Characterization of STR/microsatellite primers for the Gila monster, Heloderma suspectum screened from paired-end Illumi
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MICROSATELLITE LETTERS
Characterization of STR/microsatellite primers for the Gila monster, Heloderma suspectum screened from paired-end Illumina shotgun sequencing Michael R. Hess • Taylor Edwards • David A. Edmunds • Kevin E. Bonine
Received: 17 May 2013 / Accepted: 30 May 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract We used Illumina paired-end shotgun sequencing to characterize microsatellite loci in Heloderma suspectum. We identified over 124,000 potentially amplifiable loci and describe PCR primers for 18 variable triand tetra-nucleotide STRs. The observed number of alleles per locus ranged from 5 to 16 and heterozygosity varied from 0.64 to 0.92. In addition 13 of these loci crossamplified in the beaded lizard, Heloderma horridum. This method of microsatellite identification proved extremely efficient and cost effective. This novel marker set can be used by researchers to better understand these elusive species. Keywords Heloderma Microsatellite Illumina HiSeq PCR primers STR
The Gila monster, Heloderma suspectum, is an elusive reptile for which we currently lack basic population genetic information. It occurs in the southwestern United States and Northwestern Mexico and is considered ‘‘near-threatened’’ on the IUCN Red List, although data on conservation status M. R. Hess Department of Molecular and Cellular Biology, University of Arizona, Life Sciences South Building, 1007 E. Lowell Street, Tucson, AZ 85721, USA T. Edwards (&) Arizona Research Laboratories, University of Arizona Genetics Core, Thomas W. Keating Bioresearch Building, Room 111, 1657 E. Helen Street, Tucson, AZ 85721, USA e-mail: [email protected] D. A. Edmunds K. E. Bonine Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA
are lacking. Its biggest threats are likely habitat destruction from agriculture and urbanization. Through microsatellite markers we have the opportunity to better understand patterns of gene flow and potential threats from habitat fragmentation. Genotyping may also prove helpful in identifying the source populations of individuals that have been illegally collected for the pet trade. We performed a phenol–chloroform extraction on whole-blood collected from four individuals from Saguaro National Park, Tucson, Arizona, USA. We combined 0.5 lg of DNA from each individual (2 lg total) for paired-end shotgun sequencing on a HiSeq2000 (Illumina). We used a Covaris S220 to shear the DNA to a target length of 400 bp and followed standard protocols for the Illumina TruSeq DNA library kit. A single lane run generated 51,285,025 paired reads. To identify viable loci, we adapted the method of Castoe et al. (2012). We used SSR_Sorter (Jennings et al. 2011) to search for all possible tetra-, tri- and di-nucleotide motifs. We set the minimum number of repeats in our search to 5. In total, SSR_Sorter returned 12,956 and 13,021 sequence reads containing tetra- and tri-nucleotide motifs, respectively, and 98,935 reads containing di-nucleotide microsatellites.
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