Journal of Life Sciences 6 (2012) 1328-1333

RAPD-PCR Based Marker Approach for the Genetic Differentiation of Two Species of Cockroach (Order-Dictyoptera)
Bharat Neekhra, Divya Pandey and Subodh Kumar Jain
Molecular Biology Laboratory, Department of Biotechnology, Dr. Hari Singh Gour University, Sagar 470003, India Received: February 04, 2012 / Accepted: April 07, 2012 / Published: December 30, 2012. Abstract: Random amplified polymorphic DNA (RAPD) analysis was conducted for the differentiation of two most commonly occurring insect species Periplaneta americana and Blatella germanicana. This technique is proved to be a quick and effective to establish genetic markers to differentiate morphologically similar populations. During the study cockroach species Periplanata americana and Blatella germanicana were considered. Ten random primers were used for polymerase chain reaction (PCR). Many of such bands obtained, which differentiate between the two species. On the basis of interpretability, simplicity and reproducibility, six primers P1 (GATGACCGCC), P3 (GGCACGTAAC), P6 (GGTGCGCCTT), P7 (GTCAGAGTCG), P8 (GTCGCCGTCT) and P10 (GTGCCCGATG) were considered positive for genetic differentiation and analysis. A series of bands ranging from ~300 bp to ~1,000 bp obtained indicates that these two species are related, however they exhibit some variations. It has also been observed that the same primers also amplified some DNA fragments of the same size in both the species, which indicates the presence of conserved regions, sharing ancestral relationship. Some of the fragments were unique in both the species which may be used for diagnostic purposes. The study concludes that the RAPD-PCR technique is useful for the study of molecular taxonomy in insects. Key words: RAPD, PCR, cockroach, random primers, genetic differentiation.

1. Introduction
The distribution of insect is universal. Some of the species are easy to identify and categorize but for some other species it is a thorny task, due to their small size. The authors do identify insect species by their morphology, but there can be some small invisible changes in their morphological characteristics and distribution due to environmental effects. To solve these obstructions, the molecular techniques such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and arbitrary fragment length polymorphism (AFLP) are applied [1]. During the present investigation locally
Corresponding author: Subodh Kumar Jain, Ph.D., associate professor, research field: cell and molecular biology. E-mail: subjain@gmail.com.

available cockroaches have been considered. The RAPD-PCR technique has been used successfully to detect genetic polymorphism in plants [2, 3] for identification of barley genome segment introgressed into wheat [4] and animals [5]. RAPD markers have been used to identify subspecies and populations of Aedes aegypti [6], to differentiate closely related species and conspecific population of genus Aedes [7] and to distinguish cryptic mosquito species [8, 9], identification of sand-fly species [10], differentiate strains of Mediterranean fruitfly Ceratitis capitata [11] and to determine the origin of Israeli population of Matsucoccus josephi [12]. Little information is available on genetic variation within and between populations of cockroaches [13]. They are a cosmopolitan pest species that is obligatory commensal with human habitations. Among the

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best-known pest species are the American cockroach Periplaneta americana and the German cockroach Blatella germanicana belonging to the same order Dictyoptera share many common features like morphology, inheritance of similar habitat and nocturnal habit. Moreover they exhibit similar emergent behavior towards food and feed. Thus looking at above to overcome the difficulty to identify these two species on the basis of morphology, occurrence and behavior and feed biology, the molecular approach is an appropriate option. Keeping in view the utility and significance of RAPD-PCR technique in differentiating cell lines [14] as well as the advantage of studies done for identification of species on the basis of RAPD-PCR, the present work emphasize on the RAPD-PCR marker approach for genetic differentiation of two species of cockroach Blatella germanicana and Periplanata americana.

