GENETIC MECHANISMS AND MOLECULAR GENETIC RESOURCES FOR MAIZE

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1510	1040	40%
202	1510	1040	20%
203	1510	1040	40%

Progress 02/23/02 to 04/11/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Much greater depth of genetic knowledge and improved molecular-genetic techniques are needed to attain optimum crop efficiency, productivity, and stability, or to derive valuable new properties for maize. The tools and insights from genetics must yet fulfill the promise of new technologies, to fully integrate into plant breeding and agronomic practices. The research includes mapping and descriptions of individual genes (phenotypes) and groups of genes (quantitative traits), their regulation, and their function at specific stages of development and under specific conditions, and the genetic diversity in maize germplasm that is available for study and enhancement. As yet, extensive knowledge about these systems is limited and must be obtained. Without extensive new knowledge about genetic mechanisms, gene functions, levels of gene diversity, and tools for manipulating them, it will not be possible to increase U.S. crop production to meet the world's needs for feed and food grains. Although maize breeding maintains a steady pace of advancement in efficiency, productivity, and stability, the crop continues to require high inputs, is subject to diverse biotic and abiotic stresses, and is only slowly advancing toward its theoretical physiological ceiling. New approaches and knowledge are needed to permit feasible incorporation of desirable traits from exotic maize into mainstream maize improvement programs. Novel diversity is needed for pest resistance, yield, and enhanced kernel traits. We have initiated a new research effort to use contrasting sequence diversity in maize inbreds, exotic accessions, and teosinte accessions to identify genes of agronomic importance. It is our goal to develop tools and approaches that study and synthesize information on processes and pathways and to discover the key genes that control agronomic trait expression. As such we are at the core of the intent of NP 302- Plant Biological and Molecular Processes. We are striving to improve how we do trait analysis, not just for particular traits, but also for improving the efficiency of genetic analysis to understand any trait. 2. List by year the currently approved milestones (indicators of research progress) There have been major changes of program direction since the milestones for this plan were developed because of transfer of one scientist to another CRIS and the resignation of a second scientist in March 2004. Therefore, of the original three objectives, only accomplishments for research under Objective 2 are detailed. Objective 2: Determine the genetic mechanisms involved in regulation of metabolic pathways that control agronomic traits for maize. The four original areas of research were: Area 1) p1 diversity controlling flavone pathway genes, Area 2) use of microarray analysis to identify missing genes in the flavone pathway, Area 3) clone and characterize pal genes, and Area 4) test association analysis on flavone pathway. Year 1 (2001): Area 1) make populations; Area 2) isolate RNA, develop cDNA libraries; Area 3) clone and sequence pal genes; and Area 4) sequence flavone structural genes from association mapping population. Year 2 (2002): Area 1) develop molecular assays and measure phenotypes, Area 2) develop microarray technology, Area 3) develop gene specific probes and determine tissue specific expression, and Area 4) collect phenotypic data and complete sequencing of genes across association population. Year 3 (2003): Areas 1), 2), 3), and 4)--complete experiments and analyze results. Year 4 (2004): Area 3)--complete research; and Areas 1), 2), and 4)-- analyze results and design follow up experiments. Year 5 (2005): Areas 1), 2), and 4)--conduct follow-up experiments and publish results. 4a List the single most significant research accomplishment during FY 2006. Comparison of quantitative agronomic trait experiments in corn is hindered by the ad hoc manner in which different mapping populations are used. In collaboration with USDA colleagues in Ithaca, NY and Raleigh, NC, we have developed 25 trait mapping populations, the parents of which were chosen to maximize allelic diversity. We completed development of the populations and are currently increasing seed for release to the maize community. The individuals in the populations will be genotyping at high density with SNP markers and the genotype data will also be released to the corn research community. These populations will become the trait analyses standards and help convert quantitative trait locus analysis from a single experiment format to an integrated, genomics activity. This accomplishment aligns with National Program 302 Plant Biological and Molecular Processes and address the "Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement" component. 4b List other significant research accomplishment(s), if any. A major constraint on the use of biotechnology for crop improvement is our limited knowledge of which genes control agronomic traits. The maize genetics project of the Plant Genetics Research Unit in Columbia, Missouri, in collaboration with scientists at the University of Wisconsin and the University of California-Irvine, has developed and validated a novel approach to identifying candidate genes for the control agronomic traits by contrasting the sequence diversity present in inbred lines of maize to that present in wild teosinte accession and maize landraces. In FY2006, we extended these studies to the identification of additional genes of agronomic importance and used extended DNA sequencing to confirm the selection status of previously identified selected genes. This approach allows an efficient, systemic screen of maize genes, and our approach is being emulated by other research groups in other crop species, thereby broadly impacting crop improvement. This accomplishment aligns with National Program 302 Plant Biological and Molecular Processes and address the "Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement" component. 5. Describe the major accomplishments to date and their predicted or actual impact. The project has provided the majority of SSR, InDel (insertion/deletion) and SNP genetic marker resources in the public sector for trait mapping in maize, essentially allowing a total switch to these more efficient mapping methods. These resources impact the efficiency of genetic analysis for maize across all public and private maize projects. These markers have also been used to develop the integrated physical/genetic map of maize. Through these efforts, over half of the maize genome is now covered with anchored BAC contigs. The significant finding from the trait analysis section of the project is the demonstration of the central importance of regulatory genes as quantitative trait loci. Enzyme- encoding genes also have effects, but to a more limited extent. Related pathways of biosynthesis share intermediates. Our data show that genetic variation of regulatory or enzymatic genes can either result in shunting biochemical intermediates between pathways, or in independently-modulated flow, a process known as compartmentalization. These genetic mechanisms allow for fine control of related biochemical products and subtle variation in the corresponding agronomic traits, and guide conceptions of strategies for crop improvement. This project will continue to provide essential tools for maize improvement along with identification of novel genes for limiting traits of maize. These accomplishments align with National Program 302 Plant Biological and Molecular Processes and address the "Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement" component. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? As they were developed, new SSR, InDel, and SNP markers and the integrated genetic/physical map were transferred to researchers, both public and private, through the Maize Genome Database (MaizeGDB) iMap at www.maizemap.org, and the NSF maize diversity project website, www.panzea. org. Research findings have been published in scientific journals and presented at national meetings. Presentations were given to visiting groups and scientists interested in the impact of plant genomics on crop improvement.

