INTRODUCTION Coleus forskohlii Briq. family Lamiaceae, is the only naturally occurring species in Coleus to have fasciculated tuberous roots and it is indigenous to the Indian sub continent. It is a perennial, aromatic herb that grows wild on sun exposed arid and semiarid hill slopes of the Himalayas, Deccan Plateau, Eastern Ghats, Eastern Plateau and rain shadow regions of the Western Ghats in India. The tuberous root extracts of C. forskohlii are good source of a diterpene, forskolin which is exclusive to this species (Shah et al, 1980). Forskolin is used for the treatment of eczema, asthma, psoriasis, cardiovascular disorders and hypertension where decreased intracellular cAMP level causes disease development (Rupp et al, 1986). In Ayurveda, the tuberous roots of Coleus are used as drug for heart diseases, abdominal colic, respiratory disorder, insomnia and convulsions (Ammon and Muller, 1985). It also contains essential oil in tubers which has very attractive and delicate odour with spicy note (Misra et al, 1994). It has potential uses in food flavouring industry and can be used as an antimicrobial agent (Chowdhary and Sharma, 1998). Variation in Coleus genotypes based on forskolin and essential oil content has been reported earlier (Vishwakarma et al, 1988; Hegde, 1992; Nanaiah, 1993; Prakash and Krishnan, 1994). Since chemical characters are dependent J. Hortl. Sci. Vol. 4 (2): 143-147, 2009 Analyzing variability in Coleus forskohlii Briq. using RAPD markers C. Kavitha, E. Vadivel1, R. Sivasamy2 and K. Rajamani Horticultural College and Research Institute Tamil Nadu Agricultural University, Coimbatore - 641 003 E mail: ckavi_2k@yahoo.com ABSTRACT Coleus forskohlii Briq. is an indigenous medicinal plant with high traditional use in India. Genetic analysis of 37 diverse C. forskohlii genotypes was performed using 25 RAPD primers, which yielded 117 bands, of which 60 (51.28%) were polymorphic providing an average of 3.75 bands per primer. There were no genotype-specific products. The number of bands per primer varied from 1 (OPZ 8 & 16) to 7 (OPZ 11). Similarity matrix was constructed using Jaccard’s Coefficient and the data matrix of coefficient of similarity was subjected to cluster analysis using unweighted pair group methodology with arithmetic average (UPGMA). Cluster analysis resulted in grouping of 37 genotypes into two major clusters. The results indicated that RAPD could be used for genetic diversity analysis in C. forskohlii using higher number of primers as it is reliable, easy, rapid and cost-effective. Key words: Coleus forskohlii, genetic diversity, RAPD 1Directorate of Extension Education, TNAU, Coimbatore-641003 2Department of Biotechnology, Bharathiyar University, Coimbatore-641046 on environment, it is essential to characterize this medicinally important plant at genetic level. For any crop improvement programme, the assessment of genetic diversity and identification of superior genotypes are indispensable. Earlier, only morphological markers were used to assess the genetic diversity and for the identification of superior genotypes. Though they are important for initial genetic evaluation studies, morphological characters may change with environmental conditions. The diversity analysis of the 37 C. forskohlii genotypes by using morphological markers did not reveal any clear understanding (Kavitha et al, 2007). Hence, markers based on differences in DNA sequences between individuals, generally detecting more polymorphisms than morphological markers are preferred (Bostein et al, 1980 and Tanksley et al, 1989). Among the DNA-based markers, Randomly Amplified Polymorphic DNA (RAPD) provides excellent tool to study the genetic diversity and genetic relationship and to eliminate duplicates in germplasm (Virk et al, 1995). RAPD technique has been successfully used to genetically profile many different medicinal plant species. The present work was taken up to systematically characterize the C. forskohlii germplasm collection by RAPD markers and this is probably the first attempt to estimate genetic diversity in C. forskohlii at molecular level. 