to population-6 (populations of Dindigal, Virudhunagar and Coimbatore; Fig. I b) . UPGMA dendrogram also revealed these three populations in one cluster, and Theni (population-2) and Madurai (population- I ) were separated from the cluster (Fig. I c) .

100% 90% 80% 70% 60% I I I

1 2 3 � 4 � 5 6

Ten different (OPI- l l to OPI-20) random primers were tested with DNA samples isolated from various populations. Of the primers used OPI- 1 3 revealed a polymorphic pattern that enabled to distinguish various populations of L. orbonalis. Similarity indices and dendrogram analysis were also computed and presented (Fig. 2a, b, c). All the scorable fragments ranged from 1 450 to 480 bp. Like isozyme analysis, RAPD results were also revealed the close relationship of population-3 to population-6 (populations of Sivaganga, Dindiga1 . Virudhunagar and Coimbatore).

0.64

Rm 2

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2 0.25

3 0.25

4 0.44

5 0.44

6 0.44

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-- 0 .81 " j"-'

. : . . .. �� ,_��..::ii:k·i-:. ;.;,.- Rm 3 4

2 3 4

1 .00

0.67 1.00

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0.57 0.86 1 .00

0 .57 0.86 1.00

30% 20%

5 6

5 6 (b)

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1.00 1 .00

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(e)

Fig .. l--{a) Native PAGE analysis of esterase isozymes of tissue samples of L. orbollalis; (b) Similarity index for the eleetreophoretie

profi le of esterases; (e) Dendrogram with Homology Coefficient %: 0.0 (UPGMA) [Lane l -Madurai popUlation; Lane 2-Theni population; Lane 3-Sivaganga population; Lane 4-Dindigal population; Lane 5-Virudhunagar population; and Lane 6-Coi mbatore population I

550

2,027 1 ,904 1 ,584 1 ,375

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564

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1

2

3

4

5

6

100%

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0.20 1.00

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0.40 0.00 1 .00 1 .00

0.20 0.00 0.75 0.75

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90% 80%

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5

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0.75

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INDIAN J EXP BIOL, JUNE 2005

-- 1 ,450 -- 1 ,350 -- 960 --- 800 -- 680 -- 560

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60%

Discussion

Molecular characterization of i nsects has be<.' 11 frequently conducted on the basis of existence ( r polymorphic DNA fragments ampl ified by PCR ' , Wi l l i ams et 01. 1 0 have reported geographic I distribution of Argentine stem weevi ls , Listrollotl , '; bOllariensis ( Kuschel), by RAPD-PCR using genomi .­DNAs. Haymer and McInnis" have also pointed out . l

distinctive variation in PCR-ampl ified DNA pattel'l 1 , between laboratory-cultured and wi ld populations , I :' Mediterranean frui t tl ies, Ceratifis capital" (Weidemann) . RAPD evidence of introgression in t w. ) closely related sympatric species of Choristoneura I I�

Atlantic Canada 1 2 ; RAPD as a tool to identify genet! . variation i n ecotypes of the European corn boreri : population structure of Helicoverpa armigera usinr RAPD analysis l 4 ; RAPD markers l inked t l ) Nilaparvata lugellS ' 5 ; Genetic diversity of Iberia l l populations of Bemisia tabaci based on RAPD-PCR I ,

were some known l i terature indicating that RAPD · PCR has promise for displaying intraspecific genetil.' variations in insect species. Skoda et al. 1 7 am . Kawamura et aZ. 1 8 have attempted to clarify PCR based DNA patterns i n Cochliomyia sp. and sweel potato weevi ls, Cylas formicarius collected fron various ecotypes. Results from our study also confim the value of isozyme as a biochemical marker and the RAPD as genetic marker in distinguishing L orbonalis populations. Ident ification and discrim ination of pest populations in terms of genetil characterization would be much useful in choosing precise management strategy concerning thei l susceptib i l i ty .

Acknowledgement The authors grateful ly acknowledge the financial

assistance through research funding received from

50% 40% 30% 20% 10% 0%

2 __________________________________________________________ �-------J (c)

Fig. 2--{a) Random amplified polymorphic DNAs generated by the Primer OPI- 1 3 using genomic DNA of L.orbonalis of differenl populations; (b) Similarity index of L. orbollalis based on RAPD profi les; (c) Dendrogram with Homology Coefficient %: 0.0 (UPGMA I [Lane M-Molecular Marker ( DNA digested with EcoRI and HindIII); Lane 1 - Madura; population; Lane 2-Theni population; Lane 3 · Sivaganga population; Lane 4-Dindigal population; Lane 5 -Virudhunagar population; and Lane 6-Coimbatore population]. (Arrowhead indicates the population specific DNA fragments)

KARTHIKEY AN et at.: DETECTION OF GENETIC POLYMORPHISM IN L. ORBONALIS 55 1

Department of Science and Technology, Government of India (DO No. SP/SO/C-45-200l ).

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