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Abstract: Optimized Target Preparation Method for Single-Feature Polymorphism Detection in Maize.
- Peter Bradbury 1, Michael Gore2, René Hogers 3, Matias Kirst 4, Esther Verstege5, Jan van Oeveren5, Johan Peleman6, Edward Buckler 7 and Michiel J.T. van Eijk5, (1)USDA-ARS , Ithaca , NY , USA , (2)Cornell Univ., Ithaca , NY, (3)Keygene N.V. , 6700 AE Wageningen , Netherlands, (4)Univ. of Florida , Gainesville , FL, (5)Keygene N.V. , 6700 AE Wageningen , , Netherlands , (6)Keygene N.V., 6700 AE Wageningen, , Netherlands , (7)USDA-ARS, Cornell Univ. , Ithaca , NY
- Northeastern Branch of the ASA-SSSA (July 11, 2006)
We describe the detection of sequence validated single-feature polymorphisms (SFPs) between maize inbred lines by hybridizing RNA or complexity-reduced genomic DNA to an Affymetrix GeneChip® expression array. Direct hybridization of labeled total genomic DNA to oligonucleotide expression arrays for SFP detection was initially demonstrated in organisms with relatively small genomes, such as ~12Mb yeast and 130Mb Arabidopsis. For larger genomes such as ~2500Mb maize, however, obtaining robust hybridization signals requires a sample preparation method optimized to reduce repetitive or high-copy sequences in the target prior to array hybridization. In this study, we evaluated the efficacy of four different complexity-reduction methods for sensitive SFP detection in maize: cDNA, methyl filtration, high-Cot selection, and AFLP®. These four methods were applied to four diverse maize inbred lines (B73, Mo17, CML69, and Tzi8) with 3 replications per line (48 GeneChips®). Our results indicate that all genome reduction methodologies offer modest power to detect SFPs with the commercially available GeneChip® Maize Genome Array. Moreover, the detection of non-SNP genetic polymorphisms by probes, such as allelic non-homologies and copy number of genes, may produce some of the apparent Type I error rates.
The AFLP technology is covered by patents and patent applications owned by Keygene. AFLP® is a registered trademark of Keygene.
