Phenotypic and Genomic Analyses of a Fast Neutron Mutant Population Resource in Soybean 1[W][OA] Yung-Tsi Bolon*, William J. Haun, Wayne W. Xu, David Grant, Minviluz G. Stacey, Rex T. Nelson, Daniel J. Gerhardt, Jeffrey A. Jeddeloh, Gary Stacey, Gary J. Muehlbauer, James H. Orf, Seth L. Naeve, Robert M. Stupar, and Carroll P. Vance Plant Science Research Unit, United States Department of Agriculture-Agricultural Research Service, St. Paul, Minnesota 55108 (Y.-T.B., C.P.V.); Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (W.J.H., G.J.M., J.H.O., S.L.N., R.M.S., C.P.V.); Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, Minnesota 55455 (W.W.X.); Corn Insects and Crop Genetics Research Unit, United States Department of Agriculture-Agricultural Research Service, Ames, Iowa 50011 (D.G., R.T.N.); Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211 (M.G.S., G.S.); and Roche NimbleGen, Inc., Research and Development, Madison, Wisconsin 53719 (D.J.G., J.A.J.) Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research. The release of whole genome sequences in crops such as soybean (Glycine max; Schmutz et al., 2010) marks a new era for genomics research in crop species. Soybean is one of the most valued crops for its ability to fix nitrogen and provide seed protein and oil. Resources to study gene function in this important species are needed, and using mutagenesis to develop population resources has long proven to be a key step for identifying gene function in many organisms. A number of mutagen sources exist for introducing genomic variation. These include chemical, radiation, and transformation-induced mutagenesis of plant ge- nomes (Østergaard and Yanofsky, 2004; Waugh et al., 2006; Kuromori et al., 2009). Each of these methods results in a signature footprint of structural variation across the genome (Alonso and Ecker, 2006). Fast neu- tron (FN) radiation is a particularly promising source of mutagenesis due to the potential to create deletions in a wide range of sizes (Li and Zhang, 2002) for gene knockouts and disruptions. FN radiation has been used to induce mutations for many decades and has been shown to be an effective mutagen in plants (Koornneef et al., 1982). The major- ity of mutations that result from FN bombardment are DNA deletions that range in size from a few base pairs to several megabases (Li et al., 2001; Men et al., 2002). Precedence exists in many species, including Arabi- dopsis (Arabidopsis thaliana; Alonso et al., 2003), Med- icago truncatula (Oldroyd and Long, 2003), Glycine soja (Searle et al., 2003), barley (Hordeum vulgare; Zhang et al., 2006b), and Lotus japonicus (Hoffmann et al., 1 This work was supported by the National Science Foundation Plant Genome Program (award no. 0820769), the U.S. Department of Agriculture-Agricultural Research Service Current Research Infor- mation System (grant no. 3640–21000–024–00D), the Minnesota Soybean Research and Promotion Council, and the United Soybean Board (project no. 0288). * Corresponding author; e-mail hsie0024@umn.edu. The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Yung-Tsi Bolon (hsie0024@umn.edu), Carroll P. Vance (carroll. vance@ars.usda.gov), Robert M. Stupar (stup0004@umn.edu), and James H. Orf (orfxx001@umn.edu). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a sub- scription. www.plantphysiol.org/cgi/doi/10.1104/pp.110.170811 240 Plant Physiology Ò , May 2011, Vol. 156, pp. 240–253, www.plantphysiol.org Ó 2011 American Society of Plant Biologists Downloaded from https://academic.oup.com/plphys/article/156/1/240/6111426 by guest on 10 June 2023