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

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

“…Gly residues represent the largest fraction of the amino acids that are highly conserved in b-glucosidase enzymes, and Gly codons are very suitable targets for EMS mutagenesis in part because each Gly codon has at least two guanine bases. The observed Gly substitutions all involve changing this small amino acid to a charged amino acid such as Glu and are likely to affect enzyme structure, stability, and consequently function (Perry et al, 2009). The modeling of the observed amino acid changes on the BGD2 protein structure also suggested that these changes would affect folding and stability of the enzyme, rather than directly affecting the active sites.…”

Self-Incompatibility in the Brassicaceae

“…TILLING extends genomic resources, particularly in organisms lacking reverse-genetic tools, where mutants with a range of phenotypic severity are highly desirable. Since the inception of TILLING, this method has been applied to various organisms including Cucumis melo L. (González et al 2011 ), Solanum lycopersium (Minioa et al 2010 ), B. napus (Wang et al 2008 ;Harloff et al 2012 ), B. oleracea (Himelblau et al 2009 ), B. rapa (Stephenson et al 2010 ), Lotus japonicus (Perry et al 2009 ), Zea mays , Oryza sativa (Till et al 2007 ), Drosophila (Winkler et al 2005 ), and zebrafi sh (Wienholds et al 2003 ).…”

Functional genomics to study stress responses in crop legumes: progress and prospects

“…For example, in the model legumes, Medicago (12 000 M2 plants) (Rogers et al 2009) and Lotus (4904 M2 lines) (Perry et al 2009) mutant populations were developed for use in reverse genetics. In the case of crop legumes, over 3000 M3 lines were developed in common bean and evaluated with root nodulation tests by Porch et al (2009).…”