ECOTILLING TO DETERMINE THE EXTENT AND DISTRIBUTION OF NATURAL VARIATION IN THE BLACK COTTONWOOD, Populus trichocarpa

¹ Department of Botany, University of British Columbia, Vancouver, B.C., Canada

² Centre for Plant Research and Botanical Garden, University of British Columbia, Vancouver, B.C., Canada

³ Biotechnology Laboratory, University of British Columbia, Vancouver, B.C., Canada

⁴ Department of Forest Sciences, University of British Columbia, Vancouver, B.C., Canada

TILLING (Targeting Induced Local Lesions in Genomes) is a powerful new technique that can identify polymorphisms in a target gene by heteroduplex analysis. We are using a variation of this technique (ECOTILLING) as a means to determine the extent of natural variation in selected genes in Populus balsamifera trichocarpa (black cottonwood). For this pilot project we are examining ten different candidate genes in a total of 430 individuals from 95 different populations throughout the range of this species (southern California to Alaska), with emphasis on British Columbia populations. Initial experiments indicate that the approach works, and that allelic polymorphisms in candidate genes are frequent enough to be detected using ECOTILLING. The level of variation is not so large, however, that we need to analyse each tree individually, so we have pooled DNA samples from individuals within the same population for our primary screen for polymorphisms. Once alleles of interest in candidate genes are confirmed by sequencing, ECOTILLING data will be used for two purposes. The polymorphisms detected will be used as genetic markers and integrated into poplar linkage maps. Sequence information will also be used to establish whether or not alleles are likely to have a phenotypic effect, based on our knowledge of the gene or protein in other species. Further analysis of selected loci will be undertaken to determine whether or not the variation is neutral or under selection and whether particular alleles are correlated with specific traits or ecologies (i.e. are of adaptive significance). Ultimately, it may be possible to prove functional variation by using Arabidopsis thaliana as a model, or by comparative genomics with related species that exhibit similar traits.