Impacts
(N/A)

Publications

Yamasaki, M., Tenaillon, M., Vroh, I., Schroeder, S., Sanchez-Villeda, H., Doebley, J., Gaut, B., Mcmullen, M.D. 2005. A large scale screen for artificial selection in maize identifies candidate agronomic loci for domestication and crop improvement. The Plant Cell. 17:2859-2872.
Vroh, B., Mcmullen, M.D., Sanchez-Villeda, H., Schroeder, S., Gardiner, J., Soderlund, C., Wing, R., Fang, Z., Coe Jr, E.H. 2006. Single nucleotide polymorphisms and insertion-deletions for genetic markers and anchoring the maize fingerprint contig physical map. Crop Science. 46:12-21.
Morton, B.R., Broh Bi, I., McMullen, M.D., Gaut, B.S. 2006. An analysis of neighboring nucleotide effects on SNPs in nuclear DNA from maize (zea mays) . Genetics. 172:569-577.

Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Much greater depth of genetic knowledge and improved molecular-genetic techniques are needed to attain optimum crop efficiency, productivity, and stability, or to derive valuable new properties for maize. The tools and insights from genetics must yet fulfill the promise of new technologies, to fully integrate into plant breeding and agronomic practices. The research includes mapping and descriptions of individual genes (phenotypes) and groups of genes (quantitative traits), their regulation, and their function at specific stages of development and under specific conditions, and the genetic diversity in maize germplasm that is available for study and enhancement. As yet, extensive knowledge about these systems is limited and must be obtained. Without extensive new knowledge about genetic mechanisms, gene functions, levels of gene diversity, and tools for manipulating them, it will not be possible to increase U.S. crop production to meet the world's needs for feed and food grains. Although maize breeding maintains a steady pace of advancement in efficiency, productivity, and stability, the crop continues to require high inputs, is subject to diverse biotic and abiotic stresses, and is only slowly advancing toward its theoretical physiological ceiling. New approaches and knowledge are needed to permit feasible incorporation of desirable traits from exotic maize into mainstream maize improvement programs. Novel diversity is needed for pest resistance, yield, and enhanced kernel traits. We have initiated a new research effort to use contrasting sequence diversity in maize inbreds, exotic accessions, and teosinte accessions to identify genes of agronomic importance. 2. List the milestones (indicators of progress) from your Project Plan. There have been major changes of program direction since the milestones for this plan were developed because of transfer of one scientist (Ed Coe) to another CRIS and the resignation of a second scientist (David Willmot) in March 2004. Therefore, of the original three objectives, only accomplishments for research under Objective 2 are detailed. Objective 2 Michael D. McMullen): Determine the genetic mechanisms involved in regulation of metabolic pathways that control agronomic traits for maize. The four original areas of research were: Area 1) p1 diversity controlling flavone pathway genes, Area 2) use of microarray analysis to identify missing genes in the flavone pathway, Area 3) clone and characterize pal genes, and Area 4) test association analysis on flavone pathway. Year 1 (2001): Area 1) make populations; Area 2) isolate RNA, develop cDNA libraries; Area 3) clone and sequence pal genes; and Area 4) sequence flavone structural genes from association mapping population. Year 2 (2002): Area 1) develop molecular assays and measure phenotypes, Area 2) develop microarray technology, Area 3) develop gene specific probes and determine tissue specific expression, and Area 4) collect phenotypic data and complete sequencing of genes across association population. Year 3 (2003): Areas 1), 2), 3), and 4)--complete experiments and analyze results. Year 4 (2004): Area 3)--complete research; and Areas 1), 2), and 4)-- analyze results and design follow up experiments. Year 5 (2005): Areas 1), 2), and 4)--conduct follow-up experiments and publish results. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. p1 diversity controlling flavone pathway genes Milestone Fully Met 2. Use of microarray analysis to identify missing genes in the flavone pathway. Milestone Not Met Redirection of Research focus due to change in priorities 3. Clone and characterize pal genes Milestone Not Met Redirection of Research focus due to change in priorities 4. Test association analysis on flavone pathway. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The approved Project Plan does not extend beyond fiscal year 2006. No milestones shown beyond this date. A new Project Plan is being prepared in communication with Kay Simmons, NPL Cereal Grains. 4a What was the single most significant accomplishment this past year? A major constraint on the use of biotechnology for crop improvement is our limited knowledge of which genes control agronomic traits. The maize genetics project of the Plant Genetics Research Unit in Columbia, Missouri, in collaboration with Dr. John Doebley, University of Wisconsin, and Dr. Brandon Gaut, University of California-Irvine has developed and validated a novel approach to identifying candidate genes for the control agronomic traits by contrasting the sequence diversity present in inbred lines of maize to that present in wild teosinte accession and maize landraces. In FY2005, we extended these studies to the identification of numerous genes of agronomic importance. This approach allows an efficient, systemic screen of maize genes, and our approach is being emulated to by other research groups in other crop species thereby broadly impacting crop improvement. 4b List other significant accomplishments, if any. Comparison of quantitative agronomic trait experiments in corn is hindered by the ad hoc manner in which different mapping populations are used. In collaboration with USDA colleagues Ed Buckler, Ithaca, NY and Jim Holland, Raleigh, NC, we have developed 25 trait mapping populations, the parents of which were chosen to maximize allelic diversity. We are currently increasing and will be genotyping at high density with SNP markers and will release these populations to the corn research community. These populations will become the trait analyses standards and help convert quantitative trait locus analysis from a single experiment format to a integrated, genomics activity. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The project has provided the majority of SSR, InDel (insertion/deletion) and SNP genetic marker resources in the public sector for trait mapping in maize, essentially allowing a total switch to these more efficient mapping methods. These resources impact the efficiency of genetic analysis for maize across all public and private maize projects. These markers have also been used to develop the integrated physical/genetic map of maize. Through these efforts, over half of the maize genome is now covered with anchored BAC contigs. The significant finding from the trait analysis section of the project is the demonstration of the central importance of regulatory genes as quantitative trait loci. Enzyme- encoding genes also have effects, but to a more limited extent. Related pathways of biosynthesis share intermediates. Our data show that genetic variation of regulatory or enzymatic genes can either result in shunting biochemical intermediates between pathways, or in independently-modulated flow, a process known as compartmentalization. These genetic mechanisms allow for fine control of related biochemical products and subtle variation in the corresponding agronomic traits, and guide conceptions of strategies for crop improvement. This project will continue to provide essential tools for maize improvement along with identification of novel genes for limiting traits of maize. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? As they were developed, new SSR, InDel, and SNP markers and the integrated genetic/physical map were transferred to researchers, both public and private, through the Maize Genome Database (MaizeGDB) iMap at www.maizemap.org, and the NSF maize diversity project website, www.panzea. org. Research findings have been published in scientific journals and presented at national meetings. Presentations were given to visiting groups and scientists interested in the impact of plant genomics on crop improvement. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). There was considerable news coverage of the article published in Science (see log #0000178241 below). Articles appeared in the: ARS News, NSF homepage, L.A. Times, St. Louis Post-Dispatch, Springfield News Leader, Bradenton Herald, Charlotte Observer, Columbus Ledger-Enquirer, San Luis Obispo Tribune, Grand Forks Herald, Biloxi Sun Herald, Monterrey County Herald, Macon Telegraph, Kansas City Star, St. Paul Pioneer Press, Tallahassee.com, Centre Daily Times, Myrtle Beach Sun News, Duluth News Tribune, High Plains Journal.

Impacts
(N/A)

