Performing Department
Biological Sciences
Non Technical Summary
In many ways grapevine (the genus Vitis) is emerging as a model system in plant biology as the world wide economic impact of cultivated grapes generates an enormous volume of research across a variety of disciplines. In recent years, much of this research is genetic or molecular in nature - including an ongoing international effort to sequence the entire grapevine genome (http://www.vitaceae.org). The recent introduction of the first genome arrays and array-ready oligos derived from the cultivated grapevine, Vitis vinifera, has made it possible to apply microarray technology to related wild species. While a significant amount of baseline data will be needed for the proof of concept in grapevines, this research could eventually contribute greatly to our understanding of functional genetic differences between plant species. ! This proposal describes the opportunity presented by the development of commercially available microarray resources for grapevines to expand the PI's current research on various aspects of Vitis evolution. Comparative expression profiling of native species will not only provide new insight into the genetics of adaptation and divergence in Vitis, it will also complement two active areas of research in the PI's lab. The first is ongoing phylogenetic studies of native North American grape species, with particular emphasis on two species complexes well represented in the Southeast. The second involves bioinformatic analyses of Vitis expressed sequence tag (EST) data, examining evidence for positive selection on genes during speciation and domestication. Expression studies will certainly benefit from this work. The recent acquisition of microarray equipment by the PI's institution is another key step in achieving the goals outlined below. The overarching goal of the proposed research is to utilize the technology and data resources developed to address applied, agronomic questions, in a program of basic, evolutionary research. The general goal of this project is to show that V. vinifera microarrays can be used to study related species and achieve the same level of success and evolutionary insights as have similar comparisons between the model systems yeast, Drosophila, and Arabidopsis, and their wild relatives. ! The specific goals of this study are to create genome-wide, tissue specific expression profiles for several Southeastern grape species using genome arrays developed for the cultivated grapevine. This will include estimates of the variation in gene expression between individuals (for specific tissues) within species and populations. The relationship between transcript profiles and phenotypic differences can then be explored.
Animal Health Component
(N/A)
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Goals / Objectives
The objective of this project is to identify genes of agronomic importance in wild grapevines. To this end we will: 1) Create genome-wide, tissue specific expression profiles for wild grapevines. 2) Quantify intraspecific variation in gene expression for individual species. 3) Examine relationship between differentially transcribed genes and phenotype.
Project Methods
Preliminary sampling will focus on several pairs of species believed to represent closely related, if not sister, taxa. All living material is growing in a common environment in AAES greenhouses. Vitis mustangensis and V. shuttleworthii will be compared as will V. mustangensis from Texas and a major disjunct population in Alabama. V. palmata from Alabama and V. vulpina will be compared, as will V. rotundifolia var. rotundifolia from Alabama and V. rotundifolia var. munsoniana from Florida. ! From identical tissue types of each individual (leaf, tendril, inflorescence) Biotinylated cRNA will be produced using standard protocols (e.g. T7-Megascript kit, Ambion). cRNA will be fragmented and hybridized to a Test 3 array to determine quality. Hybridization to Affymetrix Vitis vinifera genome arrays, washing, staining, and scanning will be performed according to standard procedures and using the Affymetrix MAS software. ! In addition to the internal controls built into each chip, there are additional aspects of repeatability to be considered in this novel use of the V. vinifera DNA arrays. One important component of this proposal will be to simply demonstrate that consistent results can be obtained for other species by performing multiple biological replicates of each cRNA preparation and hybridization. It will also be important to quantify intraspecific variation in gene expression by examining multiple individuals for a given species and selected tissues. An additional consideration for using species other than cultivated V. vinifera, is that the genes themselves are diverging (in sequence), not just their level of transcription. It will therefore be necessary to distinguish the relative contributions of transcriptional regulation and sequence divergence to any apparent differences in expression observed in the interspecific comparisons by performing competitive hybridizations of genomic DNA to the same microarray chips. Bayesian, likelihood, and other statistical methods have been developed to assist in determining significant differences in expression levels.