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Pitcher Plant View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by IR@NEIST - North East Institute of Science and Technology (CSIR) Genet Resour Crop Evol DOI 10.1007/s10722-009-9444-0 RESEARCH ARTICLE PCR based molecular characterization of Nepenthes khasiana Hook. f.—pitcher plant B. S. Bhau Æ K. Medhi Æ T. Sarkar Æ S. P. Saikia Received: 25 September 2008 / Accepted: 11 May 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Nepenthes khasiana Hook. f. belonging to differs from the others with at least 40% dissimilarity. monotypic family Nepenthaceae is a rare, endan- The results show a broad range of genetic diversity gered, dioecious member of the carnivorous plant within the populations of N. khasiana Hook. f. found in North-East India. The plant is endemic to the Indian state of Meghalaya and is distributed through- Keywords Conservation Á Genetic diversity Á out the state from West Khasi hills to East Khasi hills, ISSR Á Molecular markers Á Jaintia hills and East, West to South Garo hills from Nepenthes khasiana Hook. f. Á Pitcher plant Á 1,000 to ca. 1,500 m altitude. Multi-locus analysis Polymorphism Á RAPD using PCR based Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers were used for the first time to assess the genetic diversities of N. khasiana Hook. f. collected from different parts of Meghalaya. It was Introduction observed that RAPD analysis showed more polymor- phism than ISSR fingerprinting in revealing genetic Carnivorous plants are interesting botanical oddities polymorphism in N. khasiana Hook. f. The result of that show a range of characters different from those of a cluster analysis by using UPGMA method showed normal plant. These plants typically inhabit environ- that the groups based on pooled RAPD–ISSR genetic ments where nutrients are limited, and insects trapping similarity were more similar than the groups based on may have evolved as a means of providing a supple- RAPD. Furthermore, genetic similarity reveals vari- mental source of nutrients, especially nitrogen (Givnish ability within the population at Jarain of Jaintia hills, et al. 1984;GallieandChang1997). Out of different while between populations the Baghmara region kinds of carnivorous plants spread around the world, single species of Nepenthes i.e., Nepenthes khasiana Hook. f. are found in India (Kanjilal et al. 1940). Nepenthaceae is represented by a single genus B. S. Bhau and K. Medhi contributed equally. Nepenthes which is popularly known as tropical B. S. Bhau (&) Á K. Medhi Á T. Sarkar Á S. P. Saikia pitcher plant (Mokkamul et al. 2007). Indo-Malaysia Plant Genomics laboratory, Medicinal Aromatic and is considered as the center of evolution of the genus Economic Plants (MAEP) Division, North-East Institute Nepenthes. Many species of this genus are either of Science and Technology (NEIST)-Jorhat (Formerly RRL), Jorhat, Assam 785006, India endemic to certain areas or have a very restricted e-mail: [email protected]; [email protected] distribution. 123 Genet Resour Crop Evol Some biodiversity hotspots are significant in purchased from the markets and hybridized to having their endemic species concentrated in excep- produce a diversity of pitcher characters (Mao and tionally small areas (Myers et al. 2000). Meghalaya, Kharbuli 2002). Hence the species is of great in India is one of such hotspots for Nepenthes botanical and horticultural interest (Khoshbakht and khasiana Hook. f. The species and its habitats are Hammer 2007; Mukerjee et al. 1984). unique, as it occurs as isolated populations within The fluid of the unopened pitcher of N. khasiana Meghalaya. It is a rare, endemic, endangered, dioe- Hook. f. is used by local Khasi’s and Garos as an eye cious insectivorous climbing pitcher plant (Fig. 1a) drop for redness, itching, to cure cataract and night of great biological and ecological importance (Jain blindness and is also taken for stomach troubles, and Sastri 1980; Hooker 1886; Mao and Kharbuli diabetes and for female diseases (Kumar et al. 1980; 2002). It is found growing from West Khasi hills to Rao et al. 1969; Joseph and Joseph 1986). The East Khasi hills, Jaintia hills, and east to west and unopened pitcher with its content is made into a paste south Garo hills from 1,000 to ca. 1,500 m altitude. It and applied for various skin diseases, including occurs in the Jarain area of Jaintia Hills and the leprosy (Rao et al. 1969), sometimes mixed with rice Baghmara, Bandari, Chokpot area of the Garo hills, beer and is taken to ease urinary troubles and and few more localities, such as Jarain, Bhagmara, blockages (Kharkongar and Joseph 1981). Also the Nongstoin, Mukthapur, Bhagmara, Lawbahand and pitcher along with the insects within it is made into Sonapahar (Joseph and Joseph 1986). It can be said paste mixed with water and given to cholera patients. that the species represents ancient endemic remnants Hence the species is an important ethno medicinal of older flora which usually occur in land masses of plant and needs to