Robert Stupar - US grants

PI: Wayne Parrott, Univ. of Georgia
Co-PI: Tom Clemente, Univ. of Nebraska,
Co-PI: Gary Stacey, Univ. of Missouri, Columbia
Co-PI: Carroll Vance, Univ. of Minnesota
Senior Personnel: Zhanyuan J. Zhang, Univ. of Missouri, Columbia

The soybean crop is a major source of vegetable protein and edible oil. A thorough understanding of soybean genetics is essential for the development of new soybean varieties that will meet the nutritional, environmental, and industrial requirements that soybean will fulfill over the next decades. Soybean has many research tools available that facilitate its genetic study. However, there has been one genetic tool notably absent for soybean, namely the availability of reverse genetic tools; that is, a way to identify and determine the function of soybean genes that hold academic, biological or agricultural importance. Therefore, the long-term goal of this project is to develop and distribute the resources to needed to investigate the function of soybean genes, especially those involved in soybean quality and yield. This goal will be accomplished by building and cataloging a collection of soybean deletion, insertional, and gene activation mutants derived by engineering soybeans with genes from rice, tobacco or maize that naturally move around the genome and insert themselves into other genes. Such mutants alter the appearance of the plant, or can be detected in the laboratory. Moreover, the team will investigate the use of fast neutrons to enhance their ability to find the desired mutations. The research team will then compare the relative efficiency and effectiveness of the various techniques.

Not only will soybean geneticists, breeders, farmers and consumers benefit from the information this project derives, but the set of transposon tools developed by this proposal will be available and useful for any dicot crop. Furthermore, any soybean plants that are immediately useful for additional genetic studies or for use in the development of new varieties will be immediately available to the geneticists or breeders who need and request them on a cost-recovery basis. The research will be executed in large part by graduate students and postdoctoral fellows, effectively helping train the next generation of soybean breeders and geneticists. In addition, each participating state has a series of small research projects that are specifically designed to interest and engage promising high school and undergraduate students in science and scientific careers.

Soybean oil and protein are already substrates for numerous industrial and alimentary uses. It is expected that new, designer oils and proteins can be used for everything from healthier diets to nutraceuticals to novel industrial compounds to biodiesel. The genetic knowledge and information on the available seed stocks will be made available to the public via a web site (http://digbio.missouri.edu/gmgenedb/index.php) designed for the purpose, presentations by the scientists of their work at scientific meetings, and reports of the work published in peer-reviewed journals.

The XIV National Congress of Biochemistry and Plant Molecular Biology & 7th Symposium Mexico-USA will be held in Campeche, Mexico, November 29 - December 2, 2011. The objectives of the meeting are to: 1) share the most recent research findings in plant biochemistry, genomics, and genetics between U.S. and Mexican colleagues in order that they may explore common research interests; 2) facilitate collaboration on plant biology research between US and Mexican plant scientists; and 3) ensure that there is broad participation from the US at the conference, by targeting early career, women and minority scientists. The proposed topics focus on fundamental discoveries that extend our understanding of several aspects of plant biology and address compelling and novel scientific discoveries relative to plant biochemistry, molecular biology, and food security. The proposed meeting topics include: Plant responses to the environment, Plant-Pathogen and insect interactions, Plant nutrition, Plant/crop evolution, Epigenetic regulation of plant processes, Plant molecular systematics and biodiversity, Applied biotechnology, Association mapping and crop improvement, Comparative structural and functional genomics

Broader Impacts
The international and local organizing committees will identify women, early career scientists and participants who are members of underrepresented groups. The targets for participation are: 50% for female representation, 15% for underrepresented groups and 15-20% for early career scientists.

PI: Wayne Parrott (University of Georgia)

CoPIs: Tom Clemente (University of Nebraska-Lincoln), Gary Stacey (University of Missouri-Columbia), and Carroll Vance and Robert Stupar (University of Minnesota-Twin Cities)

Soybean is the world's largest single source of vegetable protein and edible oil and thus a key crop for meeting the nutritional needs of the increasing global population. Therefore, a thorough understanding of soybean genetics will be important for the development of new soybean varieties to meet the nutritional, environmental, and industrial requirements that soybean will fulfill over the coming decades. There are many research tools available that facilitate the genetic study of soybean. However, there has been one resource notably absent for soybean - reverse genetic tools. These tolls provide a way to identify and determine the function of soybean genes of academic, biological, or agricultural importance. Therefore, the long-term goal of this project is to build upon previous funding to develop and distribute the resources to needed to investigate the function of soybean genes, especially those involved in soybean quality and yield. The primary objective of the project is to provide novel mutant plants for soybean functional genomics research. This goal will be accomplished by building and cataloguing a collection of soybean insertional and gene activation mutants derived by engineering soybeans with a transposable element from rice which naturally moves around the genome and inserts itself into other genes. When the transposable element inserts itself into soybean genes, it prevents the gene from functioning which in turn can alter the appearance of the plant, or can be detected in the laboratory. Fast neutrons will also be used to derive additional mutations, as fast neutrons delete genes altogether, also changing the plant's appearance. The change in appearance that results from disrupting or deleting a gene is a direct indication of that gene's function.

