PAG-XIV  Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA


Poster: High-throughput Methods

P90

Optimized Target Preparation Method For Single-Feature Polymorphism Detection In Maize

Michiel van Eijk1 , Peter Bradbury2 , Michael Gore3 , Rene Hogers1 , Mathias Kirst4 , Esther Verstege1 , Jan Van Oeveren1 , Edward Buckler2,3

1  Keygene N.V., Agro Business Park 90, P.O. Box 216, 6700 AE Wageningen, The Netherlands
2 USDA-ARS, Ithaca, NY 14853, USA
3 Institute for Genomic Diversity, Cornell University, Ithaca, NY 14853-2703, USA
4 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32610, USA

We describe the use of Affymetrix GeneChip® expression arrays for detecting single-feature polymorphisms (SFP; Borevitz et al., 2003) between different lines of maize. SFPs are individual oligos that constitute gene specific probes on an expression array which can behave as polymorphic markers. SFP detection is based on calculating hybridization intensity differences between samples for probes on the chip and reproducible differences reflect indel and/or SNP polymorphisms in the sequences of the probes. Although the SFP technology has been applied successfully in Arabidopsis (approx. 125 Mbp genome) using total genomic DNA as hybridization target, a limitation is that sample DNA complexity reduction is needed in order to obtain reliable hybridization signals in organisms with larger genome sizes. In fact, the need for target complexity reduction exists for highly multiplexed detection of large numbers of SNPs on DNA chips in general. cDNA has been used for SFP detection but requires RNA isolation and correction for transcription level variation (Rostoks et al., 2005).
In this study we compared four different types complexity reduction for the SFP detection: cDNA, methylation filtration, Cot DNA filtration and AFLP®. These four methods have been applied on four diverse maize lines, B73, Mo17, CML69 and Tzi8, in 3 replications per line (48 chips in total). Results will be presented.
The AFLP technology is covered by patents and patent applications owned by Keygene N.V. AFLP® is a registered trademark of Keygene N.V. GeneChip® is a trademark of Affymetrix Inc.