This project involved genetic research on maize kernel composition as part of the overall long term goal of facilitating development of value added maize hybrids. A major objective is increasing levels of oil in maize kernels, which have more calories than protein and starch, and therefore provides efficiencies in swine and poultry feeding. The other major objective involves improving the quality of the oil by increasing the levels of oleic acid (monounsaturated fat).

The specific objectives of this research outlined in the proposal were to:

Identify and define quantitative trait loci (QTL) associated with control of oil and oleic acid concentration (%) of corn grain in a set of [Illinois High Oil {IHO} x B73] x B73 backcross derived S4 lines (BC1S4 lines). Oil % is of primary interest in the overall research program.

1. Examine genetic relationships between kernel oil concentration QTL and grain yield QTL in testcross evaluations of the BC1S4 lines grown in multiple Corn Belt locations.
2. Use random mated generations of Illinois High Oil (IHO) x Illinois Low Oil (ILO) to more precisely define number and location of QTL controlling oil and fatty acid concentration than is possible with conventional mapping populations.
3. Confirm authenticity of certain QTL for higher oil and higher oleic acid % through evaluation of near-isogenic lines (NILs). These NIL confirmed QTL segments would be backcrossed into commercially elite germplasm.
4. Perform transformation with the maize fatty acid desaturase-2 (fad2) clone in antisense orientation to greatly reduce expression of FAD2 in corn embryos thus producing a much higher oleic acid % oil than is possible with present germplasm.

We accomplished or made significant progress towards all of our objectives, outlined below. More detailed, specific information and experimental results are readily available upon request.

1. We successfully evaluated the genetic relationships between kernel oil concentration QTL and grain yield. Our results indicated that most of the negative association is due to linkage drag of non-elite alleles for grain yield near the oil QTL alleles. Notably most of the oil QTL did not have a direct detectable negative effect on grain yield. This suggests that with fine mapping of favorable QTL and marker assisted selection that levels of oil could be increased to a certain point without a significant reduction in grain yield.
2. We successfully performed and completed fine mapping of oil QTL using random mated populations. We more precisely defined the locations of many QTL for oil concentration. We identified specific molecular markers (SSRs) that would be useful for marker assisted selection of oil QTL by private sector. These markers should be more effective as there would be less linkage drag of non-elite alleles for grain yield. In addition, although not a specific objective, we completed the same process for QTL controlling fatty acids, particularly oleic acid, and came to the same parallel conclusions.
3. We confirmed the major QTL for oil and oleic acid concentration on chromosome 6 using near-isogenic lines. These near-isogenic lines were developed and evaluated in a backcross to a line (B73) relevant to commercial breeding lines. Rather than backcrossing these specific segments, the mapping and confirmation information is useful to companies that have already made crosses with IHO or other high oil sources. Our research evolved to provide useful results by providing genetic information to the private sector, rather than chromosome segments.
4. We successfully demonstrated that genetic plant transformation with the gene fad2 could be used to increase levels of oleic acid to over 70%, whereas most corn is around 25% oleic acid in the oil. We also demonstrated other transgenic events that can increase levels of fatty acids to 35 to 40%. Shortly after this project started DuPont filed a patent for transformation of corn to create high oleic oil. Therefore we emphasized other aspects of the project, particularly since transformation of maize in the public sector is inefficient and slow We did examine the relationships of different classes of glycerolipids that are affected by transformation with fad2 and increasing levels of oleic has unexpected changes on the composition of different glycerolipid classes. This suggests that modification of oleic acid by transformation may have other effects, whether positive or negative remains to be determined.

Summary of Results in a more General Format

This project resulted in the mapping of genes that alter levels of oil and oleic acid concentration in the maize kernel. These genes were shown to have an effect in a backcross population that is relevant to commercial maize production. In a separate random mated population we were able to more finely map these genes. With the advent of enhanced efforts in genomics in the public and private sector in the US during this project, the information created from this project, map positions, became increasingly important to the hybrid seed industry. Map positions can be used in selection of chromosome segments within commercially elite proprietary germplasm and genomic based efforts to identify the underlying genes. Identification of underlying genes will make marker assisted selection more efficient and provides genes for future transformation strategies to alter levels of oil.

This project had an impact in the ag biotechnology area in that it has confirmed and more precisely mapped a number of chromosome regions with QTL associated with levels of oil and oleic acid. These results showed that these QTL, which had been identified in a non-elite background, could be transferred to more elite background and have the desired affect on oil. The generation evaluated, BC1S4s, is at a stage and inbreeding level similar to what a breeder would evaluate with commercial materials and donor germplasm for high oil levels. So the concept of QTL having an affect in another background is confirmed. The map information in this background is useful to private companies to perform marker assisted selection. So the work provided a proof of concept for marker assisted selection of QTL for oil and oleic acid in maize. The research also provided materials and information useful for genomics based activities designed to identify the genes underlying oil composition, and kernel composition in general.

