652 Edi Santosa (Isolating).cdr ISOLATING MICROSATELLITE FROM Amorphophallus variabilis AND ITS APPLICATION FOR POPULATION STUDY IN DRAMAGA CONSERVATION FOREST, INDONESIA *EDI SANTOSA , CHUN LAN LIAN , YOKO MINE , KEN TAKAHATA 1 2 3 3 and NOBUO SUGIYAMA 4 1 Department of Agronomy, Faculty of Agriculture, Institut Pertanian Bogor, Bogor 16680, Indonesia 2 Asian Natural Environmental Science Center, University of Tokyo, Tokyo 188-0002, Japan 3 Faculty of Agriculture, Tokyo University of Agriculture, Kanagawa 243-0034, Japan 4 Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan; Present address: Faculty of Agriculture, Tokyo University of Agriculture, Kanagawa 243-0034, Japan Received 06 May 2016/Accepted 17 July 2017 ABSTRACT Amorphophallus variabilis Blume, a member of Araceae, is a fleshy perennial tuber crop endemic in Java Island, Indonesia. The plant produces white edible corm; and it was used as food during famine time before 1960s. Rapid ecological changes and land fragmentations in Java in recent times threaten populations of A. variabilis. Here, compound microsatellite markers were developed in order to develop conservation strategies in the populations. Twelve primers pairs produced high polymorphism ranging from 5 to 22 alleles per locus. The observed and expected heterozygosities ranged from 0.191 to 0.851 and 0.380 to 0.943, respectively. This high allelic diversity indicates that these markers are suitable for the study on population genetic structure. Cross-amplification on related and non- related species was performed. Application of the markers on populations from Dramaga Conservation Forest revealed high allelic richness, high diversity within and among populations. Genetic distance among populations increased with an increase of geographic distance. Present study suggested that, it is important to study population of A. variabilis in Java in order to understand the population genetic structure and develop effective in situ conservation programs. Keywords: Araceae, genetic population, SSR, tuber crop, white iles-iles INTRODUCTION Amor phophallus variabilis Blume, synonym Brachyspatha variabilis (Blume) Schott, an Araceae, is a diploid endemic perennial tuber crop native to Java. Its distribution in Indonesia is exclusively in Java, Kangean and Madura Islands (Jansen . et al 1996; Yuzammi 2000). The plant naturally grows in less disturbed land under partial trees shading and at gap of forest trees at low altitude up to 700 to 900 m above sea level (Jansen . 1996; et al Sugiyama & Santosa 2008). is locally A. variabilis called white (Safii 1981; Wiyani 1988) iles-iles reflecting the white color of the tuber. It is easily d i s t i n g u i s h e d m o r p h o l o g i c a l l y f r o m Amorphophallus muelleri Blume (Sugiyama & Santosa 2008) known as yellow iles-iles. The plant produces a single underground corm with several cormlets; corms and cormlets exhibit dormancy during dry season (Jansen . et al 1996). The corm was utilized as staple food in Java particularly during famine situation before 1960s. Corm of contains high glucomannan A. variabilis (ca. 35% on a dry weight basis) (Ohtsuki 1968; Safii 1981; Wiyani 1988). Glucomannan is used as an important material in beverage, food and pharmaceutical industries (Jansen . 1996; et al Alonso-Sande . 2009).et al Botanically, a large genetic diversity has been observed within A. variabilis plants, including inflorescence (Santosa et al. 2004) and leaf morphologies (Sugiyama & Santosa 2008). According to Yuzammi (2000), the petioles vary from pure green to dark brown with different color and pattern of spots. Therefore, A. variabilis * Corresponding author: edisang@gmail.com BIOTROPIA 5 1 8 22 32 Vol. 2 No. , 201 : - DOI: 10.11598/btb.2018.25.1.652 22 germplasm could be used as breeding materials (Sugiyama & Santosa 2008). According to Zhang et al. (1998), several Amorphophallus species could be hybridized. The monoecious unisexual inflorescence produces unpleasant odor during anthesis (Kite & Hetterschieid 1997) to attract pollinators. Red bright mature berries develop after cross- pollination by Nitidulidae insects (Santosa . et al 2004). Seeds are dispersed long distance by birds and clonally propagules i.e. cormlets were dispersed nearby mother plants (Sugiyama & Santosa 2008). Thus, in an undisturbed population, both ramets and genets co-exist. Recently, the sliced corms of are A. variabilis used as feeds for swine (Santosa . 2004; et al Sugiyama & Santosa 2008). People collect the corms without considering the conservation. On the other hand, land fragmentations and agricultural intensifications in agroforestry system for cash crops (Santosa . 