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First published online July 29, 2009; 10.1104/pp.109.142190 Plant Physiology 151:1281-1291 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
TILLING in Lotus japonicus Identified Large Allelic Series for Symbiosis Genes and Revealed a Bias in Functionally Defective Ethyl Methanesulfonate Alleles toward Glycine Replacements1,[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)
Whole genome sequencing and transcriptome analysis have provided in-depth descriptions of the physical structure and the repertoire of gene expression in a growing number of eukaryotic organisms. However, to reveal the functions of individual genes, genetic approaches will remain of paramount importance. Forward genetics aims to identify the causative genetic change in a phenotypically interesting mutant (i.e. mutant first). In contrast, reverse genetics intends to assign a function to a gene of known sequence through phenotypic analysis of individuals in which the function of this gene is altered (i.e. gene sequence first). In higher plants, targeted gene disruption methods are not yet routine and alternative methods are required to obtain individuals in which the gene of interest is impaired. Random insertion mutagenesis using either T-DNA or transposons has been successfully used in Arabidopsis (Arabidopsis thaliana) to assemble mutant libraries, which cover the vast majority of the genes of this plant (http://signal.salk.edu). Moreover, silencing of genes of interest using RNA interference transgenesis has become a popular tool for reverse genetics (Mansoor et al., 2006  ). An alternative and well-established approach is chemical or fast-neutron mutagenesis followed by identification of mutants from a suitably sized population of mutagenized individuals. Advantages of chemical or physical mutagenesis are their applicability to organisms that are not easily transformable or in which active transposons have not been characterized and the ease of generating large independent mutant populations. Radiation typically induces deletions, which can be readily detected by PCR, using primers flanking the deletion (Li et al., 2001). However, their catastrophic nature limits the number of tolerated deletions per genome. Chemical mutagens, such as ethyl methanesulfonate (EMS), induce point mutations that have the advantage of being tolerated to a high density. This permits near saturation with a manageable number of mutant individuals (Henikoff et al., 2004). In addition, allelic series with single amino acid changes and specific phenotypes bear the potential for providing more detailed information on protein function.
Screening for point mutations without prior knowledge of the mutation is technically challenging. A technology based on mismatch recognition in heteroduplex DNA by endonucleases such as CEL I (Till et al., 2004a
The legumes, including important crop plants like bean (Phaseolus vulgaris), soybean, pea, and lentil (Lens culinaris), exhibit biological traits that are of agricultural significance but that cannot be analyzed in the model plant Arabidopsis. For example, Arabidopsis does not engage in the ecologically important arbuscular mycorrhiza symbiosis with phosphate-delivering fungi, which is formed by more than 80% of land plants (Harrison, 2005
General TILLING Population
Lotus mutant populations were generated in a continuous effort over 4 years. Following EMS mutagenesis of M1 seeds, the resulting M2 mutant population has been subdivided into several subpopulations according to phenotype. Since the initial description of the Lotus TILLING facility by Perry et al. (2003)
Mutation Frequency in the Lotus TILLING Population
We subjected GENPOP to TILLING with 84 fragments corresponding to 61 genes. For economic reasons, 53 gene fragments representing 36 genes were only tested on a subset of 2,304 plants (population 1). Upon customer request, or in cases where an insufficient quantity or quality of mutations was obtained from this subpopulation, we screened additional sets of 1,297 (population 2) or 1,303 (population 3) GENPOP plants. The total population of 4,904 plants was screened with 11 fragments representing 10 genes (Table I
). Taking into account the number of plants screened for each of the fragments, in total we obtained 576 hits from 289 Mb of amplicons (Table I). On average, we obtained two mutations per 1 Mb screened, equivalent to a mutation load in GENPOP of one mutation per 502 kb. The hit frequencies observed in the three subpopulations are not directly comparable because they were obtained with different numbers of gene fragments (Table I). In comparison, the mutation load in the coding portion of the genome after EMS mutagenesis in the Seattle Arabidopsis TILLING project (ATP) was determined on a large data set of about 1,900 independent mutations in 192 genes to be about 1 per 300 kb screened (Greene et al., 2003
Germline Genetics in Lotus
We used a single M2 individual per family as the core unit of our GENPOP. Due to Mendelian segregation and the chimeric nature of the M1 embryo that was exposed to EMS, only a proportion of the mutations present in the germline of an M1 are represented in any M2 individual of the general population, the remainder being carried by other M2 siblings (Henikoff and Comai, 2003
In the Seattle ATP population, a 2:1 segregation ratio of heterozygous versus homozygous mutants was observed after EMS mutagenesis of seeds (Greene et al., 2003
