Plant & Animal Genomes XIV Conference

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

Complexity Reduction Of Polymorphic Sequences (CRoPS): A Novel Approach For High Througput Polymorphism Discovery

Michiel J.T. Van Eijk , Nathalie Van Orsouw , Jan Van Oeveren , René Hogers , Antoine Janssen , Esther Verstege

  Keygene N.V., Agro Business Park 90, P.O. Box 216, 6700 AE Wageningen, The Netherlands

Discovery of polymorphisms, incuding single nucleotide polymorphisms (SNP) and simple sequence repeats (SSR), in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of capillary electrophoresis sequence technology. We present a novel approach for polymorphism discovery (CRoPS) in plants by combining the power of reproducible genome complexity reduction of AFLP with recently developed sequencing by synthesis (pyrosequencing) technology of 454 Life Sciences (Margulies et al., 2005. Nature 437, 376-380).
The principle or CRoPS is that tagged complexity-reduced libraries of two or more genetically diverse samples are prepared by AFLP® and sequenced at 5-10-fold redundancy in microfabricated high-density picolitre reactions. A typical sequence run yields over 200,000 sequence reads with a median length of 100 bases. Resulting sequences are clustered and sequence contigs inspected for sequence differences using bio-informatics tools. Rigorous quality measures are applied to separate sequence errors from true polymorphisms, based on redundant sequencing, sample origin information and allele frequencies. CRoPS results of SNP and SSR mining and validation in pepper and maize will be presented.
The AFLP and CRoPS technologies are covered by patents and patent applications owned by Keygene N.V. AFLP® is a registered trademark of Keygene N.V.