2005) disturbed the et al populations. Consequently, the populations of A. variabilis in Java are in danger. High exploitation has been known causing population decline and loss of genetic diversity in many species (Leimena et al. et al. 2007; Nuryanto & Susanto 2010; Santosa 2010). Therefore, in order to develop effective program on genetic conservation of , it A. variabilis is important to evaluate population size using reliable genetic markers, such as microsatellite. Microsatellite or simple sequence repeat (SSR) is widely used to develop conservation program in many species (Abdul-Muneer 2014; Nachimuthu et al. 2015). In this study, we developed microsatellite of using compound A. variabilis isolation method for the first time. Many methods have been developed for m i c r o s a t e l l i t e i s o l a t i o n s e. g. b a s e d o n sequence data (Lian . 2001; Fatimah & Sukma et al 2011), cloning, enrichment library and dual- suppression methods (Lian & Hogetsu 2002; Lian . 2006). In the present study, et al compound microsatellites are isolated according to procedure of Lian (2006). The objective et al. of this study was to develop microsatellite markers from and its application for A. variabilis genetic population study. MATERIALS AND METHODS Plant Material Plant materials were collected separately for microsatellite development and population study i.e. from Bogor Botanical Garden (BBG) and D r a m a g a C o n s e r va t i o n Fo r e s t ( D C F ) , respectively with permission from the authorities. According to Yuzammi (2000), Bogor area including the sampling sites, is the center of A. variabilis diversity. To develop microsatellite markers, 2 - 5 g fresh and healthy leaves of were collected A. variabilis during rainy season in 2005 at the BBG, Bogor, West Java Province, Indonesia. The leaves were cleaned using moist tissue paper and put into a plastic bag containing silica gel, then stored at -30 o C until being used in genomic DNA extraction. Microsatellite development was conducted in the Laborator y of Forestr y, Asian Natural Environmental Science Center, University of Tokyo, Japan from 2009 to 2010. For population study, accessions A. variabilis were sampled and characterized in the DCF (244 m asl), Bogor, West Java Province, Indonesia from 2010 to 2011. DCF is managed by the Indonesia Ministry of Environment and Forestry. Around 40 of the 60 ha DCF area is dedicated for conservation forest, locally known as the CIFOR forest (CIFOR = Center for International Forestry Research). DCF is located about 8 km northwest of Bogor Botanical Garden, 1 km west of Situ Gede Lake and 1 km north of Cisadane River. Three populations were selected for this study, i.e. A (-6.5509798, 106.7497507,17z), B (- 6 . 5 5 1 9 3 9 1 , 1 0 6 . 7 4 9 4 9 3 2 , 1 7 z ) a n d C ( - 6.5548596,106.7507271,17z) (Fig.1). In each population, plants were sampled from an area of about 0.5 to 1 ha. All plants having pseudo stems thicker than 2 cm at 10 cm above soil surface and were spaced more than 1 m apart were collected. Plants spaced less than 1 m apart were considered as a ramet (similar genet) and not sampled. Petiole colors of accessions were characterized (Table 1). Initially, 35, 25 and 38 samples were collected, and after optimizing the DNA results a set of 11, 21 and 15 23 Isolating microsatellite from Amorphophallus variabilis and its application - Santosa et al. (Novagen, USA) according to the manufacturer's instructions. The positive clones were amplified using the U19 and M13 reverse primers, and then sequenced using a Thermo Sequenase Pre-mixed Cycle Sequencing Kit (Amersham Biosciences) plus the T7 or U19 primer labeled with Texas Red (Sigma-Aldrich) on SQ-5500E sequencer (Hitachi, Tokyo). A primer (IP1) was designed from the sequenced region flanking to the SSR. The IP1 and corresponding microsatellite primers were used as the marker. In addition to microsatellites developed from A. variabilis, twenty microsatellite markers which were developed from by Santosa A. paeoniifolius et al. (2007) and stored in GenBank were evaluated for its suitability toward genotyping. A. variabilis In order to extend usefulness of the microsatellite markers, the developed markers from A. variabilis were evaluated on several Araceae members and other root crops species. PCR Amplification and Detection PCR reaction was carried out in a reaction mixture (10 µL) containing 5 - 10 ng DNA, 0.2 mM of each dNTP, 1 PCR buffer (Mg free, 2+ accessions were used for further analysis from A, B and C populations, respectively. The other samples were excluded from analysis because they failed to amplify, or produced multiple and unclear bands. Microsatellite Development A modified cetytrimethyl ammonium bromide (CTAB) method was adopted for conducting genomic DNA extraction (Zhou . 