Discovery and characterization of microsatellites for the solitary bee Colletes inaequalis using Sanger and 454 pyrosequencing
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The recent implementation of next-generation sequencing for the discovery of microsatellite markers has made this technology the most effective method for generating genetic markers in non-model organisms. Here, we report the de novo discovery of microsatellite markers for the solitary bee Colletes inaequalis using cloning/Sanger sequencing and direct 454 pyrosequencing from microsatellite-enriched genomic libraries. We identified and successfully multiplexed 18 highly variable microsatellite markers in 585 individuals. The number of alleles per locus ranged from 3 to 23, and the expected heterozygosity ranged from 0.056 to 0.912. These genetic markers will allow for the investigation of levels of inbreeding and fine-scale population structure in C. inaequalis. Our results contribute to the literature demonstrating that 454 sequencing is more time- and cost-efficient than cloning/Sanger sequencing at identifying a large number of genomic regions with microsatellite repeat motifs.
KeywordsSSRs cloning next-generation sequencing Colletidae
We would like to thank L. Duque, D. Dewey, J. Eitner, and J. Stitt for field assistance and A. Soro and two anonymous reviewers for comments on this manuscript. This work was supported by the Andrew W. Mellon Foundation at Cornell University (MMLU), the Sarah Bradley Fellowship (MMLU), and the Cornell Biology Research Fellowship Program (CKS). Additional funds were provided by National Science Foundation systematic grants (DEB-0814544 and DEB-0742998 to BND).
- Andrés, J.A., Bogdanowicz, S.M. (2011) Isolating microsatellite loci: looking back, looking ahead. In: Orgogozo, V., Rickman, M.V. (eds.) Molecular Methods for Evolutionary Genetics, pp. 211–232. Springer Protocols, SecaucusGoogle Scholar
- Batra, S.W.T. (1980) Ecology, behavior, pheromones, parasites and management of the sympatric vernal bees Colletes inaequalis, C. thoracicus and C. validus. J. Kans. Entomol. Soc. 53, 509–538Google Scholar
- Belkhir, K., Borsa, P., Chikhi, L., Raufaste, N., Bonhomme, F. (2004) GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations. Montpellier (France): Laboratoire Génome, Populations, Interactions, CNRS UMR 5171, Université de Montpellier IIGoogle Scholar
- Danforth, B.N., Ji, S., Ballard, L.J. (2003) Gene flow and population structure in an oligolectic desert bee, Macrotera (Macroteropsis) portalis. J. Kans. Entomol. Soc. 76, 221–235Google Scholar
- López-Uribe, M.M., Green, A.N., Ramírez, S.R., Bogdanowicz, S.M., Danforth, B.N. (2010) Isolation and cross-species characterization of polymorphic microsatellites for the orchid bee Eulaema meriana (Hymenoptera: Apidae: Euglossini). Conser. Genet. Resour. 1, 21–23Google Scholar
- Smit, A.F.A., Hubley, R., Green, P. (2010) RepeatMasker Open-3.0. Available from: http://www.repeatmasker.org
- Vereecken, N.J. (2008) Pollinator-mediated selection, reproductive isolation and floral evolution in Ophrys orchids. Proc. Neth. Entomol. Soc. Meet. 19, 9–21Google Scholar