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Genetic Variation in Indian Populations of Scirpophaga incertulas as Revealed by RAPD-PCR Analysis

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Scirpophaga incertulas, commonly referred to as yellow stem borer, is a predominant pest of rice causing serious losses in its yield. Genetic variation among populations of Scirpophaga incertulas collected from 28 hotspot locations in India was
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   Biochemical Genetics, Vol. 39, Nos. 1/2, 2001 Genetic Variation in Indian Populationsof   Scirpophaga incertulas  as Revealedby RAPD-PCR Analysis Lalitha Sunil Kumar, 1 , 3 Arun S. Sawant, 2 Vidya S. Gupta, 1 and Prabhakar K. Ranjekar 1  Received 24 Mar. 2000—Final 1 Sept. 2000 Scirpophaga incertulas, commonly referred to as yellow stem borer, is a predom-inant pest of rice causing serious losses in its yield. Genetic variation among populations of Scirpophaga incertulas collected from 28 hotspot locations in India was examined using the randomly amplified polymorphic DNA-polymerasechain reaction (RAPD-PCR). In all, 32 primers were used and 354 amplification products were observed. No RAPD-PCR bands diagnostic to the pest population from any specific region were identified. Cluster analysis using UPGMA showed that, with the exception of the pest population from Pattambi, all the populationscluster as one group with GD values in the range of 6–22%, suggesting that gene flowbetweenpopulationsisindependentofgeographicdistanceandappearstobeunrestricted.TherelativelyhighGDvalueof48%exhibitedbythepestpopulation from Pattambi was the only exception. KEY WORDS:  Scirpophaga incertulas ; RAPD-PCR; genetic variation; gene flow. INTRODUCTION About 90% of the world’s rice is grown in the tropics and subtropics, where thewarm and humid climate is conducive to the development of diseases and insectpopulations. The rice crop is under continuous pressure from more than a hundred 1 Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory,Pune 411008, India. 2 Entomology Division, Regional Agricultural Research Station, Konkan Krishi Vidyapeeth, Karjat,India. 3 Towhomcorrespondenceshouldbeaddressed.Fax:(020)5884032.e-mail:lalitha@dalton.ncl.res.in. 43 0006-2928/01/0200-0043$19.50/0  C  2001Plenum Publishing Corporation  44 Kumar, Sawant, Gupta, and Ranjekar species of insects that infest and feed on it. In Asia, among the insect pests, thelepidopterous stem borers, which infest the rice crop from seedling to maturity,are the most destructive and cause an annual loss of millions of tons (Herdt,1991). The yellow stem borer ( Scirpophaga incertulas ) (Lepidoptera: Pyralidae)is a predominant pest that causes rice crop losses ranging from 5 to 95% despitethe use of insecticides and the introduction of high-yielding varieties. Moreover,controlling S.incertulas withinsecticidesisuneconomicalandecologicallyunsafe.The development and use of rice varieties tolerant to  S. incertulas  have, therefore,received increasing attention in recent years.A primary step in breeding for resistance is the identification and character-ization of the most useful genes and gene combinations in the host germplasm.In addition, knowledge of the structure of the pest/pathogen population is alsoimportant for identifying and characterizing the resistance, since resistance genesare defined in terms of their effect on a pathogen/pest. When a resistance gene isdeployed in a field, it faces a population of the pest/pathogen, and therefore, it isextremely important to characterize each resistance gene in terms of its spectrumof resistance relative to the pest/pathogen population. Various parameters suchas morphological characteristics, aggressiveness toward different host genotypes,and protein and metabolite profiles are routinely employed to study the populationstructure of a number of pests (Loxdale  et al. , 1996). More recently, data on DNApolymorphisms have been used to describe the genetic variation and populationstructureofvariouspests(Loxdale etal. ,1996),andthiscanthenserveasabasisfordesigningbreedingstrategiesforanyparticulargeographicalregion.Identificationof only those resistance genes that show an “incompatible” reaction with the localpest population could be a promising strategy to reduce the losses due to pests.To date there is no report on the characterization of   S. incertulas  using DNAmarkers, in spite of it being a major rice pest. The