1999). et al DNA from 1 g dry leaves was dissolved in a final volume of 200 µL water and stored in -30 C until o use. Isolation of codominant compound microsatellite markers was performed according to the method of Lian . (2006). In brief, the et al Hae III blunt-end restriction enzyme was used to digest DNA sample. The digested DNA was then ligated to an adaptor using a DNA Ligation Kit (TaKaRa Shuzo, Japan). Fragments flanked by a microsatellite region at one end were amplified from the constructed DNA library using SSR primers, (AC) (AG) or (TC) (AC) and an adaptor 6 5 6 5 primer, AP2 (5´–CTATAGGGCACGCGT GGT–3´). The PCR products were subcloned using a pT7 Blue Perfectly Blunt Cloning Kit BIOTROPIA Vol. 25 No. 1, 2018 24 A10 A9 A8 A12 A11 A1 A3 A4 A6 A5 A2 Pop. A B2 B3 B6 B7 B8 B4 B5 B9 B10 B11 B15 B18 B17 B19 B25 B26B27 B21 B20 B22 B24 B23 B28 B16 B12 B13 B14 Pop. B C1 C13 C14 C15 C16 C18 C12 C7 C11 C8 C10 C6 C4 C20 C21 C22 C17 C19 Pop. C INDONESIA Java N Cluster I Cluster II Cluster III Mt Salak BBG DCF BOGOR AREA 5 m 5 m 5 m Road 2 km Figure 1 Site and accession positions within populations in Dramaga Conservation Forest (DCF), Bogor, Indonesia (Note: Lines represent distance and visibility between two accessions; accessions without lines represent blockage by dense trees; color represents cluster membership; bar represents 5 meter) Applied Biosystems), 2.5 mM MgCl , 0.25 U of 2 Ampli Gold (Applied Biosystems, USA), 5 Taq 0. μM of each IP1 primer and the corresponding SSR primer (labeled with Texas Red). The PCR thermal cycler (Applied Biosystems) were used with cycling profile: 9 minutes at 94 ºC, followed by 40 cycles of 30 seconds at 94 ºC, 30 seconds at the locus-specific annealing temperature (Table 2) and 1 minute at 72 ºC, and finally a 5 minutes extension at 72 ºC. The PCR products were electrophoresed on a 6% polyacrylamide gel using an SQ-5500E sequencer, and then analyzed with FRAGLYS ver. 3 software (Hitachi, Tokyo). Data Analysis Characteristics of microsatellite markers developed in the present study were tested on A. variabilis across all populations (n = 47). Two bands from an individual were considered as different alleles if the differences in molecular weight were bigger than 3 base pair (bp) for the microsatellite containing trinucleotide repeat and 2 bp for the microsatellite containing dinucleotide repeat; these bp different are matter of technical procedure not related to nucleiotide repeat motif (Santosa 2017). The numbers of alleles, observed (H ) and expected heterozygosities (H ) and O E polymorphic information content (PIC) were calculated using CERVUS ver. 3.0.3 software (Marshall et al. 1998). Hardy-Weinberg equilibrium (HWE) and linkage disequilibria between loci were tested using GENEPOP version 4.0 software on the web (Rousset 2008). Presence of null allele was estimated using CERVUS ver. 3.0.3 software (Marshall et al. 1998). Number of migration (Nm) was estimated using GENALEX software. Population Code Petiole color Population Code Petiole color A A1 Pure green B B20 Brown with small white spot A A2 Pure green B B21 Brown with large white and green spots A A3 Brown with white spot B B22 Brown with large light green spot A A4 Green with brown spot B B23 Brown with white spot A A5 Green with white spot B B25 Green with large white spot A A6 Green with brown spot B B26 Dark brown-black with white spot A A8 Pure green B B27 Brown with small white spot A A9 Brown with black spot B B28 Brown with small dark brown spot A A10 Brown with dark brown spot C C1 Pure green A A11 Brown with black spot C C4 Brown with white spot A A12 Dark green with brown spot C C6 Green with grey spot B B2 Grey with brown spot C C7 Light green with brown spot B B3 Brown with green spot C C8 Brown with pink spot B B4 Grey with green spot C C10 Pure green B B5 Brown with green spot C C11 Light green with grey spot B B6 Brown with white spot C C12 Pure green B B7 Green with small white spot C C13 Brown with white and black spot B B8 Light green with black and white spot C C14 Brown with light brown spot B B9 Light green C C16 Brown B B10 Brown with brown and black spot C C17 Dark brown with pink spot B B11 Pure green C C20 Pure green B B14 Light brown with white spot C C21 Dark brown with pink and black spo t B B16 Dark green with white spot C C22 Light green with black spot B B18 Brown with black spot Table 1 Characterization of A. variabilis accessions obtained from Dramaga Conservation Forest, Bogor, Indonesia Note: Large spot = spot width 10mm> Small spot = spot width 2.5mm< Otherwise mentioned = spot size was medium Spot was measured from 10 cm above soil surface to middle petiole length 25 Isolating microsatellite from Amorphophallus variabilis and its application - Santosa et al. Analysis of molecular variation (AMOVA) within (F ) and among population (F ) was IS ST determined from A, B and C populations separately using GENALEX software in 999 permutations. Cluster analysis was performed using NTSYST Spc 2.11p (Exeter Software, Setauket USA). Presence of allele at each locus was coded in binary form 1 (presence) or 0 (absence). Individual across populations was clustered in UPGMA dendrogram using Jaccard similarity coefficient. RESULTS AND DISCUSSION Polymorphism Test From twenty-two microsatellite loci isolated from