Isolation and characterization of tetranucleotide microsatellite markers for Pinus merkusii
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TECHNICAL NOTE
Isolation and characterization of tetranucleotide microsatellite markers for Pinus merkusii D. V. Thao • A. Y. P. B. C. Widyatmoko L. Guan • E. Gotoh • A. Watanabe • S. Shiraishi
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Received: 5 November 2012 / Accepted: 12 November 2012 / Published online: 20 November 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract Pinus merkusii is an important tree native to Southeast Asia. Thirteen tetranucleotide microsatellite makers were developed from this species using a dualsuppression polymerase chain reaction technique. Of these markers, nine loci were polymorphic. The mean number of alleles per locus was 3.18 (range 2–5) and mean expected heterozygosities were 0.525 (range 0.384–0.712). These microsatellite markers will be available to assess the genetic diversity, mating patterns and population structure which will facilitate conservation and management strategies. Keywords Pinus merkusii Tetranucleotide microsatellite marker Dual-suppression PCR
Pinus merkusii is an economically important species for pulp, paper and resin in Southeast Asia. It naturally occurs in Thailand, Burma, Laos, Cambodia, Vietnam, Philippines
D. V. Thao Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan A. Y. P. B. C. Widyatmoko Centre for Forest Biotechnology and Tree Improvement, Yogyakarta, Indonesia L. Guan Hubei Provincial Forest Tree Breeding Centre, Wuhan 430079, Hubei, China E. Gotoh A. Watanabe (&) S. Shiraishi Laboratory of Silviculture, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan e-mail: [email protected]
and Indonesia (Cooling 1968). Despite the trend of becoming one of the favorable species for reforestation in Indonesia and Vietnam, its natural areas have decreased significantly as a result of deforestation in the past decades (Bharali et al. 2012). Consequently, the species currently is in a vulnerable conservation status according to the criteria of the International Union for Conservation of Nature (IUCN 2012). Knowledge of the genetic information such as genetic diversity among and within populations and mating patterns will facilitate efforts to conserve this vulnerable species. However, so far, almost molecular markerbased studies on this species were provided by isozyme analysis (Changtragoon and Finkeldey 1995; Kartikawati 1998; Suwarni et al. 1999; Siregar and Hattemer 2004). Microsatellite markers have emerged as one of the most informative DNA-based markers for the study and conservation of genetic diversity. To date, only 5 polymorphic microsatellite markers in P. merkusii were developed, however all of them are dinucleotide microsatellites (Nurtjahjaningsih et al. 2005). Tetranucleotide microsatellites with high level of reliability in detecting multiple alleles per locus, especially, have become ideal markers for genetic analysis (Guan and Shiraishi 2011). Thus, the objective of this work was to isolate and characterize tetranucleotide microsatellite markers for
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