Leveraging the Project

Pioneer/DuPont performed fatty acid analyses in kind which enabled us to do more work than planned on oleic acid.

This research was leveraged in part towards a grant from NSF Plant Genome to perform work on more detailed fine mapping in the random mated population and performing RNA analyses to identify genes involved in oil levels.

The IMBA and NSF projects contributed to a set of information and resources that directly led to a grant to Illinois from Renessen (joint venture Monsanto and Cargill) for evaluation of a much larger, more advanced random mated population with more extensive marker coverage.

Obviously, if the project was leveraged into more support by NSF, and we now have a major project with Renessen, which has a focus on quality traits in maize, the project has been successful both in terms of academic value and relevance to the private ag sector. We demonstrated the concept and created materials and results, and then the work expanded with resources form other sources. Renessen is interested in fine mapping and gene discovery for genes underlying oil and kernel composition, so the research has evolved more towards information generation. However this information is put in conjunction with genomic resources to identify the specific genes, which come from actual cDNA libraries created in genome projects. These identified cDNA and their sequences can then used directly or indirectly for marker assisted selection and transformation to increase oil levels.

The funding of the project at Illinois by Renessen is an indication of a positive attitude towards using molecular markers in mapping and breeding to move towards value added maize hybrids.

There are acceptance concerns associated with GMOs. Molecular marker mapping is enhanced by biotechnology, but actually facilitates use of natural genetic variation. It does not create GMO products. In addition industry recognizes that not everything can be done with GMOs as sometimes unexpected problems and side effects are created. Thus the approach of molecular marker mapping for marker assisted selection and gene discovery contributes to both natural approaches and provides candidate genes for transformation. This provides two strategies. These two strategies may also be successfully combined some day in a balanced matter. This is something future research could pursue.

Suggestions for Future Research

We have developed a very comprehensive database on chromosome positions of genes affecting kernel composition (starch, protein, oil, oleic acid). This database is derived from this project in conjunction with earlier projects. We are now extracting near-isogenic lines that will be used in collaborative projects and new projects to use new microarray technologies (RNA profiling) to find the genes. The database is very valuable as it has multiple years of data so there is confidence in the results. The new technologies can be performed in short periods, a few months, but years of data collection in the field can not be performed in a few months.

Bibliography

Abstracts

Mikkilineni, Venugopal, and Torbert Rocheford. Genomic Organization of the Fatty Acid Desaturase-2 (FAD-2) EST's in Maize. 42nd Annual Maize Genetics Conference, March 16-19, 2000

Willmot, D. B., J. W. Dudley, and T. R. Rocheford. Random-mating and detection of QTL for oil in maize. Agronomy Abstracts. P. 184. 2000.

Bongard, Deverie K., Howard M. Goodman, Venugopal Mikkilineni, Torbert Rocheford, Barbara Farnworth, Jiqing Peng, and Bertrand Lemieux. P95 SNP discovery using the maize EST database. 42nd Annual Maize Genetics Conference, March 16-19, 2000.

Wong, J. C., R. J. Lambert, and T. R. Rocheford. Molecular Marker Mapping of Chromosomal Regions Associated with Carotenoids and Tocopherols in Maize. P. 36. In 42nd Annual Maize Genetics Conference Abstracts. 2000.

Damon, Steven J., and Torbert Rocheford. QTL and NIL analysis of maize kernel composition and tassel architecture. 43rd Annual Maize Genetics Conference, March 14-18, 2001.

Mikkilineni, Venugopal, Saeed Irlan, David Ash, Amanda Cebenka, Teclemariam Weldekidan, James Hawk, Torbert Rocheford, and Bertrand Lemieux. Serial Analysis of Gene Expression assay of developing kernels from the Illinois Long Term Selection Oil Strains. 43rd Annual Maize Genetics Conference, March 14-18, 2001.

Kallis, R., N. J. Engeseth, J. M. Widholm, P. Christou, D. Gahakwa, and Torbert Rocheford. Development of Transgenic Maize with Altered Linoleic/Oleic Acid Content. Maize Genetics Meeting, Lake Geneva, WI, March 2001.

Kallis, R., N. J. Engeseth, J. M. Widholm, P. Christou, D. Gahakwa, and Torbert Rocheford. Evaluation of Transgenic Maize with Altered Linoleic/Oleic Acid Content. Plant Physiology Meetings, Denver, July 2001.

Wong, J. C., R. J. Lambert, and T. R. Rocheford. Comparison of QTL in Two Populations Controlling Carotenoids and Tocopherols in Maize Kernels. To be presented at 2001 Agronomy Meeting, Charlotte, North Carolina. 2001.

Mikkilineni, Venu, Lance Davidow, Saeed Irian, Howard Goodman, David Ash, Linda Almeida, Caroline Golt, Teckle Weldikidan, James Hawk, Torbert Rocheford, and Bertrand Lemieux. Gene Discovery in Maize using Serial Analysis of Gene Expression. 44th Annual Maize Genetics Conference, March 14-17, 2002.

