INTRODUCTION Banana (Musa spp.) is one of the most important fruit crops of India, next only to mango. It is cultivated in tropical and subtropical regions of the world. India is the largest producer of banana, contributing 19.71% of the global production and a total production of 19.19mt from 0.565 mha area (Singh, 2007). Banana is the most important fruit crop of Kerala and one of the oldest cultivated fruit crops. In Kerala, several landraces are available and cultivated in different parts, known by different local names, being a highly evolved crop. Banana cultivars have a number of synonyms, resulting in a somewhat confused taxonomical status. Simmonds (1962) concluded that present day cultivars evolved by hybridization of two ancestral parents, Musa acuminata and M. balbisiana, which are considered as the main contributors of A and B, genomes respectively. All cultivars are classified into various genomic groups such as AA, AAA, AB, AAB and ABB based on morphological Diversity studies in ecotypes of banana (Musa spp.) using molecular markers and D2 analysis C. Rajamanickam and K. Rajmohan1 Horticultural College and Research Institute Periyakulam – 625 604, India E-mail : cmanickam@rediffmail.com ABSTRACT The present study was aimed at analyzing the genetic diversity of promising banana ecotypes grown in Kerala. Twenty eight ecotypes of banana were collected from different parts of Kerala. DNA isolated from these was used for RAPD analysis. Six most-promising primers viz., OPA-01, OPA-03, OPA-13, OPB-04, OPB-10 and OPB-12 were used. These yielded 46 scorable bands with an average of 7.66 bands per primer. RAPD data were analyzed statistically and a dendrogram was constructed. Twenty three characters were observed in the twenty eight banana ecotypes and were statistically analyzed as per the method proposed by Mahalanobis (D2). From RAPD dendrogram, it was found that the banana clones clustered into fourteen groups at a distance of 0.200. At a distance of 0.250, 8 out of 12 Nendran (AAB group) ecotypes formed a single cluster at the same distance. Among Palayankodan (AAB group) ecotypes PKNNR, Pisang Ceylon, Motta Poovan, Chandra Bale and Palode Palayankodan grouped together and formed a single cluster. Attu Nendran, Monthan, Robusta, Koonoor Ethan, Ilavazha and Vellapalayankodan formed individual clusters and had maximum genetic divergence. Among diploid clones, Ilavazha (BB group) had maximum genetic divergence. Among triploid clones, Attu Nendran, Robusta, Koonoor Ethan and Vellapalayankodan showed maximum genetic divergence. Among Nendran (AAB group) ecotypes, Attu Nendran and Koonoor Ethan revealed maximum genetic divergence. Among Palayankodan (AAB group) ecotypes, Vellapalayankodan recorded the highest genetic divergence. In D2 analysis too, a similar trend was observed. Key words: Banana, ecotypes, RAPD, molecular markers, D2 analysis, genetic divergence 1 Department of Plant Molecular Biology and Biotechnology, College of Agriculture, Vellayani – 695522, Thiruvananthapuram, Kerala, India scoring (Stover and Simmonds, 1987). Morphological characterization has been a major tool for classifying banana cultivars into different genomic groups. Efforts have also been made to classify bananas and plantains using quantitative traits. Variability and genetic divergence among Indian bananas were studied by Valsalakumari et al (1985). Molecular techniques have proved to be a powerful tools and have led to understanding genetic relationships among banana cultivars. Such studies are reported by Howell et al (1994), Bhat and Jarret (1995), Rekha et al (2001), Kahangi et al (2002) and Soni (2010) in Musa and Singh et al (2003) and Rai and Mishra (2005) in mango. Among various molecular characterization techniques, random amplified polymorphic DNA marker (RAPD) has been gainfully employed to investigate genetic variability (Brown et al, 1993). Hence, the present study is an attempt to characterize some of the commercially important bananas and their land races grown in Kerala using molecular markers and D2 analysis. J. Hortl. Sci. Vol. 7(1):34-40, 2012 35 MATERIAL AND METHODS PLANTING MATERIAL This study was conducted in