International Journal of Pharma and Bio Sciences … M., Arya1, S. and Bisnoi, S., Genetic...

International Journal of Pharma and Bio Sciences V1(2)2010

MOLECULAR CHARACTERIZATION OF MORPHOLOGICALLY SIMILAR FOUR

PIERIDAE BUTTERFLIES (LEPIDOPTERA: INSECTA) BY RAPD-PCR TECHNIQUE

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ASHISH D. TIPLE*1,2, SONALI V. PADWAD3 AND VISHAL P.

DESHMUKH4

1Department of Zoology, Entomology Division, RTM Nagpur University Campus, Nagpur

40033, (M. S.) India. 2 Forest Entomology Division, Tropical Forest Research Institute, Jabalpur 482021, (M. P.) India. 3 Department of Entomology, Central Institute for Cotton Research, Nagpur 440010, (M. S.) India. 4 Departments of Botany, SGB Amravati University, Amravati, 444602 (M. S.) India * Corresponding author [email protected]

ABSTRACT Molecular characterization of four morphologically similar Pieridae butterflies was carried out using

RAPD markers. Total 121 bands were scored with 8 decamer primers, of which 115 were polymorphic while 6 were monomorphic and the percentage of polymorphism was 95.04%. Dendrogram based on average similarity coefficient of 08 primers grouped four Pieridae species into two distinct clusters. Cluster-I comprised of 3 species viz. Eurema laeta, E. hecabe and E. blanda, while cluster-II consisted of E. andersonii. Cluster-I further subdivided into two sub-clusters, sub-cluster-I consisting of only Eurema laeta and sub-cluster-II comprised of E. hecabe and E. blanda. Genetic similarities between these four species were discussed on the basis of morphological characters and RAPD data. KEY WORDS

Butterflies, Genetic diversity, Molecular characterization, Pieridae, RAPD INTRODUCTION

Pieridae is one of the important and most commonly seen families of Lepidoptera. This group is characterized by high chromosome number and small size of chromosomes that make differentiation of the species difficult at cytogenetic level for high resolution genetic studies of species and populations1. This family is widely distributed throughout the world and contains about 1,200 species and 60 genera, out of which 109 species occur in India2. They are usually white or light yellow, with black patterns on the wing surface. Most of them are small or medium-sized, but some large-sized species are seldom seen especially in tropical countries. Although many studies on morphological, ecological and molecular attributes of several species of Pieridae from world over are available, very little is known about the Indian species from this family3.

When it concerns about insects, it gets more difficult, as the differences are too minute to resolve4. Morphological identification of butterflies is usually based on the wing patterns5,6,7. Generally it is attributed to the numbers and positions of spots on the wings. It has now been established that classification of closely related lepidopteran species based on morphological features can pose several difficulties on account of




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attributes that can change as function of environment and prevalence of several biotypes. These factors make morphological criteria not a preferred way for a very accurate differentiation of these species3.

In order to maintain the existing level of diversity within tropical area, sustainable use of strategies and conservation methods needs to be established. This however, turns out to be a difficult task, since basic knowledge on the distribution and dynamics of genetic diversity is mainly lacking. Recently developed molecular marker techniques provide an important tool that ease the assessment of genetic diversity and facilitate genotyping, classification, inventorying and phylogenetic studies8. RAPD9 marker is well suited for use in the large sample throughout systems required for population genetics and studies of biodiversity10.

Random amplified polymorphic DNA9 is a PCR based technique in which random primers amplify many regions of genomic DNA. RAPD has been successfully applied to study genetic structure of endangered populations11, interspecific study4 and gene flow between populations12. Earlier RAPD was successfully applied for molecular characterization of two species of butterflies belonging to family Pieridae by Sharma et al13. Present paper describes the feasibility of RAPD in discriminating four morphologically identical species of family Pieridae and the genetic diversity in these species. MATERIALS AND METHODS Butterflies

Five adult individuals of each Spotless grass yellow (Eurema laeta), One spot grass yellow (Eurema andersonii), Common grass yellow (Eurema hecabe) and Three spot grass yellow (Eurema blanda) were collected from different sites in and around Nagpur city (India). DNA from 5 individuals of each species was extracted and pooled. Extraction of DNA

Genomic DNA was extracted from the legs of freshly collected each individual by Qiagen DNeasy® extraction kit following manufacturer’s instructions (Westburg b. v; Netherlands). The concentration of DNA was determined by spectrophotometric method using UV visible scanning spectrophotometer. Random amplified polymorphic DNAs (RAPDs)