This fundamental difference between Lotus and Arabidopsis has consequences for the mutation frequency that is carried through to the M2. While in Arabidopsis 25% of the mutations are lost due to Mendelian 1:2:1 segregation, in Lotus it is at least 69.4% (25 out of 36) that are not transmitted. If we take this dilution into account when calculating the initial hit rate in the M1, we obtain one hit in 154 kb (502 kb x 30.6%), which is higher than the calculated frequency in Arabidopsis (300 kb x 75% = 225 kb; Greene et al., 2003 ). Considering that the haploid Lotus genome is approximately 472 Mb in size (Sato et al., 2008), a frequency of one mutation per 154 kb is equivalent to 3,065 heritable mutations per M1 plant. However, more relevant for users of the TILLING lines is the mutation load in the M2 and subsequent generations. The M2 generation carries 940 mutations per plant (one hit in 502 kb of diploid sequence of the 472-Mb genome), of which 10% are homozygous. Due to the loss of 25% of the heterozygous mutations during selfing, the mutation load drops to 729 and 624 mutations per plant in the M3 and M4 generations, respectively. It is important to realize that at the same time the theoretical frequency of homozygous mutations increases in each round of selfing. Therefore, second-site mutation phenotypes may only become apparent in later generations.
We observed that fertility is a more sensitive parameter for EMS mutagenesis than seed viability. While in our experiments germination was reduced to about 75% to 80%, only about 50% of these plants set seed. In practical terms, this suggests that fertility of the M1 is the most relevant bottleneck limiting the mutation load that can be obtained in the resulting M2. Similar mutation rates observed for Lotus and Arabidopsis, which have a 4-fold difference in genome size, suggest that there is an upper limit of what can be tolerated in the coding part of the genome. A conserved upper limit is consistent with observations and comparisons with animal systems (Greene et al., 2003
Of the detected mutations in GENPOP, 97.6% were G/C-to-A/T transitions typically induced by EMS (Table III
; Supplemental Table S1). A comparable analysis of mutant alleles detected after EMS mutagenesis in the Seattle ATP has revealed more that 99% G/C-to-A/T transitions (Greene et al., 2003
Successful Screens of M3 Bulked Family Seeds We increased the number of available seeds per family by collecting seeds in family bulks from M2 siblings that remained after the phenotypic screens (Fig. 1, BULKs). This collection is organized such that each seed bag can be traced back to the original M1 plant. This was done to support the anticipated user demand for seeds originating from the TILLING of GENPOP but also to support additional forward genetic screens in the background of our increasingly well-characterized mutant families. This is a significant advantage, since it allows rapid cross-referencing between the TILLING results for each family and any phenotypic information resulting from the original M2 screen or novel screen results in the structured collection of bulked M3 seeds. Our collection of 2,204 individual families in separate bulks has been made available for forward genetic screens and has been subjected to successful screens for mutants with novel symbiotic or developmental phenotypes.
A particularly rewarding screen was performed to identify genetic regulators of root nodule development (Tirichine et al., 2006b
The M3 bulked seeds were also successfully screened by the group of D. Luo at the Shanghai Institute of Plant Physiology and Ecology to isolate flower mutants, some of which have been shown to be alleles of LjLEAFY (Dong et al., 2005
As a special feature of the Lotus TILLING facility, we assembled phenotypically preselected mutant populations. Screening for phenotypes of interest allowed the assembly of three thematic mutant collections (Fig. 1). The first contained mutants with altered shoot, leaf, or flower development (DEVPOP), a second set comprised mutants with altered starch metabolism, and the third and largest population consisted of plants with defects in root nodule symbiosis (NODPOP). With these populations, we have assembled a large-scale forward and reverse genetics tool for the legume Lotus. This community resource has contributed significantly to recent advances in the field of legume research, especially root symbiosis research (Perry et al., 2003
The NODPOP was assembled from plants that did not form nodules or had fewer and/or smaller nodules by screening M2 families after infection with Mesorhizobium loti. Moreover, plants with a nodule color different from that of the wild type (pink) were included. In addition, the population contained plants that showed signs of early senescence but regreened upon the addition of nitrogen fertilizer. This latter group was expected to include plants whose nodules would be defective in nitrogen fixation. A total of 5,300 families (63,084 individuals) were screened for nodulation defects over the course of 3 years. This generated a total population of 670 individuals from 396 families (Fig. 1): 248 plants lacking nodules (123 families); 322 having fewer or smaller or white nodules (203 families); 27 root mutants (22 families); and 73 potentially unable to fix nitrogen (48 families). There were 26 additional families where individuals fell into different categories. DNA samples were collected from all mutants to assemble the NODPOP used for TILLING. Upon rescreening of the NODPOP, the original phenotype was so far confirmed in approximately 35% of lines (Fig. 1).