the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram, Kerala. Ecotypes of banana used in the study are presented in Table 1. Banana clones belonging to six genomic groups were used in the study. Five suckers (replication) of each ecotype were maintained at the Instructional Farm, College of Agriculture, Vellayani. Suckers of almost uniform size were collected from different parts of Kerala, planted and maintained here. Spacing adopted was 2.0m x 2.0m. The experiment was laid out in Completely Randomized Block (CRD) design with five replications, as per the Panse and Sukhatme (1967). Observations were recorded on plant height, pseudostem girth, number of suckers per plant, leaves per plant, leaf length, leaf width, crop duration, number of fingers per hand, fingers per bunch, bunch weight, hand weight, number of hands per bunch, length of bunch, weight; length, girth and volume of finger, pulp/peel ratio, TSS, acidity, total sugars, sugar/acid ratio and shelf life of the fruit. Data were analyzed statistically. RAPD analysis DNA extraction Emergent young leaves before they fully unfurled were used for DNA extraction in all the ecotypes of banana following the procedure of (modified) Walbot (1998). Leaves were collected in the morning hours and washed under running tap water, and then in distilled water, two to three times after chopping the leaves coarsely. After wiping off the water using tissue paper, the chopped leaves were placed in a cool, dry porcelain mortar and ground well to a fine powder in liquid nitrogen. About 1 g of emerging leaves is used for DNA extraction Powdered leaf samples were transferred to the extraction buffer (168g urea, 28ml 5M NaCl, 20ml 1M Tris HCL at, pH 8, 16ml 0.5M EDTA and 20ml phenol, made upto 400 ml with sterile water) and placed in a water-bath at a temperature of 55oC. A volume of 2.5ml of 20% SDS (5g SDS, in 25ml sterile distilled water) and a pinch of polyvinyl pyrollidone (PVP) were added and mixed gently. Then, 25ml of phenol:chloroform:isoamyl alcohol (25:24:1) solution was added and this was centrifuged at 10000rpm for 10 min at 4oC. The supernatant was mixed with equal volume of phenol:chloroform:isoamyl alcohol and Table 1. Cultivars banana types ploidy and genomic composition of banana ecotypes Sl. Clone Type Ploidy Genomic No. level composition 1. Red banana Dessert 3x AAA 2. Vellakappa Dessert 3x AAA 3. Robusta Dessert 3x AAA 4. Vellayani Nendran Dessert 3x AAB 5. Padalamurian Dessert / 3x AAB cooking 6. Myndoli Dessert / 3x AAB cooking 7. Chengazhikodan Dessert / 3x AAB cooking 8. Attu Nendran Dessert / 3x AAB cooking 9. Kaliethan Dessert / 3x AAB cooking 10. Koonoor Ethan Dessert / 3x AAB cooking 11. Mysore Ethan Dessert / 3x AAB cooking 12. Zanzibar Dessert / 3x AAB cooking 13. Quintal banana Dessert / 3x AAB cooking 14. Changanasseri Dessert / 3x AAB Nendran cooking 15. Manjeri Nendran Dessert / 3x AAB cooking 16. Palode Palayankodan Dessert 3x AAB 17. PKNNR Dessert 3x AAB 18. Chandra Bale Dessert 3x AAB 19. Pisang Ceylon Dessert 3x AAB 20. Mottapoovan Dessert 3x AAB 21. Vellapalayankodan Dessert 3x AAB 22. Monthan Cooking 3x ABB 23. Peyan Cooking 3x ABB 24. Kadali Dessert 2x AA 25. Pisang Lilin Dessert 2x AA 26. Njalipoovan Dessert 2x AB 27. Kunnan Dessert 2x AB 28 Ilavazha Leaf purpose 2x BB centrifugated at 10000rpm for 10 min. The above steps were repeated 2-3 times until the interphase disappeared. To the upper phase so collected, 1/10th volume of 3.0M sodium acetate and double the volume of 70% cold, absolute ethanol were added. The pellet was dried and dissolved in 100µl to 200µl of TE buffer and stored at 4oC. DNA quantification was done using UV-Vis spectrophotometer. DNA amplification reactions were performed in 25µl medium containing 20ng of genomic DNA, 2.5µl 1x buffer, 5pM primer, 200µM each of dNTPs (each of dATP, dTTP, dCTP and dGTP) and 0.6 units of Taq DNA polymerase. PCR was carried out with initial denaturation at 95oC for Diversity in ecotypes of banana at molecular level J. Hortl. Sci. Vol. 7(1):34-40, 2012 36 3.0 min, followed by 45 cycles of denaturation at 95oC for 1.0 min, annealing at 36oC for 1.5 min and extended at 72oC for 2.0 minutes. The synthesis step of the final cycle was extended further