The Use of Sequence-Based SSR Mining for the Development of a Vast Collection of Microsatellites in &amp;lt;i&amp;gt;Aquilegia formosa&amp;lt;/i&amp;gt;

Brandon Schlautman,Juan Zalapa,Vera Pfeiffer,Johanne Brunet

doi:10.4236/ajps.2014.515253

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 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 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, 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 for Aquilegia spp. 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.

Highlights

The western columbine, Aquilegia formosa, is a perennial herb from the family Ranunculaceae
We identified microsatellites (SSRs) and designed primers from the Nucleotide database, the Expressed Sequence Tag (EST) database, and the Genomic Survey Sequence (GSS) database through NCBI, and validated the screening by testing 45 of the primers using a wild A. formosa population
Thirty of the 45 SSR primer pairs tested amplified in the expected size range (Table 1) and displayed polymorphism across individuals (Nucleotide: 6, GSS: 10, and EST: 14 primer pairs; (Table 2))

Summary

Introduction

The western columbine, Aquilegia formosa, is a perennial herb from the family Ranunculaceae. Two independent radiation events have emerged, one leading to the North American and the other to the European clades [1] [4]. Habitat specialization may have played a greater role in the radiation of columbines in Europe while pollinator specialization has been more important in the diversification of North American species [1] [3]

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Conclusion