144 MATERIAL AND METHODS Plant material Thirty seven genotypes collected from different Coleus growing regions of Tamil Nadu and Karnataka and maintained in the Botanical Garden of Tamil Nadu Agricultural University, Coimbatore were taken for the diversity analysis (Table 1). DNA isolation and PCR amplification DNA was isolated from 40 mg of young leaf tissue following the protocol of Wilkie (1997). Mercaptoethanol (1%) and Polyvinyl Pyrrolidone (PVP) (2%) were added to the extraction buffer to remove the phenolics contaminants. To check the quality and quantity of the isolated genomic DNA, gel electrophoresis was carried out on 0.8% agarose gel. DNA concentration for PCR amplification was estimated by comparing the band intensity of a sample with the band intensities of known dilutions that gave good amplifications. The dilutions were carried out by dissolving the genomic DNA in appropriate quantity of TE buffer (pH 8.0). DNA from the 37 genotypes of C. forskohlii was amplified in PCR using a set of 25 arbitrary oligonucleotide decamer primers (Operon Technologies, Alameda, California, USA). 15 ìl reactions containing 10-20 ng of Table 1. Morphological description of Coleus forskohlii genotypes for genetic diversity analysis Genotype Salient features CF 1 Sub-erect growth, glabrous stem, pale green round small leaves, pale purple flowers, sparse roots CF 2 Sub-erect growth, glabrous stem, dark green round large leaves, pale purple flowers, dense roots CF 3 Sub-erect growth, medium pubescence, dark green leaves, violet flowers, sparse roots CF 4 Sub-erect growth, sparse pubescence, pale green leaves, violet flowers, sparse roots CF 5 Sub-erect growth, sparse pubescence, pale green large leaves, violet flowers, sparse roots CF 6 Erect growth, dense pubescence, pale green small leaves, violet flowers, dense fibrous roots CF 7 Sub-erect growth, sparse pubescence, dark green large leaves, violet flowers, dense fibrous roots CF 8 Sub-erect growth, sparse pubescence, pale green leaves, violet flowers, sparse roots CF 9 Sub-erect growth, medium pubescence, pale green small leaves, violet flowers, dense fibrous roots CF 10 Erect growth, sparse pubescence, pale green large leaves, violet flowers, sparse fibrous roots CF 11 Erect growth, medium pubescence, pale green large leaves, violet flowers, sparse fibrous roots CF 12 Sub-erect growth, sparse pubescence, pale green large leaves, violet flowers, sparse fibrous roots CF 13 Erect growth, dense pubescence, pale green small leaves, violet flowers, dense fibrous roots CF 14 Sub-erect growth, sparse pubescence, pale green large leaves, violet flowers, sparse fibrous roots CF 15 Sub-erect, medium pubescence, dark green small leaves, lilac flowers, small tubers CF 16 Sub-erect growth, sparse pubescence, pale green leaves, violet flowers, sparse fibrous roots CF 17 Erect growth, medium pubescence, pale green small leaves, violet flowers, dense fibrous roots CF 18 Erect growth, sparse pubescence, pale green leaves, violet flowers, sparse fibrous roots CF 19 Sub-erect growth, medium pubescence, variegated leaves, non flowering, small tubers CF 20 Sub-erect growth, dense pubescence, pale green large leaves, non flowering, tuberous roots CF 21 Erect growth, dense pubescence, dark green small densely packed leaves, non flowering, tuberous roots CF 22 Erect, sparse pubescence, dark green small leaves, non flowering, tuberous roots CF 23 Very erect growth, medium pubescence, pale green small leaves, violet