A. variabilis, twelve loci were polymorphic and codominant. Polymorphic information content (PIC) ranged from 0.351 to 0.932 (Table 2 ) . T h e o b s e r ve d ( H ) a n d e x p e c t e d O heterozygosities (H ) ranged from 0.191 to 0.851 E and from 0.380 to 0.946, respectively. The full length of amplified region from A. variabilis microsatellite had been deposited at GenBank for public access (www.ncbi.nlm.nih.gov/Genbank). Two of 12 loci isolated from A. variabilis, i.e. Avar01 and Avar07, could deviate from HWE proportion (p 0.001) by having an excess of heterozygosities (Table 2). Linkage disequilibria analysis indicated that the loci had high linkages (p ≤ 0.001), i.e. between Avar02 and Avar04, Avar04 and Avar08, Avar07 and Avar10, and Avar07 and Ampa10. Excluding loci Avar04 and Avar07, the other 10 loci could be used in population genetic study. According to Li et al. (2009), a set of 10 - 15 or more loci was desirable for genetic population study. Furthermore, two microsatellite primers from A. paeoniifolius, i.e. Ampa10 and Ampa15, produced clear bands in A. variabilis accessions. Therefore, in total, fourteen polymorphic microsatellite loci could be used for A. variabilis genotyping. This study is the first work on the development of polymorphic codominant microsatellite markers in . Microsatellite markers A. variabilis developed in the present study could be used to identify the polymorphism based on the standards proposed by Lian . (2006), i.e. clearness of et al bands, common annealing temperature and different allele sizes. In the present study, annealing temperature was set at 58 and 62 C. o Allele sizes among several loci had large differences in base pair size (Table 2). Therefore, loci Avar01 and Avar09, Avar05 and Avar06, and Avar03 and Avar10 could be mixed together in PCR reaction, to speed up PCR preparation and allele analysis. The loci developed from cross-A. variabilis amplified in other species (Table 3), indicating that the loci could provide a useful tool for g e n o t y p i n g i n o t h e r s p e c i e s. I n d e e d , microsatellite primers are well known for its species-specific (Lian & Hogetsu 2002; Csencsics et al. 2010), however, cross-amplifications sometimes exist (Santosa . 2007; Fatimah & et al Sukma 2011). Successful cross-amplification is probably due to the presence of homologous loci among them. Santosa (2007) have reported et al. that microsatellites isolated by dual-suppression from amplified in related species, A. paeoniifolius while Fatimah Sukma (2011) reported that and microsatellite markers obtained from sequence data could amplify across genus. Phalaenopsis However, amplification PCR products did not mean produce polymorphic allele in present study. It needs further investigation on the usefulness of the developed loci for genotyping other speciesA. variabilis . Population and Genetic Diversity of DCF The average numbers of alleles per locus was 6.5 for 14 loci. The numbers of alleles per locus varied from 3 to 11 in population A, from 3 to 16 in population B and from 3 to 14 in population C (Table 4). Locus Ampa15 produced the smallest number of alleles, whereas locus Ampa10 produced the largest number of alleles across DCF populations. Several accessions failed to amplify, i.e. locus Avar02 for A12 and C20 accessions, locus Avar04 for C6 and C10 accessions, locus Avar06 for B9 and B11 accessions, locus Avar11 for B23 and C17 accessions, locus Ampa10 for C13 accession and locus Ampa15 for A8 and C7 accessions. These accessions probably had null allele for particular loci. Point mutation in the primer annealing sites may lead to the occurrence of null alleles causing the primer failed to amplify (Lian et al. 2006). 26 BIOTROPIA Vol. 25 No. 1, 2018 T ab le 2 C h ar ac te ri st ic s o f t w el ve m ic ro sa te ll it e m ar k er s is o la te d f ro m A . va ri ab ili s L o cu s G en B an k z R ep ea t m o ti f P ri m er s eq u en ce 5 ’ to 3 ’ T a ( °C ) N A A ll el e (b p ) H O H E P IC A va r0 1 * M F 5 2 7 2 5 0 (C T ) 6 (G T ) 7 F : C T T G T T C G G A C C A C C T T C T T G A C A A T C R : (A C )7 (A G )3 6 2 5 1 2 0 -1 3 0 0 .4 8 9 0 .5 4 8 0 .4 9 2 A va r0 2 M F 5 2 7 2 5 1 (C T ) 1 2 (G T ) 7 F : C T C A A A A T C G A A T C T T C T C C A T T T T A C R : (A C )7 (A G )3 6 2 1 4 1 2 4 -1 5 4 0 .3 3 3 0 .8 7 7 0 .8 5 4 A va r0 3 M F 5 2 7 2 5 2 (C T ) 8 ..( C T T ) 1 1 ..( C T ) 5 (G T ) 7 F : C A C T C T T C C A C A C T C C C C C T G T T A C A C R : (A C )7 (A G )3 6 2 5 1 2 8 -1 4 0 0 .2 1 3 0 .3 8 0 0 .3 5 1 A va r0 4 M F 5 2 7 2 5 3 (C T ) 1 3 ... .