Large-scale genome sequencing projects in plants have yielded a wealth of sequence information that can be used to form hypotheses regarding gene function. However, in many plant species it is difficult to test these hypotheses due to a lack of reverse-genetics resources. This project will develop an Ethane methylsulfonate (EMS)-mutagenized population of maize plants and screen it using two approaches to SNP mutation detection: Targeted Induced Local Lesions IN Genomes (TILLING), which utilizes mismatch-specific endonuclease digestion to detect SNPs, and a prototype oligonucleotide microarray system, which uses a hybridization-based approach.
EMS is a chemical mutagen that causes primarily C->T to A->G transition mutations. Bioinformatic analysis can be used to predict whether these transitions result in nonsense, missense, or silent mutations. An oligonucleotide microarray can be designed to screen for the informative mutations by incorporating oligonucleotides identical to the wild-type parental sequence and oligonucleotides identical to the transition mutation. DNA from mutagenized individuals will be labeled and hybridized to the oligonucleotide microarrays to identify individuals containing a specific mutation. The advantages of this screening method are increased efficiency (more genes at lower cost), precise identification of the mutation when it is detected, and the characterized genetic background being used.
The approach employed in this project is made possible by the use of a maskless array synthesizer (MAS) device, which allows rapid construction of inexpensive oligonucleotide microarrays that can easily be customized. A single microarray can be used to screen for over 100,000 potential mutations if eight oligonucleotides are used to screen for each SNP.
The outcomes of this project will form the foundation of a publicly available reverse-genetic resource for maize that will include mutants, screening protocols, and bioinformatic tools. These resources will be valuable to researchers interested in developing reverse-genetics resources for other plant species as well as researchers interested in other SNP detection applications for plant and animal genomic DNA.
