Isolation and characterization of twelve microsatellite loci for the Japanese Devilray (Mobula japanica)
Twelve polymorphic microsatellites loci were characterized for Mobula japanica (Japanese Devilray) using an enrichment protocol. All but two loci were in Hardy–Weinberg equilibrium with no evidence of linkage disequilibrium or null-alleles for a sample of 40 individuals from two populations. The number of alleles varied from 5 to 28. Expected heterozygosity ranged from 0.2332 to 0.9589, making these microsatellite loci good candidates for population genetic studies.
KeywordsElasmobranch Mobula japanica Microsatellite Population genetics Polymorphism
The Japanese Devilray (Mobula japanica) is believed to have a circumglobal distribution throughout all temperate and tropical seas, although genetic analyses may identify separate populations or even cryptic species over such a wide range (Notarbartolo-di-Sciara 1987). The species reaches a disc width (DW, measured from wingtip to wingtip) of 310 cm. It is mainly pelagic and found inshore, offshore and, possibly, in oceanic environments (Last and Stevens 1994). M. japanica is listed as “Near Threatened” by the International Union for Conservation of Nature (IUCN: www.iucn.org/redlist), due to high (by) catch rates, increasing demand and low reproductive potential. Therefore, data regarding current genetic structure and migration patterns are needed to design effective conservation strategies (Graves 1998). Species-specific microsatellite markers provide a means of obtaining these data for threatened and endangered taxa. Here we report on the isolation and characterization of 12 novel microsatellite loci in M. japanica.
Genomic libraries enriched for microsatellite motifs were constructed by Genetic Identification Services (GIS, http://www.genetic-id-services.com; Chatsworth, CA, USA). Libraries were built using a sample containing 100 μg of genomic DNA extracted from tail tissue of a single individual M. japanica collected in El Pardito, Baja California Sur, Mexico. The sample was stored in 90% ethanol and extracted using a Qaigen Blood and Tissue DNA purification kit. Libraries were enriched for CA, CATC, TACA, TAGA motifs. GIS sequenced 54 microsatellite-containing clones using universal M13 primers, and designed primers using DesignerPCR version 1.03 (Research Genetics, Inc.).
We tested these 54 microsatellites, using a dye-labeled universal primer system (Schuelke 2000) with an M13 tagged tail (5′-CAC GAC GTT GTA AAA CGA C-3′) added to the 5′ end of the forward primer. Amplification reactions were carried out in a single nested reaction on an Applied Biosystems GeneAmp PCR 9700 in a total volume of 12 μL containing 1× PCR Mastermix (2.5 mM TAPS pH9.5, 5.0 mM KCl, 0.2 mM MgCl2, 20.0 μM of each dNTP, Taq 0.5u/μL, Thermo Scientific), 2 pmols of the M13 labeled forward primer, 9 pmol of the reverse primer, 9 pmols of the fluorescently-labeled M13 primer (Fluo, Tamra, Hex; Sigma-Genosys) and approximately 2 ng of DNA template. The following PCR temperature profile was used: 5 min at 94°C, followed by 10 cycles of 30 s at 94°C, 45 s at the primer specific Ta, 45 s at 72°C, followed by 20 cycles of 30 s at 94°C, 45 s at ((primer specific Ta) minus 2°C), 45 s at 72°C and a final extension of 72°C for 10 min. Microsatellite amplifications were mixed with Applied Biosystems GeneScan 500 Rox size standard and then run on an ABI 3100 automated sequencer, and scored using the software GENEMAPPER3.7 (Applied Biosystems). Twelve out of the original 54 loci produced successful PCR amplification. Locus-specific dye-labeled primers (6FAM, NED, PET, VIC: Applied Biosystems) were used for those 12 loci.
Characterization of twelve polymorphic microsatellite loci in Mobula japanica
Genbank acc. no.
Primer sequence (5′–3′)
(CA)8 TACGC (CA)4 CG (CA)5 CG (CA)4 (CG)2 (CA)5 (CG)2 (CA)4
(CA)3 AA (CA)30 CCT (CA)2
(GT)3 TTG (GT)14 TTATTGTGCGTATTT (GT)3 TTA (GT)4 GCTAAT (TC)2 CATTTTG (GT)3
(CA)4 TG (CA)13
(CA)3 TTCATTCAAAA (CA)2 TACATA (CA)2 CGTA (CA)2 GATATC (CA)2 GGCATAGTCATGTATA (CA)23
(GTAT)4 AT (GTAT)3
(CT)5 (ATCT)4 CT (ATCT)3 AC (CT)3 ATCTGTCTATCTT (CT)3 CCTT (CT)2
(TAGA)7 TTGACAGA (TAGA)5 CAGA (TAGA)2 (CAGA)2 TAGACAGA (TAGA)2 CAAATAGACAGATAGATAGG (TAGA)3 TTGA (CAGA)2 TAGATAAA (CAGA)2 TAGA (CAGA)2 TAGACAGA (TAGA)2 CAAATAGACAGATAGATAGG (TAGA)2
This research was funded by the Monterey Bay Aquarium and a PhD grant to MP from the University of Groningen’s TopMaster-Evolutionary Biology Program. We would like to thank Devon Pearse for advice on fluorescent labeling of PCR products; Colombo Estupiñán-Montaño for collection of samples in Ecuador; Pablo Cuevas, Felipe Cuevas and Juan Cuevas for providing help with fieldwork in Mexico; and Island Conservation for help with fieldwork. FGM thanks Instituto Politécnico Nacional (COFAA and EDI) for a fellowship.
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