OBJECTIVES: Major genome research programs are underway to isolate expressed genes of corn and to DNA sequence at least the gene-rich regions. We are developing a highly efficient method for mapping DNA sequences to chromosome and sub-chromosomal regions based on lines derived from crosses between oats and corn. Partial hybrids have been obtained that contain an entire set of oat chromosomes along with one corn chromosome; lines are available that have each chromosome of corn individually added to the oat genome. These lines are now being used to generate derivatives with only a subregion of the corn chromosome. This genetic material as well as B73xMo17 derived lines will be used to learn more about the subgenomic structure of corn and to test whether the degree of heteroallelism within and between the subgenomes contribute to heterosis. A pathogenic bacteria, called E. coli O157:H7, can contaminate hamburger and cause serious hemoraging. The contamination largely comes from the feces of cattle. Strains of E. coli have been selected that will kill the O157:H7 strain. Two genes appear to be involved and this project will test their efficacy in corn. Wild rice is a crop under domestication. The seed falls (shatters) from the panicle upon maturation and is not available to be harvested. Storms can cause major losses of wild rice yield at harvest. The shattering trait is considered a domestication trait that has been modified years ago in most major crops; the trait still exists in wild rice varieties. We have discovered major genes for shattering and have molecular genetic markers that should allow more efficient breeding for shattering resistant varieties. Methionine is an essential amino acid required in the diets of non-ruminant animals. Poultry have high demands for methionine accounting for why synthetic methionine is added to poultry rations in the U.S. A line of corn was selected that is high in methionine. Several generations of crossing, backcrossing, and selfing has resulted in relatively homozygous lines potentially high in methionine, The specific objectives are: 1. Develop efficient mapping system for corn DNA sequences. 2. Learn more about the subgenomic structure of corn and relate the information to productivity. 3. Manipulate corn to produce compounds toxic to an enterohemoraghic bacterium (E. coli O157:H7). 4. Provide molecular genetic methods to the breeding program to enhance the selection of varieties of wild rice with improved seed shattering resistance. 5. Finalize the selection and release of high methionine lines of corn.

APPROACH: We have produced lines of oats with a single corn chromosome added. These addition lines are available for all 10 corn chromosomes, individually present in the oat background. These lines are being used extensively for mapping DNA sequences to chromosome. The oat-corn addition lines are being irradiated to produce radiation hybrid (RH) lines that have only part of the corn chromosome present. These lines allow much more refined mapping of corn DNA sequences. Future research will involve continued development and characterization of the RHs, mapping ESTs, cytogenetic transmission studies of the various lines, and obtaining the transmission of the chromosome 10 addition and formation of RHs. Molecular genetic analysis of corn has shown that its genome is composed of major duplicated regions suggesting an ancient allopolyploid origin. The potential exists for many loci to not only be duplicated but to have different alleles at each locus. Maximum productivity could be the result of maximizing the number of alleles at these loci. We will screen several genetic markers in order to determine Mo17xB73 lines containing duplicate regions containing various combinations of the parental genotypes. Yield tests will ultimately be performed on selected lines with maximum heteroallelism in regions known to possess major yield QTLs. Our molecular genetics research on wild rice has resulted in the first molecular marker map and the placement of QTLs for several traits on the map. A PCR based marker has been developed that tags the most major QTL with polymorphism between shattering and non-shattering types. This QTL accounts for about 40% of the variation in seed shattering in wild rice. A marker-assisted selection program will be initiated using the molecular genetic marker for the major QTL and others as possible. Escherichia coli O157:H7 is a pathogenic strain of E. coli found in the digestive tract of beef cattle and causes food borne illness through contamination of beef during the slaughtering process. The goal of this project is to target the pathogen at the feed source or farm level to prevent contamination by developing a transgenic maize line that expresses a protein (termed "colicin") toxic to this strain of E. coli. Colicin (produced by the cea gene) degrades DNA, and an immunity protein (produced by the cei gene) is required to prevent non-specific DNA degradation. Thus both genes are required to accomplish our goal. A screening procedure was developed in our lab that provides a screen for corn with elevated levels of methionine in the endosperm. A line was discovered, called BSSS5, that is resistant to lysine-threonine inhibition as a whole kernel but is inhibited when only the embryo is cultured. The endosperm in this case possesses sufficient methionine to prevent the inhibition. BSSS 53 was found to produce a zein-2 protein with 23% of the amino acid residues as methionine. We have attempted to backcross the high-methionine trait into three different genetic backgrounds (B73, Mo17, and A632) and have some lines almost ready to release.

