EXPERIMENTAL METHOD 1 Material Prosiding INAFOR III 2015 FullIssue low rest

Bogor, 21-22 October 2015 480 Based on Peraturan Pemerintah Nomor 7 Tahun 1999, 13 tengkawang species in Kalimantan have been determined as endangered species. Therefore, in order to conserve genetic diversity of the species, genetic conservation program such as estabishment of ex-situ conservation plot is needed. Molecular markers provide an important technology for evaluating levels and patterns of genetic diversity and have been utilized in a variety of plant species Perera et al., 2000. Among the various DNA marker methods, the most informative polymorphic marker system to date is microsatellites, or SSRs simple sequence repeats. Microsatellites represent a unique type of tandemly repeated genomic sequences, which are abundantly distributed across genomes and demonstrate high levels of allele polymorphism. Finkeldey and Hattemer 2007 mentioned that microsatellite is repetitive segments of DNA two to three nucleotides in length, scattered throughout the genome in the non-coding regions between genes or within genes introns. Microsatellite markers have been used to investigate genetic diversity of some plant species, such as Teak Dirmavena, 2007, Pinus merkusii Diputra, 2013 and Instia bijuga Widyatmoko Rimbawanto, 2013 The aim of this study was to investigate genetic diversity of six populations of three Shorea producing tengkawang those have been planted in Ex-situ Conservation Plot in Kebun Raya Unmul Samarinda KRUS East Kalimantan. The information will be useful for continuing genetic conservation program of Shorea species, especially those producing tengkawang. 2. EXPERIMENTAL METHOD 2.1 Material A total of 120 samples of individual trees from 6 populations of three Shorea species distributed in West Kalimantan and West Java were used in this study. At least 19 individual trees were collected for each population. All samples were collected in ex-situ conservation plot in Kebun Raya Unmul Samarinda KRUS. Species name, population and number of sample per populations were revealed in Table 1. Tabel 1: Populations list and their number of alelles and haplotypes Species Population Ʃ samples Locus 1 Locus 2 Locus 3 ƩAllele ƩHaplo ƩAllele ƩHaplo ƩAllele ƩHaplo S. stenoptera Ketapang 20 7 26 8 35 8 35 S. pinanga Sanggau 20 6 19 7 26 7 26 S. stenoptera Sanggau 20 5 14 9 39 6 20 S. macrophylla Sanggau 21 6 19 6 18 6 20 S. stenoptera Sintang 19 6 19 6 21 6 19 S. macrophylla Haurbentes 20 8 34 4 8 4 8 2.2 Method Total genomic DNA was extracted using a modified Cetyl Trimethyl Ammonium Bromide CTAB protocol reported by Shiraishi and Watanabe 1995. PCR was performed in 10µl reaction mixture contained 10 ng10µl template DNA, 0.125µM each of primers, 1.5 mM MgCl2, 0.2 mM each of dNTPs, and 0.5 unit10µl of Amplitag Gold Polymerase Applied Biosystems. PCR was performed using a GeneAmp PCR System, Model 9700 Perkin- Elmer. The condition of amplification was as follows: 120s at 95°C, and 30 cycles of 30s at 94°C, 30s at 55°C, and 90s at 72°C, followed by 300s at 72°C. Amplified fragments were detected and sequenced using ABI 3100 genetic analyzer Applied Biosystem. Bogor, 21-22 October 2015 481 Three SSR primers developed for Shorea curtisii Ujino et al., 1998 were selected and used to analyze genetic diversity of three Shorea species producing tengkawang based on the level of heretozygosity. Those primers were Shc-02, Shc-07, and Shc-09 Table 2. Nurtjahjaningsih et al. 2012 reported the amplification of the three SSR primers for the three Shorea species. Tabel 2: Three 3 Shorea curtisiimicrosatellite primers, its motifs and primer sequences used in this study Primer Repeat motif Primer sequence 5′→3′ Shc-02 CT 2 CACT 5 F: CACGC TTTCC CAATC TG R: TCAAGA GCAGA ATCCA G Shc-07 CT 8 CACT 5 CACCCCTCA 3 CTCA 10 F: ATGTC CATGT TTGAG TG R: CATGG ACATA AGTGG AG Shc-09 CT 12 F: TTTCT GTATC CGTGT GTTG R: GCGATT AAGCG GACCT CAG Analysis of molecular variance AMOVA was used to partition the total genetic variance into components due to differences between regions, populations, and individuals. That was calculated using the software POPGENE 1.32 Yeh et al., 2000 and GenAlEx 6.5 Peakall Smouse, 2012. 3. RESULT AND DISCUSSION 3.1 Genetic diversity