Plant & Animal Genomes XV Conference

January 13-17, 2007
Town & Country Convention Center
San Diego, CA

Development And Exploitation Of A Lettuce (Lactuca sativa) 6.6 Million Feature Affymetrix Genechip For Massively Parallel Genotyping And Gene Expression Analysis

David G Caldwell¹ , Hans van Leeuwen¹ , Kevin Stoffel² , Fallon Chen¹ , Alex Kozik¹ , María José Truco¹ , Richard W Michelmore¹ , Allen Van Deynze²

¹  UC Davis Genome Center, University of California, Davis, CA, 95616, USA

²  Seed Biotechnology Center, University of California, Davis, CA, 95616, USA

Currently, there is the need for high-resolution genetic and physical maps in many crops to characterize the genetic mechanisms controlling agriculturally important traits. Although the discovery of sequence-based markers has become relatively straightforward, their identification and implementation into mapping populations or germplasm screens currently remains laborious and expensive. Single Feature Polymorphisms (SFPs) based on microarray hybridization is emerging as a powerful marker technology because of its highly parallel nature. Current gene expression arrays rarely provide redundancy for any one SFP, leading to both false positive and negative scoring of SFPs and subsequently to incorrect mapping or associations. Furthermore, a limited proportion of each gene is represented on expression chips, limiting SFP detection in species with low polymorphism such as lettuce. We have developed a 6.6 million feature Affymetrix GeneChip® for both robust SFP discovery, genotyping and gene expression analysis. Probes from ~29,000 cultivated lettuce (L. sativa) unigenes and an additional ~6,000 unique sequences from four other related lettuce species (L. serriola, L. saligna, L. virosa and L. perennis) were synthesized on the GeneChip®. Each probe set contains an average of 173 probes per transcript (6,415,642 total probes). A technical replicate control block of 100 probes is synthesized 169 times across the chip to facilitate signal normalization and post processing. To validate our SFP discovery scripts, approximately 2,000 manually curated regions, each containing 1-3 putative SNPs, were tiled in 2 bp increments. We are currently assessing various target preparation and hybridization methodologies including cDNA, total genomic DNA and High-Cot DNA.