INTRODUCTION

Since its introduction, Polymerase chain reac-tion (PCR) (MULLIS and FALOONA 1987) has rev-olutionized molecular biology and has become auseful tool in numerous applications. A variantof basic PCR developed by WILLIAMS et al.(1990), where random arbitrary primers areused, has eliminated the need for prior knowl-edge of specific DNA sequences. This resulted instudy of Random Amplified Polymorphic DNA(RAPD) useful in differentiating various plantand animal spcies and their resistant and non-resistant strains. RAPD-PCR has been success-

fully applied to identify different populationswithin a species.

As majority of the lepidopterans are pests andhave high chromosome number, the study ofgenetic diversity in this group is very difficult onthe basis of number and size of chromosomes(which is very small). So, in the present investiga-tions an attempt has been made to study the genet-ic relationship in two species of lepidopteraninsects belonging to family Pieridae by RAPD-PCR technique.

MATERIAL AND METHODS

Two species of butterflies belonging to genus Catop-silia, namely C. pyranthe and C. crocale collected from

CARYOLOGIA Vol. 56, no. 2: 223-226, 2003

RAPD-PCR in two species of Catopsilia (Pieridae,Lepidoptera)VIJAY LAKSHMI SHARMA1, MAMTESH1, TAJINDER KAUR GILL1, SIDDHARATH SHARMA2, ADNAN-AL-BADRAN2 and RANBIR CHANDER SOBTI2

1 Department of Zoology, Panjab University, Chandigarh 160 014, India.2 Department of Biotechnology, Panjab University, Chandigarh 160 014, India.

Abstract - RAPD-PCR (random amplified polymorphic DNA-polymerase chainreaction) technique was used to analyze the polymorphism among the individualsof the two species of butterflies, namely Catopsilia pyranthe and Catopsilia crocale,which were captured from their natural habitat. Three decanucleotide primers viz.,primer A(AAGAGCCCGT), primer B(AACGCGCAAC) and primerC(CCCGTCAGCA) were used to amplify the gDNA from the adult individuals ofboth the species. A series of bands ranging from below 125 bp to 1500 bp were pro-duced by these primers. Standard curves were prepared and used to calculate thenumber of base pairs present in all the amplified fragments. The results obtainedwith these primers revealed sufficient amount of relatedness and variations in thesetwo species. Primer A amplified some common DNA fragments of the same size inboth the species, revealing the presence of conserved regions and thereby indicat-ing their ancestral relationship. Some of the fragments were unique to the individ-uals of both the species which could be used to generate their diagnostic profiles.Primer B produced single bands but of different sizes in the invididuals of both thespecies revealing the presence of intraspecific genetic variation. Primer C did notamplify the gDNA of the individuals of C.pyranthe but produced 4 bands rangingfrom 480 to 1160 bp length in C.crocale. These results indicate that the RAPD-PCRtechnique is useful in the studies on molecular taxonomy of Lepidoptera.

Key words: Catopsilia, RAPD, PCR, Primers.

* Corresponding author: fax +91 172541873; e-mail:rcsobti@pu.ac.in

Chandigarh (India) constituted the material for the pre-sent research work. They belong to family Pieridae. TheDNA isolation method of DOYLE and DOYLE (1990)was employed with minor modifications.

Isolation of Genomic DNAFor DNA extraction, adult individuals of both the

species were used. Single individual, after the removalof wings, was homogenized in chilled Tris-EDTAbuffer (8.0 pH). This homogenized mass was trans-ferred in 1.5 ml Eppendorf and centrifuged at 10,000rpm for 8 min at 4oC. The upper layer was discardedand 500µl of PBS (pH 8.0) was added to the pellet andmixed well. The mixture was again centrifuged at10,000 rpm for 8 min and 500µl of Tris-EDTA wasadded to the pellet. To this solution 70µl of 10% SDSand then 6µl proteinase K was added and mixed well.The whole solution was incubated at 65oC in waterbath for 1 hr. and then 100µl of 5M sodium chloridewas added followed by the addition of 80µl CTAB(Cetyl trimethyl ammonium bromide). It was mixedwell and again incubated at 65oC for 10 min. Then

750µl solution of chloroform: isoamyl alcohol (24:1)was added and solution was centrifuged at 10,000rpm for 8 min at 4oC. The upper layer was carefullytransferred to another Eppendorf and 550µl of chilledisopropanol was added to it. The mixture was gentlyshaken to precipitate DNA and was kept overnight infreezer. After that it was centrifuged at 10,000 rpm for10-15 min. Supernatant was discarded and the pelletwas dissolved in 100µl of Tris-EDTA and stored at –20oC till use.

The concentration of DNA was calculated by spec-trophotometric method using UV-visible scanningspectrophotometer.

Amplification by PCRPCR was carried out according to the method of

WILLIAMS et al. (1990) with certain modifications.Three random primers namely, PA(AAGAGCCCGT),PB(AACGCGCAAC) and PC(CCCGTCAGCA) wereused. Each reaction mixture of 30µl consisted of 200µMeach of dATP, dCTP, dGTP and dTTP, 10µM primer,100µg/ml of BSA, 1 unit of taq polymerase and 30

224 SHARMA, MAMTESH, GILL, SHARMA, BADRAN and SOBTI

Fig. 1 – L1-bands of HindIII marker. L2, L3, L4 and L5-RAPD profiles of C. crocale with PA, PB, PB and PC respec-tively; L6-negative control; L7 and L8-RAPD profiles ofC. pyranthe with PA and PB.