DNA of 43 symbiosis-defective mutants with unidentified genetic defects from the mutant collections of Krzysztof Szczyglowski (Southern Crop Protection and Food Research Centre, Ontario; Murray et al., 2006
M3 progeny of a subset of the nodulation-defective mutants were subjected to a microscopic screen for infection thread-deficient (itd) mutants, and four lines (SL0295-2 [itd1], SL1450-5 [itd2], SL1947-2 [itd3], and SL3055-2 [itd4]) that exhibited root hair curling but aborted infection thread formation were characterized in more detail (Lombardo et al., 2006 M3 progeny of nodulation-defective M2 individuals were also scored for their ability to form structurally intact arbuscular mycorrhiza symbiosis. To date, we have identified 36 families that were defective in both nodulation and in arbuscular mycorrhiza formation. For the majority of these common symbiosis mutants, it was possible to assign them to one of the previously identified common symbiosis loci (Fig. 2 ; Supplemental Fig. S3).
Several of our NODPOP individuals did not produce seeds, so we attempted to rescue the corresponding mutants by rescreening sibling seeds available as bulked M3 seeds (Fig. 1, BULKs). We screened the bulked M3 seeds of 97 families (each containing approximately 50 plants) for the segregation of symbiosis mutants. In 55 of those 97 families, no mutant plants could be identified in the bulked M3 seeds. In the remaining 42 families (43%), individual mutant plants were recovered. In 12 of these families, all mutant representatives died during development. Twenty-two families segregated mutants with root defects and therefore may represent pleiotropic phenotypes.
Subsequent to the identification of the NODULE INCEPTION (NIN) gene (Schauser et al., 1999
In parallel to the TILLING effort, we established a high-throughput mapping pipeline for systematic assignment of genomic map positions to all mutant loci not identified by TILLING. Mutant lines confirmed in the M3 were crossed to the polymorphic mapping parent MG-20 (Kawaguchi et al., 2001 The complete allelic series of symbiosis genes identified through both TILLING and mapping approaches are listed in Supplemental Figure S3. Detailed phenotypic descriptions of these allelic series have been or will be published elsewhere.
We identified 97 mutant alleles in 12 genes required for symbiosis (Supplemental Table S2) by screening NODPOP. Since the requirement of the 12 genes for root symbiosis had been demonstrated previously, causative alleles should be sufficient, on their own, to cause symbiosis-defective phenotypes. Upon rescreening, 19 lines did not exhibit a nodulation defect; therefore, these 19 mutant alleles do not have an effect on gene function (Supplemental Fig. S2). Of the remaining 78 nodulation-deficient lines, 47 could harbor mutations that are responsible for the observed phenotype, since they are homozygous and lead to missense or nonsense mutations, to frame shifts, or affect splice sites (Table IV ; Supplemental Fig. S2). However, the evaluation of the identified 27 homozygous missense mutations (Supplemental Fig. S2; Supplemental Table S3) requires a more detailed analysis because it is difficult to make reliable a priori predictions about the consequences of a particular amino acid substitution for protein function.
