MATERIAL AND METHOD 1 Leaves samples

Bogor, 21-22 October 2015 518 The diversity of the dipterocarp family is under assault from deforestation and habitat alteration. Effective in-situ and ex-situ conservation strategy are required to conserve the existing genetic resources. To conserve genetic resources it is essential not only to maintain the existing diversity but also to understand ecological and evolutionary processes that have been responsible for the origin, evolution, and maintenance diversity at intraspecific and higher taxonomic level. It is assumed that a better understanding of diversity and the mechanisms maintaining diversity may be helpful in developing effective strategies for conservation of genetic resources. The distinct genetic characteristics maintain by each species would determine the proper conservation strategy of the species. A case study for the case was taken to two lowland endemic Shorea species, Shorea javanica and Shorea selanica by using direct sequencing of the nuclear gene. 2. MATERIAL AND METHOD 2.1 Leaves samples Samples of 15 to 16 mature trees were collected from 5 populations of. S. javanica along the west coast of Sumatera Lampung and South Sumatera. While samples of S. selanica were taken from 8-10 individuals in 3 population originated from different islands of Moluccas. 2.2 Loci studied For both S. javanica and S. selanica, three nuclear genes, GapC, PgiC and GBSSI, were partially sequenced. The GapC was amplified and sequenced using primers designed by Ishiyama et al. 2003. We newly designed primers for PgiC and GBSSI based on DNA sequences of Shorea species deposited in GenBank Kamiya et al. 2005: Kamiya et al. unpublished. 2.3 DNA isolation, amplification and sequencing Genomic DNA was isolated from adult leaves using modified CTAB method Muray and Thomson 1980. Partial regions of the three nuclear genes were amplified for each individual by performing nested PCR. Prior to sequencing, the PCR products were purified using rAPid Alkaline Phosphatase TM Roche, Germany and exonuclease I New England Biolabs, Massachusetts, USA. Purified products were directly sequenced for both strands using ABI Prism 3100 automatic sequencer Applied Biosystems. 2.4 Data analyses DNA sequences were checked visually and forward and backward traces assembled using the ATGC program Genetyx Corporation, Japan. Because the PCR products of nuclear regions were directly sequenced, it was not possible to determine the genotype if there were more than two heterozygous sites. However, genetic diversity parameters, such as the number of segregating sites and nucleotide diversities could be estimated without determination of individual haplotype. To assess levels of nucleotide polymorphism, nucleotide diversity π Nei 1987 and haplotype diversity H d Nei 1987 for each of the investigated loci were estimated. To test for deviation from selective neutrality and other assumptions constant population size with no mig ration, Tajima’s D Tajima 1989 test was performed for individual loci. For this test, the 95 confidence interval of Tajima’s D statistics of individual loci was obtained using 10,000 replicates of coalescent simulations under standard neutral model Hudson 1990. Population structure was examined using ARLEQUIN ver. 3.5 Excoffier et al. 2005. Genetic differentiation, F ST of nuclear loci was estimated through analysis of molecular variance AMOVA. We also calculated pairwise F ST to know the population structuring among pair of populations. Bogor, 21-22 October 2015 519 3. RESULT AND DISCUSSION 3.1