PCR-SSCP and Sequence Analysis of Three Populations of Microtermes
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` ESSENCE - International Journal for Environmental Rehabilitation and Conservation Volume V: No. 1 2014 [9 – 21] [ISSN 0975 - 6272] [www.essence -journal.com] PCR-SSCP and sequence analysis of three populations of Microtermes obesi (Order: Isoptera; Family: Termitidae) from Chandigarh (India) on the basis of partial 16Sr RNA and ND1 gene Mamtesh Kumari 1, Vijay Lakshmi Sharma 2, Monika Sodhi 3, Manishi Mukesh 3, Yogesh Kumar Shouche 4 and Ranbir Chander Sobti 5 Received : January 12, 2014 Accepted : May 17, 2014 Online : July 07, 2014 Abstract In the present work three populations of to 0.7 %. Thus, in this study ND1 gene was Microtermes obesi collected from Chandigarh, found to be evolving faster than 16Sr RNA. India were studied. PCR-SSCP and sequencing Keywords: 16Sr RNA tRNA leu ND1 techniques have been applied to characterize PCR - SSCP analysis the partial sequences of two mitochondrial mitochondrial genes genes i.e. , 16Sr RNA and 16Sr RNA tRNA leu conformational patterns ND1 of these populations. SSCP analysis percent diversity sequence revealed two types of conformational patterns variations for each gene. Types-I and –II were found in 419 bp long 16Sr RNA and Types -A and -B in Introduction 532 bp long 16Sr RNA tRNA leu ND1. A+T Termites of the genera Odontotermes and content was seen to be high for each gene Microtermes cause the heaviest destruction of which was above 60%. Stretches of As were seasoned timber both within buildings and more in 16S r RNA, while in 16S rRNA tRNA extramurally. Losses due to termites run to leu ND1, stretches of Ts were more. For 16Sr several millions of rupees in agricultural crops RNA the percent diversity was found to be alone. About 10-25 per cent loss is estimated in zero within M. obesi individuals. In case of most field and forest crops. Severe l oss in 16Sr RNA tRNA leu ND1 it ranged between 0.2 different regions of India has been recorded on highly susceptible crops such as wheat and For correspondence: sugarcane in northern India, maize, 1Deptt. of Zoology, R.C.U. Government P.G College, Uttarkashi. 2Deptt. of Zoology, Panjab University, Chandigarh. India groundnuts, sunflower and sugarcane in 3National Bureau of Animal Genomic Resources, Karnal, India southern India, tea in North -Eastern India and 4National Centre of Cell Sciences, Pune. India cotton in western India. Termite pro blems in 5Babasahib Bhimrao Ambedkar Univer sity, Lucknow. India E-mail: [email protected] agriculture in Southeast Asia largely affect 9 Kumari et al. /Vol. V [1] 2014 /9 – 21 perennial tree crops. The most economically this level. They also suggested that 16S in important genera throughout Southeast Asia combination with another gene would give a are Microtermes, Coptotermes, Odontotermes, more fully resolved tree. In 2010, Yeap et al. Macrotermes, Trinevitermes and Heterotermes. (2010) used the information from partial 16S, 12S and Though a good deal of work has been carried sequence of mitoc hondrial genes COII out on the taxonomy of Indian termites based together with morphometric on their morphological aspects, identifying measurements to determine the relationship Coptotermes heimi C. gastroi workers and separating soldiers of different between and and species is very difficult and in spite of using found these species to be conspecific. precise measurements, overlap may occur CP analysis of genome is another approach (Scheffrahn and Su, 1994). They, thereby, need introduced by Orita et al . (1989). The to be characterized at molecular level. technique can detect single base pair mutations Nowadays, molecular methods have in a PCR product without any prior sequence revolutionized insect systematics (Roderick, knowledge beyond that needed for the PCR 1996; Caterino et al ., 2000), and they are amplification. The product is denatured to form increasingly being applied to diverse groups of single stranded DNA and snapcooled to form insects. folded structure, w hich affects the mobility of The population structure of Coptotermes the strands in a non-denaturing gel. Hence two gastroi (Wasmann) was characterized by using bands are expected from homozygotes and four microsatellite markers by Yeap et al. (2011). from heterozygotes. Two identically migrating Mitochondrial DNA is a valuable marker that bands cannot be assumed to have identical is being used to study the insects phylogeny. sequences, because not all mutations will affec t Sharma et al. (2013) and Singla et al. (2013) mobility. used the partial fragments of mt. genes COI Molecular diagnosis, therefore, is just one way and COII and 12S to determine the that might gain insight as to the origin of newly phylogenetic positions of various Indian introduced species of termites, so that termites. Similarly, partial fragments of two intervention may be directed in the most mitochondrial genes viz ., 16SrRNA and ND1 economically effective manner. The were used in present study to find the ex tent of information so obtained can fu rther be used to genetic relatedness/variations in three denitrify the source of introduction of termites, populations of Microtermes obesi (Isoptera: to discriminate the species for the application Termitidae). 