by 6.0 min. Finally, products of amplification were cooled to 4oC. Forty one decamer primers were screened for efficiency using DNA isolated from ‘Attu Nendran’ as a representative sample. Of the 41 decamer primers used, 34 yielded amplification products. These primers produced 123 bands, of which 116 bands (94.31%) were polymorphic and seven bands (5.61%) were monomorphic. Twenty five primers showed a high level of polymorphism. Finally, six most-polymorphic primers were used for RAPD analysis of all the 28 banana clones. PCR amplification was made by six different random primers (OPA-01, OPA-03, OPA-13, OPB-04, OPB-10 and OPB- 12 (Operon Technologies, USA) and these were selected for further amplification. Amplification products were mixed with loading buffer containing bromophenol blue, separated electrophoretically on 1.4% agarose gel containing 0.5µg/ ml ethidium bromide. For data analysis, only amplification products reproducible over two amplifications were included. PCR products were Difenoconazoled as discrete variable: a Difenoconazole ‘+’ for presence and ‘-‘for absence of a homologous band. A genetic Similarity Matrix was constructed using the Jaccard’s Co-efficient method (Jaccard, 1908). Based on similarity coefficient, the distance between clones was computed with the help of a software package NTSYS (version 2.02i). A dendrogram was constructed by UPGMA method and, association between various genotypes was estimated from the dendrogram (Fig. 1). D2 analysis Morphological observations were recorded from 28 banana ecotypes based on the method of Stover and Simmonds (1987). Data on plant morphology, quantitative yield, fruit and quality parameters were recorded. D2 statistics, a measure of distance, based on multiple characters proposed by Mahalanobis (1936) was used. Grouping of varieties was done by Tocher’s method (Rao, 1952). Relative contribution of characters to divergence at the cluster as well as genotype level was assessed on the basis of coefficient of variation of individual traits. Average intracluster distance was calculated using the following formula. D2 is defined as: k D2 = d i 2 = (Yi1 – Yim)2, (1± m) i = 1 where Yi1 and Yim are uncorrelated means of the 1th and mth clones for the ith character. Average intercluster distance was calculated by taking each cluster and its distance from another cluster. Cluster diagram was drawn with the help of square root of D2 values showing relationships within and between clusters (Fig. 2). RESULTS AND DISCUSSION D2 analysis Analysis of Variance showed highly significant differences between genotypes for each of the twenty three characters studied. From the present study, all the 28 genotypes can be grouped into six clusters (Table 2). Fig 1. Dendrogram of 28 banana ecotypes of Kerala using RAPD marker (for clone number, refer Table 1) Fig 2. Cluster diagram showing relationship between different clusters Rajamanickam and Rajmohan J. Hortl. Sci. Vol. 7(1):34-40, 2012 37 Maximum number of genotypes are included in Cluster II (14 genotypes), viz., Red banana, Vellakappa, Robusta, Padalamurian, Chengazhikodan, Kaliethan, Vellayani Nendran, Attu Nendran, Mysore Ethan, Manjeri Nendran, Changanasseri Nendran, Monthan, Peyan and Ilavazha. This is followed by Cluster I (9 genotypes), namely, Palode Palayankodan, PKNNR, Chandra Bale, Pisang Ceylon, Mottapoovan, Kadali, Pisang Lilin, Njalipoovan and Kunnan. Cluster IV (Quintal banana), Cluster V (Vellapalayankodan) and Cluster VI (Koonoor Ethan) formed individual clusters. Cluster III contained only two genotypes, namely, Myndoli and Zanzibar. Ecotypes occurring in Cluster I were Palode Palayankodan, PKNNR, Mottapoovan, Chandra Bale and Pisang Ceylon. Another member of Palayankodan, Vellapalayankodan, fell under Cluster V. Table 2. Group constellations in twenty eight ecotypes of banana Cluster No. of Ecotype/s ecotypes C 1 9 Palode Palayankodan, PKNNR, Chandra Bale, Pisang Ceylon, Mottapoovan, Kadali, Pisang Lilin, Njalipoovan, Kunnan C 2 14 Red banana, Vellakappa, Robusta, Padalamurian, Chengazhikodan, Kaliethan, Myndoli, Attu Nendran, Mysore ethan, Manjeri Nendran, Changanasseri Nendran, Monthan, Peyan, Ilavazha C 3 2 Vellayani