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First published online September 16, 2009; 10.1104/pp.109.142919 Plant Physiology 151:1077-1086 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Deletion-Based Reverse Genetics in Medicago truncatula1,[W],[OA]Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom (C.R., G.O.); and Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 (J.W., R.C.)
The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants. T-DNA and transposon-based mutagenesis has been widely employed but is limited to species in which transformation and tissue culture are efficient. In other species, TILLING (for Targeting Induced Local Lesions IN Genomes), based on chemical mutagenesis, has provided an efficient method for the identification of single base pair mutations, only 5% of which will be null mutations. Furthermore, the efficiency of inducing point mutations, like insertion-based mutations, is dependent on target size. Here, we describe an alternative reverse genetic strategy based on physically induced genomic deletions that, independent of target size, exclusively recovers knockout mutants. Deletion TILLING (De-TILLING) employs fast neutron mutagenesis and a sensitive polymerase chain reaction-based detection. A population of 156,000 Medicago truncatula plants has been structured as 13 towers each representing 12,000 M2 plants. The De-TILLING strategy allows a single tower to be screened using just four polymerase chain reaction reactions. Dual screening and three-dimensional pooling allows efficient location of mutants from within the towers. With this method, we have demonstrated the detection of mutants from this population at a rate of 29% using five targets per gene. This De-TILLING reverse genetic strategy is independent of tissue culture and efficient plant transformation and therefore applicable to any plant species. De-TILLING mutants offer advantages for crop improvement as they possess relatively few background mutations and no exogenous DNA.
Due to advances in sequencing technology, the generation of genomic sequence data is no longer a limiting factor in the genetic dissection of plant development and physiology. Identification of new genes and verification of gene structure have also been facilitated by high-throughput characterization of RNA transcripts. Attempts to complement the massive availability of sequence data with automated computational annotation has been of only limited value, identifying only a proportion of functional gene products and producing high levels of inaccurate annotation (Yamada et al., 2003  ; Haas et al., 2005). Descriptive genomic approaches, such as gene expression analysis, massively parallel signature sequencing, and serial analysis of gene expression, supply basic information about gene expression but fall short of allowing us to assign gene function. Therefore, the development of these technologies has fuelled demand for reverse genetic platforms. The generation and analysis of mutants remains central to contemporary genetics.
The ability to infer gene function through homology and expression analysis leads biologists to directly test hypotheses by disrupting the activity of genes known only by their sequence. Forward genetics, starting from phenotypic screens, has historically underpinned plant genetics and remains a central and unbiased approach to genetic questions. However, even with the availability of dense genetic maps anchored to genomic sequence data, cloning genes on the basis of phenotype is still not a trivial task. Systematic reverse genetic platforms, allowing researchers to obtain plants mutated at any identified locus, have streamlined functional genomics in well-resourced model species. These platforms are generally based on insertional mutagenesis using T-DNA (Azpiroz-Leehan and Feldmann, 1997 Fast neutrons are a form of high-energy radiation that has been shown to induce a broad range of deletions and other chromosomal mutations in plants. Several sources of fast neutrons are potentially available for mutagenesis, including particle accelerator spallation sources and nuclear research reactors, the latter type being used in this study. Fast neutrons produced by nuclear fission reactors are accompanied by gamma radiation, but the contribution is adjustable. The emission rate achieved with nuclear reactors is in general much higher compared to spallation sources, reducing irradiation time from days to hours. Importantly, the neutron energy should be in the range of approximately 500 keV to 5 MeV to generate the short-range secondary particles within the cell nucleus that mediate strand breakage (J. Palfalvi, personal communication).
Bruggemann et al. (1996)
Despite a long history of use as a mutagen in forward genetics, fast neutron bombardment has not been exploited extensively in the development of reverse genetic platforms. One exception to this is the work of Li et al. (2001) Here, we describe the development of a novel reverse genetic strategy in the model legume M. truncatula, which exploits a large population of plants harboring chromosomal deletions and a highly efficient screening strategy for the discovery of deletions within targeted regions. Deletion based TILLING (De-TILLING) combines fast neutron mutagenesis with PCR-based screening and a three-dimensional (3D) pooling strategy for the efficient recovery of knockout mutants. This method can provide a useful alternative strategy for species in which T-DNA or transposon tagging resources are limited and for providing a targeted approach for identifying mutants in smaller or otherwise untagged genes of all plant species.
