Molecular Ecology Notes (2002) 2, 38 – 41 © 2002 Blackwell Science Ltd Blackwell Science, Ltd PRIMER NOTE Twenty-seven new microsatellites for the migratory Asian catfish family Pangasiidae ZEB S. HOGAN* and BERNIE P. MAY† *Department of Wildlife, Fish, and Conservation Biology, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA, †Department of Animal Science, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA Abstract In this paper, we describe primers for 27 new microsatellite loci tested on five species of migratory Asian catfish: Pangasius krempfi, P. bocourti, P. conchophilus, P. pleurotaenia, and Helicophagus waandersii. These primers were developed from a (GATA) n library cre- ated from the pooled DNA of three species of pangasiid catfish. All 27 loci are polymorphic in at least one species. Fifteen loci are polymorphic in at least three species. The primers described in this paper are thus shown to be useful in several species within the catfish family Pangasiidae, and may prove useful in additional species in future tests. Keywords: catfish, Mekong River, microsatellites, Pangasiidae, Pangasius, Helicophagus Received 14 May 2001; revision accepted 13 September 2001 The Asian catfish family Pangasiidae comprises more than 20 species, including at least 12 species found in the Mekong River Basin, Southeast Asia (Pouyard et al. 2000). Within this region, pangasiid catfish are one of the dominant food fish, popular in aquaculture and frequently the basis of local wildcapture fisheries. Well known for large and sometimes spectacular migrations, pangasiid catfish move extensively between Vietnam, Cambodia, Laos, and Thailand (Lieng et al. 1995; Baird et al. 2001). Pangasiid catfish fisheries are probably the largest and most complex catfish fisheries in the world. Today, over- fishing, dam building, and habitat degradation threaten several of these species (Dudgeon 1992; Hogan 1998). Although DNA technology is an important tool in the management of many fisheries, the application of popu- lation genetic data to management in the developing countries of Southeast Asia is only now beginning. For this reason, almost no information exists about the genetic population structure or current levels of gene flow occur- ring within Mekong species. Here, we describe 27 new primer pairs designed to amplify microsatellite loci. These primers were developed for population genetic studies of several species of pangasiid catfish of the Mekong River Basin. Two subgenomic libraries were constructed by Genomic Identification Services (Chatsworth, California, USA) using a mixture of equal masses of DNA of three species: Pangasius larnaudei, P. conchophilus, and P. pleurotaenia. DNA was extracted from dried fin using a standard TNES- urea procedure of Belfiore & May (2000), quantified and diluted, then mixed 1:1:1 in a buffer solution (final concen- tration 100 μg/100 μL in 25 mm Tris plus 2.5 mm EDTA, pH 8.0; 125 μL total). We mixed DNA of three species to minimize the cost of library construction per species and thus ultimately created a single multispecies library. We also suspected that pooling DNA would decrease the chance of sequencing identical clones during the screening process. Over 90% (115 of a total 126) of the clones from this multispecies library contained unique sequences, whereas single species libraries in our laboratory typically average about 60 –70% unique sequences. Aside from the use of pooled DNA, the library construction followed the proced- ure described in (Belfiore & May 2000). Of the clones initially screened, 100% (n = 5) (GATA) n and 100% (n = 7) (CA) n contained microsatellites. In addition, approximately 300 recombinant clones from the (GATA) n library were sampled and amplified in 15 μL polymerase chain reactions (PCR) containing 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 5 mm MgCl 2 , 2.5 mm dNTPs, 0.3 μL pUC forward and reverse sequencing primers, and 0.5 U Taq DNA polymerase (Gibco). The PCR reactions were amplified using an MJ Research PTC-100 96 V thermocycler according to the fol- lowing themocycler profile: 94 °C for 4 min 30 s, 25 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 30 s, and 72 °C for Correspondence: Zeb S. Hogan. Fax: 1 530 752 0175; E-mail: zshogan@ucdavis.edu