16Sr RNA is specific to a given of corrective treatment measures and for species. Black and Piesman (1994) using 16Sr cataloguing species with the intent to correctly RNA investigated the phylogeny of ticks at the classify them that would likely be discovered family level and concluded that this gene is later . Molecular diagnostics are expected to useful for phylogenetics of ticks at or below yield genetic information from the collections, which will further be used as an integral 10 Kumari et al. /Vol. V [1] 2014 /9 – 21 component of phylogenetic studies. 65 °C for 1 hour in water bath. After the Materials and Method addition of 120 µl of phenol : chloroform : isoamyl alcohol (25 : 24 : 1), the tubes were Collection and storage vortexed for 30 seconds and then centrifuged Termites were collected from three locations for 5 m in at 10,000 rpm in cooling centrifuge. separated by about 10-15 km of distance within The upper aqueous layer was carefully Chandigarh and its surrounding areas from transferred to fresh tube and 500 µl of North-Western part of India (T able 1) and isopropanol was added to it and stored at 4 °C preserved in 100% ethanol. Voucher for overnight and then centrifuged at 7000 rpm specimens were put in 70% ethanol mixed with for 10 minutes. The supernatant was discarded a few drops of glycerol and maintained in th e and the pellet was washed with 70% ethanol. Department of Zoology, Panjab University, The alcohol was drained out, the pellet dried Chandigarh (UT), India. and dissolved in 30 µl of TE and stored at – Identification of termites 20 °C after checking in 0.8% agarose gel. The Soldier specimens of all the populations concentration of DNA was then quantified by collected (packed in scintillation vials in 70% using-UV visible scanning spectr ophotometer. ethanol) were sent to the Isoptera Section of Amplification of 16SrRNA gene the Zoological Survey of I ndia (ZSI), Kolkata, The region between nucleotides 7902 and 8321 for their identification. The details of collection in Bombyx mori (GenBank, AB070264) site, date of collection, source and name of the (Yukuhiro et al ., 2002) of 16SrRNA was authors were mentioned on each vial. amplified by using the universal primers LR -J- Isolation of genomic DNA 13007 and LR-N-13398 in the same termites to Genomic DNA was extracted from termites give a 419 bp long fragment ( Table 2, Fïg.1). regardless of their castes by u sing standard Amplification of 16Sr RNA -tRNA leu-ND1 phenol: chloroform extraction method gene (Sambrook et al., 1989). The whole insect was A 532 bp long fragment of 16Sr RNA-tRNA homogenized in 1.5 µl eppendorf tube in 500 leu-ND1 gene from nucleotides 136 to 668 in µl of TE (Tris EDTA, pH 8) with hand pestle Reticulitermes banyulensis (GenBank, and the homogenate was centrifuged at 7000 - AY510537) (Kutnik et al ., 2004) was 10,000 rpm for 10 minutes in cooling amplified by using the appropriate primers centrifuge (4 °C). The supernatant was (Table 2, Fig. 2). discarded and the pellet dissolved in 500 µl of Single strand conformation polymorphism lysis buffer (400 µl of TE and 100 µl of 10% for 16Sr RNA AND ND1 genes of termites SDS) followed b y the addition of 6 µl of Proteinase K and the solution was incubated at SSCP analysis was carried out by using protocol of Vega et al. (1997) with slight 11 Kumari et al. /Vol. V [1] 2014 /9 – 21 modifications. Five microlitres of PCR product the upper lid was removed and the shark was resuspended in an equal volume of tooth comb was inserted between the glass formamide loading buffer in separate 0.2ml plates taking care that it just pierces the top of PCR tubes. the gel and complete wells were formed. The samples were denatured by placing them in Electrophoresis to detect SSCPs thermocycler at 95°C for 5 minutes and the Electrophoresis was carried out in a vertical tubes were cooled by keeping them unit (BioRad Protean II system) by casting immedi ately on the ice. Denatured samples non-denaturing polyacrylamide gels using well (3.0 µl each) were loaded in each well and the clean glass plates. Gels were made from 12% apparatus was run at 350V for 12 hours at acrylamide solution and 0.5X TBE; 15°C. These were ideal conditions for polymerization was initiated by adding 40µl of detection of variations in 16SrRNA and ND1 TEMED and 400µl of ammonium per sulphate genes. These conditions in fact allowed (10% w/v). Gels were electrophoresed in 1X detection o f fragments up to 550 bp length.