tubes for 20-25 times. Spin it down for 10 min at 8,000 rpm. Transfer the supernatant phase in another fresh tube. To supernatant add equal amount of chloroform + IAA (1:1), i.e., 250 L each. Mix it gently for 10 min; transfer the supernatant phase in another fresh tube. Add 1/10 3M Sodium acetate (50 L) and 0.8th volume of isopropanol, i.e., 400 L. Mix gently to allow DNA to clump. Spin it down for 10 min at 10,000 rpm at 4 C. Discard supernatant, add 500 L of 70% ethanol, keep it for 10 min and spin down at 12,000 rpm for 10 min at 4 C. Discard supernatant and allow the pellet to dry at room temperature at 37 C under laminar hood. Resuspend the pellet in 50 L of TE. Dissolve the pellet in water bath at 55 C for overnight and store at 4 C. The genomic DNA was checked on 0.8% agarose gel and stored at -20 C. The concentration of DNA was determined using UV spectrophotometer (Cole Parmer Ins. Company, USA). 2.2 Random Amplified Polymorphic DNA The amplification reaction was carried out in a 50 L reaction volume containing sterile water 39.0 L, 10 Taq Buffer A 5.0 L, 10 mM dNTP mix 2.0 L, RAPD Primer 2.0 L, DNA Template (10 ng/L) 1.0 L and Taq DNA Polymerase (3 U/L) 1.0 L. The random sequence 10-mer primers were purchased from Genei, Bangalore (India). For each primer examined, negative control was maintained which contained all the components except the genomic DNA. The DNA was amplified in a PCR machine thermocycler (Techne, UK) using the programme for initial denaturation: 94 C for 5 min, followed by first loop of 10 cycles denaturation at 94 C for 45 seconds, annealing of primer at 35 C for 1 min, and extending primer at 72 C for 1.5 min and in second loop, 40 cycles denaturation at 94 C for 45 seconds, annealing of primer at 37 C for 45 seconds, and extending primer at 72 C for 1 min and final extension at 72 C for 10 min. The amplified products were separated according to molecular size on 2% agarose gels in TE

2. Materials and Methods

Specimens of cockroach species were morphologically identified and collected from leaking pipes under the sinks, toilets, shower stalls and dishes from the dark areas of the university hostels, food store-room and restaurant. Most of the collections were done at night usually between 7-8 p.m. 2.1 Isolation of Genomic DNA Total cockroach DNA was isolated by phenol:chloroform:isoamylalcohol method. Adult individuals were crushed using pestle mortal in 500 L of lysis buffer. After adding 30 L proteinase K, incubated at 37 C over night (see that the tissues get dissolved properly). Add equal amount of phenol: chloroform: isoamylalcohol (IAA)25:24:1 and mix it gently by inverting the tubes for 20-25 times. Spin it down for 10 min at 8,000 rpm. Remove the supernatant in another fresh tube. Add equal amount (500 L) of phenol: chloroform: isoamylalcohol in supernatant phase and mix gently by inverting the

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RAPD-PCR Based Marker Approach for the Genetic Differentiation of Two Species of Cockroach (Order-Dictyoptera)

buffer and detected by staining with ethidium bromide. Gels were photographed on Gel Documentation system (MultiDoc-lt, Labmate). Six oligonucleotide primers were selected for study P1 (GATGACCGCC), P3 (GGCACGTAAC), P6 (GGTGCGCCTT), P7 (GTCAGAGTCG), P8 (GTCGCCGTCT) and P10 (GTGCCCGATG).

3. Results and Discussion

The genomic DNA of two species of cockroach Periplanata americana and Blatella germanicana (order-Dictyoptera) have been subjected to RAPD-PCR analysis with six decamer oligonucleotide primers (P1, P3, P6, P7, P8 and P10). RAPD patterns were visually analyzed and scored from photographs. All the primers produced a large number of discrete bands with different intensity
L 2 3 5 6

suggesting that the amplified fragments repeated in the genome in varying degrees (Figs. 1-3). For the analysis and comparison of these patterns, a set of distinct and well separated bands were selected while neglecting the weak and unresolved bands were not considered. As a result of initial RAPD analysis of pooled DNA, six primers were chosen for further analysis, on the basis of the criteria of band pattern quality, reproducibility and the presence of the diagnostic bands. These primers were then applied to study the suborder of the two species of the cockroaches. It is noted that white arrows represent base pair length of species specific bands. The bands grouped under c are an example of a complex pattern for which homologous bands can not be reliably be assigned in the two species (primer P1).
L 2 3 5 6