Nendran, Zanzibar C 4 1 Quintal banana C 5 1 Vellapalayankodan C 6 1 Koonoor Ethan Inter and intra cluster distance among the six clusters was variable (Table 3). Intercluster D values express diversification among groups of genotypes resembling each other, based on 23 characters under the study, while intra- cluster D values express the magnitude of divergence between clones within a cluster. Intra-cluster genetic distance, D values, ranged from 96 (Cluster I) to 159 (Cluster III) indicating wide divergence. Maximum inter-cluster distance was observed between Cluster VI and Cluster V (803), followed by Cluster VI and Cluster I (762), while, minimum inter-cluster distance D (182) was between Cluster IV and Cluster III. Intercluster distances and their mutual relationship are depicted in Fig. 2. Intercluster distance is higher than intracluster distance in all the cases indicating greater divergence of genotypes between clusters. Similar relationships were also observed by Valsalakumari et al (1985) in banana and Balasubramanyan et al (2009) in mango. Table 3. Estimation of average intra and inter cluster D for six clusters constructed from 28 ecotypes of banana Cluster Number C 1 C 2 C 3 C 4 C 5 C 6 C 1 96 244 460 408 202 762 C 2 112 269 210 310 553 C 3 159 182 536 336 C 4 0 427 382 C 5 0 803 C 6 0 Bold figures in diagonals are intra-cluster distances Contribution of individual character towards divergence (Table 4) revealed that maximum contribution to total differences was made by number of fingers per hand (15.48%), shelf life of fruit (14.88%), number of fingers per bunch (13.69%) and finger girth (8.93%). Lowest contribution of frequency was recorded in leaf width, TSS, pseudostem girth, number of leaves per plant, number of suckers per plant, hand weight, pulp/peel ratio, fruit volume and level of acidity. Table 4. Contribution of various characters to divergence S. No. Character Frequency % Contribution 1. Plant height (cm) 5 2.98 2. Psendostem girth (cm) 2 1.19 3. No. of leaves 2 1.19 4. Leaf length (cm) 1 0.60 5. Leaf width (cm) 4 2.38 6. No. of suckers 2 1.19 7. Crop duration (days) 3 1.79 8. Bunch weight (kg) 11 6.55 9. No. of hands per bunch 13 7.74 10. No. of fingers per bunch 23 13.69 11. No. of fingers per hand 26 15.48 12. Finger length (cm) 3 1.79 13. Finger girth (cm) 15 8.93 14. Finger weight (g) 5 2.98 15. Bunch length (cm) 5 2.98 16. Hand weight (kg) 2 1.19 17. Pulp-peel ratio 2 1.19 18. Fruit volume (cc) 2 1.19 19. TSS (°Brix) 1 0.60 20. Acidity (%) 2 1.19 21. Total sugars (%) 7 4.17 22. Sugar /acid ratio 7 4.17 23. Shelf life (days) 25 14.88 RAPD analysis Six random primers that were used for amplification (OPA-01, OPA-03, OPA-13, OPB-04, OPB-10 and OPB- 12) gave scorable polymorphic bands. Number of Diversity in ecotypes of banana at molecular level J. Hortl. Sci. Vol. 7(1):34-40, 2012 38 polymorphic and monomorphic bands obtained from these primers are presented in Table 5. Number of bands produced by each primer varied with genotype. The primers yielded 46 scorable bands with an average of 7.66 bands per primer. Nucleotide sequence of the above - mentioned primers is presented in Table 6. A total of 46 bands were analyzed and a dendrogram was constructed (Fig. 1). From the dendrogram, at a distance of 0.20 it is seen that diploid groups were different from triploid groups. Among the diploids, AB genomic clones, namely, Kunnan and Njalipoovan were found to be grouped together. AB genotypes are different from the BB genotype. Kadali and Pisang Lilin (belonging to AA group) Ilavazha (of BB group) formed three separate groups, at a distance of 0.20. Ecotypes belonging to AA genotype formed a single cluster at a distance of 0.25. In D2 analysis too, ecotypes belonging to AA group fell under the same cluster. Ilavazha (BB) was different from both AA and AB genotypes with respect to molecular profile. Ilavazha (BB group) formed a single cluster and also had maximum genetic divergence among diploid clones. Ilavazha is different in geographical origin and is morphologically too different from other diploid clones. In D2 analysis of diploid clones, Kadali, Pisang Lilin, Njalipoovan and Kunnan formed a cluster, while, Ilavazha formed a separate