Publications

Wright, S., Vroh Bi, I., Schroeder, S., Doebley, J., Yamasaki, M., Mcmullen, M.D., Gaut, B. 2005. The genomic extent of artificial selection [2005]. Maize Genetics Conference. No. T24, p. 32.
Szalma, S.J., Buckler Iv, E.S., Snook, M.E., Mcmullen, M.D. 2005. Association analysis of candidate genes for maysin and chlorogenic acid accumulation in maize silks. Journal of Theoretical and Applied Genetics. 110(7):1324-1333.
Wright, S., Vroh, I., Schroeder, S., Yamasaki, M., Doebley, J., Mcmullen, M.D., Gaut, B. 2005. The genomic extent of artificial selection in maize. Science. 308(5726):1310-1314.
Zhao, Q., Mcmullen, M.D., Doebley, J. 2005. Molecular population genetics of maize domestication [abstract]. Maize Genetics Conference. Paper No. 176. p. 130.
Johal, G., Penning, B., Mcmullen, M.D. 2005. Identifying a natural suppressor of cell death in maize: implications for gene discovery, diversity evaluation and beyond [abstract]. Maize Genetics Conference. Paper No. 207. p.141.
Yamasaki, M., Tenaillon, M., Vroh, I., Schroeder, S., Sanchez-Villeda, H., Doebley, J., Gaut, B., Mcmullen, M.D. 2005. Genomic screen for domestication and improvement genes in maize [abstract]. Plant and Animal Genome Conference. p. 393.
Yamasaki, M., Tenaillon, M., Vroh Bi, I., Schroeder, S., Sanchez-Villeda, H., Doebley, J., Gaut, B., Mcmullen, M.D. 2005. Genomic screening for domestication and improvement genes in maize [abstract]. Maize Genetics Conference. Paper No. 161. p. 118.
Briggs, W., Buckler Iv, E.S., Canaran, P., Doebley, J., Fulton, T., Gaut, B., Goodman, M., Holland, J.B., Kresovich, S., Mcmullen, M.D., Stein, L., Ware, D., Wright, S., Zhao, W. 2005. Molecular and functional diversity in the maize genome [abstract]. Maize Genetics Conference. Paper No. 177. p. 126.
Svabek, C., Boddu, J., Cortes-Cruz, M., Mcmullen, M.D., Chopra, S. 2005. Isolation and functional characterization of a flavonoid 3' hydroxylase corresponding to the red aleurone 1 locus of maize [abstract]. Maize Genetics Conference. Paper No. 168. p. 122.
Flint Garcia, S.A., Houchins, K.E., Yamasaki, M., Doebley, J., Mcmullen, M. D. 2005. Genetic diversity and selection for amino acid genes and content in diverse maize [abstract]. Maize Genetics Conference. Paper No. 199. p. 137.
Briggs, W., Vroh, B., Yamasaki, M., Pressoir, G., Mcmullen, M.D., Gaut, B., Kresovich, S., Buckler Iv, E.S., Doebley, J. 2005. Snp genotyping for diversity and mapping studies in maize and teosinte [abstract]. Maize Genetics Conference. Paper No. 182. p. 129.
Frost, J., Snook, M., Houchins, K.E., Rector, B.G., Widstrom, N.W., Mcmullen, M.D. 2005. The genetic basis of recurrent selection gains for maysin in maize silks [abstract]. Plant and Animal Genome Conference. p. 395.
Meyer, J., Snook, M.E., Houchins, K.E., Rector, B.G., Widstrom, N.W., Mcmullen, M.D. 2005. The genetic basis of increased maize silk maysin levels through recurrent selection [abstract]. Maize Genetics Conference. No. T7, p. 24.

Progress 10/01/03 to 09/30/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Much greater depth of genetic knowledge and molecular-genetic technique are needed to attain optimum crop efficiency, productivity, and stability, or to derive valuable new properties for maize. The tools and insights from genetics must yet fulfill the promise of new technologies, to join fully into plant breeding and agronomic practices. The research includes mapping and descriptions of individual genes (phenotypes) and groups of genes (quantitative traits), their regulation, and their function at specific stages of development and under specific conditions, and the genetic diversity in maize germplasm that is available for study and enhancement. As yet, extensive knowledge about these systems is limited and must be obtained. Without extensive new knowledge about genetic mechanisms, gene functions, levels of gene diversity, and tools for manipulating them, it will not be possible to increase U.S. crop production to meet the world's needs for feed and food grains. Although maize breeding maintains a steady pace of advancement in efficiency, productivity, and stability, the crop continues to require high inputs, is subject to diverse biotic and abiotic stresses, and is only slowly advancing toward its theoretical physiological ceiling. New approaches and knowledge are needed to permit feasible incorporation of desirable traits from exotic maize into mainstream maize improvement programs. Novel diversity is needed for pest resistance, yield, and enhanced kernel traits. We have initiated a new research effort to use contrasting sequence diversity in maize inbreds, exotic accessions, and teosinte accessions to identify genes of agronomic importance. 