900 700 500

b d

1000 700 500

400

c b

c a

100

100

Fig. 1 RAPD banding pattern amplified by primer P1 and P3. Lane L: 100 bp DNA ladder. Lane 2 and 3: Periplanata americana and . Lane 5 and 6: Blatella germanicana and .

Fig. 2 RAPD banding pattern amplified by primer P6 and P7. Lane L: 100 bp DNA ladder. Lane 1 and 2: Periplanata americana and . Lane 3 and 4: Blatella germanicana and . White arrows represent base pair lengths of species specific bands.

RAPD-PCR Based Marker Approach for the Genetic Differentiation of Two Species of Cockroach (Order-Dictyoptera)

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Fig. 3 RAPD banding pattern amplified by primer P8 and P10. Lane L:100 bp DNA ladder. Lane 1 and 2: Periplanata americana and . Lane 4 and 5: Blatella germanicana and . White arrows represent base pair lengths of species specific bands. Table 1 RAPD analysis with six arbitrary primers to differentiate Periplanata americana and Blatella germanicana. Nucleotide sequence (5 to 3) GATGACCGCC GGCACGTAAC GGTGCGCCTT GTCAGAGTCG GTCGCCGTCT GTGCCCGATG Size range of amplified bands (~) P.a 430-> 1000 500-> 1000 300-900 350-900 550-1000 550-800 5 3 4 3 3 1 Total number of bands P.a 5 3 3 3 3 1 B.g 5 3 4 3 3 1 B.g 5 3 4 3 3 1 Number of selected species-specific RAPD fragments P.a P.a B.g B.g 1 2 3 1 1 1 2 3 1 1 1 2 1 2 2 1 1 2 1 2 2 1

Primer P1 P3 P6 P7 P8 P10

P.a: Periplanata Americana; B.g: Blatella germanicana.

RAPD analysis has to become a valuable tool for the analysis of genetic variation, estimating genetic distance among populations and generating molecular markers for economic traits. The varietal-specific amplification of distinct bands, permit their use in genetic fingerprinting. RAPD therefore, appears to be useful in differentiating species, subspecies and strains of different insects [1, 15]. RAPD-PCR technique is cost effective, take less time, the results can be directly inferred from the gel and it reveals large amount of genetic variations, therefore it finds various entomological applications [16-18]. Six decanucleotide primers were used to amplify the genomic DNA from the adult individuals of both the species. A series of bands ranging from 300 bp to > 1,000 bp were produced by these primers (Table 1). The results obtained with these primers revealed

sufficient amount of relatedness and variations in these two species. Primer P1 amplified some common bands of the same molecular weight in the species, indicating their ancestral relationship. Many bands amplified primers were unique to the individuals of both the species which could be used to generate their genetic marker profiles. Primer P10 produced single bands but of different sizes in the individuals of both the species suggesting the presence of intraspecific genetic variation. Primer P8 did not amplify the genomic DNA of the individuals of P. americana but reveal good banding pattern with B. germanicana thus primer P8 can be treated as clear genetic marker for B. germanicana species. Primer P6 produced an additional band d (500 bp) in case of P. americana but not in P. americana female as well as other species. Therefore this specific band may be considered as sex specific for P. americana. Primer P3 and P7 produced