flower, dense fibrous roots CF 24 Sub-erect growth, dense pubescence, dark green small densely packed leaves, non flowering, tuberous roots CF 25 Erect growth, medium pubescence, pale green small densely packed leaves, non flowering, tuberous CF 26 Sub-erect growth, dense pubescence, dark green small leaves, non flowering, tuberous roots CF 27 Sub-erect, sparse pubescence, pale green large leaves, non flowering, tuberous roots CF 28 Erect growth, dense pubescence, pale green leaves, non flowering, tuberous roots CF 29 Sub-erect growth, dense pubescence, pale green leaves, non flowering, tuberous roots CF 30 Erect growth, sparse pubescence, pale green leaves, non flowering, tuberous roots CF 31 Erect growth, dense pubescence, dark green leaves, non flowering, tuberous roots CF 32 Sub-erect growth, dense pubescence, dark green leaves, non flowering, tuberous roots CF 33 Erect, sparse pubescence, pale green large leaves, non flowering, tuberous roots CF 34 Sub-erect, medium pubescence, pale green leaves, non flowering, tuberous roots CF 35 Sub-erect, dense pubescence, pale green leaves, non flowering, tuberous roots CF 36 Sub-erect growth, medium pubescence, pale green leaves, non flowering, tuberous roots CF 37 Sub-erect growth, dense pubescence, pale green leaves, non flowering, tuberous roots J. Hortl. Sci. Vol. 4 (2): 143-147, 2009 Kavitha et al 145 genomic DNA, 1.5 mM of assay buffer. 0.5 mM each of dATP, dTTP, dGTP and dCTP, 1.5 ìM of primer, 0.25 mM MgCl 2 and 0.03 units of Taq DNA polymerase (Bangalore Genei Pvt Ltd, Bangalore). Amplifications were performed in PTC Thermal Cycler (MJ Research Inc.,) programmed for an initial denaturation at 94º C for 5 min., 44 cycles of 1 min. denaturation at 94ºC, 1 min. annealing at 37º C and 2 min. extension at 72ºC and a final extension of 10 min. at 72ºC and then at 4ºC till storage. PCR amplified products (15ìl) were subjected to electrophoresis in a 1.2 % agarose gel in 1X TBE buffer at 100 volts for 3.5h using Aplex submarine electrophoresis unit. The ethidium bromide stained gels were documented using Alpha Imager TM 1200 – Documentation and Analysis system (Alpha Innotech Corportion, USA). Data analysis RAPD bands were scored by considering only the clear and unambiguous bands. Markers were scored for the presence and absence of the corresponding band among the different genotypes. The scores ‘1’ and ‘0’ were given for the presence and absence of bands, respectively. Polymorphism information content (PIC) or expected heterozygosity scores for each RAPD marker were calculated based on the formula Hn = 1- “pi2, where pi is the allele frequency for the ith allele (Nei, 1973). The data obtained for RAPD profiling was subjected to cluster analysis. Similarity matrix was constructed using Jaccard’s coefficient for RAPD profiling and the similarity values were used for cluster analysis. Sequential agglomerative hierarchical non – overlapping (SAHN) clustering was done using unweighted pair group methodology with arithmetic averages (UPGMA) and data analysis was done using NTSYSpc version 2.02 (Rohlf, 1994). RESULTS AND DISCUSSION In the present study, C. forskohlii germplasm collection was assessed for diversity at DNA level, to define its core diversity and identify individual genotypes. As the first step, the RAPD analysis was carried out using a set of 25 decamer random primers for DNA amplifications through PCR. This profiling showed only 16 out of the 25 primers yielding unambiguous markers detectable as distinct bands. In the subsequent analysis, all the 16 primers produced polymorphic bands, a total of 117 distinct bands from 37 DNA templates of different genotypes, among which 60 (51.28%) bands were polymorphic (Table 2, Fig 1 and 2). The range of polymorphic bands in C. forskohlii genotypes was 