INTRODUCTION

The Diamondback Moth (DBM), Plutella xylostella (Linnaeus) (Yponomeutidae: Lepidoptera) is known for developing resistance to almost all insecticides used for its control (Sannaveerappanavar and Viraktamath, 2006). It causes significant economic damage to vegetable crops belonged to cruciferous family in tropical and sub-tropical countries grown throughout the year. The crop losses due to DBM may often go up to 100% (Calden and Hare, 1986). The world production of cruciferous crops is estimated to be around 42.2 million tonnes and the cost of control of DBM alone is about one billion US dollars per annum (Talekar and Shelton, 1993). In India, cabbage and cauliflower are grown in an area of 0.438 million hectares with a production of 6.34 million tonnes per annum. The annual loss due to the menace of DBM in India was estimated to be 16 million US dollars (Mohan and Gujar, 2003). Despite its economic importance, population structure of this pest is not known from India.

Different types of molecular approaches to characterize variability/diversity of species are available now. Development of molecular markers provides an excellent knowledge/information on the genetic diversity of DBM and helps to identify pesticide resistant populations in order to evolve appropriate management strategies (Roehrdanz and Degragillier, 2000). Apart from management aspects, these also provide the frame work to understand the taxonomy, population structure, races/biotypes etc. Among the different molecular tools, RAPD technique has been widely used in the determination of population structure of an insect, where prior knowledge of DNA sequences are not known and aids in fingerprinting of the genotypes and to develop DNA markers (RAPD markers) specific to races and biotypes (Harry et al., 1998). This technique has several advantages when compared to other techniques with respect to handling, simplicity and cost effectiveness. With this existing scenario, in the present study an attempt was made to study the genetic variability of P. xylostella population from six geographical regions representing the south and north Indian cruciferous ecosystem using RAPD markers.

MATERIALS AND METHODS

Insect populations: Larvae and pupae of DBM were collected from six locations of the south and north Indian cruciferous ecosystem viz., Bangalore, Hassan, Shimoga and Belgaum (Karnataka); Delhi and Ludhiana (Punjab) and a laboratory reared susceptible population. These were reared separately in the laboratory on mustard seedlings by adopting the mass rearing technique developed by Liu and Sun (1984) with suitable modifications to get the progeny.

Chemicals: All of the chemicals and reagents were of molecular grade. Primers, Taq polymerase, dNTPs, Magnesium chloride, 10x genie buffer, Agarose, Ethidium bromide were from Bangalore Genie Ltd, Bangalore, India.

DNA extraction: DNA isolations were performed as in the method described by Saghai-Maroof et al. (1984). In brief, 20 larvae of first generation progeny from each location were randomly picked, dissected and the gut contents were completely removed to avoid any contamination of plant DNA. Resulting skin and legs were used to prepare genomic DNA. These samples were stored at -20°C until further use. The larval sample was quickly frozen in liquid nitrogen and ground into a fine powder using mortar and pestle. The fine powder was transferred to a sterile tube containing 750 μL of lysis buffer (1% Cetyl Trimethyl Ammonium Bromide (C-TAB), 5% Polyvinyl Pyrrolidone (PVP), 1.4 M NaCl, 20 mM 2-mercaptoethanol) and mixed gently. To above mixture an equal volume (750 μL) of phenol: chloroform: isoamyl alcohol (25:24:1) was added and mixed well by inversion. The mixture was centrifuged at 8000 rpm for 10 min at 4°C and the upper aqueous layer containing the DNA was carefully transferred to another 1.5 mL centrifuge tube and equal volume of ice cold isopropanol was added. The mixture was gently shaken by inversion to precipitate the DNA. The precipitated DNA was again centrifuged at 8000 rpm for 5 min and the resultant supernatant/upper layer was discarded. The pellet was washed with 500 μL of 70% ethanol, dried and re-suspended in appropriate quantity of 10xTE buffer (10 mM Tris HCl (pH 8.0)+1mM EDTA).

Table 1:	Primer sequences used for RAPD-PCR analysis in P. xylostella L.

The isolated DNA was purified and quantified according to the protocol described by Maniatis et al. (1982). The concentration of DNA was assessed by a spectrophotometer method using UV visible spectrophotometer. Finally, the DNA was stored at -20°C for further use.

PCR amplification: The genomic DNA from P. xylostella collected from six different places was subjected to Polymerase Chain Reaction (PCR) using five selected decamer random primers (Table 1). PCR was carried out for each primer in 25 μL standard reaction mixture consisting of 40 ng of template DNA, 0.2 mM primer, 3.5 mM magnesium chloride, 10x Bangalore Genie buffer, 3 U Taq polymerase and 10 mM each dATP, dTTP, dGTP and dCTP. PCR amplification was carried out using thermocycler (Eppendorf AG, Germany) in 0.2 mL micro-centrifuge tubes. Cycle conditions were 1 min initial denaturation at 94°C followed by 40 cycles of 1 min at 94°C for denaturation, 40 sec at 35°C for annealing, 1 min 30 sec at 72°C for extension and ended with 10 min final extension at 72°C.

Electrophoresis of PCR products: PCR products were separated on 1.0% agarose gels by using electrophoresis at 5 V cm^-1 for 2.0 h. The banding pattern was visualized using ethidium bromide staining. All the 18 primers were tested at least twice for the reproducibility of banding pattern. A set of 5 primers producing reproducible banding patterns were selected for the present study. The molecular size of the amplified products was estimated using 1 kb DNA marker (MBI Fermentas). The gel pictures acquired through a gel documentation system into a computer were processed and scored to get binary data.

Scoring of bands and statistical analysis: PCR amplification products of the seven samples were scored as presence (1) or absence (0) of bands. Genetic similarity matrix was computed for each individual population using Jacquard similarity co-efficient (Sneath and Sokal, 1973). The resultant similarity matrix was used to generate/construct dendogram using unweighted pair group method with arithmetic average (UPGMA) statistical software.