2. List the milestones (indicators of progress) from your Project Plan. There have been major changes of program direction since the milestones for this plan were developed because of transfer of one scientist (Ed Coe) to another CRIS and the resignation of a second scientist (David Willmot) in March 2004. Therefore, of the original three objectives, only accomplishments for research under Objective 2 are detailed. Objective 2 (Michael D. McMullen): Determine the genetic mechanisms involved with regulation of metabolic pathways that control agronomic traits for maize. The four original areas of research were: Area 1) p1 diversity controlling flavone pathway genes, Area 2) use of microarray analysis to identify missing genes in the flavone pathway, Area 3) clone and characterize pal genes, and Area 4) test association analysis on flavone pathway. Year 1 (2001): Area 1) make populations; Area 2) isolate RNA, develop cDNA libraries; Area 3) clone and sequence pal genes; and Area 4) sequence flavone structural genes from association mapping population. Year 2 (2002): Area 1) develop molecular assays and measure phenotypes, Area 2) develop microarray technology, Area 3) develop gene specific probes and determine tissue specific expression, and Area 4) collect phenotypic data and complete sequencing of genes across association population. Year 3 (2003): Areas 1), 2), 3), and 4)--complete experiments and analyze results. Year 4 (2004): Area 3)--complete research; and Areas 1), 2), and 4)-- analyze results and design follow up experiments. Year 5 (2005): Areas 1), 2), and 4)--conduct follow-up experiments and publish results. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. The milestones for the entire 5-year plan for areas 1), 3), and 4) have been completed. The publication for areas 1) and 4) was submitted in January and is currently under revision for resubmission. The experiments for area 3) are completed and will be published by inclusion with an addition project not listed in the project plan. Research on area 2 was not performed to allow initiation of a new research area: Using sequence diversity to identify genes selected during domestication or crop improvement (see Question 4). 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004: A major constraint on the use of biotechnology for crop improvement is our limited knowledge of which genes control agronomic traits. The maize genetics project of the Plant Genetics Research Unit in Columbia, Missouri, in collaboration with Dr. John Doebley, University of Wisconsin, and Dr. Brandon Gaut, University of California-Irvine have developed and validated a novel approach to identifying candidate genes for the control agronomic traits by contrasting the sequence diversity present in inbred lines of maize to that present wild teosinte accession and maize landraces. In FY2004, we demonstrated that genes with low sequence diversity in inbred lines are greatly enriched for domestication and improvement genes. This approach allows an efficient, systemic screen of maize genes, and will provide the maize research community with numerous candidate genes for directed improvement of agronomic traits. B. Other Significant Accomplishment(s), if any: Recurrent selection is a routine method used by plant breeders, however, the nature of the genes selected during trait improvement is unknown. In research largely performed by University of Missouri graduate student Jenelle Frost, we conducted quantitative trait locus analyses on four populations for which the parents were developed by recurrent selection for high maysin levels. We demonstrated that genetic variation of both regulatory and structural genes was selected to lead to enhance maysin levels. Our data indicate that much of the genetic variation selected is involved in enhancing the flow of intermediates into the flavone pathway. These results form a conceptual basis to model results of selection for other metabolic pathways. C. Significant Accomplishments/Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The project has provided the majority of SSR, InDel (insertion/deletion) and SNP genetic marker resources in the public sector for trait mapping in maize, essentially allowing a total switch to these more efficient mapping methods. These resources impact the efficiency of genetic analysis for maize across all public and private maize projects. These markers have also been used to develop the integrated physical/genetic map of maize. Through these efforts, over half of the maize genome is now covered with anchored BAC contigs. The most significant finding from the trait analysis section of the project is the demonstration of the central importance of regulatory genes as quantitative trait loci. Enzyme-encoding genes also have effects, but to a more limited extent. Related pathways of biosynthesis share intermediates. Our data show that genetic variation of regulatory or enzymatic genes can either result in shunting biochemical intermediates between pathways, or in independently- modulated flow, a process known as compartmentalization. These genetic mechanisms allow for fine control of related biochemical products and subtle variation in the corresponding agronomic traits, and guide conceptions of strategies for crop improvement. This project will continue to provide essential tools for maize improvement along with identification of novel genes for limiting traits of maize. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? As they were developed, new SSR, InDel, and SNP markers and the integrated genetic/physical map were transferred to researchers, both public and private, through the Maize Genome Database (MaizeGDB), and iMap at www.maizemap.org. Research findings have been published in scientific journals and presented at national meetings. Presentations were given to visiting groups and scientists interested in the impact of plant genomics on crop improvement.

