Plant & Animal Genome VI Conference

P35

CLONING AND MAPPING OF A PUTATIVE POLYPHENOL OXIDASE GENE IN WHEAT, Triticum aestivum L.

252 Emerson Hall, Plant Breeding Dept., Cornell University, Ithaca, NY 14853

Polyphenol oxidases (PPO) are involved in the browning response observed in many species' fruits and leaves upon wounding. Discoloration in fresh doughs and alkaline noodles manufactured from wheat flour has also been associated with PPO activity. PPO DNA sequences from tomato, potato, apple, grape, pokeweed and spinach were compared and PCR primers (PPOF4.1 and PPOR4.1) developed that could amplify the region between the copper-binding sites in all of these sequences using PCR simulation software. This approach relied on the possibility that the copper-binding sites are highly conserved across species and was high risk due to its application of sequence information to such widely divergent species (ie. from dicotyledonous species to a monocotyledonous species). Amplification of wheat genomic DNA using PPOF4.1 and PPOR4.1 generated a fragment of approximately the predicted size, based on the average amplicon size resulting from the computer simulations. The TA cloning method was used to recover possible PPO sequences from the amplification procedure and several of these submitted for nucleotide sequencing. A wheat genomic library was screened using the recovered clones as well as a potato PPO cDNA clone as probes. Secondary library screening was performed on plaques positive for either of these probes. The resultant genomic clones were mapped to chromosome arms using ditelosomic genetic stocks.