( C T ) 4 (G T ) 7 F : C T T C C C T A T G C A G G T G A G T C R : (A C )7 (A G )3 5 8 8 1 1 3 -1 5 5 0 .3 3 3 0 .4 2 7 0 .3 9 6 A va r0 5 M F 5 2 7 2 5 4 (G T ) 5 (G A ) 7 F : G T G A A G G A G G T G G G C G T T T T G R : (T C )7 (A C )3 5 8 5 1 7 7 -1 8 7 0 .4 6 8 0 .5 2 0 0 .4 6 0 A va r0 6 M F 5 2 7 2 5 5 (G T ) 1 0 (G A ) 7 F : C T A A C G A C T A A G G A C T T A A G C R : (T C )7 (A C )3 5 8 2 2 6 3 -1 4 1 0 .5 1 1 0 .9 4 3 0 .9 2 8 A va r0 7 * M F 5 2 7 2 5 6 (G T ) 1 0 (G A ) 7 F : C G C T G A T G T A C T T G T T G A C A T T G R : (T C )7 (A C )3 5 8 1 1 1 3 9 -1 7 1 0 .7 2 3 0 .7 6 1 0 .7 1 9 A va r0 8 M F 5 2 7 2 5 7 (G T ) 8 (G A ) 7 F : C A T G G T C C A T G G G T T T A G C T T T G C R : (T C )7 (A C )3 6 2 2 0 1 6 6 -2 3 6 0 .7 4 5 0 .9 3 3 0 .9 1 8 A va r0 9 M F 5 2 7 2 5 8 (G T ) 5 (G A ) 7 F : G G T G T A C G A C T A G A G T T T T G T C G R : (T C )7 (A C )3 6 2 7 1 7 5 -1 8 3 0 .4 2 6 0 .6 7 6 0 .6 2 0 A va r1 0 M F 5 2 7 2 5 9 (G T ) 8 (G A ) 7 F : G A T G T C A T T C T C C G C C A C C G A G T A G R : (T C )7 (A C )3 6 2 1 0 1 9 6 -2 2 8 0 .8 5 1 0 .7 6 9 0 .7 2 6 A va r1 1 M F 5 2 7 2 6 0 (G T ) 6 (G A ) 7 F : G A T A C T G C T A T A C C G A G T G T C C T A T G R : (T C )7 (A C )3 6 2 7 1 5 3 -1 8 5 0 .2 0 0 0 .6 5 5 0 .6 0 7 A va r1 2 M F 5 2 7 2 6 1 (T C ) 8 G (C T ) 2 (G T ) 5 (C T ) 5 (G T ) 7 F : G G A A C A C T A G C G A G T A C A A T G T A T C R : (A C )7 (A G )3 6 2 5 1 3 5 -1 4 9 0 .1 9 1 0 .7 8 6 0 .7 4 1 N o te : = G en B an k r ep o si to ry c o d e o f p ri m er s an d f ra gm en ts s eq u en ce z * = s ig n if ic an t d ev ia ti o n f ro m H ar d y– W ei n b er g E q u il ib ri u m ( < 0 .0 1 ) p T = an n ea li n g te m p er at u re a N A = n u m b er o f a ll el e H O = o b se rv ed h et er o zy g o si ty H E = ex p ec te d h et er o zy g o si ty P IC = P o ly m o rp h ic I n fo rm at io n C o n te n t E ac h r ev er se p ri m er ( R ) w as t ai le d w it h a n a d d it io n al 1 9 n u cl eo ti d e (U 1 9 ) to t h e 5 ' e n d 27 Isolating microsatellite from Amorphophallus variabilis and its application - Santosa et al. 28 T ab le 3 C ro ss -s p ec ie s am p li fi ca ti o n o f t w el ve m ic ro sa te ll it e p ri m er s d ev el o p ed f ro m A . va ri ab ili s N o S p ec ie sz F am il y O ri gi n o f t h e sp ec im en L o ci A V A R 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 0 1 1 1 2 1 A gl ao ne m a pi ct um ( R o x b .) K u n th . A ra ce ae B B G + + + + - + + + + + + + + + + + + + + 2 A lo ca si a al ba S ch o tt . A ra ce ae B B G + + + + + + + + + + - + + + - + + + + + 3 A nt hu ri um s p p . A ra ce ae B B G + + + + + - + + - + + + - + + + + + 4 A m or ph op ha llu s k on ja c K . K o ch A ra ce ae T an as h i F ar m , U T + + + + + + + + + + + + + + + + + + + + 5 A m or ph op ha llu s m ue lle ri B lu m e A ra ce ae IP B + + + + + + - + + + + + + + + + - + + 6 A m or ph op ha llu s pa eo ni ifo liu s (D en n st .) N ic o ls o n A ra ce ae K u n in g an , In d o n es ia + + + + + + + + + + + + + + + + + + + + + + + + 7 A m or ph op ha llu s ti ta nu m ( B ec c. ) B ec c. E x . A rc an g A ra ce ae B B G + + + + - - - + - + + + + 8 C al ad iu m b ic ol or ( A it o n ) V en t. A ra ce ae IP B + + + + + + + + + + + + - - + + + + + + 9 C ol oc as ia e sc ul en ta ( L .) S ch o tt A ra ce ae IP B + + + + - + + + + + + - + + - + + + 1 0 D ie ff en ba ch ia f ou rn ie ri N .E . B r. A ra ce ae B B G + + + + + + + + + - - - - + + + + + 1 1 D ra co nt iu m g ig as ( S ee m .) E n gl . A ra ce ae B B G + + + + + + + + + + - + - - + - + + 1 2 H om al om en a pe nd ul a (B lu m e) B ak h . f. A ra ce ae B B G + + + + + + + + + + - + + + + + + + + + 1 3 Ip om oe a ba ta ta s (L ) L am . C o n vo lv u la ce ae B B G + + + + + + + + + + - + + + - + - + 1 4 M an ih ot e sc ul en ta C ra n tz E u p h o rb ia ce ae B B G + + - + + + - - + + - + - + 1 5 N el um bo s p p . A ra ce ae T an as h i F ar m , U T + + + + + + + + + + + + - - + + + + 1 6 S ch is m at og lo ti s ca ly pt ra te ( R o x b .) Z o ll . & M o ri tz i A ra ce ae B B G + + - + + + + + + + + + + + + + + + + + + 1 7 S pa th ip hy llu m c an na ef ol iu m S ch o tt A ra ce ae B B G + + + + + + + + + + + + + + + - + + + + 1 8 S ol an um l yc op er si cu m L . S o la n ac ea e T an as h i F ar m , U T + + + + + + - - + + + + + + + + 1 9 S ol an um t ub er os um L . S o la n ac ea e C ia n ju r, I n d o n es ia + + + + + + + + + + - + + + + + + + + + 2 0 T ac ca s p p . A ra ce ae B B G + + + + + + + + + + + + + - - + - + 2 1 X an th os om a sa gi ti fo liu m ( L .) S ch o tt . A ra ce ae B B G + + + + + + + + + + + + + + + - + + + + + + 2 2 Z am io cu lc as z am ii fo lia E n gl . A ra ce ae B B G + + + + + + + + - + + + + - + + + + + z N o te : = O n e in d iv id u al p er s p ec ie s B B G = B o g o r B o ta n ic al G ar d en U T = U n iv er si ty o f T o k yo -J ap an IP B = In st it u t P er ta n ia n B o g o r '– ' = n o a m p li fi ca ti o n '+ ' = am p li fi ca ti o n s in gl e b an d '+ + ' = m u lt ip le b an d s A m p li fi ca ti o n b an d d id n o t a lw ay s p ro d u ce m ic ro sa te ll it e al le le BIOTROPIA Vol. 25 No. 1, 2018 Number of effective alleles of A, B and C populations were 5.43, 7.36 and 6.71, respectively. B population showed higher allelic richness as compared to A and C populations. More than one