Molecular Characterization of Five Medicinally Important Species of Typhonium (Araceae) through Random Amplified Polymorphic DNA (RAPD) Laxmikanta Acharyaa, Arup Kumar Mukherjeea, Pratap Chandra Pandab,*, and Premananda Dasc a DNA Fingerprinting Laboratory, Plant Biotechnology Division, Regional Plant Resource Centre, Nayapalli, Bhubaneswar 751 015, Orissa, India b Taxonomy and Conservation Division, Regional Plant Resource Centre, Nayapalli, Bhubaneswar 751 015, Orissa, India. E-mail: [email protected] c Indian Institute of Technology, Kharagpur, West Bengal, India * Author for correspondence and reprint requests Z. Naturforsch. 60c, 600Ð604 (2005); received January 7/February 16, 2005 The interrelationship of five medicinally important species of Typhonium (Araceae) in- cluding T. venosum, which was previously placed under the genus Sauromatum, was inferred by analysis of random amplified polymorphic DNA (RAPD). DNA from pooled leaf samples was isolated and RAPD analysis was performed using 20 decamer oligonucleotide primers. Out of a total of 245 bands amplified, 12 were found to be monomorphic while 233 bands were polymorphic including 86 species-specific bands. The genetic similarities were analyzed from the dendrogram constructed by the pooled RAPD data using a similarity index. The dendrogram showed two distinct clades, one containing T. roxburghii, T. trilobatum and T. venosum and the other containing the remainder two species, i.e. T. diversifolium and T. flagelliforme. Both the clusters shared a common node approx. at 23.7% level of similarity. The maximum similarity of 31.2% was observed between T. venosum and T. trilobatum. In view of its close genetic similarity with other members of Typhonium, transfer of Sauroma- tum venosum to the genus Typhonium and merger of the two genera was supported. Key words: Molecular Taxonomy, Genomic Relationship, RAPD, Typhonium

Introduction eaten with banana for stomach complaints, applied externally to the bite of venomous snakes and the The genus Typhonium (Araceae) is comprised same time given orally. The tribals also feed the of about 30 species (Mabberly, 1997) and is distrib- raw tubers of T. trilobatum to the cattle against uted throughout the tropical and subtropical re- worm infection. It is also used as poultice in the gions of the world with greater concentration of treatment of tumors and haemorrhages. The rhi- species in Southeast Asia, Indo-Malaysia and zomes of T. flagelliforme have traditionally been Northeastern Australia. In India, it is represented used as an expectorant for coughs and as treat- by 16 species (Santapau and Henry, 1973). Among ment for other pulmonary ailments. In the Philip- the species found in India, T. trilobatum (L.) pines, the floral inflorescence is used to arrest Schott is the most common and grows wild in a bleeding and help in wound healing (Parry, 1980). wide range of habitats. Besides, T. divaricatum (L.) This plant has been used extensively as one of the Decne, T. flagelliforme (Lodd.) Bl., T. bulbiferum components of traditional herb for combating Dalz., T. venosum (Dryand ex Ait.) Hett. & Boyce, breast, lung, colon and liver cancer. and T. roxburghii Schott are the other species Being the source of food and medicine against found in abundance in the country. In Orissa, the a spectrum of ailments, the wild populations of the occurrence of three wild species namely, T. triloba- species have been severely eroded due to over- tum (Haines, 1921Ð1925), T. venosum (syn. Sauro- exploitation. This necessitated conservation of the matum venosum) (Panda et al., 1995) and T. flagel- species in the wild and assessment of the genetic liforme (Panda, 2002) has been reported. diversity of the genus Typhonium through molecu- Tuber and leaves of T. trilobatum, T. flagelli- lar techniques. No report, however, is available on forme, and T. venosum are used by tribals as the molecular characterization and determination source of food and medicine. The tubers of many of genomic relationship among the species except species are used as stimulant and for cure of piles, that of Sriboonma et al. (1993).