Impacts
(N/A)

Publications

Mcmullen, M.D., Kross, H., Snook, M.E., Cortes-Cruz, M., Houchins, K.E., Musket, T.A., Coe Jr, E.H. 2004. Salmon silk genes contribute to the elucidation of the flavone pathway in maize (zea mays l.). Journal of Heredity. 95(3):225-233.
Wei, F., Nelson, W., Goicoechea, J.L., Engler, F., Lee, S., Butler, E., Kim, H., Schroeder, S., Fang, Z., Mcmullen, M.D., Bi, I., Davis, G., Sanchez-Villeda, H., Yim, Y., Havermann, S., Bowers, J., Paterson, A., Polacco, M.L., Gardiner, J., Cone, K., Coe Jr, E.H., Soderlund, C., Wing, R.A. 2004. An integrated comprehensive map of the maize genome [abstract]. Plant and Animal Genome Conference. 12:75.

Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Much greater depth of genetic knowledge and molecular-genetic techniques are needed to attain optimum crop efficiency, productivity, and stability, or to derive valuable new properties for maize. The tools and insights from genetics must yet fulfill the promise of new technologies, to join fully into plant breeding and agronomic practices. The research includes mapping and descriptions of individual genes (phenotypes) and groups of genes (quantitative traits), their regulation, and their function at specific stages of development and under specific conditions, and the genetic diversity in maize germplasm that is available for study and enhancement. As yet, extensive knowledge about these systems is limited and must be obtained. We have initiated a new research effort to use contrasting sequence diversity in maize inbreds, exotic accessions, and teosinte accessions to identify genes of agronomic importance. 2. How serious is the problem? Why does it matter? Without extensive new knowledge about genetic mechanisms, gene functions, levels of gene diversity, and tools for manipulating them, it will not be possible to increase U.S. crop production to meet the World's needs for feed and food grains. Although maize breeding maintains a steady pace of advancement in efficiency, productivity, and stability, the crop continues to require high inputs, is subject to diverse biotic and abiotic stresses, and is only slowly advancing toward its theoretical physiological ceiling. New, valuable properties remain to be discovered as well. Commercial corn in the U.S. consists of only an estimated 5% of the germplasm in the world. New approaches and knowledge are needed to permit feasible incorporation of desirable traits from exotic maize into mainstream maize improvement programs. Novel diversity is needed for pest resistance, yield, and enhanced kernel traits. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 302, Plant Biological and Molecular Processes (100%). This research addresses the goal of understanding mechanisms, both genetic and biochemical, by which traits controlled by single genes, and by multiple genes, result in visible (phenotypes) and measurable (growth and production efficiency) differences. This research also defines the diversity available in maize germplasm resources. Novel approaches on using diversity to identify genes of agronomic importance will be developed. Multiple-gene traits relevant to yield, desirable plant structure, and insect resistance will be mapped, candidate genes identified, and the biochemical interrelationships and regulation of groups of genes controlling these traits will be defined. Understanding the nature of genetic variation for traits is key to developing rational approaches for biochemical pathway modifications related to crop improvement. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2003: A major constraint on the use of biotechnology for crop improvement is our limited knowledge of which genes control agronomic traits. The maize genetics project of the USDA-ARS Plant Genetics Research Unit, Columbia, Missouri, in collaboration with John Doebley, University of Wisconsin, and Brandon Gaut, University of California-Irvine, have developed and validated a novel approach to identifying candidate genes for the control agronomic traits (See Vigouroux et al., publications list). We have demonstrated that by contrasting levels of sequence diversity among maize inbreds, exotic maize accessions, and teosinte (wild maize) accessions, we can identify genes that have undergone selection at maize domestication or improvement, based on their importance to selected agronomic traits. This approach, when applied in a systemic screen of maize genes, will provide the maize research community with numerous candidate genes for manipulation of agronomic traits. B. Other significant accomplishment(s), if any: none. C. Significant activities that support special target populations: none. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The project has provided the majority of simple sequence repeat (SSR), insertion-deletion (InDel) and single nucleotide polymorphism (SNP) genetic marker resources in the public sector for trait mapping in maize, essentially allowing a total switch to these more efficient mapping methods. These resources impact the efficiency of genetic analysis for maize across all public and private maize projects. These markers have also been used to develop the integrated physical/genetic map of maize. Through these efforts, over half of the maize genome is now covered with anchored Bacterial artificial chromosome (BAC) contigs. The most significant finding from the trait analysis section of the project is the demonstration of the central importance of regulatory genes as quantitative trait loci. Enzyme-encoding genes also have effects, but to a more limited extent. Related pathways of biosynthesis share intermediates. Our data show that genetic variation of regulatory or enzymatic genes can either result in shunting biochemical intermediates between pathways, or in independently modulated flow, a process known as compartmentalization. These genetic mechanisms allow for fine control of related biochemical products and subtle variation in the corresponding agronomic traits, and guide conceptions of strategies for crop improvement. This project will continue to provide essential tools for maize improvement along with identification of novel genes for limiting traits of maize. Additionally, this project will broaden the germplasm base of U.S. maize by identifying novel genes from exotic germplasm to overcome limiting factors for maize production. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2004: Finish data collection and analysis for a selective genotyping project for CRW resistance. Finish the second year of a corn rootworm (CRW) mapping population project. Release one CRW population and two European corn borer resistant inbreds. Genotype lines divergent for protein content and present the results. Pioneer the use of MITES markers for quantitative trait loci (QTL) detection. Complete a Design II experiment to estimate protein level genetic components. Complete a SNP development project with the release of at least 3000 mapped SNPs for the maize research community. Extend the current physical map of maize from 50 to 75% genome coverage. Use sequence diversity by "signatures of selection" to identify candidate genes for agronomic traits. FY2005: Complete final year of QTL identification in our CRW3 population. Complete the backcross conversion of a stalk-rot QTL into two sources with horizontal resistance and validate the QTL with near-isogenic-line tests. Determine a comprehensive genome map of Myb genes for maize and identify candidate traits regulated by these transcription factors. Demonstrate that modification of the expression of genes identified as having "signatures of selection," alters agronomic traits. FY2006: Continue to identify and test candidate genes for a wide range of agronomic traits. Define functions for many of the maize Myb genes. Complete the QTL study with Cuba 164. Complete the first year of a QTL study in a stalk-rot/multiple-blight-resistant recurrent selection population. Complete marker-assisted selection trials to validate putative QTL in previous projects. Complete conversion of several high or altered protein lines with a transfer RNA (tRNA) miscoding in a high- lysine transgenic. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? As they were developed new SSR, InDel, and SNP markers and the integrated genetic/physical map were transferred to researchers, both public and private, through the Maize Genome Database (MaizeDB), and iMap at www. maizemap.org. Research findings have been published in scientific journals and presented at national meetings. Presentations to visiting groups and scientists interested in impact of plant genomics on crop improvement. Organized cooperators in seven states for a 12 location, two-year trial: "Genotype by environment interactions for reaction to European corn borer" which will also serve to guide the design of mapping population(s).

