HE Moore Inferred by RAPD Markers

A comparative genetic diversity in natural population and cultivated individuals in Satakentia liukiuensis (Hatus.) H. E. Moore inferred by RAPD markers

Joko Ridho Witono1 and Katsuhiko Kondo2

1Center for Plant Conservation-Bogor Botanical Garden, Indonesian Institute of Sciences (LIPI) Jalan Ir. H. Juanda 13, Bogor 16003, Indonesia; 2Laboratory of Plant Genetics and Breeding Science, Department of Agriculture, Faculty of Agriculture, Tokyo University of Agriculture, 1737 Funako, Atsugi City, Kanagawa Prefecture 243-0034, Japan

1Author for correspondence ([email protected]) Received June 20, 2010; accepted July 21, 2010

ABSTRACT. Genetic diversity of Satakentia liukiuensis in some cultivated individuals in Bogor Botanical Garden (Indonesia) and Nong Nooch Tropical Garden (Thailand) compared with natural population was analyzed by RAPD markers. Satakentia liukiuensis is a monotypic genus and an endemic palm species in Japan and it distributed in very restricted area in Ishigaki Island and Iriomote Island in Yaeyama Group of the Ryukyu Islands. The ten of RAPD primers (Operon Tech. Inc.) were used in RAPD analysis generated 82 ampliﬁ ed bands, which is 27 bands (32.9%) were polymorphic. A phenogram generated by UPGMA clustering analysis shows all accessions from natural population were separated from cultivated individuals with genetic distance 0.104. Genetic variation among accessions in natural population was lower than cultivated individuals. The genetic uniformity of S. liukiuensis in natural population may due to the result of the inbred nature of its small population size for a long time. In our opinion, the existence of S. liukiuensis will be sustainable, since the natural population of the species in Ishigaki Island was established as protected areas and managed by Satake Corporation and also the corporation announced and built `The Toshihiko Satake Memorial Palm Museum` in natural locality sites of the species. RAPD method is a very fast way of obtaining genetic information and the methods require no prior knowledge of the DNA sequence to generate genetic markers.

KEYWORDS: Cultivated individuals, Genetic diversity, Natural population, RAPD markers, Satakentia liukiuensis

Palm (Palmae/Arecaceae) consists of six subfamilies, 14 together and were placed in Hydriastele (Baker and Loo tribes, 37 subtribes, 190 genera and 2364 accepted species 2004). (Uhl and Dransfi eld 1987; Govaerts and Dransfi eld 2005). Gulubia liukiuensis was recognized until H.E. Moore Six species (six genera) such as Livistona chinensis var. Jr., (1969) established the species into a new genus subglobossa, Trachycarpus fortunei, Rhapis humilis, Nypa Satakentia because it had distinctive morphological fruticans, Arenga engleri and Satakentia liukiuensis are characters comparing other closely related genera such as native to Japan (Meyer and Walker, eds. 1965; Hatusima Gulubia and Clinostigma (both Pacifi c palm genera). The 1975; Govaerts and Dransfi eld 2005). Satakentia liukiuensis name of Satakentia is proposed to honor H. E. Moore Jr.’s is a monotypic genus and an endemic palm species in longtime correspondent and ardent student of palms, Mr. Japan and distributed in very restricted area on hillslopes Toshihiko Satake and Hatusima 18500 (KAG) was chosen or more rarely nearly sea level in Yonehara Village in as the lectotype for the species. The palm is becoming a Ishigaki Island and in the hamlet of Somai and Nakama popular species for cultivation in countries in the River on clay sandstone slopes in Iriomote Island and subtropical and tropical climates, especially in the southern Yaeyama group of the Ryukyu Islands (Moore 1969). U.S.A., Indonesia and its native country (Jones 1995). Satakentia liukiuensis is fi rstly described as Gulubia liukiuensis by Hatusima (1964) based on the two herbarium MATERIALS AND METHODS specimens collected in Iriomote Island such as S. Hatusima Plant materials. Plant materials were obtained from the 18500 (fl owering type) and K. Kuroshima s.n. (fruiting natural population in Ishigaki Island, Japan (22 individuals) type). The new species was most closely related to Gulubia and certain cultivated individuals in Bogor Botanical cylindrocarpa Becc. from New Hebrides, G. moluccana Garden, Indonesia (three individuals) and Nong Nooch Becc. from Moluccas, and G. hombronii Becc. from Tropical Garden, Thailand (fi ve individuals). Solomon Isl. Gulubia was established by Beccari (1885) and it consisted of ten species, until Essig (1982) revised DNA extractions. Total genomic DNA of Satakentia and reduced the genus into nine species. Therefore, liukiuensis was extracted from silica gel dry leaf tissues Gulubia and other closely related genera within subtribe (Chase and Hills 1991) using CTAB protocol described Arecinae (Siphokentia and Gronophyllum) were combined by Ban (1995) with some modifi cations. 28 WITONO AND KONDO

