|Gore, Michael - CORNELL UNIVERSITY|
|Hogers, Rene - KEYGENE|
|Kirst, Matias - UNIVERSITY OF FLORIDA|
|Verstege, Esther - KEYGENE|
|Van Oeveren, Jan - KEYGENE|
|Peleman, Johan - KEYGENE|
|Van Eijk, Michiel - KEYGENE|
Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 12, 2007
Publication Date: July 16, 2007
Citation: Gore, M., Bradbury, P., Hogers, R., Kirst, M., Verstege, E., Van Oeveren, J., Peleman, J., Buckler Iv, E.S., Van Eijk, M. 2007. Evaluation of Target Preparation Methods for Single Feature Polymorphism Detection in Large Complex Plant Genomes. Crop Science. 47:S135-S148. Interpretive Summary: High-density microarrays designed to measure gene expression levels have been shown to be effective for detecting large numbers of single feature polymorphisms (SFPs) in species with relatively small genomes. The microarray used in this study, the Affymetrix Maize GeneChip, contains a grid of probes that bind to DNA sequence from approximately 18,000 different maize genes, with each gene represented by fifteen probes. When DNA sequence does not exactly match that of an individual probe, the binding intensity is reduced. This phenomenon provides an opportunity to detect sequence polymorphisms by finding probes with different binding intensities in different inbred lines. This technique is less effective for organisms like maize with complex genomes containing large amounts of non-genic, repetitive DNA because this extra DNA binds weakly to many probes masking the signal from the genes targeted by those probes. This study examined four methods used to reduce the size of the genome so that problems from non-specific binding would be reduced. The ability of these methods to identify approximately 1500 known polymorphisms in three maize inbred lines (B73, Mo17, and CML69) was evaluated. The best of the methods still were only moderately effective. Our findings suggest that improved gene-enrichment methods combined with custom designed microarrays will be needed to effectively use this technology for SFP detection in organisms with large genomes.
Technical Abstract: For those genomes low in repetitive DNA, hybridizing total genomic DNA to high-density expression arrays offers an effective strategy for scoring single feature polymorphisms (SFPs). Of the ~2.5 Gb that constitute the maize genome (Zea mays L.), only 10-20% are genic sequences, with large amounts of repetitive DNA intermixed throughout. As a result, a target preparation method engineered to generate a high genic-to-repetitive DNA ratio is essential for SFP detection in maize. To that end, we tested four gene enrichment and complexity reduction target preparation methods for scoring SFPs on the Affymetrix GeneChip Maize Genome Array (“Maize GeneChip”). Methylation filtration (MF), Cot filtration (CF), mRNA-derived cRNA, and AFLP methods were applied to three maize inbred lines (B73, Mo17, and CML69) with three replications per line (36 Maize GeneChips). Our results indicate that these particular target preparation methods offer only modest power to detect SFPs with the Maize GeneChip. Most notably, CF and MF are comparable in power, detecting more than 10,000 SFPs at a 20% false discovery rate. Although reducing sample complexity to ~125 Mb by AFLP improves SFP scoring accuracy over other methods, only a minimal number of SFPs are still detected. Our findings of residual repetitive DNA in labeled targets and other experimental errors call for improved gene-enrichment methods and custom array designs to more accurately array genotype large, complex crop genomes.