Impacts
(N/A)

Publications

BUSHMAN, B.S., SNOOK, M.E., GERKE, J.P., SZALMA, S.J., BERHOW, M.A., HOUCHINS, K.E., MCMULLEN, M.D. TWO LOCI EXERT MAJOR EFFECTS ON CHLOROGENIC ACID SYNTHESIS IN MAIZE SILKS. CROP SCIENCE. 2002. v. 42. p. 1669-1678.
CHOPRA, S., COCCIOLONE, S.M., BUSHMAN, B.S., SANGAR, V., MCMULLEN, M.D., PETERSON, T. THE MAIZE UNSTABLE FACTOR FOR ORANGE1 IS A DOMINANT EPIGENETIC MODIFIER OF A TISSUE SPECIFICALLY SILENT ALLELE OF PERICARP COLOR1. GENETICS. 2003. v. 163. p. 1135-1146.
CORTEZ-CRUZ, M., SNOOK, M.E., MCMULLEN, M.D. THE GENETIC BASIS OF C- GLYCOSYL FLAVONE B-RING MODIFICATION IN MAIZE (ZEA MAYS L.) SILKS. GENOME. 2003. v. 46. p. 182-194.
DIAS, A.P., BRAUN, E.L., MCMULLEN, M.D., GROTEWOLD, E. RECENTLY DEVELOPED MAIZE R2R3 MYB GENES PROVIDE EVIDENCE FOR DISTINCT MECHANISMS OF EVOLUTIONARY DIVERGENCE AFTER DUPLICATION. PLANT PHYSIOLOGY. 2003. v. 131. p. 610-620.
FLINT-GARCIA, S.A., MCMULLEN, M.D., DARRAH, L.L. GENETIC RELATIONSHIP OF STALK STRENGTH AND EAR HEIGHT IN MAIZE. CROP SCIENCE. 2003. v. 43. p. 23- 31.
SZALMA, S.J., SNOOK, M.E., BUSHMAN, B.S., HOUCHINS, K.E., MCMULLEN, M.D. DUPLICATE LOCI AS QTL: THE ROLE OF CHALCONE SYNTHASE LOCI IN FLAVONE AND PHENYLPROPANOID BIOSYNTHESIS IN MAIZE. CROP SCIENCE. 2002. v. 42. p. 1679- 1687.
VIGOUROUX, Y., MCMULLEN, M.D., HITTINGER, C.T., HOUCHINS, K.E., SCHULZ, L., KRESOVICH, S., MATSUOKA, Y., DOEBLEY, J. IDENTIFYING GENES OF AGRONOMIC IMPORTANCE IN MAIZE BY SCREENING MICROSATELLITES FOR EVIDENCE OF SELECTION DURING DOMESTICATION. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES. 2002. v. 99. p. 9650-9655.
YIM, Y.S., DAVIS, G.Y., DURU, N., MUSKET, T., LINTON, E.W., MESSING, J.W., MCMULLEN, M.D., SODERLUND, C., POLACCO, M.L., GARDINER, J.M., COE JR, E.H. CHARACTERIZATION OF THREE MAIZE BAC LIBRARIES AND ANCHORING OF THE PHYSICAL MAP TO THE GENETIC MAP USING HIGH-DENSITY BAC FILTER HYBRIDIZATION. PLANT PHYSIOLOGY. 2002. v. 130. p. 1686-1696.

