In situ and Ex situ Conservation of Commercial Tropical Trees - ITTO

378it is preferred to have higher genetic gain and uniform quality. It is also importantto keep genetic diversity as high as possible. It is, however, sometimes difficultto obtain both requirements. Therefore, estimating the genetic diversity ofmaterials for the operational plantings is necessary for designing better schemesfor sampling and selection.In this study, DNA analysis using RAPDs (random amplifiedpolymorphic DNAs, Williams et al. 1990) and microsatellites were conductedto evaluate the genetic diversity in one of the hedge-orchards in the DipterocarpCenter, East Kalimantan.Materials and MethodsPlant materials and DNA extractionLeaf materials were collected from 28 trees of S. leprosula planted in a hedgeorchardin the Dipterocarp Center, Inhutani I, Batuampar, East Kalimantan,Indonesia. They were dried and preserved in silica gel. Total genomic DNAwas extracted from ca. 100mg of leaves using a modified protocol (Shiraishi &Watanabe 1995) of the CTAB method (Murray & Thompson 1980). For RAPDanalysis, DNA was purified using Wizard Clean Up System (Promega).Microsatellite analysisFour microsatellites (Shc01, Shc03, Shc04, Shc09) developed for S. curtisii(Ujino et al. 1998) were applied to S. leprosula. For each microsatellite,fluorescence labeled forward primers and non-labeled reverse primers weresynthesized according to the reported primer sequences (Ujino et al. 1998) andused for PCR amplification. Each 10 µL reaction contained 1x PCR buffer(supplied with AmpliTaq Gold DNA polymerase, Perkin Elmer), 2.0 mM MgCl 2,200 µM each dNTP, 0.5 µM each Primer, 0.5 unit AmpliTaq Gold DNApolymerase (Perkin Elmer), and 25 ng template DNA. PCR amplification wasperformed at 94°C for 10 min, followed by 35 cycles at 94°C for 30 s, 50-60°Cfor 30 s, 72°C for 60 s, followed by 1 min at 72°C using a GeneAmp PCRSystem 9700 (Perkin Elmer). Annealing temperature at the initial cycle was60°C, and it was decreased 1°C / cycle for the following 9 cycles. After theinitial 10 cycles, annealing temperature was fixed at 50°C. PCR productswere electrophoresed using ABI 310 Genetic Analyzer (PE AppliedBiosystems). The lengths of PCR products were determined using GeneScanand Genotyper software (PE Applied Biosystems).