be conserved (Mao and Kharbuli geological antiquity (Paleoendemics), (Bramwell and 2002). The genus has also been studied for their Valentine 1972). staining properties of chemical nepenthoquinone. The species is under major threats due to many Plumbagin, one kind of nepenthoquinone has been human activities, such as road construction, agricul- reported from Nepenthes of Nepenthaceae (Cannon ture, deforestation; coal mining, animal grazing, et al. 1980). Other possible roles of Nepenthoquinone landslides etc., destroying natural habitats of this are as allelopathic substance (Harbone 1982; Rey- rare endemic plant. The plant is also being collected nolds 1987; Jayaram and Prasad 2005), or as insec- and exported by local plant collectors to other states ticidal, molluscicidal or antifeedant chemicals of India. On account of its fascinating beauty of (Thomson 1987). The species is officially classified pitcher (Fig. 1b), N. khasiana Hook. f. is often as a threatened species and is included in the list of Fig. 1 a Nepenthes khasiana plant growing in the natural habitat; b Close-up of a pitcher 123 Genet Resour Crop Evol Fig. 2 Collection sites within Meghalaya rare and threatened taxa of India (Jain and Baishya generalizations have not always been supported by the 1977; Jain and Sastri 1980). It is also included in the available information. In rare plant species (Young Appendix I of the Convention of International Trade et al. 1996; Gitzendanner and Soltis 2000; Hoebee and of Endangered Species (CITES) of the world, which Young 2001). Hence, there is a need to develop means that it is a protected species and any type of empirical information about the extent and distribution collection from the wild and its sale is forbidden of genetic diversity in species that are targeted for except when the purpose of the import is not conservation. In this paper, we provide baseline data commercial, e.g., for international scientific exchange. on RAPD and ISSR variations within and among the In view of the importance to conserve this beau- seven populations of N. khasiana Hook. f. and attempt tiful, endemic and endangered pitcher plant, knowl- to identify factors that may have influenced the edge of genetic diversity within the genus is essential partitioning of genetic diversity. On the basis of this for establishing effective and efficient conservation information, we then can recommend a conservation practices. Traditionally, morphological characters programme for this species. have been used to characterize levels and patterns of diversity. Since these traits represent only a small portion of the plant genome and are influenced by Materials and methods environmental factors, plant age, phenology, they have limited utility for describing the potentially Study sites and sample collection complex genetic structure, which may exist within and in between taxa (Avise 1994). These facts make Six natural populations located in different parts of difficult the use of such descriptions in plant identi- Meghalaya were selected for the evaluation of RAPD fication and screening the genetic relationships. analysis. Young leaves of 45 individuals of N. Various molecular approaches have been devised to khasiana Hook. f. were collected from populations overcome these constraints (Soltis and Soltis 1991). in Bhagmara (BM), Jarain (JR), Rongsai (RSN), The present study was aimed at determining the extent Baghmara Massighat (BMG), Chokpot (CP) and of genetic diversity within and among populations. Bandari (BD). The localities are shown on the map Knowledge of genetic variation is fundamental to (Fig. 2). Another site of collection was at the North- designing strategies for conservation, since the East Institute of Science and Technology (NEIST), primary goal of conservation is to preserve the Jorhat campus. Leaf material were immediately dried prevalent spectrum of genetic diversity and thus the using self-indicating silica gel, then transported to the evolutionary potential (Holsinger and Gottlieb 1991). lab, and stored at -45°C until DNA extraction. A number of generalizations have been made about relationships among the level and distribution of DNA isolation and PCR amplification genetic variation of a species and its ecological and biological traits (Loveless and Hamrick 1984; Lewontin Leaves were freeze-dried using a lyophilizer. DNA and Hubby 1966; Heubl et al. 2006). However, such extraction of the samples was done by modified 123 Genet Resour Crop Evol CTAB method (Doyle and Doyle 1990). Genomic Data analysis DNA was quantified against a known quantity of unrestricted Lambda (k) DNA by running it in a 0.8% Each gel of RAPD and ISSR was analyzed by scoring agarose gel. present (1) or absent (0) bands. The RAPD, ISSR and pooled data matrices were entered into the NTSYS RAPD amplification version 2.02 K package. Pair wise comparisons of populations were made and genetic diversity param- To optimize the PCR amplification conditions, eters were determined.
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