Screening Strategy
In a deletion-based system, pooling of plants means that wild-type sequences are preferentially amplified over rare deletion-containing alleles, even at relatively low pooling depths. To increase efficiency of mutant detection in a fast neutron mutagenized population, it was desirable to screen large pools of mutants. This creates the challenge to identify specific deletion alleles in a large background of wild-type alleles and, therefore, the need to suppress amplification from wild-type sequences. This was achieved by Li et al. (2001)
In spite of the success of target detection in the delete-a-gene mock, we were unable to discover deletions in target genes in a population of fast neutron-mutagenized M. truncatula using this strategy. We hypothesized that this may reflect the unlikely event of designing primers that sufficiently spanned these large deletions to allow discovery. It was thus desirable to establish a detection system that would allow discovery of smaller deletions. We set up a second recapitulation of delete-a-gene using a much smaller deletion, the nsp2-1 mutant, which possesses a 435-bp deletion (Oldroyd and Long, 2003 ). A secondary PCR using nested primers L7, R7, L5, and R5 (Fig. 2A
) shows the 1,561-bp nsp2 to one amplicon barely able to compete for amplification with the 1,996-bp wild-type fragment at a ratio of one mutant in 25 wild types (Fig. 2A).
We therefore attempted to develop a system that could detect these small deletions in large pools of wild-type plants. Poison primer suppression was first described for detecting deletions induced by the mutagen trimethylpsoralen in mutant populations of Caenorhabditis elegans (Edgley et al., 2002 ). In addition to the nested PCR assay described above, a third poison primer is included in the first round of PCR (Fig. 2B). A small PCR product, known as the suppressor fragment, is produced between the poison primer and one of the external primers. The suppressor fragment is amplified more efficiently than the full-length wild-type product due to its smaller size. Amplification from a mutant template present within the DNA pool, in which the poison primer binding site has been deleted, produces a single amplicon from the external primers. During the second round of nested PCR, the suppressor fragment, lacking one of the external primer binding sites, cannot act as a template. Only the deletion allele and wild-type allele will now be amplified. Because the production of the wild-type amplicon has been limited by competition from the suppressor fragment in the first round, the mutant amplicon is able to successfully compete for amplification. A poison primer strategy that included the L7B poison primer in the first round of PCR allowed detection of the nsp2-1 mutant at a pooling level of 1:1,000 in the second round of PCR (Fig. 2B). This is consistent with the pooling depth used in the original C. elegans study (1:1,200), although for a small target of 701 bp, a detection sensitivity of 1:5,000 was demonstrated (Edgley et al., 2002). To further enhance the sensitivity of deletion detection, we assessed the capability of restriction enzymes to suppress the production of wild-type amplicons. In this strategy, a nested PCR assay is designed centered upon restriction sites unique within the amplified region. Predigesting the DNA pool with this restriction enzyme will destroy a majority of the wild-type template, allowing the mutant allele, in which this restriction site has been deleted, to successfully compete for amplification. Predigesting the nsp2-1 pooled templates with EcoRV and amplifying using the standard nested PCR protocol increased the detection sensitivity to 1:4,000 (Fig. 2C). Amplification from the wild-type allele is not completely suppressed, and the mutant allele is not reliably amplified in the more highly pooled templates. We assessed how integrating both restriction suppression and poison primers impacted on deletion detection sensitivity. Amplifying from a predigested template and including the poison primer, designed to bind within 30 bp of the restriction site, we achieved much greater detection sensitivities. The nsp2 deletion removes only 20% of the amplified region, yet using the poison primer and restriction suppression we were able to detect the deletion mutant in pools containing a 24,000-fold excess of wild-type sequences (Fig. 2D).
To create a population for De-TILLING, wild-type M. truncatula seeds were mutagenized by exposure to fast neutron radiation. The most effective mutagenic dose of fast neutron radiation was determined to provide a maximum number of deletions per line while retaining a practical level of plant survival and fertility. A 50% survival in the treated M1 plants represents a reasonable balance between mutagenesis and fertility. The segregation of albino phenotypes in the M2 progeny of mutagenized seed has also been used as an indicator of mutagenic rate. For a fast neutron-mutagenized population of Arabidopsis, an albino frequency of 2% has been equated with around 10 induced deletions per line (Koornneef et al., 1982 The M1 seeds were grown to maturity in groups of five plants in a single container and seed pooled from each container. DNA was isolated from 25 seedlings representing each pool, and this DNA was normalized in a 96-well format. A stack of five 96-well plates was considered a tower and represented 12,000 M2 seedlings from 2,400 M1 plants. Thirteen towers were produced to give a total population of 156,000 M2 plants, derived from an original population of 31,200 mutagenized M1 plants. Each tower was pooled in rows, columns, and plates to create 25 3D pools per tower (Fig. 3 ).