0939Ð5075/2005/0700Ð0600 $ 06.00 ” 2005 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D L. Acharya et al. · Molecular Characterization of Typhonium 601

Molecular markers like RAPD, RFLP, ISSR and RAPD analysis AFLP are stable, reliable and not influenced by For RAPD analysis, PCR amplification of 25 ng the environment and thus being used nowadays of genomic DNA was carried out using 25 stan- for assessment of the genetic diversity and estab- dard decamer oligonucleotide primers out of lishing genomic relationship among different plant which 20 primers [OPA02, OPA03, OPA04, taxa. The present work was undertaken to assess OPA10, OPA16, OPA18, OPD02, OPD03, the genetic diversity among 5 medicinally impor- OPD05, OPD07, OPD08, OPD10, OPD18, tant species of Typhonium and to understand the OPD20, OPN02, OPN04, OPN05, OPN06, OPN08 genomic relationship among them using RAPD and OPN16 (Operon Technologies, Alameda, marker. USA)] produced satisfactory amplification. The RAPD analysis was performed by the methods of Materials and Methods Williams et al. (1990). Each amplification reaction Plant material mixture of 25 µl contained 20 ng of template µ Five species of Typhonium (T. diversifolium, DNA, 2.5 l of 10X assay buffer (100 mm Tris- T. flagelliforme, T. roxburghii, T. trilobatum and HCl, pH 8.3, 0.5 m KCl and 0.01% gelatin), 1.5 mm µ T. venosum) were studied. Except for T. diversifo- MgCl2, 200 m each of dNTPs, 20 ng of primer and lium and T. venosum, the other three species were 0.5 U Taq DNA polymerase (Bangalore Genei collected from the premises of Regional Plant Re- Pvt. Ltd., Bangalore, India). The amplification was source Centre, Bhubaneswar, Orissa, India. T. di- carried out in a thermal cycler (Perkin Elmer, versifolium was collected from Darjeeling hills and 9600). The first cycle consisted of denaturation of T. venosum from Sanaghagra in Keonjhar district, template DNA at 94 ∞C for 5 min, primer anneal- Orissa, India. Leaves from ten different plants of ing at 37 ∞C for 1 min and primer extension at the same species were collected at random and 72 ∞C for 2 min. In the subsequent 42 cycles, the leaf samples of each species were pooled together period of denaturation was reduced to 1 min while and genomic DNA was isolated. the primer annealing and primer extension time were maintained as in the case of the first cycle. Genomic DNA isolation The last cycle consisted of only primer extension at 72 ∞C for 7 min. The PCR products were sepa- DNA was isolated from freshly collected young rated on a 1.5% agarose gel containing ethidium leaves by the CTAB method as described by bromide solution (0.5 µg/ml of gel solution). The Doyle and Doyle (1990). RNA was extracted with size of the amplicons was determined using stan- µ RNaseA (Quiagen) treatment: @ 60 g for 1 ml dards (100 bp ladder plus, MBI Fermentas, Grai- of crude DNA solution at 37 ∞C followed by two ciuno, Vilnius, Lithuania). The DNA fragments washings with phenol/chloroform/iso-amyl-alcohol were observed under UV light and photographed. (25:24:1 v/v/v) and subsequently two washings with chloroform/iso-amyl-alcohol (24:1 v/v). After cen- trifugation, the upper aqueous phase was sepa- Data analysis rated, 1/10 volume of 3 m sodium acetate (pH 4.8) The presence/absence of bands in RAPD analy- was added and DNA precipitated with 2.5 volume sis was recorded in binary (0, 1) form. All the of pre-chilled absolute ethanol. The extracted bands (polymorphic and monomorphic) were DNA was dried and then dissolved in 10 mm Tris- taken into account for calculation of similarity HCl [tris(hydroxy methyl) amino methane]/1 mm with a view to avoid over-/underestimation of the EDTA (ethylene diamine tetra acetic acid diso- distance (Gherardi et al., 1998). Jaccard’s coeffi- dium salt) (T10E1 buffer, pH 8). Quantification cient of similarity (Jaccard, 1908) was measured was made by running the dissolved DNA in 0.8% and a dendrogram based on similarity coefficients agarose gel along side uncut λ DNA of known con- generated by the un-weighted pair group method centration. The DNA was diluted to 25 ng/µl for using arithmetic averages (UPGMA) (Sneath and RAPD. Sokal, 1973) and SHAN clustering. The statistical analysis was done using the computer package NTSYS-PC (Rohlf, 1997). Resolving power (Rp) of the RAPD primer was calculated according to Prevost and Wilkinson (1999): Rp = ΣIB, where IB 602 L. Acharya et al. · Molecular Characterization of Typhonium