Fig. 1. Satekentia liukiuensis (Hatushima) H. E. Moore (Source: http://fern.la.coocan.jp/Arecaceae/Satakentia%20liukiuensis/Satakentia%20liukiuensis. htm) RAPD (random amplifi ed polymorphic DNA) method has been widely employed in many plant groups representing a diverse selection of families and classes. The method is a fi ngerprinting method using single short primers with arbitrary sequences to search for variation in the entire genomid DNA (Williams et al. 1990; Welsh and McClelland 1990). A discrete PCR (polymerase chain reaction) product is generated when the primer binds to site on opposite strand of genomic DNA that are within an amplifi able distance. The presence and absence of the specifi c PCR product is assumed the represent mutations in the primer binding sites of the genomic DNA (Wolfe and Liston 1998). In many cases, RAPD markers have proved useful for studying genetic variation at low taxonomic level. In the present study, genetic diversity of Satakentia liukiuensis (‘Satake palm’) from the natural population and certain cultivated individuals were compared using RAPD markers in order to identify genetic relationships among individuals and offer genetic resource informations for conservation program.

Amplifi cation and electrophoresis. PCR mixtures were Genetic distance among accessions was calculated using performed in a volume of 20 μl containing sterilized Nei (1972) coeffi cient. A clustering analysis of all distilled water, 250 μM primer (Operon Tech. Inc.), 10x accessions were performed using the UPGMA (unweighted Taq Buffer (Promega) per reaction, 125 μM dNTP pair-group method with an arithmetic average). Mixtures (Takara), 0.5 U Taq DNA Polymerase (Promega), and 20 ng template DNA. The thermocycler programme RESULTS AND DISCUSSION was predenaturation at 940°C for 5 min; 45 cycles of 940 The ten primers used in RAPD analysis generated 82 °C for 1 minute, 360°C for 45 seconds, 720°C for 2 min; amplifi ed bands, which is 27 bands (32.9%) were fi nal extension 720°C for 5 min and followed by soaking polymorphic and 55 bands (67.1%) were monomorphic at 40°C until use. among Satakentia liukiuensis accessions. The RAPD The fragments produced by PCR amplifi cation were banding patterns generated by OPA 7 and OPB 8 are separated by electrophoresis on 1.5 % (w/v) agarose gel in shown in Figure 2. Each primer produced 6 to 12 bands 1x TAE Buffer and stained with a Ethidium Bromide (1.0 with an average amplifi ed and polymorphic were 8.2 μg/ml). The fragments pattern was visualized and bands and 2.7 bands, respectively (Table 1). photographed using a gel documentation system (UVP Inc., UK) and molecular weight were estimated using 10 The genetic distance among accessions ranged from kbp DNA Standard Marker (Novagen). 0.000 to 0.151, in which among accessions within natural Data analysis. All RAPD bands were scored for their population ranged from 0.000 to 0.037 and among presence (1) or absence (0). The number of bands and the cultivated accessions ranged from 0.015 to 0.079 (Table 2). percentage of polymorphic bands were calculated A phenogram generated by UPGMA clustering analysis manually and then compiled into a matrix for clustering shows all accessions from natural population were separated analysis using NTSYSpc (Numerical Taxonomy and from cultivated accessions with genetic distance 0.104 Multivariate Analysis System) version 2.1 (Rohlf, 2000). (Fig. 3). Within natural population, the accessions were GENETIC DIVERSITY OF SATAKENTIA LIUKIUENSIS ANALYZED BY RAPD MARKERS 29

