Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa

Abstract

Ecotilling was used as a simple nucleotide polymorphism (SNP) discovery tool to examine DNA variation in natural populations of the western black cottonwood, Populus trichocarpa, and was found to be more efficient than sequencing for large-scale studies of genetic variation in this tree. A publicly available, live reference collection of P. trichocarpa from the University of British Columbia Botanical Garden was used in this study to survey variation in nine different genes among individuals from 41 different populations. A large amount of genetic variation was detected, but the level of variation appears to be less than in the related species, Populus tremula, based on reported statistics for that tree. Genes examined varied considerably in their level of variation, from PoptrTB1 which had a single SNP, to PoptrLFY which had more than 23 in the 1000-bp region examined. Overall nucleotide diversity, measured as (Total), was relatively low at 0.00184. Linkage disequilibrium, on the other hand, was higher than reported for some woody plant species, with mean r2 equal to 0.34. This study reveals the potential of Ecotilling as a rapid genotype discovery method to explore and utilize the large pool of genetic variation in tree species.

A protocol for TILLING and Ecotilling in plants and animals

“…Detection of dozens of polymorphisms in a single fragment is possible because only a small fraction of heteroduplexes are cleaved at any single position by the CEL I nuclease used for TILLING 10,17 . Ecotilling can be used to discover and catalog common polymorphisms without sample pooling, as was done for Arabidopsis and the black cottonwood (Populus trichocarpa) 18 . To detect homozygous polymorphisms, an equal amount of DNA from a reference sample is added to each assay well.…”

“…For Ecotilling, the expected frequency of nucleotide polymorphism will influence the level of desired pooling. When cataloging common nucleotide changes, pooling is not appropriate because of the additional work needed to determine which individuals in a pool harbor the polymorphism 17,18 . However, for rare polymorphisms, pooling reduces effort but will obscure the identification of very common polymorphisms.…”

Updates and perspectives on the utilization of molecular makers of complex traits in rice

“…Additionally, targeting induced local lesions in genomes (TILLING) or eco-TILLING is a powerful approach for identifying allelic variation in natural populations at target loci and making them available for association mapping (Gilchrist et al, 2006). This approach has proven to be useful for the identification and functional validation of candidate genes for complex traits governing disease resistance (Manosalva et al, 2009).…”

Nuevas herramientas en la lucha contra las virosis del pimiento

“…El EcoTILLING también se ha desarrollado en el cacahuete silvestre Arachis duranensis K. & G., para buscar nuevos alelos de un gen relacionado con reacciones alérgicas (Ramos et al, 2009). Adicionalmente, esta metodología puede utilizarse para la detección de SNPs (Single Nucleotide Polymorphisms) en poblaciones naturales (Gilchrist et al, 2006;Barkley et al, 2008;Elias et al, 2009;Galeano et al, 2009;Till et al, 2010). En melón, el EcoTILLING se ha utilizado con éxito en la detección de variantes alélicas del factor de iniciación a la traducción eIF4E, implicado en la resistencia a virus de distintas familias que ocasionan enfermedades de importancia económica en diferentes cultivos (Nieto et al, 2007).…”

“…Así, para buscar entradas que presenten variaciones en el carácter de interés hay que conocer qué genes están controlando o están implicados en ese carácter. Si la elección es incorrecta, la variación nucleotídica encontrada no estará relacionada con la variabilidad de nuestro carácter y la búsqueda habrá sido infructuosa.El primer estudio en una especie cultivada con la técnica EcoTILLING se realizó Gilchrist et al, 2006;Barkley et al, 2008;Elias et al, 2009;Galeano et al, 2009;Till et al, 2010). En melón, el EcoTILLING se ha utilizado con éxito en la detección de variantes alélicas del factor de iniciación a la traducción eIF4E, implicado en la resistencia a virus de distintas familias que ocasionan enfermedades de importancia económica en diferentes cultivos (Nieto et al, 2007).…”