cluster. Among triploids, clones belonging to AAA group were different from AAB or ABB as seen in the dendrogram. Vellakappa and Red banana, both belonging to the AAA group, grouped together. In D2 analysis also, these two clones clustered together. Shanmugavelu et al (1992) reported that Vellakappa was probably a mutant of Red banana, as these two naturally resembled each other in several aspects. Robusta, falling under AAA group formed a separate cluster in the dendrogram. This might be due to the dwarf stature of Robusta, which is a distinct character of the Cavendish group. However, in D2 analysis, all three clones belonging to the AAA genome (Red banana, Vellakappa and Robusta) fell under a single cluster. Rekha et al (2001) also observed that Robusta did not group together in the dendrogram with other clones belonging to AAA genome, namely, Red banana, Grand Naine and Dwarf Cavendish. In the present study, eight out of 12 Nendran ecotypes belonging to AAB genome grouped together with respect to molecular profile. Ecotypes like Chengazhikodan, Myndoli, Kaliethan, Vellayani Nendran, Zanzibar Mysore Ethan, Changanasseri Nendran and Manjeri Nendran formed a single group D2. Koonoor Ethan and Attu Nendran formed independent clusters. The same trend was seen in RAPD profiles too. Simi (2001) reported Manjeri Nendran, Myndoli, Chengazhikodan, Attu Nendran, Changanasseri Nendran and Mysore Ethan as falling under the same cluster. Koonoor Ethan morphologically varied from the rest of Nendran ecotypes and was also of a geographically different origin. Fruit characters like length, girth and weight of finger, shelf life and TSS in this clone were higher, compared to other Nendran ecotypes. Padalamurian and Quintal banana grouped together at a distance of 0.25. Quintal banana, considered Giant Plantain, recorded higher plant height, pseudostem girth, days from flowering to harvest, crop duration and bunch weight. Hence, in morphological clustering, it is under a different cluster from Padalamurian, although the two fell under the same cluster as per RAPD analysis. From RAPD profiles, among the six Palayankodan ecotypes, five clones (Mottapoovan, Pisang Ceylon, PKNNR, Chandra Bale and Palode Palayankodan) are grouped together, the exception being Vellapalayankodan. In D2 analysis too, Palode Palayankodan, Mottapoovan, PKNNR, Pisang Ceylon and Chandra Bale formed a single cluster while Vellapalayankodan formed a separate cluster. Vellapalayankodan belonging to this particular clone is unique, characterized by robust growth characters, with higher value for crop duration, pseudostem girth, plant height, number of fingers per bunch, bunch length, number of Table 5. Number of polymorphic and monomorphic bands obtained with different primers Primer Number of Number of polymorphic monomorphic bands bands OPA-01 9 - OPA-03 8 - OPA-13 5 1 OPB-04 9 - OPB-10 9 1 OPB-12 6 1 Total 46 3 Table 6. Nucleotide sequence of promising primers and number of informative RAPD markers Primers Sequence Number of informative RAPD markers OPA-01 5’CAGGCCCTTC3’ 9 OPA-03 5’AGTCAGCCAC3’ 8 OPA-13 5’CAGCACCCAC3’ 5 OPB-04 5’GGACTGGAGT3’ 9 OPB-10 5’CTGCTGGGAC3’ 9 OPB-12 5’CCTTGACGCA3’ 6 Rajamanickam and Rajmohan J. Hortl. Sci. Vol. 7(1):34-40, 2012 39 suckers per plant, number of hands per bunch, finger length, sugar/acid ratio and shelf life of fruit. It has an ashy-white fruit skin which is uncommon in Palayankodan ecotypes. This result indicated that including cultivars with desirable characters, and traits such as disease resistance and high inter-cluster distance, would result in a highly segregating generation in breeding programmes. In selecting cultivars for hybridization, considerable care should be taken to select specific clusters and specific cultivars from selected clusters. Our study showed that cultivars belonging to the same genomic group was highly variable, as, these belonged to different clusters. In D2 analysis, Monthan and Peyan came under a single cluster and were similar in 21 characters out of 31. Clones belonging to ABB genomic group (Monthan and Peyan) at a distance of 0.20 formed an independent cluster and were different from other triploid genomic clones (AAA