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TILLING in Lotus japonicus Identified Large Allelic Series for Symbiosis Genes and Revealed a Bias in Functionally Defective Ethyl Methanesulfonate Alleles toward Glycine Replacements^1,[W],[OA]

Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom (J.P., M.C., K.M., M.P.); John Innes Centre, Norwich NR4 7UH, United Kingdom (J.P., T.W., T.L.W.); and University of Munich, Biocenter, Genetics, 82152 Martinsried, Germany (A. Brachmann, A. Binder, M.C., M.G., K.H., K.M., M.P.)

We have established tools for forward and reverse genetic analysis of the legume Lotus (Lotus japonicus). A structured population of M2 progeny of 4,904 ethyl methanesulfonate-mutagenized M1 embryos is available for single nucleotide polymorphism mutation detection, using a TILLING (for Targeting Induced Local Lesions IN Genomes) protocol. Scanning subsets of this population, we identified a mutation load of one per 502 kb of amplified fragment. Moreover, we observed a 1:10 ratio between homozygous and heterozygous mutations in the M2 progeny. This reveals a clear difference in germline genetics between Lotus and Arabidopsis (Arabidopsis thaliana). In addition, we assembled M2 siblings with obvious phenotypes in overall development, starch accumulation, or nitrogen-fixing root nodule symbiosis in three thematic subpopulations. By screening the nodulation-defective population of M2 individuals for mutations in a set of 12 genes known to be essential for nodule development, we identified large allelic series for each gene, generating a unique data set that combines genotypic and phenotypic information facilitating structure-function studies. This analysis revealed a significant bias for replacements of glycine (Gly) residues in functionally defective alleles, which may be explained by the exceptional structural features of Gly. Gly allows the peptide chain to adopt conformations that are no longer possible after amino acid replacement. This previously unrecognized vulnerability of proteins at Gly residues could be used for the improvement of algorithms that are designed to predict the deleterious nature of single nucleotide polymorphism mutations. Our results demonstrate the power, as well as the limitations, of ethyl methanesulfonate mutagenesis for forward and reverse genetic studies. (Original mutant phenotypes can be accessed at http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus Access to the Lotus TILLING facility can be obtained through http://www.lotusjaponicus.org or http://revgenuk.jic.ac.uk)

² Present address: John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom.

³ Present address: Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark.

The author 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) is: Trevor L. Wang (trevor.wang{at}bbsrc.ac.uk).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.142190

^* Corresponding author; e-mail parniske{at}lmu.de.

Received May 29, 2009; accepted July 21, 2009; published July 29, 2009.

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