Table 1. Primer sequences with the number of scorable ampliﬁ ed and polymorphic bands Primer Sequence No. of ampliﬁ ed No. of Polymorphic % Polymorphic Bands bands bands OPA 3 AGTCAGCCAC 7 2 28.6 OPA 4 AATCGGGCTG 12 5 41.7 OPA 7 GAAACGGGTG 9 5 55.6 OPA 9 GGGTAACGCC 6 2 33.3 OPA 18 AGGTGACCGT 6 2 33.3 OPB 4 GGACTGGAGT 11 1 9.1 OPB 6 TGCTCTGCCC 8 1 12.5 OPB 8 GTCCACACGG 6 1 16.7 OPB 12 CCTTGACGCA 11 3 27.3 OPB 15 GGAGGGTGTT 6 5 83.3 Total 82 27 32.9

Fig. 2. RAPD banding patterns of Satakentia liukiuensis generated by (a) OPA 7 and (b) OPB 8 primers. M=DNA standard marker, I=Accessions from Ishigaki Island, N=Accessions from Nong Nooch Tropical Garden-Thailand and B=Accessions from Bogor Botanical Gardens-Indonesia. grouped into four clusters with genetic distance ranged subfamily Arecoideae, S. liukiuensis belongs to tribe from 0.007 to 0.027. Iguanurinae whereas A. engleri is a member of tribe Caryotoideae (Uhl and Dransfi eld 1987). Our hypothesis Genetic variation generated by the ten RAPD markers suggested that hybridization does not occur between S. among accessions in natural population is lower than liukiuensis and A. engleri. On the other hand, in botanical cultivated individuals in the botanical gardens. The genetic garden, the species was cultivated artifi cially sympatry uniformity of S. liukiuensis in natural population may due with other palm species are exposed to conspecifi c or may to the result of the inbred nature of its small population congeneric, accordingly, the opportunities of hybridization size for a long time. The surviving individuals of S. may occur (Maunder et al. 2001). liukiuensis are the remnants of a possibly former larger The dispersal mechanisms of S. liukiuensis is very population and wider natural distribution, which, through scanty, whether the palm is naturally occur in resticted human activity by land clearing and over-exploitation, distribution is diffi cult to determine, though low dispersal population of the species has declined. The ‘cabbage’ of ability, profound habitat specifi city or sensitivity natural the species is said to have been eating during World War disturbance suggested that these may be so. The genetic II (Uhl and Dransfi eld 1987). Moreover, the species were issues have been addressed by the determination of growing sympatrically with other palm species Arenga existing variation within the population, aspect of habitat, engleri that dominate in the forest understorey which is and environmental issues by examination of natural sites taxonomically they were placed in different tribe within (Dowe et al. 1997). Information on genetic variation of 30 WITONO AND KONDO

Table 2. Genetic distance among accessions used in the study according to Nei (1972) coefﬁ cients