Progress 10/01/01 to 09/30/02

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Much greater depth of genetic knowledge and molecular-genetic technique is needed to attain optimum crop efficiency, productivity, and stability, or to derive valuable new properties for maize. The tools and insights from genetics must yet fulfill the promise of new technologies, to join fully into plant breeding and agronomic practices. The research includes mapping and descriptions of individual genes (phenotypes) and groups of genes (quantitative traits), their regulation, and their function at specific stages of development and under specific conditions, and the genetic diversity in maize germplasm that is available for study and enhancement. As yet, extensive knowledge about these systems is limited and must be obtained. 2. How serious is the problem? Why does it matter? Without extensive new knowledge about genetic mechanisms, gene functions, levels of gene diversity, and tools for manipulating them, it will not be possible to increase U.S. crop production to meet the World's needs for feed and food grains. Although maize breeding maintains a steady pace of advancement in efficiency, productivity, and stability, the crop continues to require high inputs, is subject to diverse biotic and abiotic stresses, and is only slowly advancing toward its theoretical physiological yield ceiling. New, valuable properties remain to be discovered as well. Commercial corn in the United States consists of only an estimated 5% of the germplasm in the world. New approaches and knowledge are needed to permit feasible incorporation of desirable traits from exotic maize into mainstream maize improvement programs. Novel diversity is needed for pest resistance, yield, and enhanced kernel traits. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 302, Improving Plant Biological and Molecular Processes (100%). This research addresses the goal of understanding mechanisms, both genetic and biochemical, by which traits controlled by single genes, and by multiple genes, result in visible (phenotypes) and measurable (growth and production efficiency) differences. This research also defines the diversity available in maize germplasm resources. Novel approaches on using diversity to identify genes of agronomic importance will be developed. Multiple-gene traits relevant to yield, desirable plant structure, and insect resistance will be mapped, candidate genes identified, and the biochemical interrelationships and regulation of groups of genes controlling these traits will be defined. Understanding the nature of genetic variation for traits is key to developing rational approaches for biochemical pathway modifications related to crop improvement. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002: The application of molecular markers as a routine step in crop improvement requires high-throughput genotyping tools. The maize genetics project of Plant Genetics Research Unit in Columbia, MO, in collaboration with other scientists in the NSF-funded Maize Mapping Project, have initiated the first large-scale single-nucleotide polymorphism (SNP) discovery project in the maize public sector. Single nucleotide polymorphisms have been identified in 1,300 genes in this first year of the project. This resource will provide the public research community with the first truly high-throughput markers in maize allowing great improvement in marker-assisted selection efficiency. B. Other Significant Accomplishment(s), if any: Novel genes are needed to remove limiting factors for corn production and utilization. The Plant Genetics Research Unit, Columbia, MO, in collaboration with L.M. Pollak, USDA-ARS Ames, IA, and B.E. Hibbard, USDA- ARS, Columbia, MO, has identified corn germplasm sources that segregate broadly for yield, protein content and corn rootworm (CRW) resistance. We have confirmed these materials in the field and have begun genotyping to identify quantitative trait loci (QTLs) and candidate genes. The impact will be the acquisition of the knowledge required for the genetic dissection and use of molecular markers for improvement of these traits. C. Significant AccomplishmentsActivities that Support Special Target Populations: none. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? This is a new project based on a Project Plan certified by the OSQR 2/7/02, replacing 3622-21000-019-00D, "Genetic mechanisms and molecular genetic resources for corn." Current research activity follows from the previous project. The project has provided simple sequence repeat (SSR) resources for trait mapping in maize, essentially allowing a total switch to these more efficient mapping methods. This resource impacts the efficiency of genetic analysis for maize across all public and private maize projects. The bacterial artificial chromosome (BAC) libraries of maize produced under this project are in wide use in the maize research community. The most significant finding from the trait analysis section of the project is the demonstration of the central importance of regulatory genes as quantitative trait loci. Enzyme-encoding genes also have effects, but to a more limited extent. Related pathways of biosynthesis share intermediates. Our data show that genetic variation of regulatory or enzymatic genes can either result in shunting biochemical intermediates between pathways, or in independently-modulated flow, a process known as compartmentalization. These genetic mechanisms allow for fine control of related biochemical products and subtle variation in the corresponding agronomic traits, and guide conceptions of strategies for crop improvement. This project will continue to provide essential tools for maize improvement along with identification of novel genes for limiting traits of maize. Additionally, this project with broaden the germplasm base of U.S. maize by identifying novel genes from exotic germplasm to overcome limiting factors for maize production. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2003: Present a physical map for maize with a majority of the genome in ordered BAC contigs and anchored to the maize genetic map. Release SNP mapping resources to the maize community. Define QTLs for flavone synthesis based on gene expression effects. Define the utility of association analysis in the detection and characterization of QTLs for agronomic traits using maysin and chlorogenic acid synthesis as model traits. Determine the exotic maize accession contribution to Germplasm Enhancement of Maize (GEM) lines. Identify novel genes controlling protein levels in maize kernels using diverse germplasm for the GEM project. Characterize storage protein and amino acid profile variability among key accessions representing diverse races of maize. FY2004: Determine genes for resistance to CRW and use information to select for improved CRW-resistant populations and lines. Complete a SNP development project with the release of at least 3,000 mapped SNPs for the maize research community. FY2005: Define and utilize novel genes for yield and other agronomic traits from the exotic line Cuba 164 to improve elite maize germplasm. Determine a comprehensive genome map of Myb genes for maize and identify candidate traits regulated by these transcription factors. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? The new SSR and SNP markers we have developed were transferred to researchers, both public and private, through the Maize Genome Database (MaizeDB) as they were developed. Research findings have been published in scientific journals and presented at national meetings. Presentations to visiting groups and scientists interested in impact of plant genomics on crop improvement were made. Organized cooperators in seven states for a 12 location, two-year trial: "Genotype by environment interactions for reaction to European corn borer" which will also serve to guide the design of mapping population(s).

Impacts
(N/A)

Publications

McMullen, M.D., Lee, E.A., Szalma, S.J., Bushman, B.S., Snook, M. The role of quantitative trait locus analysis in gene discovery. Proceedings of the 56th Annual Corn & Sorghum Research Conference. 2001. p. 237-245.
Vroh, I., McMullen, M.D., Schroeder, S., Sanchez-Villeda, H., Houchins, K., Fang, Z., Polacco, M.L., Gardiner, J., Morgante, M., Coe E.H. Single nucleotide polymorphism discovery and mapping in maize. 44rd Annual Maize Genetics Conference. 2002. Abstract. p. 83.
Sharopova, N., McMullen, M.D., Schultz, L., Schroeder, S., Sanchez-Villeda, H., Gardiner, J., Bergstrom, D., Houchins, K., Melia-Hancock, S., Musket, T., Duru, N., Polacco, M.L., Edwards, K., Ruff, T.G., Register, J.C., Brouwer, C.R., Thompson, R.D., Velasco, R., Chin, E., Lee, M., Woodman- Clikeman, W., Long, M.J., Liscum, E., Cone, K.C., Davis, G., Coe, E.H., Jr. Development and mapping of SSR markers for maize. Plant Molecular Biology. 2002. v. 48. p. 463-481.