Isolation and characterization of 16 microsatellite markers in Intsia palembanica , a high-value tropical hardwood speci
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MICROSATELLITE LETTERS
Isolation and characterization of 16 microsatellite markers in Intsia palembanica, a high-value tropical hardwood species Chai Ting Lee • Soon Leong Lee • Lee Hong Tnah Kevin Kit Siong Ng • Chin Hong Ng • Shawn Cheng • Naoki Tani
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Received: 9 November 2013 / Accepted: 25 November 2013 / Published online: 8 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Intsia palembanica is a high-value tropical hardwood species of the family Leguminosae. A total of 16 microsatellite markers were developed for this important timber species using an enrichment approach. These markers were screened on 24 samples from a natural population and subsequently tested for reproducibility, ease of scoring and locus-specificity, across five populations. The observed and expected heterozygosities ranged from 0.026 to 0.976 and 0.026 to 0.928, respectively, while the mean number of alleles per locus per population was 8.73. These microsatellite markers are informative and will be useful for conservation genetic study, individual identification and timber tracking. Keywords Intsia palembanica Merbau Microsatellite Tropical timber
Intsia palembanica is a highly sought after tropical timber species marketed under the trade name Merbau. Overexploitation due to high demand of this heavy hardwood in the international market has raised the concern over its
Electronic supplementary material The online version of this article (doi:10.1007/s12686-013-0100-9) contains supplementary material, which is available to authorized users. C. T. Lee (&) S. L. Lee L. H. Tnah K. K. S. Ng C. H. Ng S. Cheng Genetics Laboratory, Forest Research Institute Malaysia, 52109 Kepong, Selangor, Malaysia e-mail: [email protected] N. Tani Forestry Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Ohwashi, Tsukuba, Ibaraki 305-8686, Japan
dwindling population. Therefore, there is an urgent need to carry out conservation genetic study of this species using versatile molecular markers such as microsatellites. The total genomic DNA was extracted from 5 g of leaves from an I. palembanica tree from the Forest Research Institute Malaysia (FRIM, Selangor; 3°140 N 101°380 E) using a modified cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson 1980) and subsequently purified using CsCl ethidium bromide gradient. A microsatellite-enriched library was then constructed via the magnetic bead hybridization selection approach using 50 biotinylated (CT)15 and (GT)15, as described in Tnah et al. (2012). Based on 121 unique microsatellitecontaining sequences of the 271 clones sequenced, 37 primer pairs of 19–24 bp were designed using OLIGO 6.67 Software (Molecular Biology Insights). From the initial screening using four unrelated individuals [PCR amplifications were conducted following the protocol reported in Tnah et al. (2012)], 32 primer pairs which gave specific amplification products were selected for fluorescent labeling (HEX or 6-FAM) and further screened using 24 samples
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