All of the genetic stocks developed by this work will be made available to the soybean research and breeding communities upon request. To aid in identifying and distributing mutant plants, interested researchers will attend workshops where they will search for mutants that are relevant to their research programs. Besides the basic research community, soybean breeders also will benefit directly, by gaining the ability to make informed decisions about which genes to select for in order to more easily develop higher-yielding, disease and stress resistant soybean varieties. Genotype and phenotype information will be available via SoyBase (www.soybase.org), Soybean Knowledge Base (SoyKB; http://soykb.org), and/or the project website at http://soymutants.uga.edu.

PI: Nevin Young (University of Minnesota)

Co-PIs: Michael Sadowsky, Robert Stupar, and Peter Tiffin (University of Minnesota), Maria Harrison (Boyce Thompson Institute for Plant Research), Betsy Martinez-Vaz (Hamline University), Jason Rafe Miller (J. Craig Venter Institute), Joann Mudge (National Center for Genome Resources)

This project uses functional assays, de novo genome assembly and bioinformatic data-mining to characterize symbiosis genes in the model legume Medicago truncatula. Legumes are noteworthy for the sophisticated symbioses they form with rhizobial bacteria and arbuscular mycorrhizal (AM) fungi. However, existing knowledge about symbioses comes primarily from knockout mutants, an approach that often misses genes of subtle yet significant effect, especially genes likely to be important in contemporary evolution. In earlier work, several strongly supported candidate symbiosis loci were discovered through genome-wide association analysis (GWAS) and support for candidate genes often included independent evidence like expression profile, correlation with multiple traits or co-localization with known symbiotic phenotypes. The current project will test ~100 of these candidate loci through reverse genetic experiments involving Tnt1 insertion and RNAi "knockdown" plant lines. Promising genes will be examined through interaction assays involving previously defined Sinorhizobium and AM strains and tested for "gene-for-gene" relationships using a panel of 48 sequenced Sinorhizobium strains. Earlier GWAS mapping only targeted SNP variation, even though structural variants (SVs) and copy number variants (CNVs) are known to have major impacts on genome variation. This is especially relevant to the genomics of symbiosis because the large gene families such as the NB-ARC domain-containing genes and nodule cysteine rich peptides (NCRs) play critical roles in symbiosis. This project will deeply sequence and de novo assemble 30 nodal M. truncatula accessions, in order to discover SVs and CNVs. SVs and CNVs will be imputed genome-wide, leading to a new round of GWAS to discover symbiotic loci missed in the earlier phase of mapping. The primary outcomes of this project will be the identification of genes associated with the contemporary evolution of symbiosis as well as the architecture of M. truncatula genomic diversity.

This research effort will be extended by involving undergraduates from Hamline University, a four year institution located near the University of Minnesota, and from the University of Puerto Rico (UPR). Students at Hamline will work throughout the academic year and then together with UPR students during the summer as part of Minnesota's Life Sciences Summer Undergraduate Research Program (LSSURP) program. Their work will target the important but largely uncharacterized Sinorhizobial enzyme, ACC deaminase, and students will also participate in the screening of reverse genetic mutants. These experiences will provide the students the opportunity to develop their own hypothesis-driven projects. Through joint mentoring by project PIs, student training will also assist the undergraduate research program at Hamline to become more competitive for its own future external research initiatives. Genomic sequence resources will be available for public through medicagohapmap.org, and the Short Read Archive, dbSNP and FTP sites at Genbank. Importantly, the underlying Medicago Hapmap GWAS platform, available at medicagohapmap.org, will provide a long-term resource for the broader research community to discover legume genes controlling quantitative variation of agricultural and biological interest, especially trait variation in alfalfa (Medicago sativa), the fourth most widely cultivated crop in the U.S.

PI: Peter LaFayette (University of Georgia)
coPIs: Robert Stupar (University of Minnesota), Minviluz Stacey (University of Missouri), Thomas E. Clemente (University of Nebraska), and C. Nathan Hancock (University of South Carolina - Aiken)

Soybeans provide the single greatest source of vegetable protein and oil worldwide, and uses of this important crop now range from human food to alternatives for petroleum-derived chemicals. The goal of this project is to create molecular tools to make it easy for geneticists and breeders to identify and study the function of genes associated with desirable agronomic traits. The project will provide laboratory and field research opportunities for postdoctoral fellows and undergraduate students, including students from a predominantly undergraduate institution. In addition, yearly workshops will bring together soybean researchers from across the nation to participate in hands-on training in precision genomics and in screening plant populations to identify new traits of interest. The resources developed by this project will advance soybean improvement, which in turn can lead to designer oils and proteins for healthier diets, industrial compounds, and biodiesel to meet a growing global demand.

This research will add to the current set of forward and reverse genetics resources available to the plant research community. A key activity will be to develop and characterize the publicly available soybean fast neutron (FN) mutagenesis population with a goal of attaining a biologically relevant amount of genomic coverage. Concurrently, individual genes within FN deleted regions that alter traits of importance to the soybean community (e.g., yield, oil, protein, stress tolerance, and nitrogen fixation) will be mutated using CRISPER/Cas9 technology to pinpoint the genes responsible for specific phenotypes. In addition, a set of complementary, transposon-based mutagenesis populations will be made to specifically tailor gene discovery for the highly duplicated genome of soybean. TheTnt1 insertional mutagenesis population will result in traditional loss-of-function phenotypes, the Ac/Ds activation tagging population will cause gene mis-expression, and the mPing transposon will be used for both gene silencing and overexpression. These strategies are expected to result in recovery of novel phenotypes not previously observed in soybean. Data, phenotypes, seeds and other information will be available via the project website at http://soymutants.uga.edu and at SoyBase.org.