About 10 random primers were used in single primer PCR. The PCR reaction mixture (25 µl) contained 25-50 ng of DNA, 1.25 U Taq DNA polymerase enzyme (Fermentas, USA), 0.4 µM each dNTPs (10 mM), 2.5 mM MgCl2 (15 mM), 1X Taq DNA polymerase buffer supplied with enzyme and 0.4 µM decamer primer (Operon, USA). The DNA was amplified in a programmable thermocycler (T Personal Biometra, GMBh, Gottingen, Germany) using the following conditions: Initial denaturation at 940C for 5 min, followed by 45 cycles denaturation at 940C for 1 min, annealing of primer at 380C for 1 min, and extending primer at 720C for 3 min and final extension at 720C for 7 min. The amplified PCR products were fractionated on 1.2% agarose gel using 1X TAE buffer containing 5µg/ml ethidium bromide. Gel was photographed on Gel documentation system (Alpha Innotech, USA).

For RAPD analysis data was scored as ‘1’ for the presence and ‘0’ for the absence of band of each species. The similarity matrix was prepared by using Nei and Li14 formula. The average of similarity matrix was used to generate a tree for cluster analysis by UPGMA (Unweighted Pair Group Method with Arithmetic Average) method using NTSYS 2.115.




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RESULTS AND DISCUSSION

In present investigation total 10 decamer RAPD primers were tested of which, 8 primers (Table 1) produced reproducible banding pattern and were selected for analysis.

Table 1 RAPD primers, their sequences and % polymorphism.

Primer Seq 5’ to 3’ Total bands

Polymorphic bands

Monomorphic bands

Polymorphism (%)

OPP-5 CCCCGGTAAG 07 07 00 100 OPP-8 ACATCGCCTA 17 16 01 94.11 OPP-10 TCCCGCCTAC 11 10 01 90.90 OPP-12 AAGGGCGAGT 14 13 01 92.85 OPP-15 GGAAGCCAAC 16 15 01 93.75 OPP-17 TGACCCGCCT 17 16 01 94.11 OPP-18 GGCTTGGCCT 19 19 00 100 OPP-19 GGGAAGGACA 20 19 01 95

Total 121 115 06 95.04

In all 121 bands were produced of which 115 were polymorphic and 06 were monomorphic (Fig 1). On an average 15.12 bands per primer were scored and the average percentage polymorphism was 95.04%. Primer OPP-19 produced maximum 20 bands, while primer OPP-5 produced minimum 7 bands.




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RAPD banding pattern of four Pieriedae butterfly species

Figure 1 RAPD banding pattern of four Pieridae species with primer OPP-12. (Lane1 Lambda DNA/Eco RI + Hind III, Lane 2 Eurema laeta, Lane 3 Eurema andersonii, Lane 4 Eurema hecabe, Lane 5 Eurema blanda)

Table 2 Similarity matrix of four Pieridae species based on RAPD data.

Eurema laeta

Eurema andersonii Eurema hecabe Eurema blanda

Eurema laeta

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Eurema andersonii 0.303 1 Eurema hecabe 0.352 0.241 1 Eurema blanda 0.430 0.450 0.436 1




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In all 2 primers (OPP-5 and OPP-18) produced 100% polymorphism, whereas primer OPP-10 produced minimum 90.90 % polymorphism. Similarity matrix (Table 2) revealed highest (0.450) similarity coefficient between Eurema andersonii and Eurema blanda, whereas minimum (0.241) similarity coefficient was observed between Eurema andersonii and Eurema hecabe. Dendrogram generated (Fig 2) from average similarity coefficient of 08 primers grouped four Pieridae species into two distinct clusters.

Dendrogram of four Pieridae species based on RAPD data.

Figure 2 Dendrogram of four Pieridae species based on 8 RAPD primer data.

Cluster-I comprised of 3 species viz. Eurema laeta, E. hecabe and E. blanda, while cluster-II consisted of E. andersonii. Cluster-I further subdivided into two sub-clusters, sub-cluster-I consisting of only Eurema laeta and sub-cluster-II comprised of E. hecabe and E. blanda. The dendrogram based on RAPD data showed much similarity with morphological differentiation of these 4 species. E. blanda and E. hecabe forming one cluster and it is supported by morphological similarities between these two species. E. blanda is very difficult to differentiate from E. hecabe, the only difference is, former species having three black spots while latter species with two black spots on under-side of FW5,6,16. Though Eurema laeta is placed in another sub-cluster of Cluster I, it shows similarities with E. hecabe, as both species having black band on apex of upper side of forewing. Isolated position of E. andersonii on dendrograms was attributed to minor variation in wing colour, as it having rich greenish yellow wing colour. E. andersonii was placed in cluster-II but it showing morphological similarities with E. hecabe, both species having upper side of forewing with marginal black border. According to Kunte7 and Wynter Blyth6 it is difficult to identify E. andersonii due to its similarity with Eurema hecabe. Clustering of all four Pieridae species was firmly supported by morphological characters.

