Molecular Ecology Resources (2008) doi: 10.1111/j.1755-0998.2008.02246.x © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd M E R 2246 Operator: Luo Xiaohua Dispatch: 15.07.08 PE: Crystal Chan Journal Name Manuscript No. Proofreader: Chen Xiaoming No. of Pages: 4 Copy-editor: Apple Rosales 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES New polymorphic tetranucleotide microsatellites improve scoring accuracy in bottlenose dolphin Tursiops aduncus ALEXANDER NATER, ANNA M. KOPPS and MICHAEL KRÜTZEN Anthropological Institute and Museum, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Abstract We isolated and characterized 19 novel tetranucleotide microsatellite markers in the Indo- Pacific bottlenose dolphin (Tursiops aduncus) in order to improve genotyping accuracy in applications like large-scale population-wide paternity and relatedness assessments. One hundred T. aduncus from Shark Bay, Western Australia, were screened for polymorphism. Cross-amplification was tested on four other small odontocete species. The new tetranucleotide microsatellite loci showed a more than fourfold higher scoring accuracy and significantly lower stutter formation compared to eight dinucleotide loci, although overall allelic diversity was significantly reduced. Keywords: bottlenose dolphin, genotyping accuracy, stutter bands, tetranucleotide microsatellites, Tursiops aduncus Received 5 March 2008; revision received 28 March 2008; revision accepted 16 April 2008 Bottlenose dolphins (Tursiops sp.) are among the best- studied inshore cetaceans worldwide. Detailed genetic studies from different bottlenose dolphin populations (e.g. Möller et al. 2001; Krützen et al. 2004; Natoli et al. 2004) have led to insights into the evolution of their intricate social system, mating strategies and population demography. With a few exceptions (Palsboll et al. 1999), almost all microsatellite markers employed in cetacean studies feature dinucleotide repeat motifs, such as (CA) n . This is surprising, as problems associated with polymerase chain reaction (PCR) artefacts, such as incorrect allele scoring due to stutter bands (Litt & Luty 1989; Hauge & Litt 1993; Litt et al. 1993), have been shown to have a significant impact on the accuracy of genotyping studies in natural populations (Hoffman & Amos 2005). Simulations have demonstrated that error rates as low as 1% may result in a rate of incorrect paternity exclusion exceeding 20% (Hoffman & Amos 2005). Because of the problems associated with the interpretation of stutter bands, the forensic community has long switched to tetranucleotide motifs, such as (GATA) n , in which stuttering is highly reduced (Edwards et al. 1991). Here we describe the development of 19 novel tetranucleotide microsatellite loci from the Indo- Pacific bottlenose dolphin. We successfully tested these loci for cross-amplification in four other odontocete species. We also compare the scoring error rate of our new loci with that of eight dinucleotide loci, which we employed previously (e.g. Krützen et al. 2005) and specifically retyped for this study. Tissue samples were collected by biopsy sampling (Krützen et al. 2002) in Shark Bay, Western Australia, and stored in a saturated NaCl/20% (v/v) dimethyl sulphoxide solution (Amos & Hoelzel 1991) until further processing in the laboratory. Total genomic DNA was isolated with the Gentra Puregene DNA Purification Kit (QIAGEN) and quantified with an ND-1000 spectrophotometer (NanoDrop). Ten micrograms of genomic DNA from a male individual was digested with NheI and RsaI (both New England Biolabs) and size selected for fragment sizes between 400 bp and 1200 bp. DNA fragments were processed with mung bean nuclease and calf intestine phosphatase (both New England Biolabs), followed by ligation of an SNX linker to both ends (Hamilton et al. 1999). Enrichments were performed by hybridization with biotinylated oligonucle- otide probes [(GACA) 7 (GATA) 7 and (GATC) 7 ] containing a 3′-dideoxy nucleotide to prevent probes from acting as primers in subsequent PCRs (Koblizkova et al. 1998). M- 280 streptavidin-coated Dynabeads (Dynal) were used to capture biotinylated oligos. After PCR and clean-up with a QIAquick PCR Purification Kit (QIAGEN), the enrichment procedure was repeated in order to enhance enrichment efficiency. Ligation of the enriched fragments into a pGEM-3Z Correspondence: Alexander Nater, Fax: +41 44 635 68 04; E-mail: a.nater@aim.uzh.ch