title: TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements.
abstract: 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).
authors.affiliation: Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom.
authors.forename: Jillian
authors.initials: J
authors.lastname: Perry
authors.forename: Andreas
authors.initials: A
authors.lastname: Brachmann
authors.forename: Tracey
authors.initials: T
authors.lastname: Welham
authors.forename: Andreas
authors.initials: A
authors.lastname: Binder
authors.forename: Myriam
authors.initials: M
authors.lastname: Charpentier
authors.forename: Martin
authors.initials: M
authors.lastname: Groth
authors.forename: Kristina
authors.initials: K
authors.lastname: Haage
authors.forename: Katharina
authors.initials: K
authors.lastname: Markmann
authors.forename: Trevor L
authors.initials: TL
authors.lastname: Wang
authors.forename: Martin
authors.initials: M
authors.lastname: Parniske
mesh_terms: D000483:Alleles; D017360:Arabidopsis / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D030541:Databases, Genetic; D005020:Ethyl Methanesulfonate / Q000494:pharmacology*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D018745:Genome, Plant; D000070116:Lotus / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D055170:Plant Root Nodulation / Q000235:genetics*; D013559:Symbiosis / Q000235:genetics*
chemical_list: D018744:DNA, Plant; D005020:Ethyl Methanesulfonate