(band informativeness) = 1 Ð [2 ¥ (0.5 Ð P)], P the minimum (73) in case of T. roxburghii.The being the proportion of the 5 species containing highest number of 27 unique bands was detected the band. in T. diversifolium and 10, the lowest, for T. rox- burghii.InT. trilobatum, the highest number of Results 102 polymorphic bands was generated and the A total of 245 numbers of loci were amplified least (61) for T. roxburghii (Table II). The ampli- in the RAPD analysis using 20 decamer oligo- cons were amplified in the range of 100 to 3000 nucleotide primers. Out of these, 12 bands were base pairs. The maximum (19) and minimum (7) monomorphic while 233 bands were polymorphic number of bands were amplified with the primer including 86 species-specific bands (Table I). The OPD18 and OPA16, respectively (Table I). RAPD banding pattern of the five species of Ty- Among the primers used, OPD18 generated maxi- phonium is represented in Fig. 1. The maximum of mum polymorphic bands but the highest number 114 loci was amplified in case of T. trilobatum and of unique bands was noted with the primer

Table I. Details of RAPD analysis in five species of Typhonium.

Primer Nucleotide sequence Total Polymorphic Monomorphic Unique Resolving bands bands bands bands power

OPA02 5ЈTGCCGAGCTG3 12 11 1 3 12.8 OPA03 5ЈAGTCAGCCAC3Ј 8 8 0 3 6.4 OPA04 5ЈAATCGGGCTG3Ј 11 11 0 3 10.8 OPA10 5ЈGTGATCGCAG3 10 9 1 2 9.6 OPA16 5ЈAGCCAGCGAA3Ј 7 7 0 1 7.2 OPA18 5ЈAGGTGACCGT3Ј 10 10 0 4 8 OPD02 5ЈGGACCCAACC3Ј 13 13 0 2 13.2 OPD03 5ЈGTCGCCGTCA3Ј 14 13 1 4 13.2 OPD05 5ЈTGAGCGGACA3Ј 11 11 0 3 10.4 OPD07 5ЈTTGGCACGGG3Ј 11 11 0 5 6.8 OPD08 5ЈGTGTGCCCCA3Ј 10 10 0 3 7.6 OPD10 5ЈGTGTGCCCCA3Ј 11 10 1 3 8.4 OPD18 5ЈGAGAGCCAAC3Ј 19 19 0 5 14.4 OPD20 5ЈACCCGGTCAC3Ј 15 15 0 7 11.2 OPN02 5ЈACCAGGGGCA3Ј 12 12 0 5 8 OPN04 5ЈGACCGACCCA3Ј 12 12 0 6 10.8 OPN05 5ЈACTGAACGCC3Ј 17 15 2 8 12.8 OPN06 5ЈGAGACGCACA3 12 9 3 7 9.2 OPN08 5ЈACCTCAGCTC3Ј 15 15 0 7 10.8 OPN16 5ЈAAGCGACCTG3 15 12 3 5 12

Total 245 233 12 86

1 2345M12345123451234 51 2345

Fig. 1. RAPD banding pattern of five species of Typhonium as revealed through primer OPD20, OPN02, OPN04, OPN06, OPN08. The lanes represent: 1, T. roxburghii;2,T. trilobatum;3,T. flagelliforme;4,T. diversifolium;5, T. venosum. L. Acharya et al. · Molecular Characterization of Typhonium 603

Table II. Banding pattern in five species of Typhonium.