or AAB). In clustering based on D2 analysis, Monthan and Peyan were found to belong to a single group. However, as per Rekha et al (2001) found that all the cultivars of ABB group (Monthan, Cuba, Muthia, Karpooravally and Klue Teparod) came under a single cluster. The above molecular characterization tallied with association of genotypes based on genomic classification of Simmonds. Some aspects needed further investigation as there was confusion regarding grouping of cultivars such as Quintal banana, Vellapalayankodan, Myndoli, Attu Nendran, Koonoor Ethan, Robusta and Ilavazha. These clones formed separate clusters which might be due to less number of decamer primers used in this study. ACKNOWLEDGEMENT The authors gratefully acknowledge Kerala Agricultural University, Kerala, India, for providing funds and facilities to carry out doctoral research work. REFERENCES Balasubramanyan, S., Manivannan, M.I., Vani, V., Saraswathy, S. and Rajamanickam, C. 2009. Genetic divergence in mango. Paper presented in National Seminar on Production, postharvest technology and marketing of mango, Horticultural College and Research Institute, TNAU, Periyakulam, Tamil Nadu, India, p. 28-29 Bhat, K.V. and Jarret, R.L. 1995. Random amplified polymorphic DNA and genetic diversity in Indian Musa germplasm. Genet. Resource Crop Eval., 42:107-118 Brown, P.T.H., Lang, F.D., Kranz, E. and Lorz, H. 1993. Analysis of single protoplasts and regenerated plants by PCR and RAPD technology. Mol. Gen. Genet., 237:311-317 Howell, E.C., Newbury, H.J., Swennen, R.L., Withers, L.A. and Ford-Lloyd, B.V. 1994. The use of RAPD for identifying and classifying Musa germplasm. Genome, 37:328-332 Jaccard, P. 1908. Nouvelles rescherchers sur la distribution, Florale Bull. Soc. Vaudoise de Sciences Naturelles, 44:223-270 Kahangi, E.M., Lawton, M.A. and Kumar, C.A.C.Y. 2002. RAPD profiling of some banana varieties selected by small scale farmers in Kenya. J. Hortl. Sci. Biotech., 77:393-398 Mahalanobis, P.C. 1936. On the generalized distance in statistics. Proc. Nat’l. Acad. Sci. India 2:49-55 Panse, V.G. and Sukhatme, P.V. 1967. Statistical Methods for Agricultural Workers. 2nd Edition. Indian Council of Agricultural Research, New Delhi, 356pp. Rao, C.R. 1952. Advanced Statistical Methods in Biometrical Research. John Wiley and Sons, New York, 390pp. Rekha, A., Ravishankar, K.V., Anand, L. and Hiremath, S.C. 2001. Genetic and genomic diversity in banana (Musa spp. and cultivars) based on D2 analysis and RAPD markers. INFOMUSA, 10:29-34 Rai, D. and Mishra, K.K. 2005. Studies on genetic divergene in bael (Aegle marmelos Correa.). Ind. J. Hort., 62:152-154 Shanmugavelu, K.G., Aravindakshan, K. and Sathiyamurthy, S. 1992. Banana taxonomy, breeding and production technology. Metropolitan Bank Co. Pvt. Ltd., New Delhi, 459pp. Simi, S. 2001. Molecular characterization of banana (Musa AAB plantain subgroup) clones. M.Sc. (Hort.) thesis, Kerala Agricultural University, Thrissur, 72pp. Simmonds, N.W. 1962. The Evolution of the Bananas. Longmans, Green and Company Limited, London, 170pp. Singh, H.P. 2007. Research and development of banana and Diversity in ecotypes of banana at molecular level J. Hortl. Sci. Vol. 7(1):34-40, 2012 40 plantain in India. Souvenir - National Conference on Banana, 25-28 October, 2007. NRCB, Trichy, Tamil Nadu, India, pp 1-19. Singh, R., Meena, K.K. and Singh, S.K. 2003. Genetic divergence for yield and its component traits in pomegranate (Punica granatum L.). Ind. J. Pl. Genet. Resources, 16:133-134 Soni, K.B., Manju, R.V., Priya, A.O., Shimi, S. and Suseela, K.G. Molecular characterization of banana clones using RAPD markers. 2010 Global Conference on Banana, NRCB, Trichy, Tamil Nadu, India, 10-13 December, 2010, pp.1-19 Stover, R.H. and Simmonds, N.W. 1987. Bananas. Third Edition. Longman Scientific and Technical, Harlow, Esse, England, 445pp. Valsalakumari, P.K., Nair, P.C.S. and Prabhakaran, P.V. 1985. Genetic divergence in banana. Agril. Res. J. Kerala, 22:146-149 Walbot, V. 1988. Preparation of DNA from single rice seedlings. Rice Genet. Newslett., 5:149-151 (MS Received 28 September 2011, Revised 3 March 2012) Rajamanickam and Rajmohan J. Hortl. Sci. Vol. 7(1):34-40, 2012