PCR-based RAPD technique has revolutionized the field of molecular biology. Because of robustness and simplicity this technique is extensively used in DNA typing of necrophageous insects17, gene flow between




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populations12, evaluation of genetic population structure11, determination of genetic relationships4, phylogenetics in mites and ticks18. Sharma et al13 had demonstrated the application of RAPD in discrimination of two Pieridae butterfly species at sex level. Since no DNA sequence information is required to perform RAPDs, it can be widely used for species differentiation1.

CONCLUSION

In present study RAPD is found to be most easy and robust technique to discriminate four morphologically similar Pieridae species and it can be easily applied to those butterfly species where morphological characters are creating doubt in species identification. Beside this, it will provide an additional support to taxonomist, as it revealing information based on genetic constitution.

ACKNOWLEDGEMENT Authors are thankful to Dr. A. M. Khurad, Head of the Department of Zoology, RTM Nagpur

University Campus for kind help. REFERENCES 1. Sharma, V. L., Bhatia, S., Gill, T. K., Badran, A. A., Kumari, M., Singh, J. J. and Sobti, R. C.,

Molecular characterization of two species of butterflies (Lepidoptera: Insecta) through RAPD-PCR technique. Cytologia 71(1): 81–85, (2006).

2. Kehimkar, I., The Book of Indian Butterflies. Bombay Natural History Society, (2008). 3. Sobti, R. C., Sharma,V. L., Kumari, M., Gill, T. K., Singh, J., Sodhi, M., Mukesh, M., Bansal, M., Arya1, S. and Bisnoi, S., Genetic relatedness of six North-Indian butterfly species (Lepidoptera: Pieridae) based on 16S rRNA sequence analysis. Mole Cellular Bioche 295: 145–151, (2007). 4. Tiple A. D., Khurad A. M. and Padwad, S. V., Genetic relationships among some Lycaenidae butterflies as revealed by RAPD analysis. Cytologia 74(2): 165–169, (2009). 5. Evans, W. H., The identification of Indian butterflies. Bombay Natural History Society. Bombay, (1932). 6. Wynter-Blyth, M. A., Butterflies of the Indian Region. p. 523. Bombay Natural History Society.

Bombay, (1957). 7. Kunte, K., Butterflies of Peninsular India. Universities Press (Hyderabad) and Indian Academy of Sciences, Bangalore, (2000).

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plant breeding, molecular biology and genetics. Belgium J. Bot. 129: 107-117, (1997).




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9. Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafaiski, J. A. and Tingey, S. V., DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res. 18:

6531–6535, (1990).

10. Waugh, R. and Powell, W., Using RAPD markers for crop improvement. Trends in Biotech 10: 186-191, (1992).

11. Vandewoestijne, S. and Baguette, M., The genetic structure of endangered populations in the Cranberry Fritillary, Boloria aquilonaris (Lepidoptera, Nymphalidae): RAPDs vs allozymes. Heredity 89(6): 439-445, (2002).

12. Hoole, J. C., Joyce, D. A. and Pullin, A. S., Estimates of gene flow between populations of the swallowtail butterfly Papilio machaon in Broadland, UK and implications for conservation. Biol Cons 89(3): 293-299, (1999).

13. Sharma, V. L., Gill, T. K., Sharma, S., Badran, A. L. A. and Sobit, R. C., RAPD-PCR in two species of Catopsilia (Pieris, Lepidoptera). Caryologia 56(2): 219–222, (2003).

14. Nei, M. and Li, W., Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci 76: 5269-5273, (1979). 15. Rohlf, F. J., NTSYS-PC numerical taxonomy and multivariate analysis system. Version 2.1 Exeter

Software, Setauket, NY, (1999). 16. Haribal, M., The Butterflies of Sikkim Himalaya and their Natural History. Sikkim Nature Conservation Foundation (SNCF), Sikkim, (1992). 17. Benecke, M., Random amplified polymorphic DNA (RAPD) typing of necrophageous insects (diptera, coleoptera) in criminal forensic studies: validation and use in practice. Forensic Sci

Interna 98: 157–168, (1998). 18. Cruickshank, R. H., Molecular markers for the phylogenetics of mites and ticks. System Appl Acarol 7:

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