RRC ID 4683
Author Perry J, Brachmann A, Welham T, Binder A, Charpentier M, Groth M, Haage K, Markmann K, Wang TL, Parniske M.
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.
Journal Plant Physiol.
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).
Volume 151(3)
Pages 1281-91
Published 2009-11
DOI 10.1104/pp.109.142190
PII pp.109.142190
PMID 19641028
PMC PMC2773058
MeSH Alleles Arabidopsis / genetics DNA, Plant / genetics Databases, Genetic Ethyl Methanesulfonate / pharmacology* Gene Expression Regulation, Plant Genes, Plant Genome, Plant Lotus / genetics* Molecular Sequence Data Mutagenesis* Mutation Phenotype Plant Root Nodulation / genetics* Symbiosis / genetics*
IF 6.305
Times Cited 53
Lotus / Glycine Lotus japonicus