A problem intrinsic to any PCR-based screening strategy is the generation of false positives due to production of spurious PCR products. In many circumstances, sequencing the spurious product would be sufficient to separate genuine from spurious products. Characterization of 31 spurious products produced using the De-TILLING screening strategy (e.g. Fig. 4B ) demonstrated that these products invariably originated from the target sequence and in addition were structurally identical to deletion alleles. These possessed deletions ranging from 249 bp to 1.7 kb with an average internal deletion size of 1,261 bp representing 55% of the amplified region. These amplicons were not reproduced in subsequent PCR reactions. This phenomenon was also noted in C. elegans deletion detection platforms by Jansen et al. (1997) and Liu et al. (1999). Lui noted a similar number of false positive amplicons of this type using unmutagenized genomic DNA and suggested they may arise from polymerase slippage across gaps formed by secondary loops in the DNA template. To address this problem, the 3D pools were finally combined to create four reciprocal half tower pools (HTPs) for each tower (Fig. 3). The entire population consists of 54 HTPs in which each of the 13 towers is represented twice. Detection of a mutation therefore results in an identical pair of PCR fragments from two of the HTPs. When a mutant is detected within the HTPs, both PCR amplicons are sequenced to validate and characterize the deletion allele. Subsequent PCR screening of 25 3D pools for that tower allows the identification of a single seed packet from which the mutant originated. This packet of seeds can then be screened for individual plants harboring the characterized deletion.
Detecting Novel Deletion Mutants in the Population We searched for deletions in a LysM receptor-like kinase. Five restriction sites, unique within regions of approximately 2.4 kb, were identified within and closely adjacent to the 1.9-kb coding region (Fig. 4A). De-TILLING assays were designed centered upon each restriction site, resulting in nested PCR amplicon sizes of 2.0 to 2.3 kb. A single deletion allele was detected possessing a 422-bp deletion (Fig. 4B). This was uniquely identified by the StyI-based assay as none of the other targeted restriction sites were removed by the deletion. Following detection, the pool from which the mutation originated was identified by screening the 3D pools of tower 4 (Fig. 4C). We then recovered the mutant from a screen of 29 M2 plants from the identified pool (Fig. 4D). Despite the deletion only removing 18.1% of this 2.3-kb nested product, we were able to detect this mutant using the De-TILLING method.
To demonstrate the utility of the De-TILLING strategy for recovering mutations in small genes, we targeted the transcription factor EFD (for ethylene response factor required for nodule differentiation; Vernie et al., 2008
We attempted to recover deletions in 14 genes using a minimum of five well-distributed targets per locus. Target sizes ranged from 1.2 to 3.2 kb with an average target amplicon size of 2,335 bp. The sizes of deletions that we detected using the De-TILLING method were 422, 1,270, 1,570, and 1,723 bp with target amplicons sizes of 2.3 kb (18.2% deletion), 1.9 kb (67.9%), 2.9 kb (54.1%), and 2.6 kb (67.5%), respectively (Table I ). All mutations completely removed the targeted restriction site and the poison primer binding site.
Here, we describe a combination of detection strategies that greatly enhances the utility of deletion detection in mutant populations to that previously described. De-TILLING can be used to recover deletion mutants for the majority of plant species and offers several advantages over conventional TILLING. A standard TILLING population of 4,000 lines requires amplification, CelI digestion, and analysis of fluorescently labeled PCR products for 500 samples. To reach acceptable levels of cost and efficiency, small, heavily mutagenized populations are essential. Mutants recovered from these populations will possess a very large number of nontarget mutations. For an Arabidopsis TILLING population, conservative estimates suggest the density of mutations in exons to be approximately three per megabase (Colbert et al., 2001 ). The impact of this is around 20 to 25 profoundly affected genes per EMS-mutagenized genome (Henikoff and Comai, 2003). This can affect the viability of mutants and confound and delay downstream analyses. To achieve saturation, fast neutron-mutagenized populations need to be larger than those created using EMS; however, the mutants recovered will be knockouts and will possess far fewer nontarget mutations. The large population sizes in De-TILLING are offset by the high level of pooling employed, which allows great efficiencies of time and cost in comparison to the standard 8-fold pooling of TILLING.