specific allele per locus existed in each population. Out of 147 alleles generated by 14 loci across populations, 61 alleles were specific to particular population. A, B and C populations had 16 (26.2%), 27 (44.3%) and 18 (29.5%) specific alleles, respectively. H value was the highest in A O population, while H value was the highest in C E population. Fixation indices (F ) averaged for IS each population across all loci had high values ranging from 0.240 to 0.294 (Table 4). AMOVA analysis showed low F values both ST within and among populations. F value across all ST populations was 0.026, whereas F values among ST A-B, A-C and B-C populations were 0.022 ( = p 0.009), 0.034 ( = 0.003) and 0.025 ( = 0.001), p p respectively, suggesting that each population was slightly conserved. On the other hand, variation among individuals within population was high (64.0%). According to Jansen (1996) and et al. Yuzammi (2000), has large phenotypic A. variabilis variations in leaf size, petiole and inflorescence color. The populations exhibited low genetic distance based on Nei (1972), i.e. 0.151 for population A to population B, 0.210 for population A to population C and 0.149 for population B to population C. Individuals across all populations were clustered in three groups (Fig. 2). Group I composed of accessions from A and C populations. Group II composed of accessions from A, B and C populations. Group III contained only one accession from population C, i.e. C22. Population B was exclusively clustered under subgroup II. In each population, with exception for B28, C1, C4, C6 and C7, accessions clustered in the same group were generally located close to each other (Fig. 1). Accessions A10, A11 and A12 from A population were clustered in subgroup II with those from B population. These accessions were geographically close to each other (Fig. 1). This suggests that genetic exchange via pollen occurred between accessions located at the peripheral zone of a population, as stated by Pasquet . (2008). Sugiyama dan Santosa (2008) et al stated that Nitidulidae insect becomes important pollinator in .A. variabilis Morphological variations in petiole (Table 1) was unlikely correlated with microsatellite profiles. Accessions with green petiole without spot, i.e. A1, A2, A8, B11, C1, C10, C12 and C20 were clustered into two different groups, i.e. Group I and Group II. This finding was in disagreement with the results of Santosa et al. (2012), where flower size, leaf size and the presence of petiole spot were tightly linked with AFLP grouping. The discrepancy between the result of Santosa et al. (2012) and this study could be due to the fact that AFLP is a dominant marker. In A. paeoniifolius, petiole color, shape and color of spot and petiole roughness are affected by soil fertility (Sugiyama & Santosa 2008). In the previous work, Santosa et al. (2004) grouped A. variabilis accessions from West Java Province, Indonesia based on inflorescence morphology into four groups. Therefore, it is important to conduct further study to investigate whether petiole and peduncle colors in A. variabilis are also affected by soil fertility. Table 4 Description for three Amorphophallus variabilis populations collected from Dramaga Conservation Forest, Bogor, Indonesia Population Number of allele HO HE FIS Effective Richness Specific A (n=11) 5.43 3.56 1.14 0.481 0.645 0.240 B (n=21) 7.36 4.58 1.93 0.464 0.667 0.282 C (n=15) 6.71 4.20 1.29 0.465 0.681 0.294 Note: F = Fixation Index calculated using formula: (H - H ) / H or 1 - (H / H )IS E O E O E n = number of sample, evaluated using 10 microsatellite loci from A. variabilis and two loci from A. paeoniifolius 29 Isolating microsatellite from Amorphophallus variabilis and its application - Santosa et al. Conservation Strategy Three populations evaluated in DCF exhibited unique genetic feature. According to an interview with administrative staff members of DCF, there was no intercropping program, harvesting or any man-made disturbance on the A. variabilis populations. Unexpectedly, during field survey, several A. paeoniifolius plants were found at DCF. According to local officer, the plants were introduced from Yogyakarta by farm laborers in year 2000s. Many bushes, weeds and tree seedlings grew densely inside DCF, forming shady condition and blocking more than 75% of sunshine. According to Sugiyama and Santosa (2008), Amorphophallus species grow well under shading up to 75% shading level. It was possible that heavy shading disturbed the growth of , resulting to A. variabilis low density of the studied-population in DCF. Sugiyama and Santosa (2008) also stated that single mother corms of produced 14.3 A. variabilis cormlets per year. Cormlets were detached from mother corm after dormancy release. Two to seven cormlets usually grew close (0 - 13 cm) to mother corms, unlike seedlings from seeds which usually grew apart (> 60 cm) from the mother plant. Considering