Characterization of 13 microsatellite loci for the tope shark, Galeorhinus galeus, discovered with next-generation sequencing and their utility for eastern Pacific smooth-hound sharks (Mustelus)
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- Chabot, C.L. & Nigenda, S. Conservation Genet Resour (2011) 3: 553. doi:10.1007/s12686-011-9402-y
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The tope shark, Galeorhinus galeus, is a commercially important member of the Triakidae that has been exploited globally for the past 80 years. Here we describe 13 microsatellite loci for G. galeus discovered by next-generation sequencing (Roche 454 pyrosequencing) and their utility for eastern Pacific smooth-hound sharks (Mustelus). These loci were polymorphic (3–12 alleles) with observed heterozygosity between 0.11 and 0.86 and expected heterozygosity between 0.24 and 0.87. Several loci (7 of 13) amplified consistently for Mustelus californicus and M. henlei. These loci are the first to be characterized explicitly for G. galeus and should be useful in the investigation of population structure of this vulnerable elasmobranch.
KeywordsGaleorhinus galeus Microsatellite Mustelus californicus Mustelus henlei Triakidae
The tope shark, Galeorhinus galeus (Triakidae), has been commercially exploited for greater than 80 years with populations demonstrating historic collapses (Ebert 2001). Continued exploitation of the species has resulted in a classification of vulnerable by the IUCN (Walker et al. 2006) and a need to determine the connectivity of globally distributed individuals in order to generate conservation strategies. Nuclear microsatellites have been used to reveal patterns of population connectivity in numerous taxa. Therefore, we have set out to generate a library of microsatellite markers for G. galeus using next-generation sequencing technology (Roche 454 pyrosequencing) in order to elucidate the patterns of population structure and gene flow in G. galeus.
DNA used for the generation of the microsatellite library was extracted from the fin clip of an Australian sample using the DNeasy blood and tissue extraction kit (Qiagen, Valencia, USA) following the manufacturer’s protocols. 500 ng of DNA was prepared for whole genome shotgun sequencing on the Roche Genome Sequencer FLX instrument utilizing the GS FLX Titanium Rapid Library Preparation Kit (Roche Applied Sciences, Indianapolis, USA) following the manufacturer’s protocol. The library was quantified for DNA fragment size distribution and concentration (Agilent 2100 Bioanalyzer) and then processed with the GS FLX emulsion polymerase chain reaction (PCR) and sequencing kits. Sequencing was performed using 1/16th of a picotiterplate and yielded 40,156 sequences.
The sequences were screened for potential microsatellite loci by MSATCOMMANDER (Faircloth 2008) under the default settings. Of the 40,156 sequences, 1,344 contained putative microsatellite loci. Similar to a previous study of the Australian gummy shark (Boomer and Stow 2010), the majority of microsatellite motifs identified were dinucleotide in nature (~80%). Primers for dinucleotide (minimum repeat number (mrn) = 8), tetranucleotide (mrn = 4), and pentanucleotide (mrn = 4) loci were designed by the PRIMER3 software (Rozen and Skaletsky 2000) embedded in MSATCOMMANDER using the default settings. In total, 32 primer pairs were used for amplification trials consisting of 18 dinucleotide, 11 tetranucleotide, and 2 pentanucleotide loci. For all loci, the forward primer was synthesized with an M13F(-20) sequence (GTAAAACGACGGCCAG) added to the 5′ end to incorporate a 5′ fluorescent label per the technique of (Boutin-Ganache et al. 2001). Initially, eight samples from four subpopulations (2 samples per population: North America (California), South Africa, Australia (Australian Bight and Tasmania), and the U.K. (Irish Sea)) were used to test amplification of loci and evaluate polymorphic content. The PCR protocol was as follows: A 10 μL touchdown PCR was performed using an Eppendorf Mastercycler epgradient S thermal cycler and the following reaction conditions: 10–100 ng template DNA, 0.2 μM reverse primer, 0.01 μM forward primer, 0.01 μM dye labeled M13 primer, 0.4 mg/mL BSA, and 5.0 μL of Qiagen Multiplex Mastermix (Qiagen, Valencia, USA). Initial denaturation was at 95°C for 15 min followed by 25 cycles of denaturation (94°C for 30 s), annealing (59°C for 90 s), extension (72°C for 60 s) and another 20 cycles of denaturation (94°C for 30 s), annealing (53°C for 90 s), extension (72°C for 60 s), and terminating with a final extension (60°C for 30 min). All PCR products were then electrophoresed on an Applied Biosystems (ABI) 3730xl DNA Analyzer. Allele sizes were determined by using an internal lane standard LIZ 500 (ABI) and GeneMapper® 3.7 (ABI). Out of the 32 primer pairs tested, 13 were successfully amplified by PCR and further characterized using additional samples from the Australian Bight and Tasmania (n = 28). In order to validate the dataset, 30% of our samples were reanalyzed at all loci producing identical genotypes between reads.
Characteristics of 13 microsatellite loci for Galeorhinus galeus
Forward primer 5′–3′
Reverse primer 5′–3′
PCR results of the 13 loci for Mustelus californicus and M. henlei
We would like to thank John Pollinger for the preparation of the 454 library and Robert K. Wayne for laboratory equipment and reagents. Funding was provided by the Southern California Academy of Sciences and the U.S. Department of Education (GAANN Fellowship).
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