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Characterization of tetranucleotide microsatellite

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Molecular Ecology Notes (2003) 3, 244–246 doi: 10.1046/j.1471-8286.2003.00412.x© 2003 Blackwell Publishing LtdBlackwell Publishing Ltd.PRIMER NOTECharacterization of tetranucleotide microsatellite loci in the African Savannah Elephant (Loxodonta africana africana)E. A. ARCHIE,* C. J. MOSS† and S. C. ALBERTS**Biology Department, Duke University, Box 90338, Durham, NC 27708, USA, †Amboseli Elephant Research Project, PO Box 15135, Langata 00509, Nairobi, Kenya AbstractMost African elephant (Loxodonta africana africana) populations are isolated and thusthreatened by a loss of genetic diversity. As a consequence, genetic analysis of African ele-phant populations will play an increasing role in their conservation, and microsatellite lociwill be an important tool in these analyses. Previously published sets of polymorphic micro-satellites developed for African elephants are all dinucleotide repeats, which are prone totyping error. Here, we characterize 11 tetranucleotide microsatellite loci in the African ele-phant. All loci were polymorphic in 32 faecal samples and two tissue samples from 33 indi-vidual African savannah elephants.Keywords: African elephant, Loxodonta africana, tetranucleotide microsatellites Received 23 November 2002; revision accepted 17 January 2003African elephants (Loxodonta africana africana) are acharismatic, economically important, endangered species.Their populations currently exist in small isolated pocketsof habitat, and this threatens elephant genetic diversity(Georgiadis et al. 1994). Effective management of elephantpopulations requires efficient, noninvasive typing of indi-viduals at a reliable set of polymorphic genetic markers,in order to measure genetic diversity within and amongpopulations.All previously published microsatellite markers forAfrican elephants are dinucleotide repeats (Nyakaana &Arctander 1998; Comstock et al. 2000). These markers aresubject to mistyping due to polymerase slippage duringpolymerase chain reaction (PCR) (Taberlet et al. 1999). Thisproblem is especially acute when template DNA is oflow quality or concentration, as with faecal samples ordegraded tissue samples (Taberlet et al. 1999; Morin et al.2001). Consequently, tetranucleotide microsatellites aremore reliable markers for noninvasive genotyping (forexample see Fernando et al. 2001). In this paper we describethe isolation and characterization of 11 polymorphic tetra-nucleotide markers for African elephants.Whole blood samples were collected from two Africanelephants at the North Carolina State Zoo. Genomic DNAwas extracted from both individuals using a Puregenegenomic DNA isolation kit (Gentra). The resulting DNAwas digested with four restriction enzymes, Sau96I, FokI,HinCIII and DraIII, pooled, and sent to Genetic Identifica-tion Services (GIS, Chatsworth, CA, USA). GIS constructedsix libraries each enriched for one of the following repeatsequences: AAAG, TACA, ATG, GGAA and CATC. Positivecolonies were amplified with universal M13 primers,and clones with inserts between 350 and 700 bp weresequenced. Thirty-seven sequences provided by GIS and60 additional clones sequenced in our laboratory yielded 49unique sequences with repeat motifs. Two CATC enrichedlibraries were the richest sources of lengthy (greaterthan 10 repeats) tetranucleotide microsatellites. Primerswere designed for 23 unique loci using the web-basedprograms web primer (http://genome-www2.stanford.edu/cgi-bin/SGD/web-primer) and primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_http://www.cgi).Each pair of primers was tested on template DNAderived from both elephant tissue (n = 2) and elephant fae-ces (n = 32), representing a total of 33 different individualsfrom Amboseli National Park, Kenya. One individual wasrepresented by both tissue and faeces. We also includedfive known mother–daughter pairs for Mendelian checks.These individuals, and replication, provided checks offaecal genotypes.Correspondence: E. A. Archie. Fax: (919) 660–7293; E-mail:[email protected] NOTE 245© 2003 Blackwell Publishing Ltd, Molecular Ecology Notes, 3, 244–246Faecal samples were collected within 10 min of defeca-tion and stored in 95% ethanol. Tissue samples were col-lected from animals found dead of natural causes. DNAwas extracted from tissue using a DNeasy Tissue Kit (Qia-gen). Faecal DNA was extracted using a QIAamp DNAStool Mini Kit (Qiagen). The following modifications weremade to the protocol for the Stool Kit: (i) the initial samplevolume was approximately 800 µL; (ii) samples weredigested overnight in 1.0 mL of buffer ASL with 1 mg ofproteinase K in a 55 °C incubator shaking at 225 r.p.m.; (iii)the samples were not digested with proteinase K in bufferAL; and (iv) a combined volume of 1200– 1800 µL of super-natant fluid, buffer AL and 95% ethanol was centrifugedthrough each spin column. DNA was eluted in 100 µL ofbuffer AE.Microsatellites were amplified in 10 µL reaction volumeseach containing 1.5 mm MgCl2, 200 µm dNTP, 10 µg ofbovine serum albumin (BSA), 2.5 pm of each primer (for-ward primer labelled with fluorescent dyes FAM, VIC orNED; Applied Biosystems), 0.4 U of Amplitaq Gold DNApolymerase (Applied Biosystems) and 1 µL of extractedDNA. PCRs were performed in an MJ Research PTC-200Peltier Thermal Cycler. The thermal profile for PCR ampli-fication for LaT05, 07, 08, 13, 17 and 24 was 95 °C for10 min, followed by one cycle of 95 °C for 30 s, 66 °Cannealing temperature for 30 s and 72 °C for 30 s. In thesubsequent 10 cycles, all conditions were the same exceptthat the annealing temperature decreased by one degreeper cycle. This was followed by 30 cycles of 95 °C for 30 s,56 °C for 30 s and 72 °C for 30 s. The thermal profile endedwith a single extension of 72 °C for 5 min. For LaT06, 16, 25and 26 the thermal profile for PCR amplification was 95 °Cfor 10 min, then 40 cycles of 95 °C for 30 s, 52 °C for 30 s and72 °C for 30 s, followed by a single extension of 72 °C for5 min.Of 26 tested pairs of primers (designed for 23 loci),11 pairs amplified polymorphic tetranucleotide micro-satellites (Table 1). PCR products were separated usingan ABI PRISM® 3700 DNA Analyser. Allele sizes weredetermined using genescan (ABI) software. Polymorphismranged between six and 15 alleles (Table 1).Of the resulting 374 genotypes (34 DNA extracts at 11loci), 282 (75.40%) were replicated between one and fourtimes. There was an exact correspondence between the faecaland tissue genotypes for


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