1-7. The average number of bands per primer was 3.75. There was no genotype specific product. RAPD data from the 16 primers was used for cluster analysis. The PIC values ranged between 0.06 and 0.42 (Table 2). The mean PIC score for all loci was 0.24. The mean PIC score was greater than 0.24 for 50% of the RAPD primers. The PIC value provides an estimate of the discriminatory power of a marker by taking into account not only the number of alleles at a locus but also the relative frequencies of these alleles. Cluster analysis was performed on Jaccard’s similarity coefficient matrices calculated from RAPD markers to generate a dendrogram of 37 genotypes of C. Table 2. Primers used for RAPD analysis and outcome from PCR amplification S.No. Primer Name Primer sequence Polymorphic bands Total number of bands % polymorphism PIC 1 OPZ 01 5’-TCTGTGCCAC-3’ 4 10 40.00 0.38 2 OPZ 02 5’-CCTACGGGGA-3’ 2 6 33.33 0.16 3 OPZ 03 5’-CAGCACCGCA-3’ 6 8 75.00 0.24 4 OPZ 04 5’-AGGCTGTGCT-3’ 3 8 37.50 0.14 5 OPZ 05 5’-TCCCATGCTG-3’ 4 5 80.00 0.25 6 OPZ 06 5’-GTGCCGTTCA-3’ 2 7 28.57 0.41 7 OPZ 07 5’-CCAGGAGGAC-3’ 4 11 36.36 0.28 8 OPZ 08 5’-GGGTGGGTAA-3’ 1 5 20.00 0.06 9 OPZ 09 5’-CACCCCAGTC-3’ 4 7 57.14 0.42 10 OPZ 10 5’-CCGACAAACC-3’ 3 6 50.00 0.17 11 OPZ 11 5’-CTCAGTCGCA-3’ 7 12 58.33 0.29 12 OPZ 12 5’-TCAACGGGAC-3’ 5 8 62.50 0.33 13 OPZ 13 5’-GACTAAGCCC-3’ 7 9 77.77 0.18 14 OPZ 15 5’-CAGGGCTTTC-3’ 2 5 40.00 0.07 15 OPZ 16 5’-TCCCCATCAC-3’ 1 4 25.00 0.10 16 OPAW03 5’-CCATGCGGAG-3’ 5 6 83.33 0.37 Mean 60 117 51.28 0.24 Analyzing variability in Coleus using RAPD markers J. Hortl. Sci. Vol. 4 (2): 143-147, 2009 146 forskohlii (Fig. 3). All the genotypes could be placed in the range of 0.07 to 0.97 of similarity indices. The dendrogram, based on the similarity index, is indicative of considerable level of polymorphism among the genotypes at DNA level. The dendrogram obtained through cluster analysis revealed two major clusters. The dendrogram clearly depicted the variability present in the C. forskohlii germplasm and it separated 37 genotypes into two major clusters. The first cluster consisted of two genotypes CF 1 and CF 2 which were distinctly different from the rest of genotypes by possessing round shaped leaves and being non tuberous in nature. The second cluster enclosed the remaining 35 genotypes and was again grouped into two sub clusters. The sub cluster I consisted of tuberous and non tuberous genotypes whereas the sub cluster II consisted of only tuberous genotypes. The genotypes in these sub clusters were also again divided into sub sub clusters and there was not a clear discrimination between the genotypes. The reason might be the gene responsible for the tuber bearing habit might not have been amplified with the primers used for the study. Moreover, higher percentage of similarity of almost 50 per cent among the genotypes could be attributed to the lot of missing data points Fig 1. A section of RAPD profile of C. forskohlii genotypes for primer OPZ 11 Fig 2. A section of RAPD profile of C. forskohlii genotypes for primer OPZ 13 Fig 3. Dendrogram of 37 Coleus forskohlii genotypes based on 60 RAPD markers constructed using UPGMA based on Jaccard’s coefficient J. Hortl. Sci. Vol. 4 (2): 143-147, 2009 Kavitha et al 147 as the number of primers and amplified products analysed were relatively less. Similar report in Andrographis paniculata was also reported (Padmesh et al, 1999). Pejie et al (1998) reported that 150 polymorphic bands make possibly a reliable estimate of genetic similarities among genotypes within the same species. In the present study, a total of 60 distinct bands were produced by the 16 primers out of 25 primers used. Hence, it is suggested that further genetic diversity analysis in C. forskohlii with more number of primers for RAPD or the advanced