Spotted bat ( Euderma maculatum ) microsatellite discovery using illumina sequencing
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Spotted bat (Euderma maculatum) microsatellite discovery using illumina sequencing Faith M. Walker • Jeffrey T. Foster • Kevin P. Drees • Carol L. Chambers
Received: 3 December 2013 / Accepted: 25 December 2013 Ó Springer Science+Business Media Dordrecht 2014
Abstract The spotted bat (Euderma maculatum) is a rarely-encountered species for which behavior and population attributes are largely unknown. Using next-generation sequencing, we identified and characterized 17 microsatellite loci, which were screened for 31 individuals from northern Arizona. Allelic diversity, observed heterozygosity, and power of discrimination were high (NA: 5–8 alleles per locus; HO: 0.55–0.90; PID: 1.2 9 10-15). All loci were in HWE, there was no evidence of null alleles or linkage disequilibrium, and five loci amplified and were variable in another Vespertillionid (Eptesicus fuscus). We will use these loci to evaluate gene flow and genetic diversity across the range of the spotted bat and determine population size in northern Arizona. The latter information is important to resource managers, who attempt to set mortality thresholds for bats at wind energy facilities in this region. Keywords Euderma maculatum Spotted bat Next-generation sequencing Microsatellite
The spotted bat (Euderma maculatum) is a charismatic species patchily distributed across western North America. Because spotted bats are cryptic (nocturnal, volant, solitary), much of their natural history and population biology F. M. Walker (&) C. L. Chambers School of Forestry, Northern Arizona University, 200 East Pine Knoll Dr., Flagstaff, AZ 86011, USA e-mail: [email protected] F. M. Walker J. T. Foster K. P. Drees Center for Microbial Genetics and Genomics, Northern Arizona University, Bldg. 56, 3rd floor, 1298 S Knoles Dr., Flagstaff, AZ 86011-4073, USA
is unknown; E. maculatum has been designated a species of concern in Canada and the United States, in part because of this lack of information. Using high-throughput sequencing, we generated a suite of microsatellite markers to elucidate aspects of E. maculatum biology. Genomic DNA was extracted from an E. maculatum wing punch with a DNEasy Blood and Tissue Kit (Qiagen, Hilden, DEU). DNA was fragmented with a SonicMan sonicator (Brooks Life Science Systems, Spokane, WA). Whole genome sequencing libraries were prepared and quantified with qPCR using KAPA reagents (KAPA Biosystems, Woburn, MA). Fragments 500 bp long were selected with Agencourt AMPure magnetic beads (Beckman Coulter, Brea, CA). Sequencing was performed on an illumina MiSeq with v2 reagents, yielding 13 million paired 250 bp reads. We used ABySS 1.3.2 (Simpson et al. 2009) for de novo genome assembly. Microsatellites 4–6 bp in length with at least six repeats were discovered in this assembly and primers for the loci were designed with msatcommander-1.0.8-beta (Faircloth 2008). We tested 56 primer pairs using the universal tail PCR labeling system of U’Ren et al. (2007). DNA from heart and kidney tissue samples of five
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