Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom.
Plant Physiol. 2009 Nov;151(3):1281-91. doi: 10.1104/pp.109.142190. Epub 2009 Jul 29.
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).
我们已经建立了用于豆类植物百脉根（Lotus japonicus）正向和反向遗传学分析的工具。通过使用 TILLING（靶向诱导基因组局部突变）方案，可对 4904 个乙基甲磺酸诱变的 M1 胚胎的 M2 后代进行结构化群体中单核苷酸多态性突变检测。扫描该群体的子集中，我们发现扩增片段每 502kb 就有一个突变。此外，我们在 M2 后代中观察到纯合突变和杂合突变的比例为 1:10。这揭示了百脉根和拟南芥（Arabidopsis thaliana）之间在生殖系遗传学上的明显差异。此外，我们在三个主题子集中组装了 M2 同胞，他们在整体发育、淀粉积累或固氮根瘤共生方面表现出明显的表型。通过在一组已知对根瘤发育至关重要的 12 个基因的 M2 个体的缺陷型群体中筛选突变，我们为每个基因鉴定了大量等位基因系列，生成了一个独特的数据集，结合基因型和表型信息，有利于结构-功能研究。该分析显示，功能缺陷等位基因中甘氨酸（Gly）残基的替换存在显著的偏向性，这可能可以用 Gly 的特殊结构特征来解释。Gly 允许肽链采用氨基酸替换后不再可能的构象。这种以前未被认识到的 Gly 残基处蛋白质的脆弱性可用于改进旨在预测单核苷酸多态性突变有害性的算法。我们的结果证明了甲基磺酸乙酯诱变在正向和反向遗传学研究中的强大功能，同时也证明了它的局限性。（原始突变表型可在 http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus 上访问，可通过 http://www.lotusjaponicus.org 或 http://revgenuk.jic.ac.uk 获得对百脉根 TILLING 设施的访问权限）。