Fig. 3. A phenogram of Satakentia liukiuensis accessions based on RAPD markers generated by Nei (1972) coeffi cient and the UPGMA clustering method. In our opinion, both in situ and ex situ conservation are necessary to conserve the genetic diversity of Satakentia liukiuensis. The Biodiversity Convention states that ex situ conservation should be undertaken as far as possible and as appropriate and predominantly for the purpose of complementing in situ conservation. GENETIC DIVERSITY OF SATAKENTIA LIUKIUENSIS ANALYZED BY RAPD MARKERS 31 particular species in natural population has already been Taxon 40: 215-220. indicated as an essential for genetic resources conservation, Dowe, J. L., Benzie, J. and Ballment, E. 1997. Ecology and genetics of Carpoxylon macrospermum H. Wendl. & especially for the species which has restricted distribution Drude (Arecaceae): an endangered palm from Vanuatu. areas in small population like S. liukiuensis. For in situ Biol. Cons. 79: 205-216. conservation purposes, habitat protection is necessary to Dransfi eld, J. and Beentje, H. 1995. Palms of Madagascar. maintain genetic variation within the existing populations Royal Botanic Gardens Kew and The International Palm Society. Lawrence-Kansas. and to prevent loss of genetic diversity. Essig, F. B. 1982. A synopsis of the genus Gulubia. Principes Ex situ conservation should include the collection of 26: 159-173. germplasm from separate populations with signifi cant Govaerts, R. and Dransfi eld, J. 2005. World Checklist of Palms. Royal Botanic Gardens, Kew. genetic differences (Martin et al. 1997), but in practical, it Hatusima, S. 1964. A new palm from the Ryukyus. Mem. of is diffi cult due to constraints of space. However, the the Fac. of Agric. Kagoshima Univ. 51: 39-42. proven ability of S. liukiuensis to adapt to wide range of Hatusima, S. 1975. Flora of the Ryukyus. Okinawa Biological environmental conditions in botanical gardens make the Research Institute (in Japanese). Jones, D. L. 1995. Palms throughout the World. Smithsonian ex situ approach to conservation of the species is important. Institution Press, Washington. The cultivated palms in botanical gardens are partly Martin, C., Gonzales-Benito, M. E. and Iriondo, J. M. 1997. derived from natural population, being of increasing value Genetic diversity within and among populations of a threatened species: Erodium paularense Fern. Gonz. and as the natural population continue to decline especially a Izco. Mol. Ecol. 6: 813-820. number of highly threatened palms, such as Dypsis Maunder, M., Lyte, B., Dransfi eld, J. and Baker, W. 2001. The cabadae which is widely cultivated in botanical garden conservation value of botanic garden palm collections. but it has never been recorded as a wild species (Dransfi eld Biol. Cons. 98: 259-271. Ohwi, J. 1965. Flora of Japan. In: F. G. Meyer and E. H. and Beentje 1995). Walker, Eds., Smithsonian Institution, Washington. D.C. Moore, Jr., H. E. 1969. Satakentia-A new genus of Palmae- ACKNOWLEDGEMENTS. We thank to the the Representative Arecoideae. Principes 13: 3-12. Toshiko Satake, Satake Corporation, Higashi-Hiroshima, Japan for Uhl, N. W. and Dransfi eld, J. 1987. Genera Palmarum: A fi nancial support and the Directors of Bogor Botanical Garden, classifi cation of palms based on the work of H. E. Moore, Indonesia and Nong Nooch Tropical Garden, Thailand for supplying Jr. The L.H. Bailey Hortorium and the International Palm plant materials. Society. Lawrence-Kansas. Welsh, J. and McClelland, M. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acids Res. 18: LITERATURE CITED 7213-7218. Baker, W. J. and Loo, A. H. B. 2004. A synopsis of the genus Williams, J. G. K., Hanafey, M. K., Rafalski, J. A. and Tingey, Hydriastele (Arecaceae). Kew Bull. 59: 61-68. S. V. 1993. Genetic analysis using random amplifi ed Ban, Y. 1995. DNA-RNA extraction methods. In: I. Shimamoto polymorphic DNA markers. Methods Enzym. 218: 704- and T. Sasaki Eds., PCR Research protocols of plants. 740. Shujunsha. Tokyo (in Japanese). Wolfe, A. D. and Liston, A. 1998. Contributions of PCR-based Beccari, O. 1885. Annals Jardin Botanic Buitenzorg 2: 128- methods to plant systematics and evolutionary biology. 131. pp.43-86. In: D. E. Soltis, P. S. Soltis and J. J. Doyle, Eds.. Chase, M. W. and Hills, H. H. 1991. Silica gel: an ideal material Molecular systematics of plants II: DNA Sequencing. for fi eld preservation of leaf samples for DNA studies. Kluwer Academic Publishers. Massachusetts.