that mature plant of A. variabilis is able to produce 25 - 280 seeds (Sugiyama & Santosa 2008), the population density could increase steadily yearly in undisturbed condition. It is interesting to conduct further study on population dynamic of A. variabilis in isolated condition, such as in DCF. In the present study, several accessions failed to amplify resulting to low number of sampling size in each population (Table 4) that might cause an underestimation of population genetic. According to Li (2009), the numbers of et al. effective alleles were determined by the number of sampling size and the numbers of loci used. A set of 40 samples per population is necessary for population study. During DNA extraction, several leaf samples contained large amount of glucomannan. Thus, it is important to improve 30 Figure 2 Dendrogram of UPGMA based on Jaccard similarity index of A. variabilis constructed using microsatellite data obtained (Note: A, B, and C codes represent A, B and C populations, respectively) BIOTROPIA Vol. 25 No. 1, 2018 DNA extraction method in the near future, to enhance the accuracy of population genetic evaluation. Genetic distance between A and C populations was significantly larger than those of other population pairs. However, it was still unclear why these populations were distantly separated in terms of genetic profile. Geographically, A and C populations were separated by about 200 meters. The large genetic distance suggested that exchange of genetic materials among A and C populations was restricted. Interestingly, within C population, C22 seemed to be out of group (Fig. 2). This might be caused by the fact that C22 was an introduced seed from other distant populations by birds as primary agent for long-distance seeds dispersal of (Sugiyama & Santosa A. variabilis 2008). The number of estimated genetic exchange or migration among A-B, A-C and B-C populations were 11.23, 7.21 and 9.81, respectively, and were considered as high. Average number of estimated migrant (Nm) based on the number of specific alleles across all populations was high, i.e. 9.39. Smaller degree of genetic differentiation (F ) ST among A and B, and B and C populations, indicated populations located closely to each other mostly underwent intense genetic exchange through cross-breeding. Finding in DCF indicated that the A. variabilis existed as a meta-population. Santosa (2012) et al. has suggested to conserve single large population in conservation strategy of , however, A. variabilis it should be further clarified by evaluating accessions from distant populations across Java Island using codominant microsatellite primers. Many researchers, students and professional workers stated that a large number of A. variabilis plants existed in various agroecological condition in West Java and Yogyakarta provinces. Therefore, it is interesting to evaluate population A. variabilis from different sites in Java Island to develop conservation strategy for A. variabilis. CONCLUSIONS Microsatellite primers developed in the present study were applicable for the population study of A. variabilis. Three A. variabilis populations in Dramaga Conservation Forest exhibited low genetic diversity among populations, but high genetic distance within a population. Increasing number of specific allele with the increasing geographical distances among populations implies the importance to study genetic population from the larger geographical range in Java Island to determine the best conservation strategy for A. variabilis. ACKNOWLEDGEMENTS Part of this work was supported by Grant-in- Aid from the Japan Society for the Promotion of Science (JSPS), University of Tokyo, Japan and Institut Pertanian Bogor (IPB), Indonesia. We thank Mr Yuzammi from Bogor Botanical Garden, Indonesia, Dr Kazuhike Nara and Dr Kimura Megumi from Laboratory of Forest Science, and Dr O New Lee from Laboratory of Horticulture Sciences, University of Tokyo, Japan, for their kind technical assistances. We thank Bogor Botanical Garden and Center for International Forestry Research (CIFOR), Bogor, Indonesia for providing plant materials. REFERENCES Abdul-Muneer PM. 2014. Application of microsatellite markers in conservation genetics and fisheries management: Recent advance in population structure analysis and conservation strategies. G e n e t Re s I n t e r n a t 2 0 1 4 : I D 6 9 1 7 5 9 . doi:10.1155/2014/ 691759 Alonso-Sande M, Teijeiro-Osorio D, Remuñán-López C, Alonso MJ. 2009. Glucomannan, a promising polysaccharide for biopharmaceutical purposes. Eur J Pharm Biopharm 72:453–62. Csencsics D, Brodbeck S, Holderegger R. 2010. Cost- e f f e c t i ve , s p e c i e s - s p e c i f i c m i c r o s a t e l l i t e development for the endangered dwarf bulrush ( ) using next-generation sequencing Typia minima t e c h n o l o g y. J H e r e d 1 0 1 ( 6 ) : 7 8 9 – 9 3 . doi:10.1093/jhered/esq069 Fatimah, Sukma D. 2011. Development of sequence-based microsatellite marker for orchid. Phalaenopsis HAYATI J Biosci 18(2):71–6. Jansen PCM, van der Wilk C, Hetterscheid WLA. 1996. Amorphophallus Blume ex Decaisne. In: Flach M, Rumawas F, editors. PROSEA 9: Plant Yielding Non-seed Carbohydrates. Leiden (NL): Backhuys Publication. p. 45 50.