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RAPD-PCR Based Marker Approach for the Genetic Differentiation of Two Species of Cockroach (Order-Dictyoptera) Theobroma cacao, L.J. Am. Soc. Hort. Sci. 120 (1995) 681-686. R.J. Schnell, C.M. Ronning, R.J. Knight, Identification of cultivators and validation of genetic relationships in Mangifera indica using RAPD markers, Theor. Appl. Genet. 90 (1995) 269-274. J.D. Shermon, L.Y. Smith, T.K. Black, L.E. Talbert, Identification of barley genome segments introgressed into wheat using PCR markers, Genome 44 (2001) 38-44. E.A. Rose, Applications of the polymerase chain reaction to genome analysis, FASEBJ 5 (1991) 46-54. M.E. Ballinger-Crabtree, W.C. Black, B.R. Miller, Use of genetic polymorphism detected by the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) for differentiation and identification of Aedes aegypti subspecies and populations, Am. J. Trop. Med. Hyg. 47 (1992) 893-901. S. Kambhampati, W.C. Black, K.S. Rai, Randomly amplified polymorphic dna of mosquito species and populations (Diptera: Culicidae): Techniques, statistical analysis and application, J. Med. Entomol. 29 (6) (1992) 939-945. R.C. Wilkerson, T.J. Parsons, D.G. Albright, T.A. Klein, K.J. Braun, Random amplified polymorphic DNA (RAPD) markers readily distinguish Cryptic mosquito species (Diptera:Culicidae:Anopheles), Insect. Mol. Biol. 1 (4) (1993) 205-211. R.C. Wilkerson, T.J. Parsons, T.A. Klein, T.V. Gaffigan, E. Bergo, J. Consolim, Diagnosis by random amplified polymorphic DNA polymerase chain reaction of four cryptic species related to Anopheles allitarsis (Diptera: Culicidae) from Paraguay, Argentina and Brazil, J. Med. Entomol. 32 (1995) 697-704. R.E. Adamson, R.D. Ward, M.D. Feliciangeli, R. Malignon, The application of random amplified polymorphic DNA for Sandfly species identification, Med. Vet. Entomology 7 (1993) 203-207. D.S. Haymer, D.O. Mc Innis, Resolution of population of the Mediterranean fruit fly at the DNA level using random primers for the polymerase chain reaction, Genome 37 (2) (1994) 244-248. Z. Mendel, D. Nestel, R. Gafny, Examination of the origin of the Israeli population of Matsucoccus josephi (Homoptera: Matsucoccidae) using random amplified polymorphic DNA-polymerase chain reaction method, Ann. Entomol. Soc. Am. 87 (1994) 165-169. W. Booth, S.M. Bogdanowicz, P.A. Prodohl, R.G. Harrison, C. Schal, E.L. Vargo, Identification and characterization of 10 polymorphic microsatellite loci in the German cockroach, Blatella germanicana, Mole. Eco. Not. 7 (2007) 648-650. X. Lery, B. La Rue, J. Cossette, G. Charpentier,

a number of distinct ban pattern which may be considered as species-specific precise diagnostic marker for Periplanata americana and Blatella germanicana. The number and size of amplified products varied depending upon the sequence of random primers and genotypes used; a total of 75 discrete amplified products were obtained out which 34 products exhibited diagnostic as well as species-specific pattern. On an average 12.5 bands per primer were scored. RAPD-PCR technique is extremely useful for rapid identification of genetic polymorphisms in Dictyoptera because of the reproducibility of the results for each of the species. Four primers out of six primers generated clear genetic markers for the genomic DNA of both species. RAPD differences have been found between geographically isolated populations of other insects including Parasitoid wasps [19] and Argentine stem weevil [20]. RAPD can find wide use in their identification and differentiation populations of within closely species related [21, species 22]. and

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polymorphism revealed is of great importance in species diagnostics [23-27] as the pattern of bands revealed by RAPD-PCR are often species specific.
[10]

4. Conclusion
The diverse nature of bands indicates the genetic distance while the presence of common bands indicates evolutionary relationship between both the species. It is also concluded that some of the fragments are unique in both the species which might be used for diagnostic purposes according the RAPD-PCR analysis.
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