Virus-induced Gene Silencing as a Tool for Functional Analyses in the Emerging Model Plant Aquilegia (Coumbine, Ranunculaceae)

Background: The lower eudicot genus Aquilegia, commonly known as columbine, is currently the subject of extensive genetic and genomic research aimed at developing this taxon as a new model for the study of ecology and evolution. The ability to perform functional genetic analyses is a critical component of this development process and ultimately has the potential to provide insight into the genetic basis for the evolution of a wide array of traits that differentiate flowering plants. Aquilegia is of particular interest due to both its recent evolutionary history, which involves a rapid adaptive radiation, and its intermediate phylogenetic position between core eudicot (e.g., Arabidopsis) and grass (e.g., Oryza) model species. Results: Here we demonstrate the effective use of a reverse genetic technique, virus-induced gene silencing (VIGS), to study gene function in this emerging model plant. Using Agrobacterium mediated transfer of tobacco rattle virus (TRV) based vectors, we induce silencing of PHYTOENE DESATURASE (AqPDS) in Aquilegia vulgaris seedlings, and ANTHOCYANIDIN SYNTHASE (AqANS) and the B-class floral organ identity gene PISTILLATA in A. vulgaris flowers. For all of these genes, silencing phenotypes are associated with consistent reduction in endogenous transcript levels. In addition, we show that silencing of AqANS has no effect on overall floral morphology and is therefore a suitable marker for the identification of silenced flowers in dual-locus silencing experiments. Conclusion: Our results show that TRV-VIGS in Aquilegia vulgaris allows data to be rapidly obtained and can be reproduced with effective survival and silencing rates. Furthermore, this method can successfully be used to evaluate the function of early-acting developmental genes. In the future, data derived from VIGS analyses will be combined with large-scale sequencing and microarray experiments already underway in order to address both recent and ancient evolutionary questions. Background The genus Aquilegia is comprised of approximately 70 species distributed across temperate areas of North America, Europe, and Asia, with several ornamental varieties sold commercially [1]. These species have undergone a very recent and rapid adaptive radiation in response to biotic and abiotic factors, resulting in low sequence variation among species [2-4]. Thus, they are ideal for evolutionary studies in that they display a wide range of ecological and morphological diversity but retain high levels of crosscompatibility between species, allowing for genetic dissection of traits [5]. Aquilegia possesses a small diploid Published: 12 April 2007 Plant Methods 2007, 3:6 doi:10.1186/1746-4811-3-6 Received: 12 March 2007 Accepted: 12 April 2007 This article is available from: http://www.plantmethods.com/content/3/1/6 © 2007 Gould and Kramer; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.