Species Total no. of bands Polymorphic bands Monomorphic bands Unique bands

T. roxburghii 73 61 12 10 T. trilobatum 114 102 12 18 T. flagelliforme 109 97 12 19 T. diversifolium 100 88 12 27 T. venosum 113 101 12 12

OPN05. Maximum similarity was seen among the mon node at approx. 23.7% level. In the first species with the primer OPA02. While the highest clade, of the three species, T. trilobatum formed a number of unique loci got amplified in OPN05, cluster with T. venosum having 31.2% similarity the lowest numbers of species-specific loci were and together shared a common node with T. rox- found in the primer OPA16 i.e. 1 in each case. burghii at 25.9% level of similarity. In the second Among the RAPD primers used, the resolving cluster, T. flagelliforme and T. diversifolium had a power ranged from 6.4 for OPA03 to 14.4 for similarity index of approx. 26%. OPD18. T. venosum showed maximum similarity (31.2%) with T. trilobatum and the latter with T. roxburghii at 28.10% (Table III). Similarly, Discussion T. diversifolium and T. flagelliforme were close to each other exhibiting 28.57% similarity. The con- The study was limited to RAPD analysis of only structed dendrogram (Fig. 2) showed two distinct five important species of Typhonium namely, clades, one containing T. roxburghii, T. trilobatum T. roxburghii, T. trilobatum, T. venosum, T. flagelli- and T. venosum and the other containing the re- forme and T. diversifolium. Most of these species mainder two species, i.e. T. diversifolium and are important as medicinal and food plants in In- T. flagelliforme. Both the clusters shared a com- dia and elsewhere and due to over-exploitation, their wild populations are declining rapidly. This necessitates assessment of the genetic diversity of T. roxburghii the genus through molecular characterization and evaluation of their medicinal properties. Hooker (1894) recognized Sauromatum and Ty- T. trilobatum phonium as two separate genera under the tribe Arineae distinguishable on the basis of emergence T. venosum of leaves either after or before flowering. Of the remaining 4 species of the genus Typhonium, T. flagelliforme based on the characters of the limb of the spathe, he placed T. trilobatum and T. roxburghii under T. diversifolium one group along with others and T. diversifolium and T. flagelliforme in two separate groups. The 0.00 0.13 0.25 0.38 0.50 close similarity between T. trilobatum and T. rox- Similarity Coefficient burghii has also been established by RAPD analy- Fig. 2. Dendrogram as revealed from RAPD data sis in the present study as they had a similarity of through SHAN clustering. more than 28%.

Table III. Similarity values between different species of Typhonium.

T. roxburghii T. trilobatum T. flagelliforme T. diversifolium T. venosum

T. roxburghii 100 T. trilobatum 28.10 100 T. flagelliforme 24.39 27.78 100 T. diversifolium 19.33 19.31 28.57 100 T. venosum 23.20 31.21 28.87 21.99 100 604 L. Acharya et al. · Molecular Characterization of Typhonium

As remarked by Sriboonma et al. (1993), the in- T. venosum, earlier included under Sauromatum fra-generic classification of Typhonium has not yet and known as S. venosum, was quite closer to been established and as a result, the placement of T. trilobatum showing a similarity coefficient of species in one group or the other has been based about 30% and formed a single clade along with on personal judgments. Engler (1920) recognized T. roxburghii. Unlike other two species, T. veno- two sections under the genus, namely, section Het- sum even did not form a separate node by itself to erostalis Engl. characterized by the clavate sterile be treated as a genetically very distant species so to say a separate genus. Therefore, the taxonomic flowers and section Eutyphonium Engl. without merger of the genus Sauromatum with Typho- clavate sterile flowers. He placed T. flagelliforme nium, as has been done by Hetterscheid and under the former section and T. trilobatum under Boyce (2000), appears justified. the latter. The data obtained in the present study also supports this view. However, inclusion of both Acknowledgements the species under one group based on shoot mor- Thanks to the Director, Regional Plant Re- phology (Murata, 1990) could not be established source Center, Bhubaneswar, Orissa, India for from data obtained by RAPD analysis. providing laboratory facilities.

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