– Kite GC, Hetterscheid WLA. 1997. Inflorescence odors of Amorphophallus Pseudodracontium and (Araceae). Phytochemistry 46(1):71–5. 31 Isolating microsatellite from Amorphophallus variabilis and its application - Santosa et al. Leimena HEP, Subahar TS, Adianto. 2007. Population structure of topshells ( ) in Saparua Trochus niloticus Island, Central Moluccas, Indonesia. BIOTROPIA 14(2):52-61. doi:10.11598/btb.2007.14.2.18 Li O, Zhao Y, Guo N, Lu C, Sun X. 2009. Effects of sample size and loci number on genetic diversity in wild population of grass carp revealed by SSR. Zool Res 30(2):121-30. Lian C, Hogetsu T. 2002. Development of microsatellite markers in black locust ( ) using a Robinia pseudoacacia dual-suppression-PCR technique. Mol Ecol Notes 2:211–3. Lian CL, Wadud MA, Geng QF, Shimatani K, Hogetsu T. 2006. An improved technique for isolating codominant compound microsatellite markers. J Plant Res 119:415–7. Lian C, Zhou Z, Hogetsu T. 2001. A simple method for developing microsatellite markers using amplified fragment of inter-simple sequence repeat (ISSR). J Plant Res 114:381–5. Marshall TC, Slate J, Kruuk L, Pemberton JM. 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Mol Ecol 7:639–55. Nachimuthu VV, Muthurajan R, Duraialaguraja S, Sivakami R, Pandian BA, Ponniah G, ... Sabariappan R. 2015. Analysis of population structure and genetic diversity in rice germplasm using SSR markers: An initiative towards association mapping of agronomic traits in . Rice 8:30. doi:10.1186/212284-Oryza sativa 015-0062-5 Nei M. 1972. Genetic distance between populations. Amer Nat 106:283–91. Nuryanto A, Susanto AH. 2010. Genetic variability of Polymesoda erosa population in the Segara Anakan Cilacap. BIOTROPIA 17(1):22-30. Ohtsuki T. 1968. Studies on reserve carbohydrates of four Amorphophallus species, with special reference to mannan. Bot Mag Tokyo 81:119–26. Pasquet RS, Peltier A, Hufford MB, Oudin E, Saulnier J, Paul L, ... Gepts P. 2008. Long-distance pollen flow assessment through evaluation of pollinator foraging range suggests transgene escape distance. PNAS 105(36):13456–61. Rousset F. 2008. GENEPOP'007: A complete re- implementation of the GENEPOP software for windows and Linux. Mol Ecol Resources 8:103–6. Safii I. 1981. Study on extraction of mannan flour of iles-iles ( ) [Undergraduate Thesis]. Retrieved A. variabilis from Faculty of Agricultural Technology, Institut Pertanian Bogor. 81 p. (in Indonesian) Santosa E. 2017. DNA preparation of Amorphophallus paeoniifolius for SSR evaluation. Available from: h t t p : / / w w w. p r o t o c o l . i o. d o i : 1 0 . 1 7 5 0 4 / protocols.io.jjdcki6 Santosa E, Sugiyama N, Hikosaka S, Takano T. 2004. Classification of in West Amorphophallus variabilis Java, Indonesia based on mor phological characteristics of inflorescences. Jpn J Trop Agric 48:25–34. Santosa E, Sugiyama N, Hikosaka S, Takano T, Kubota N. 2005. Intercropping patterns in cacao, rubber, and timber plantations in West Java, Indonesia. Jpn J Trop Agric 49(1):21–9. Santosa E, Lian CL, Pisooksantivatana Y, Sugiyama N. 2007. Isolation and characterization of polymorphic microsatellite markers in Amorphophallus paeoniifolius (Dennst.) Nicolson, Araceae. Mol Ecol Notes 7:814–7. Santosa E, Mine Y, Nakata M, Lian C, Sugiyama N. 2010. Genetic diversity of cultivated elephant foot yam (Amorphophallus paeoniifolius) in Kuningan, West Java as revealed by microsatellite markers. J Appl Hort 12(2):125–8. Santosa E, Sugiyama N, Kawabata K, Hikosaka S. 2012. Genetic diversity of using Amorphophallus variabilis AFLP. J Agron Indonesia 40(1):62–8. Sugiyama N, Santosa E. 2008. Edible in Amorphophallus Indonesia: A potential crop in agroforestry. Yogyakarta (ID): Gadjah Mada University Press. 125 p. Wiyani L. 1988. Extraction and characterization of mannan from white corm iles-iles ( Bl.) A. variabilis [Undergraduate Thesis]. Retrieved from Faculty of Agricultural Technology, Institut Pertanian Bogor. 129 p. (in Indonesian) Yuzammi. 2000. A taxonomic revision of the terrestrial and aquatic (Araceae) in Java [Thesis]. Retrieved Aroid from School of Biological Science, Faculty of Life Science, University of New South Wales. 359 p. Zhang SL, Liu PY, Sun YM. 1998. Artificial adjustment of flower period and hybridizing techniques of Amor phophalus konjac. Acta Bot Yunn 0. Suppl(5):62–6. Zhou Z, Miwa M, Hogetsu T. 1999. Analysis of genetic structure of a population in a Suillus grevillei Larix kaempferi stand by polymorphism of inter-simple sequence repeat (ISSR). New Phytol 144:55–63. 32 BIOTROPIA Vol. 25 No. 1, 2018 Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9 Page 10 Page 11