Ausubel 9729599 Experiments are described which are designed to develop and map 350 PCR-based DNA mapping markers for Arabidopsis thaliana. Each marker will correspond to a mapped single nucleotide polymorphism (SNP) from the Columbia and Landsberg erecta ecotypes. These SNPs will be detected using specially designed allele-specific PCR primers that will generate allele specific patterns (i.e., detectable amplification product only from one ecotype) that can be rapidly and reliably scored by a variety of analytical methods. The acronym SNAP has been coined to designate single nucleotide amplified polymorphism's. The proposed experiments involve the synthesis of 350 sets of SNAP primers (each consisting of two primer pairs, one for each ecotype), each of which preferentially amplifies a single, mapped DNA sequence from Columbia or Landsberg erecta DNA. Using these co-dominant SNAPs, it will be possible to map any Arabidopsis gene with only a few hours of labor to an accuracy of approximately 2cM, using DNA isolated from a single leaf, harvested from a limited number of F2 (Columbia X Landsberg erecta) progeny. The specific aims are: 1) To use 350 previously identified sequences that contain single nucleotide polymorphism's between the Columbia and Landsberg erecta ecotypes of Arabidopsis to generate SNAP primers that will preferentially amplify either the Columbia or the Landsberg erect allele 2)To map the SNAP markers generated using DNA from 96 recombinant inbred lines from a Columbia X Landsberg erecta cross 3) To develop methods that will allow many genes to be mapped in an efficient manner using SNAPs. This will include the re-design of the reverse primers to adjust the sizes of the products to allow amplification of several markers in the same amplification reaction. The usefulness of SNAP markers for detecting polymorphisms in DNA pooled from several individual F2 mutants will also be investigated. )4 To test the feasibility of adapting SNAP mapping to automation using DNA micro array analysis by modeling a DNA microarray using a traditional dot blot hybridization procedure. The proposed approach will be the fastest, simplest and potentially the least expensive of all the whole-genome mapping methods developed to date. The sequences of the primers, as they are designed and mapped, will be provided to the Arabidopsis community. Additionally, agreements with a commercial firm will be arranged to synthesize sets of primers as inexpensive "mapping kits".
