Molecular Ecology Notes (2006) 6, 30–32 doi: 10.1111/j.1471-8286.2005.01127.x © 2006 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE Development and characterization of ten new microsatellite markers in a mangrove tree species Bruguiera gymnorrhiza (L.) M. S. ISLAM,*† C. L. LIAN,† N. KAMEYAMA,‡ B. WU* and T. HOGETSU* *Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan, †Asian Natural Environmental Science Center, The University of Tokyo, Midori-cho 1-1-8, Nishitokyo-shi, Tokyo 188-0002, Japan, ‡ Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, 903-0213, Japan Abstract Bruguiera gymnorrhiza is an ecologically and somewhat economically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from B. gymnorrhiza using a dual-suppression polymerase chain reaction (PCR) technique. These loci provided microsatellite markers with polymorphism of two to five alleles per locus within 216 individuals from nine natural populations of B. gymnorrhiza on Iriomote Island, the Sakishima Islands, Japan. The expected and observed heterozygosities ranged from 0.220 to 0.720 and from 0.104 to 0.447, respectively. Keywords: mangrove, population genetics, Rhizophoraceae, SSR (simple sequence rapeod) marker Received 5 May 2005; revision received 6 June 2005; accepted 11 July 2005 Mangroves are the plant communities that develop on mud flats in the intertidal zone, i.e. between the highest and the lowest tide levels. These salt marsh forests cover approximately 181 000 km 2 along the coastline of tropical and subtropical regions in the world (Spalding et al. 1997). The importance of mangrove forests in the marine food web, their role in stabilizing sediments and protecting shorelines against erosion, and their utility to local human communities are now well recognized (Dodd & Rafii 2002). Recently, microsatellite markers have been developed and used to investigate the population genetics of mangrove trees, such as Avicennia alba (Teixeira et al . 2003), Avicennia germinans (Nettel et al . 2005) and Rhizophora stylosa (Islam et al . 2004). Bruguiera gymnorrhiza (L.), of the family Rhizophoraceae, is the most widely distributed mangrove tree species. A few microsatellite markers with low polymorphism have been developed from B . gymnorrhiza (Sugaya et al . 2003). However, to investigate the population genetics and mating patterns of this species precisely, we also developed 10 new microsatellite markers from B . gymnorrhiza . We collected leaf samples from 216 individual trees from nine natural populations of B . gymnorrhiza (24 individuals per population) on Iriomote Island of the Sakishima Islands, in the southernmost part of Japan. The leaf samples were dried with silica gel and preserved at room tempera- ture until use. Total genomic DNA was extracted from a sample leaf using a modified cetyltrimethyl ammonium bromide (CTAB) method (Zhou et al . 1999). We isolated microsatellite regions from B . gymnorrhiza using a dual-suppression polymerase chain reaction (PCR) technique (Lian & Hogetsu 2002; Islam et al . 2004). Briefly, DNA was digested separately with six blunt-end restriction enzymes ( Afa I, Alu I, Eco RV, Hae III, Hin cII and Ssp I), and then the frag- ments were ligated with a special adaptor using a DNA ligation kit (Takara Shuzo). In the first step, fragments flanked by a microsatellite at one end were amplified from the DNA library using microsatellite primers and adaptor primer AP2 (5 ′ -CTATAGGGCACGCGTGGT-3 ′ ). In the second step, primer IP1, designed from the sequenced region flanking the microsatellite, and, for nested PCR, another primer (IP2) based on the sequence between IP1 and the microsatellite were prepared. As adaptor primers for nested PCR, AP1 (5 ′ -CCATCGTAATACGACTCACTATAGGGC-3 ′ ) and AP2 were also prepared. The primary PCR was con- ducted with each constructed DNA library using primers IP1 and AP1. The secondary PCR was conducted with Correspondence: Md. Sajedul Islam, Asian Natural Environmental Science Center, The University of Tokyo, Midori-cho 1-1-8, Nishitokyo-shi, Tokyo 188-0002, Japan Fax: +81-424-65-5601; E-mail: sajed@anesc.u-tokyo.ac.jp or sajed97@yahoo.com