marker system producing a large number of informative polymorphic markers per primer pair that are highly reliable and reproducible (Mueller and Wolfenbarger, 1999 ; Mullis et al, 1986) has to be attempted. Although the data presented here are not conclusive to infer the phenetic relationship between the various genotypes, they reflect the utility of RAPD in the analysis of genetic variability distribution within this important indigenous medicinal plant. This is probably the first report which deals with the analysis of genetic diversity at the molecular level in C. forskohlii. REFERENCES Ammon, H.P.T. and Muller, A.B. 1985. Forskolin: from an ayurvedic remedy to a modern agent. Planta Medica, 46: 473-477 Bostein, D., White, R. M., Skolnick and Davis, R.W. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Amer. J. Hum. Genet., 32: 314 -331 Chowdhary, A.R. and Sharma, M.L. 1998. GC-MS investigations on the essential oil from Coleus forskohlii Briq. Indian Perfumer, 42 : 15-16 Hegde, L.N. 1992. Studies on germplasm evaluation, induced autotetraploidy and hybridization in Coleus forskohlii (Willd.) Briq. (Syn. C. barbatus Benth.). Ph.D. Thesis, University of Agricultural Sciences, Bangalore Kavitha, C., Vadivel, E., Rajamani, K. and Thangamani, C. 2007. Analysis of variability for qualitative and quantitative traits in Coleus forskohlii Briq. J. Hort. Sci., 2: 44-46 Misra, L.N., Tyagi, B.R., Ahmad, A. and Bahl, J.R. 1994. Variablity in the chemical composition of the essential oil of Coleus forskohlii genotypes. J. Essent. Oil Res., 6: 243-247 Mueller, U.G. and Wolfenbarger, L.1999. AFLP genotyping and fingerprinting. Trends Ecol. Evol., 14: 389-394 Nanaiah, K.M. 1993. Studies on hybridization, chromosomal doubling, grafting and leaf anatomy in Coleus forskohlii Briq. Ph.D., Thesis, University of Agricultural Sciences, Bangalore Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci., USA. 70: 3321- 3323 Padmesh, Sabu, P.K.K., Seeni, S. And Pushpangadan, P. 1999. The use of RAPD in assessing genetic variability in Andrographis paniculata Nees., a hepatoprotective drug. Curr. Sci., 76:833-835 Pejie, I., Ajmone-Marsan, P., Morgante, M., Kozumplick, V., Castiglioni, P., Taramino, G. and Motto, M.1998. Comparitive analysis of genetic similarity among maize inbred lines detected by RFLPs, RAPDs, SSRs and AFLPs. Theor. Appl. Genet., 97: 1248-1255 Prakash and Krishnan, R. 1994. Comparative performance of accessions and intervarietal hybrids in Coleus forskohlii Briq. J. Root Crops, 20: 70-73 Rohlf, F.J. 1994. NTSYS-PC: Numerical taxonomy and multivariate analysis system. version 2.2. State University of New York, Stony Brook, New York Rupp, R.H., De Souza, N.J. and Dohadwalla, A. N. 1986. Proceedings of the International Symposium on Forskolin: Its chemical, biological and medical potential. Hoechst India Limited, Bombay. pp. 19-30 Shah, V., Bhat, S.V., Bajwa, B.S., Dornaeur, H.and De Souza, N.J. 1980. The occurrence of forskolin in Labiatae. Planta Medica, 39: 183-185 Tanksley, S.D., Young, N.D., Paterson, A.H. and Bonierbale, M.W. 1989. RFLP mapping in plant breeding: New tools for an old science. Bio -Tech., 7: 257-264 Virk, P.S., Brian, V.F.L., Jackson, M.T. and Newbury, J.1995. Use of RAPD for the study of diversity within plant germplasm collections. Heredity, 74: 170-179 Vishwakarma, R.A., Tyagi, B.R., Ahmed, B. and Hussain, A.1988. Variation in forskolin content in the roots of Coleus forskohlii. Planta Medica, 54: 471-472 Wilkie, S. 1997. Genomic DNA isolation, Southern blotting and hybridization. In: Plant Molecular Biology- A Laboratory Manual. (Clark, M.S. ed.) Springer- Verlag, New York.pp. 3-53 (MS Received 20 December 2008, Revised 2 July, 2009) J. Hortl. Sci. Vol. 4 (2): 143-147, 2009 Analyzing variability in Coleus using RAPD markers