objective of the present studyis, therefore, to assess the level of genetic diversity between morphologicallyindistinguishable populations of   S. incertulas  from various hotspot locations inIndia where high-intensity borer infestation is favored by multiple rice cropping,culturalpractices,andclimaticconditions.Oursisthefirstdetailedmolecularstudyon diversity analysis of   S. incertulas  using randomly amplified polymorphic DNA(RAPD) markers. MATERIALS AND METHODSPest Collection Larvae of   S. incertulas  in the third- and fourth-instar stage were collected fromrice fields from the following regions (Figs. 1 and 2).1. Tehsils of Karjat (A), farm of the Konkan Krishi Vidyapeeth at Karjat(BB), Khalapur (B), Pali (C), Panvel (D), and Pen (E) (Raigad district,Maharashtra State).  Genetic Variation in Indian  Scirpophaga incertulas  45Fig. 1.  The four districts of the Konkan stretch in Maharashtra State, showingthe geographic distribution of populations used in the present study. The lettersrefer to the collection sites as listed under Materials and Methods. I, II, III, andIVrepresenttheThane,Raigad,Ratnagiri,andSindhudurgdistricts,respectively.  46 Kumar, Sawant, Gupta, and RanjekarFig. 2.  Geographic distribution of populations used in the present study. The letters refer to thecollection sites as listed under Materials and Methods. 2. TehsilsofMurbad(F),Shahapur(G),Wada(H),andBhiwandi(I)(Thanedistrict, Maharashtra State).3. Tehsils of Chiplun (J), Khed (K), Rajapur (L), and Sangamneshwar (M)(Ratnagiri district, Maharashtra State).4. Tehsils of Malvan (N), Kudal (O), and Vengurla (P) (Sindhudurg district,Maharashtra State).5. Sambalpur (Q) and Bhuvaneshwar (R) (Orissa State).6. Chinsurah (S) (West Bengal State).  Genetic Variation in Indian  Scirpophaga incertulas  47 7. Pattambi (T) (Kerala State).8. Aduthurai (U), Aliyarnagar (Y), and Coimbatore (Z) (Tamil Nadu State).9. Mandya (V) (Karnataka State).10. Warangal(W),Nellore(X),andHyderabad(AA)(AndhraPradeshState).(A tehsil represents a division of a district in a state.) DNA Extraction Live larvae of   S. incertulas  obtained by dissecting them out of rice stems werefrozen in liquid nitrogen, and DNA was extracted by the CTAB method of Rogersand Bendich (1988) as follows. Larvae were ground in liquid nitrogen and 5 mlCTAB extraction buffer [2% hexadecyltrimethyl ammonium bromide, 100 m  M  Tris–HCl(pH8.0),20m  M   EDTA(pH8.0),1.4  M   NaCl,1%polyvinylpyrrolidone]was added to the ground larvae. The homogenate was incubated at 60 ◦ C for 30 to40minandthencooledtoroomtemperature.Thehomogenatewasemulsifiedwithan equal volume of a chloroform:isoamylalcohol mixture (24:1), and the wholewascentrifugedatroomtemperatureand10,000rpmfor15min.Anequalvolumeof CTAB precipitation buffer [1% CTAB, 50 m  M   Tris–HCl (pH 8.0), 10 m  M  EDTA (pH 8.0)] was added to the supernatant, and the solution was mixed gentlyand allowed to stand at room temperature for an hour. The pellet was dissolvedin high-salt TE buffer [1  M   NaCl, 10 m  M   Tris–HCl (pH 8.0), 1 m  M   EDTA]and precipitated with twice the volume of chilled ethanol. The precipitate waswashed twice with chilled 70% ethanol and redissolved in TE buffer. The DNAsolution was treated with RNase A (Sigma) at a final concentration of 30  µ g/ml at37 ◦ C for 1 h, followed by deproteinization with chloroform: isoamylalcohol andprecipitation with 2 vol of chilled ethanol. The DNA was finally dissolved in TEbuffer. PCR Amplification and Agarose Gel Electrophoresis PCR amplification was performed with oligonucleotide primers from kits A, B, F,G,H,J,K,andLsynthesizedbyOperonTechnologies(Almeda,CA).Thereactionmixture (25  µ l) contained 10 m  M   Tris–HCl (pH 8.3), 50 m  M   KCl, 1.5 m  M  MgCl 2 , 0.0015% gelatin, a 200  µ M concentration each of dATP, dTTP, dCTP,and dGTP (USB), 5 pmol of primers, 25 ng of genomic DNA, and 0.6 U of Taqpolymerase (Bangalore Genie, India). Each reaction was overlaid with 50  µ l of mineral oil (Sigma) to prevent evaporation. Amplification was carried out in a MJDouble Engine PCR machine. The initial denaturation step of 5 min at 94 ◦ C wasfollowedby45cyclesconsistingofadenaturationstepof1minat94 ◦ C,annealingat36 ◦ Cfor1min,andanextensionstepof2minat72 ◦ Cusingthefastestavailabletemperature transitions. The last cycle was followed by 5 min at 72 ◦ C to ensurethat the primer extension reaction was completed. To reduce the possibility of 
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