|SCHLAUTMAN, BRANDON - UNIVERSITY OF WISCONSIN|
|PFEIFFER, VERA - UNIVERSITY OF WISCONSIN|
Submitted to: American Journal of Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2014
Publication Date: 7/16/2014
Publication URL: http://handle.nal.usda.gov/10113/59548
Citation: Schlautman, B., Pfeiffer, V., Zalapa, J., 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(15):2402-2412.
Interpretive Summary: Western Columbine, Aquilegia formosa, is a perennial herb from the family Ranunculaceae. It belongs to the genus Aquilegia, which includes approximately 75 species with similar number of species occurring in North America, Asia and Europe. As a result of its variation in ecology, floral morphology, pollination, floral development and mating system, the genus Aquilegia is ascending to the status of model species for studies in ecology, evolution and plant development. To improve its usefulness as a model species, genomic tools are being developed for this genus, including the recent release of A.coerulea Goldsmith whole genome sequence. Despite the recent development of nuclear tools for Aquilegia, the availability of microsatellite loci remains limited. Therefore, an extensive number of microsatellite primers located in transcribed and genomic regions were developed for A.formosa. This collection is the most comprehensive set of microsatellite primer-pairs developed in Aquilegia to date. Moreover, our approach illustrates how sequence-based SSR mining results in the identification of many more microsatellites of more diverse motif and repeat types than enrichment based on SSR capture. This collection of microsatellites contributes to the various molecular tools being developed for Aquilegia. Given the extensive knowledge of the ecology, pollination biology, floral biology and mating system of some Aquilegia species, these molecular tools will benefit researchers mapping functional genes or studying selection, population genetic structure, or gene flow in Aquilegia species.
Technical Abstract: Numerous microsatellite markers were developed for Aquilegia formosa from sequences deposited within the Expressed Sequence Tag (EST), Genomic Survey Sequence (GSS), and Nucleotide databases in NCBI. Microsatellites (SSRs) were identified and primers designed for 9 SSR containing sequences in the Nucleotide Database, 3,803 sequences in the EST database and 2,226 sequences in the GSS database. For validation purposes, 45 primer-pairs were used to amplify DNA from 16 A. formosa individuals 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. A high level of heterozygosity was observed at the majority of these loci. The use of 10 of these polymorphic loci was sufficient to individually separate the 16 individuals using a principal coordinate analysis. This comprehensive collection of primers significantly increases the availability of microsatellite primers for Aquilegia spp. and provides ample material for future studies that require highly variable SSRs such as mapping and association studies and investigation of plant mating system and gene flow.