Comparison of effectiveness of RAPD , ISSR and SSR markers for analysis of coconut (Cocos nucifera L.) germplasm accessions

The effectiveness of Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Simple Sequence Repeat (SSR) markers was investigated to identify polymorphism among worldwide collection of coconut germplasm accessions. RAPD (ten primers), ISSR (ten primers) and SSR (ten primer pairs) techniques produced 86, 97 and 92 polymorphic markers, respectively in 33 coconut germplasm accessions. RAPD and ISSR produced 76.7% and 82.9% polymorphic markers, respectively whilst SSR markers exhibited 100% polymorphism. Mean Polymorphism Information Content (PIC) value was highest for SSR (0.78). ISSR markers exhibited relatively high PIC (0.31) compared to RAPD markers (0.23). SSR markers exhibited a wide range of similarity (0.000-0.857) compared to RAPD and ISSR markers. Between the two multilocus markers viz., RAPD and ISSR, the latter was more efficient based on PIC value, percentage of polymorphism and better reproducibility. The Mantel test gave r=0.510 between RAPD and ISSR similarity matrices revealed high correlation between them. There was no correlation between RAPD:SSR and ISSR:SSR similarity matrices. As there was a good correspondence between RAPD and ISSR similarity matrices, ISSR may be used as an alternative to replace RAPD in the genetic diversity assessment. INTRODUCTION Coconut (Cocos nucifera L.) is the most extensively grown and used nut in the world, playing a significant role in the economic, cultural and social life of over 80 tropical countries. In India, it is grown in 1.9 million ha and production is 9.5 million nuts (FAOSTAT, 2004). Improvement of this crop is a strategically long-term process. Markers based on differences in DNA sequence between individuals generally detect more polymorphism than morphological and protein based markers (Tanksley et al., 1989). Molecular markers play a vital role in easing the breeding processes aiming for the heterosis. Molecular marker techniques have been proven powerful in estimation of genetic diversity. Thus, PCR based multiple loci marker techniques which include Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Inter Simple Sequence Repeat (ISSR), and Simple Sequence Repeat (SSR) or microsatellites (Gupta and Varshney, 2000) are playing important role in crop improvement (Budak et al., 2004). 1 Centre for Plant Molecular Biology, TNAU, Coimbatore-641003 India. Manimekalai, Nagarajan & Kumaran SSR detects the allelic variation by way of repeat numbers with in a locus and one pair of SSR primer deals with one locus (Weising et al., 1992) where as RAPD detects both allelic and locus variations in a single assay and deals with insertions, deletions, point mutations which influence the base sequence of primer binding sites, allowing polymorphism to be detected (Williams et al., 1990). ISSR uses microsatellite, usually 16-25 bp long as primers in a single primer PCR reaction targeting multiple genomic loci to amplify mainly the inter-SSR sequences of different sizes (Zietkiewicz et al., 1994). Various molecular marker techniques such as Restriction Fragment Length Polymorphism (RFLP) (Lebrun et al., 1998), RAPD (Everard, 1999; Upadhyay et al., 2004; Manimekalai and Nagarajan, 2006a), AFLP (Perera et al., 1998), ISTR (Rohde et al., 1995), SSR (Perera et al., 1999; Meerow et al., 2003) and ISSR (Manimekalai and Nagarajan, 2006b) have been used for analysis of coconut germplasm in different regions. Though comparison of molecular markers for their efficiency, effectiveness and informativeness in germplasm analysis has been carried out in many other crops (Katsiotis et al., 2003 in pepper; Souframanien and Gopalakrishna, 2004 in blackgram; Archak et al., 2003 in cashew; Prakash et al., 2005 in coffee), such studies are limited only to a single comparison between AFLP and SSR in coconut (Teulat et al., 2000). Therefore, in the present investigation, three marker systems viz., RAPD, ISSR and SSR were compared for their informativeness and effectiveness in coconut germplasm analysis. MATERIALS AND METHODS Leaf samples Leaf samples were collected from thirty-three germplasm accessions (one palm per accession) obtained from International Coconut Gene Bank for South Asia, Central Plantation Crops Research Institute (Research Centre), Kidu, Karnataka, India. The germplasm accessions consisted of 29 tall accessions, two dwarf accessions and two intermediate accessions. Out of 33 accessions, eight belong to South East Asia, nine accessions belong to South Pacific, four accessions belong to Atlantic and America, eleven accessions belong to South Asia and one accession belongs to Africa. DNA was extracted from 2 g of fresh leaf material from 33 coconut accessions using Nucleon phyto pure Plant DNA extraction kit (Amersham Bioscience). RAPD analysis RAPD analysis was carried out as described in Manimekalai and Nagarajan (2006a). Ten random primers were used to amplify the DNA. The PCR products were subjected to electrophoresis through a 1.2% agarose gel using 1X TBE buffer at 90 volts for 2 h in Bio-Rad submarine electrophoresis unit. The ethidium bromide stained gels were documented using the Alpha Imager 1200 Documentation and Analysis system (Alpha Innotech Corporation, USA). ISSR analysis ISSR analysis was carried out as described in Manimekalai and Nagarajan (2006b). Ten primers targeting the microsatellite regions were used to amplify the DNA. Primers were purchased from University of British Columbia, Canada). The PCR products were Comparison of Molecular Markers in Coconut subjected to electrophoresis through a 1.8% agarose gel using 1X TBE buffer at 90 volts for 2 h in Bio-Rad submarine electrophoresis unit (Bio-Rad Laboratories, USA). The ethidium bromide stained gels were documented using the Alpha Imager 1200. (Alpha Innotech Corporation, USA and purchased through JH Bio Innovations Pvt. Ltd., Bangalore, India). SSR analysis SSR analysis was carried out as described in Perera et al. (2000). Primer sequences were obtained from CIRAD (Centre de Coopération Internationale en Recherche Agronomique pour le Développement), Montpellier, France. Initially, Gradient PCR was used to standardize the annealing temperature for 10 primer pairs used in this study. The PCR products were separated in 3% Agarose gel (Agarose 1000 , Invitrogen). Data analysis Data were scored as 1 for the presence and 0 for the absence of a DNA band of each accession. Only the clear, unambiguous and reproducible bands were considered for scoring. The average Polymorphism Information Content (PIC) and Marker Index (MI) were calculated by applying the formulas given by Powell et al. (1996) and Smith et al. (1997). The binary data matrices were entered into the NTSYS PC package (Exeter Software, New York). The data were analyzed using Qualitative routine to generate Jaccard’s similarity coefficient. Matrix comparison The similarity matrix based on RAPD, ISSR and SSR markers were compared by the MXCOMP routine of NTSYS pc. The normalized Mantel statistic Z (Mantel, 1967) was used to determine the level of association between the matrices. Cophenetic correlation between the original similarity matrix and cophenetic matrix was calculated.