Willmot, D. B., J. W. Dudley, T. R. Rocheford, and A. Bari. Effects of random-mating on QTL detection in an Illinois high by low oil strain cross. Poster submission to the Long-Term Selection Symposium, Urbana, IL. 2002.

Proceedings

DeBaene, Jan. Detection of Kernel Oil Concentration QTL in Backcross Derived Lines and Testcrosses. Department of Crop Sciences, University of Illinois. Thirty-Fifth Annual Illinois Corn Breeders School, March 1-2, 1999.

Mikkilineni, Venugopal and Torbert Rocheford, Genomic organization of fatty acid desaturase-2(FAD2) ESTs in maize. Department of Crop Sciences, University of Illinois. Thirty-Sixth Annual Illinois Corn Breeders' School, March 6-7, 2000.

Willmot, David, John Dudley, Torbert Rocheford, and Al Bari. Effect of Random-mating on Marker-QTL Associations in an IHO x ILO cross. Department of Crop Sciences, University of Illinois. Thirty-Seventh Annual Illinois Corn Breeders' School, March 5-6, 2001.

Damon, Steven J. and Torbert R. Rocheford. Near-Isogenic Line Analysis of Maize Kernel Oil QTL on Chromosome 6, and Related Kernel Composition Studies. Department of Crop Sciences, University of Illinois. Thirty-Seventh Annual Illinois Corn Breeders' School, March 5-6, 2001.

Wong, J. C., C. O. Egesel, R. J. Lambert, and T. R. Rocheford. Comparing QTL and Candidate Genes for Carotenoids and Tocopherols in Two Maize Populations. Department of Crop Sciences, University of Illinois. Thirty-Eighth Annual Illinois Corn Breeders School, March 4-5, 2002.

Publications

Rocheford, T. R., J. Wong, C. Egesel, R. Lambert. 2002. Enhancement of Vitamin E levels in Corn. Journal of American College of Nutrition 21:191-198S

Mikkilineni, V., and T.R Rocheford. 2003.Genomic Organization of Fatty Acid Desaturase-2 (fad2) and Fatty Acid Desaturase-6 (fad6) EST's in Maize. Theor. Appl. Genet. (In Press).

Egesel, C, J. Wong, T. Rocheford, R. Lambert. 2003. Combining Ability of Maize inbreds for Carotenoids and Tocopherols (In Press)

Wong, J., T. Rocheford, and R. Lambert. 2003. Idnetification of QTL for levels of Vitamin E in Corn. Crop Science (submitted)

Wassom, J. J. Wong, V. Mikkilineni, J. DeBaene, T. Rocheford. 2003. QTL associated with maize kernel composition and related traits among Illinois High Oil x B73 backcross-derived lines. (In final stages editing and preparation)

Mikkilineni, V. J. Wassom, T. Rocheford. 2003. Identification of QTL for levels of oleic acid in a backcross population, and near-isogenic line evaluation (In final stages editing and preparation)

Kallis, R., N. J. Engeseth, J. M. Widholm, P. Christou, D. Gahakwa, and Torbert Rocheford. 2003. Evaluation of Transgenic Maize with Altered Linoleic/Oleic Acid Levels and Effects on Glycerolipid Classes. (In final stages of preparation)

Willmot, D. B., J. W. Dudley, T. R. Rocheford, and A. Bari. Effects of random-mating on detection of kernel trait QTL in an Illinois high by low oil strain cross. Theoretical and Applied Genetics. In preparation. 2003.

A number of other publications will come from this work, some in preparation.

Theses

Venugopal Mikkilinenini Ph.D. Thesis. Genetic Analysis of fatty acid desaturase2 and levels of oil and oleic acid in maize. University of Illinois at Urbana-Champaign, 2000.

Steven John Damon. Detection and Near-isogenic Line Evaluation of Kernel Composition QTL in Maize. M.S. Thesis. University of Illinois at Urbana-Champaign, 2001.

Jeffrey Cleighton Wong. Genetic Analysis of Variation for Carotenoids and Tocopherols in Maize. Ph.D. Thesis. University of Illinois at Urbana-Champaign, 2002.

Jeremy Johnson, M.S. Thesis. University of Illinois at Urbana-Champaign, Expected June, 2003. He already has interviews with Pioneer and Golden Harvest

Personnel

The IMBA project supported all or part of graduate students and postdoctoral associates or their research. Some have been placed in good positions and are contributing to the competitiveness of US Agriculture. Examples include:

Jan DeBaene, Postdoctoral Associate, now working as a breeder for KWS, ID

Venu Mikkilinenini, Ph.D, now Postdoctoral Associate at University of Delaware

Steve Damon, M.S., now Research Specialist working with Syngenta, WI

Jeff Wong, Ph.D, now Assistant Professor in Department of Crop Sciences at California Polytechnic State University, San Luis Obispo

David Willmot, Postdoctoral Associate, now Research Geneticist, USDA-ARS, Department of Agronomy, University of Misouri, Columbia