KEYWORDS: corn; plant nutrition; plant genetics; cytogenetics; gene mapping; chromosome mapping; wild rice; transgenic plants; escherichia coli; molecular genetics; plant improvement; oats; genomes; gene expression; dna sequences; chromosomes; hybrids; alleles; heterosis; crop yields; seed shattering; domestication; genetic markers; gene analysis; food nutritive value; animal nutrition; dna insertion; disease prevention

PROGRESS: 2006/01 TO 2006/12
Oat-corn addition lines have been developed for every corn chromosome. These strains allow - in a single experiment - the rapid mapping of corn DNA sequences to their respective chromosome. These strains resulted from more than 100,000 oat x corn crosses and isolation of over 7300 immature embryos. We extended the available genetic backgrounds by recovering oat-corn addition lines with B73 or Mo17. Between the two inbreds, we have a complete addition line set; the B73 addition lines will be useful in assembling the corn genome. The DOE Joint Genome Institute will sequence chromosome10 using our Mo17 chromosome 10 oat-maize addition line. We also have added to our collection of radiation hybrid lines for each chromosome. These lines allow the placement of corn genes to specific regions of the chromosome. Corn gene expression/silencing in the oat-corn addition lines is being tested using the DNA chip technology. Out of 17,000 tested sequences, 890 were expressed in the chromosome 5 addition. About half of these sequences are of known position and are either placed on chromosome 5 or in a duplicated region of the genome. We further pursued the possibility of introducing C4 photosynthesis to oat with these materials. Addition lines with both chromosomes 6 and 9 (carrying genes for key C4 enzymes) were tested for an effect on CO2 compensation point. A statistically significant change occurred in compensation point but not an impressive one. We will next investigate a third chromosome that has an effect on leaf cell morphology (known as Kranz Anatomy) involved in photosynthetic efficiency. NIR analyses of self-pollinations of a new high oil accession indicate that the kernels contain 20% oil. A QTL analysis is underway. There is considerable interest in this material as a source of biodiesel and ethanol. Using the molecular genetic map we published for wild rice, a PCR-based marker system has been identified for use in marker assisted breeding for the seed shattering trait. Genetic tests indicate a close marker/trait linkage. The recent outbreak of the pathogenic E. coli 0157:H7 on spinach emphasizes the importance of having control measures to minimize such occurrences. We are developing what might be considered a prototype approach of using corn feed to alter the microflora in the intestinal tract as a means to reduce the presence of this pathogenic bacterium at its source. Whole corn plants were regenerated from appropriate calli and found to carry the colicin gene, express the RNA and protein, and have anti-microbial activity against E. coli O157:H7. An extensive bioinformatics analysis has been performed of 1100 DNA sequences involved in human cancers to determine if they are present in plants. P53 is a protein involved in more than 50% of human cancers. We found a p53 binding sequence in the promoter of the MAD1 (Mitosis Arrest Deficiency) gene in human; MAD1 acts as a mitotic checkpoint to ensure chromosome stability. A MAD1 homolog was found in maize. Laboratory analyses of knockout mutants of MAD1 in Arabidopsis are underway.

IMPACT: 2006/01 TO 2006/12
Crop productivity is at least half due to genetic advances. Continued understanding of genetics and the application of molecular genetics will allow continued progress in food production. In addition, additional health benefits and identification of unique biofuel resources will be forthcoming.

PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
1. Odland, W., A. Baumgarten, and R. Phillips. 2006. Ancestral rice blocks define multiple related regions in the maize genome. The Plant Genome 1: 541-548 (Supplement to Crop Sci. 46).
2. Phillips, R.L. 2006. Genetic tools from nature and the nature of genetic tools. In: CSSA Golden Anniversary Symposium. Ed. C. Stuber. Crop Sci. 46: 2245-2252.
3. Rines, H.W., S.J. Molnar, N.A. Tinker, and R.L. Phillips. 2006. Oat. In: Kole, C. (ed.). Genome Mapping and Molecular Breeding in Plants: Cereals and Millets Vol. 1. Springer, Inc., NY, USA. pp. 211-242.
4. Phillips, R.L., W.E. Odland, and A.L. Kahler. 2006. Rice as a reference genome and more. In: 5th Intl. Rice Genetics Symp., Eds. D.S. Brar, D. Mackill, and B. Hardy. In press.

PROJECT CONTACT:
Name: Phillips, R. L.
Phone: 612-625-1213
Fax: 612-625-1268
Email: phill005@umn.edu