Despite the functional preselection of NODPOP, the stochastic distribution of mutations dictates that nonconsequential alleles will be recovered as well. It is important, therefore, to determine the frequency at which mutations are expected to occur by chance alone. We TILLed approximately 53.7 kb of genomic sequence corresponding to 26.8 kb of coding and 26.9 kb of noncoding sequence of the 12 symbiosis genes in Figure 2. By inspection of 224 families with a confirmed nodulation phenotype (Fig. 1), we screened a total of 12.0 Mb of diploid sequence. Taking into account the observed rate of one hit per 502 kb, we expected approximately 24 mutations to occur by chance. We obtained more than three times as many, indicating that enrichment occurred through preselection. From the total of 78 alleles, 31 mutations were heterozygous, in a noncoding region, or silent (Table IV) and so likely to be present by chance. In these cases, the nodulation phenotype should be caused by an additional mutation in the genome. This is consistent with the finding that nine families within NODPOP (SL0317, SL0355, SL0456, SL0605, SL0820, SL1719, SL1913, SL5369, and SL5426) carry mutations in more than one symbiosis gene. To confirm a causative connection between each of the missense mutations and the symbiosis phenotype, complementation tests would be required, which are beyond the scope of the current analysis. A summary of all potentially causative mutations in nodulation-deficient NODPOP lines is provided in Supplemental Table S3.
Almost equal numbers of G-to-A and C-to-T transitions were observed among GENPOP alleles as expected for a random distribution. In contrast, there were 3.4 times as many G-to-A than C-to-T base changes in symbiosis genes in the 47 potentially causative NODPOP alleles (Table III). This enrichment is caused by two additive components. First, five of the six splice site mutations identified in NODPOP are G-to-A transitions (Supplemental Table S3); the remaining one is a G-to-T transversion. Second, we observed that deleterious amino acid exchanges showed strongly biased distributions, both in the amino acids that were affected and in the underlying base changes. Surprisingly, mutations that replace Gly are well overrepresented in potentially functionally defective alleles (Fig. 3 ; Supplemental Tables S3 and S4). The overrepresentation by 11 mutations affecting Gly is contributing to an increase in the ratio of G-to-A versus C-to-T transitions, since all nonsynonymous positions in Gly codons are occupied by G.
We analyzed the distribution of amino acids that were replaced by EMS mutagenesis in our collection of missense and nonsense alleles in functionally impaired mutant lines. We asked the question whether replacements in some amino acids are more likely to result in a nonfunctional protein than others. To detect such compositional biases in our allele collection, we first determined the expected distribution of hits between the amino acids occurring in the genes under study. For this, we analyzed the susceptibility to EMS mutagenesis of each of the codons in the genetic code. Assuming random mutagenesis and by taking into account the codon usage of the genes under study and the total number of mutations, we could calculate an expected number of hits for each of the codons for a particular amino acid. This expectation value was compared with the observed frequency in 102 EMS alleles of 14 genes with a variety of functions TILLed in the GENPOP (excluding mutations leading to splice site or frame shift mutations) and showed no significant deviation (Fig. 3; Supplemental Table S4). In contrast to this, TILLING of 12 genes in the NODPOP resulted in 39 nonsynonymous potentially causative alleles (also excluding mutations leading to splice site or frame shift mutations) with a strongly biased distribution (Fig. 3; Supplemental Table S4). The number of hits in TGG encoding Trp was 5-fold higher than expected by chance (six versus 1.2). Mutations affecting this codon are likely to be overrepresented in a series of functionally impaired alleles, since mutations of either of the two Gs to A results in a (premature) stop codon, TGA or TAG. Likewise, we observed a significant accumulation of mutations (five versus 1.6) in the CAA and CAG codons for Gln, leading to the stop codons TAA and TAG (Fig. 3). Surprisingly, codons for Gly, the smallest amino acid, were hit more than twice as often as expected by chance (11 versus 5.3), whereas codons for Ala, the second smallest amino acid, are slightly underrepresented (two versus 5.6).