Fig. 2 – L9-bands of HindIII marker. L10-bands of C. pyran-the with PC; L11 and L12-bands of C. crocale and C. pyran-the respectively with PB; L13-negative control.

Table 1 – Base pair lengths of bands obtained with primer A in C.crocale and C.pyranthe.

Species Lane No. No. of bands Band Base pairproduced length (bp)

C. crocale 2 7 1 15002 13203 11604 9005 5646 4807 320

C. pyranthe 7 6 1 9002 5643 4804 3205 below 1256 below 125

ng/ml of gDNA, overlaid with 50µl of mineral oil. PCRwas carried out for an initial denaturation period at94oC for 5 min, followed by 35 repetitions of followingcycle: denature 94oC for 1 min, anneal at 35oC for 1 minand extend at 72oC for 5 min. Following the last cyclean additional 5 min at 72oC was allowed for completionof extension. PCR products can be stored at 4oC. Theywere visualized by electrophoresing 10µl of amplifiedproduct on 1.5% agarose gel in 1XTAE buffer for 4hrs. The gels were stained with ethidium bromide andphotographed under UV illumination.

Scoring RAPDThe size of PCR products was estimated by com-

parison with that of standard run in the same gel.(Hind IIIdigest). For this, standard curve was plottedwith number of base pairs (bp) vs relative mobility forvarious DNA fragments present in Hind III digestmarker. The bands present in both the species, presentonly in C. pyranthe and in C. crocale were scored.

RESULTS

The concentration of DNA extracted fromindividuals of two species ranged between 200-300 ng/µl as determined by spectrophotometricmethod.

RAPD-PCR analysisThe RAPD-PCR products of C. crocale and

C.pyranthe amplified with the 3 primers namely,PA, PB, and PC produced a series of bandsranging from < 125 bp to 1500 bp having dif-ferent intensities. Some of them were polymor-phic, while others were monomorphic (Figs. 1and 2).

A total of nine RAPD-PCR products fromtwo species revealed 21 bands with 3 primers(Table 1, 2 and 3). The primer A produced 7bands in C. crocale and 6 in C. pyranthe. Howev-er, the former had these bands in the range of1500 bp to 320 bp length, while latter had bandsfrom 900 bp to < 125 bp length. The lower fourbands of C. crocale corresponded to upper fourbands in C. pyranthe. With primer B, single bandwas produced in both the species but of varyingbase pair lengths (Table 2). The primer C pro-duced four bands in C. crocale ranging from 1160bp to 480 bp length but it produced no band inC. pyranthe revealing genetic differences betweentwo species.

DISCUSSION

RAPD-PCR identified the genetic similaritiesand differences between the two species at genet-ic level. Based on number of bands shared incommon, the two species show similarities. Dif-ferences have also been observed as a result ofcertain fragments present in one species andabsent in others and vice-versa.

The study of RAPD-PCR products from twospecies of genus Catopsilia with three primersrevealed that PA produced maximum bands inboth the species and some of them were commonto both, showing conserved regions in them, as aspecific primer amplifies discrete segments ofDNA and closely related species are more likelyto share conserved regions than distantly relatedone. At the same time, it (PA) showed certain

RAPD-PCR IN TWO SPECIES OF CATOPSILIA 225

Table 2 – Base pair lengths of bands obtained with primer B in C.crocale and C.pyranthe.

Species Lane No. No. of bands Band Base pairproduced length (bp)

C. crocale 3 1 1 4804 - - -11 1 1 1280

C. pyranthe 8 1 1 Below 12512 1 1 720

Table 3 – Base pair lengths of bands obtained with primer C in C.crocale and C.pyranthe.

Species Lane No. No. of bands Band Base pairproduced length (bp)

C. crocale 1 11605 4 2 900

3 5644 480

C. pyranthe 10 - - -

bands specific to each species revealing theiruniqueness in respective species. So, it showsrelated and species specific DNA differentiationin two species. Similar studies have been carriedout in 5 species of genus Aedes using two primers(KAMBHAMPATI et al. 1992). The PCR productsamplified by PC show amplification in C. cro-cale, but not in C. pyranthe, revealing species spe-cific amplification with this primer.

The study of RAPD-PCR product with PBreveal differences in different individuals of twospecies. In two individuals of C. crocale singlebands of 480 bp and 1280 bp length respective-ly were obtained with the primer PB, while suchbands of inter individual variation in C. pyranthewere of <125 bp and 720 bp. This indicated theexistence of genetic polymorphism in them. So,the use of this technique provides informationregarding inter and intra-specific variations.

RAPD differences have been found betweengeographically isolated populations of otherinsect species including Aedes mosquitoes(KAMBHAMPATI et al. 1992) Mediterranean fruitfly (HAYMER and Mc INNIS 1994) Pine bast scales(MENDEL et al. 1994), Parasitoid wasps (EDWARDS

and HOY 1993) and Argentine stem weevil(WILLIAMS et al. 1994).

RAPD can find wide use in the identificationand differentiation of closely related species andpopulations within a species (XU et al. 1998; LI etal. 2000; ZHOU et al. 2000; SEBASTIANI et al.2001). Genetic polymorphism revealed is of greatimportance in species diagnostics (BABCOCK andHERATY 2000; POZDNYAKOV et al. 2000) as thepattern of bands revealed by RAPD-PCR areoften species specific.

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Received July 12, 2002; accepted February 3, 2003

226 SHARMA, MAMTESH, GILL, SHARMA, BADRAN and SOBTI