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Open Access Highly Accessed Research article

TILLING to detect induced mutations in soybean

Jennifer L Cooper1, Bradley J Till12, Robert G Laport1, Margaret C Darlow1, Justin M Kleffner3, Aziz Jamai4, Tarik El-Mellouki4, Shiming Liu4, Rae Ritchie5, Niels Nielsen5, Kristin D Bilyeu6, Khalid Meksem4, Luca Comai27 and Steven Henikoff1*

  • * Corresponding author: Steven Henikoff steveh@fhcrc.org

  • † Equal contributors

Author Affiliations

1 Fred Hutchinson Cancer Research Center, Seattle, WA 98107, USA

2 Department of Biology, University of Washington, Box 355325, Seattle, WA 98195, USA

3 National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA

4 Department of Plant Soil and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USA

5 USDA-ARS Crop Production and Pest Control Research Unit, Purdue University, West Lafayette, IN 47907, USA

6 USDA-ARS Plant Genetics Research Unit, Columbia, MO 65211, USA

7 Current address: Department of Plant Biology and Genome Center, UC Davis, Davis, CA 95616, USA

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BMC Plant Biology 2008, 8:9  doi:10.1186/1471-2229-8-9

Published: 24 January 2008

Abstract

Background

Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).

Results

We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (~1/140 kb), while another EMS population had a mutation density of ~1/250 kb. The remaining population had a mutation density of ~1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.

Conclusion

The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.