The Use of Sequence-Based SSR Mining for the Development of a Vast Collection of Microsatellites in Aquilegia formosa


Numerous microsatellite markers were developed forAquilegia formosafrom sequences deposited within the Expressed Sequence Tag (EST), Genomic Survey Sequence (GSS), and Nucleotide databases in NCBI. Microsatellites (SSRs) were identified and primers were designed for 9 SSR containing sequences in the Nucleotide database, 3803 sequences in the EST database, and 2226 sequences in the GSS database. For validation purposes, 45 primer pairs were used to amplify DNA from 16A. formosaindividuals from the H. J. Andrews experimental forest in Oregon, a Long Term Ecological Research (LTER) site. Genetic polymorphisms were identified at 30 of the 45 microsatellite loci with an average of 13.2 alleles per locus, and the observed level of heterozygosity was greater than 0.8 for 21 of the 30 loci. The use of these polymorphic loci was sufficient to individually separate the 16 individuals using a principal coordinate analysis. This comprehensive collection of primers significantly increased the availability of microsatellite primers forAquilegiaspp. and provided ample material for future studies that required highly variable SSRs such as mapping and association studies and investigation of plant mating system and gene flow.

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Schlautman, B. , Pfeiffer, V. , Zalapa, J. and Brunet, J. (2014) The Use of Sequence-Based SSR Mining for the Development of a Vast Collection of Microsatellites in Aquilegia formosa. American Journal of Plant Sciences, 5, 2402-2412. doi: 10.4236/ajps.2014.515253.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Bastida, J.M., Alcantara, J.M., Rey, P.J., Vargas, P. and Herrera, C.M. (2010) Extended Phylogeny of Aquilegia: The Biogeographical and Ecological Patterns of Two Simultaneous but Contrasting Radiations. Plant Systematics and Evolution, 284, 171-185.
[2] Hodges, S.A. and Arnold, M. (1994) Columbines: A Geographically Widespread Species Flock. Proceedings of the National Academy of Sciences, 91, 5129-5132.
[3] Whittall, J.B. and Hodges, S.A. (2007) Pollinator Shifts Drive Increasingly Long Nectar Spurs in Columbine Flowers. Nature, 447, 706-710.
[4] Fior, S., Li, M., Oxelman, B., Viola, R., Hodges, S.A., Ometto, L. and Varotto, C. (2013) Spatiotemporal Reconstruction of the Aquilegia Rapid Radiation through Next-Generation Sequencing of Rapidly Evolving cpDNA Regions. New Phytologist, 198, 579-592.
[5] Brunet, J. and Eckert, C.G. (1998) Effect of Floral Morphology and Display on Outcrossing in Blue Columbine, Aquilegia caerulea (Ranunculaceae). Functional Ecology, 12, 596-606.
[6] Fulton, M. and Hodges, S.A. (1999) Isolation between Aquilegia formosa and Aquilegia pubescens. Proceeding of the Royal Society of London B, 266, 2247-2252.
[7] Herlihy, C.R. and Eckert C.G. (2005) Evolution of Self-Fertilization at Geographical Range Margins? A Comparison of Demographic, Floral, and Mating System Variables in Central vs. Peripheral Populations of Aquilegia canadensis (Ranunculaceae). American Journal of Botany, 92, 744-751.
[8] Brunet, J. and Sweet, H.R. (2006) Impact of Insect Pollinator Group and Floral Display Size on Outcrossing Rate. Evolution, 60, 183-195.
[9] Herlihy, C.R. and Eckert, C.G. (2007) Evolutionary Analysis of a Key Floral Trait and Its Effect on the Mating System in Aquilegia canadensis (Ranunculaceae). Evolution, 61, 1661-1674.
[10] Brunet, J. (2009) Pollinators of the Rocky Mountain Columbine: Temporal Variation, Functional Groups and Associations with Floral Traits. Annals of Botany, 103, 1567-1578.
[11] Brunet, J. and Larson-Rabin, Z. (2012) The Response of Flowering Time to Global Warming in a High-Altitude Plant: The Impact of Genetics and the Environment. Botany, 90, 319-326.
[12] Van Etten, M.L. and Brunet, J. (2013) The Impact of Global Warming on Floral Traits That Affect the Selfing Rate in a High-Altitude Plant. International Journal of Plant Sciences, 174, 1099-1108.
[13] Chase, V.C. and Raven, P.H. (1975) Evolutionary and Ecological Relationships between Aquilegia formosa and A. pubescens (Ranunculaceae), Two Perennial Plants. Evolution, 29, 474-486.
[14] Kramer, E.M., Holappa, L., BillieGould, M., Jaramillo, A., Setnikov, D. and Santiago, P.M. (2007) Elaboration of B Gene Function to Include the Identity of Novel Floral Organs in the Lower Eudicot Aquilegia. The Plant Cell, 19, 750-766.