A closer analysis revealed that this bias is, at least partially, the consequence of the rather narrow mutational spectrum that is explored by EMS mutagenesis. For example, the five amino acids Phe, Ile, Lys, Asn, and Tyr are not EMS targets, because of the lack of G and C at nonsynonymous positions in their codons. Of the 410 theoretical amino acid and nonsense interconversions (21 coding/noncoding options x 20 alternatives), only 170 can be achieved by single nucleotide exchanges. And of these, only 26 are possible through EMS mutagenesis (Supplemental Fig. S4). To obtain an approximate quantitative measure for the degree of conservation of these 26 possible changes, we projected them onto the BLOSUM62 matrix (Henikoff and Henikoff, 1992
To test whether this bias toward Gly hits is due to the particular genes studied here, we analyzed the set of EMS-induced RPM1 alleles (Tornero et al., 2002
We observe a significant overrepresentation of Gly replacements in functionally defective EMS alleles in two independent studies, with a total of 94 missense mutations (39 from the nodulation screen and 55 from the RPM1 study; Supplemental Table S4). We suggest that this particular propensity of EMS-induced Gly replacements to impair protein function might be a general phenomenon that has not been recognized previously. Using different mutagens, more than 4,000 amino acid replacements were tested in a large-scale structure-function analysis of the lac repressor (Markiewicz et al., 1994 In conclusion, we have generated a unique forward and reverse genetics resource for legume research and uncovered several allelic series for genes important in symbiotic interactions. The detailed analysis of a large number of functionally defective EMS alleles has revealed a hitherto unobserved bias in the amino acid replacements leading to defective gene products. This information is important not only for prediction of the deleterious effects of the commonly used mutagen, EMS, on genes subjected to TILLING but may also be used to optimize algorithms designed to predict the deleterious effect of naturally occurring mutations detected in human genome sequences.
Plant Material and EMS Mutagenesis Seeds of Lotus (Lotus japonicus) ecotype B-129 ‘Gifu’ were a kind gift of Jens Stougaard (Aarhus University). All other Lotus ecotypes were obtained from the National Agricultural Research Center (Toyohira, Sapporo, Japan) and Legumebase, Japan (http://www.shigen.nig.ac.jp/lotusjaponicus/index_e.html). Mutagenesis was performed in 10 successive batches over the course of 3 years starting in spring 1999. Typically, 3.5 g of dry seeds was imbibed per batch, rinsed, and, after removal of washing water, treated overnight with 60 µL of EMS suspended in 10 mL of distilled water. M2 seeds from the resulting fertile M1 plants were sown in individual families (Fig. 1). In the first 2 years, a total of 45,600 M2 individuals were subjected to phenotypic screens to assemble thematic subpopulations.
During the first year of building the populations (families SL0001–SL3552), M2 individuals that exhibited interesting developmental abnormalities were isolated and photographed, and the resulting data were made available in an online database (Perry et al., 2003
M2 families were screened for nodulation mutants over a 3-year period. Initial findings for the first 2 years were presented previously (Perry et al., 2003
After the removal of nodulation and other phenotypically interesting mutants from the family, a single healthy-looking M2 individual, which scored wild-type by phenotypic criteria, was chosen to represent each M2 family in GENPOP. DNA from each GENPOP plant was extracted, and seeds were collected in individual seed bags.
In the first year, a representative from each family was taken to form the GENPOP, any nodulation mutants within the families were removed to generate the NODPOP, and finally, phenotypically impaired mutants were removed to provide a source for future trait-specific or theme-based TILLING. The remaining individuals from each family were planted outside in the field, and the family bulk was harvested. This generated 2,084 bulked family lines. In the second year, family lines were only bulked if they contained a starch mutant, resulting in 120 bulked lines. These were grown under glasshouse conditions. This collection of bulked family seeds is accessible in 2,204 seed bags, each representing M3 progeny of a single M1 individual.
Starch content in Lotus aerial tissue was determined using iodine staining as described by Harrison et al. (1998)
Initially, the Lotus TILLING facility utilized an ABI377 sequencer (Perry et al., 2003 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AB162016, AM230792, AP009158, AJ575247, AJ575254, AJ238956, EF012819, DQ665943, AP009253, AJ890252, AB162017, and AP004579.
The following materials are available in the online version of this article.
We thank Soizic Cheminant, Jodie Pike, Iain McRobbie, Paul Bishop, and Ruth Pothecary for technical assistance, Jens Stougaard, Krzysztof Szczyglowski, and Judith Webb for supplying seeds or DNA samples for some lines in NODPOP+, and the National BioResource Project LegumeBase in Japan for providing the MG ecotypes. We thank Paul Schulze-Lefert for inspiring reverse genetics based on single nucleotide polymorphism detection and Brande Wulff for the NODPOP idea. Received May 29, 2009; accepted July 21, 2009; published July 29, 2009.
1 This work was supported by two consecutive grants from the United Kingdom Biotechnology and Biological Sciences Research Council, one John Innes Centre joint research grant to M.P. and T.L.W., and a grant from the European Union Marie Curie research training network INTEGRAL. 
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.
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2 tests.
and 
, respectively. Gly can explore areas in the two-dimensional Ramachandran plot of 