[15] Voelckel, C., Borevitz, J.O., Kramer, E.M. and Hodges, S.A. (2010) Within and between Whorls: Comparative Transcriptional Profiling of Aquilegia and Arabidopsis. PLoS ONE, 5, e9735.
[16] Kramer, E.M. (2009) Aquilegia: A New Model for Plant Development, Ecology, and Evolution. Annual Review of Plant Biology, 60, 261-277.
[17] Kramer, E.M. and Hodges, S.A. (2010) Aquilegia as a Model System for the Evolution and Ecology of Petals. Philosophical Transactions of the Royal Society B, 365, 477-490.
[18] Fang, G.C., Blackmon, B.P., Henry, D.C., Staton, M.E., Saski, C.A., Hodges, S.A., Tomkins, J.P. and Luo, H. (2010) Genomic Tools Development for Aquilegia: Construction of a BAC-Based Physical Map. BMC Genomics, 11, 621.
[19] Yang, J.Y., Counterman, B.A., Eckert, C.G. and Hodges, S.A. (2005) Cross-Species Amplification of Microsatellite loci in Aquilegia and Semiaquilegia (Ranunculaceae). Molecular Ecology Notes, 5, 317-320.
[20] Gallagher, K.G., Milligan, B.G. and White, P.S. (2004) Isolation and Characterization of Microsatellite DNA Loci in Aquilegia sp. Molecular Ecology Notes, 4, 686-688.
[21] Li, L.F., Pang, D., Liao, Q.L. and Xiao, H.X. (2011) Genomic and EST Microsatellite Markers for Aquilegia flabellata and Cross-Amplification in A. oxysepala (Ranunculaceae). American Journal of Botany, 98, e213-e215.
[22] Brunet, J., Larson-Rabin, Z. and Stewart, C.M. (2012) The Distribution of Genetic Diversity within and among Populations of the Rocky Mountain Columbine: The Impact of Gene Flow, Pollinators, and Mating System. International Journal of Plant Sciences, 174, 1099-1108.
[23] Garrido, J.L., Fenu, G., Mattana, E. and Bacchetta, G. (2012) Spatial Genetic Structure of Aquilegia Taxa Endemic to the Island of Sardinia. Annals of Botany, 109, 953-964.
[24] Brunet, J. and Holmquist, K.G.A. (2009) Influence of Distinct Pollinators on Female and Male Reproductive Success in the Rocky Mountain Columbine. Molecular Ecology, 18, 3745-3758.
[25] da Maia, L.C., Palmieri, D.A., de Sonza, V.Q., Kopp, M.M., de Carvalho, F.I. and de Oliveira, A.C. (2008) SSR Locator: Tool for Simple Sequence Repeat Discovery Integrated with Primer Design and PCR Simulation. International Journal of Plant Genomics, 2008, 412696-412705.
[26] Martins, W.S., Lucas, D.C.S., Neves, K.F.S. and Bertioli, D.J. (2009) WebSat—A Web Software for MicroSatellite Marker Development. Bioinformation, 3, 282-283.
[27] Schuelke, M. (2000) An Economic Method for the Fluorescent Labeling of PCR Fragments. Nature Biotechnology, 18, 233-234.
[28] Zalapa, J., Cuevas, H., Zhu, H., Steffan, S., Senalik, D., Zeldin, E. McCown, B., Harbut, R. And Simon, P. (2012) Using Next-Generation Sequencing Approaches to Isolate Simple Sequence Repeat (SSR) Loci in the Plant Sciences. American Journal of Botany, 99, 193-208.
[29] Peakall, R. and Smouse, P.E. (2006) GENALEX 6: Genetic Analysis in Excel. Population Genetic Software for Teaching and Research. Molecular Ecology Notes, 6, 288-295.
[30] Kalinowski, S.T., Taper, M.L. and Marshall, T.C. (2007) Revising How the Computer Program CERVUS Accommodates Genotyping Error Increases Success in Paternity Assignment. Molecular Ecology, 16, 1099-1006.
[31] Fu, Y.B., Peterson, G.W., Richards, K.W., Tarn, T.R. and Percy, J.E. (2009) Genetic Diversity of Canadian and Exotic Potato Germplasm Revealed by Simple Sequence Repeat Markers. American Journal of Potato Research, 86, 38-48.
[32] Mujaju, C., Sehic, J. and Nybom, H. (2013) Assessment of EST-SSR Markers for Evaluating Genetic Diversity in Watermelon Accessions from Zimbabwe. American Journal of Plant Sciences, 4, 1448-1456.
[33] Yang, J.Y. and Hodges, S.A. (2010) Early Inbreeding Depression Selects for High Outcrossing Rates in Aquilegia formosa and Aquilegia pubescens. International Journal of Plant Sciences, 171, 860-871.
[34] Hamblin, M.T., Warburton, M.L. and Buckler, E.S. (2007) Empirical Comparison of Simple Sequence Repeats and Single Nucleotide Polymorphisms in Assessment of Maize Diversity and Relatedness. PLoS ONE, 2, Article ID: e1367.
[35] Adam-Blondon, A.F., Roux, C., Claux, D., Butterlin, G., Merdinoglu, D. and This, P. (2004) Mapping 245 SSR Markers on the Vitis vinifera Genome: A Tool for Grape Genetics. Theoretical and Applied Genetics, 109, 1017-1027.

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