The size of the population of plants needed to saturate a genome depends firstly on the rate at which loci are deleted from the genome and secondly on the number of deletions that can be detected using the screening method. For Arabidopsis lines exposed to fast neutron radiation at a standard dose of 60 Gy, Koornneef et al. (1982)
PCR-based methods do not recover deletions of all sizes. Only a subset of the induced deletions will be detected by any screening method. Deletions must be small enough to be flanked by the nested PCR primers and large enough to produce mutant amplicons whose amplification can be separated from the wild type. Our screening was carried out using an average target size of 2.3 kb and ranged from 1.2 to 3.2 kb. Detected deletions removed from 18% to 68% of the target region, although we were able to model detection of a 14% deletion using the nsp2-1 mutant. We can therefore estimate that we would be targeting deletions within the range of 0.2 to 2.2 kb using this method. The estimate of 10 deleted coding regions per line would include many deletions outside this range. While limiting detection to small deletions is highly desirable, this increases the population size needed to achieve saturation. The number of lines (n) needed to increase the probability of recovering a mutant (F) to any level is related to the observed frequency of detectable deletions (P) through the formula:
Screening 13 towers (31,200 M1 plants) enabled us to recover mutants in four out of the 14 genes we targeted. A population of 125,000 M1 would therefore give an 80% probability of recovery. Given that the relationship of diminishing returns exists for any reverse genetics screening platform, a combination of approaches will always be the most effective strategy. Increasing the size range of detectable deletions to 4 kb may improve the recovery of mutants. Extending this strategy to include large deletions to increase recovery would, however, lead to the problems highlighted by Li et al. (2001)
Recovering a deletion removing an 85-bp exon of the EFD transcription factor demonstrated the utility of De-TILLING for the targeting of small genes. Mutagenizing small genes is problematic for reverse genetic strategies based on insertional and point mutation inducing mutagens. The probability of identifying an insertion is dependent upon the size and structure of the targeted gene. The probability of finding a mutant possessing an insertion in a particular Arabidopsis gene can be calculated using the formula:
). Therefore, for an Arabidopsis collection containing 100,000 insertions, there is only a 55% probability of identifying an insertion mutant for a 1-kb target. For a similar Medicago collection, with a genome size of 400 Mb, the probability is approximately 22%. Using deletion mutagenesis, the probability of recovering a mutant is entirely independent of the size of the target sequence. It is far easier to hit a small target with a large deletion than an insertion or point mutation. The structure of the gene is also significant. Genes possessing small exons and large introns will be more difficult to mutagenize using TILLING or insertional techniques. Insertions are unlikely to be found within small exons, and those that fall within introns or intergenic regions are likely to have no effect on protein function. TILLING relies on the identification of approximately 1-kb regions with a high probability of introducing deleterious point mutations. Where a gene is structured as small exons and large introns, it is difficult to identify useful target regions for TILLING. Deletion-based reverse genetic platforms do not carry these limitations.
Deletion-based reverse genetic systems have the ability to inactivate multiple genes. Plant genomes are highly redundant, and it is estimated that <10% of the genes tagged in Arabidopsis are likely to generate a phenotypic change (Meinke et al., 2003 In addition to its use as a research tool, deletion mutagenesis has the potential to find application in crop improvement programs. The use of fast neutron mutagenesis is applicable to any plant species. It is conducted on large batches of dry seed at very low cost and is therefore ideally suited to applications in crop improvement. Fast neutron-mutagenized lines with lower levels of nontarget mutations and an absence of any foreign DNA sequences may be more acceptable to consumers concerned with the perceived dangers of genetic modification. In comparison with the well-established TILLING method, De-TILLING can be used to isolate mutants at a fraction of the time and cost. Fast neutron mutagenesis generates complete knockout mutants that do not possess the very high number of background mutations that are typical for TILLING mutants. De-TILLING can also address the problems of targeting small genes, a problem that is intrinsic to all methods based on insertion and point mutation. As the cost of sequencing continues to fall, the low cost, scalability, and technical simplicity of De-TILLING has the potential to become a valuable tool in a wide variety of plant species.
Fast Neutron Population Wild-type Medicago truncatula seed (A17, Jemalong) was exposed to fast neutron radiation at a dose of 32.5 to 35 Gy at the Atomic Energy Research Institute (Budapest). The rate of mutagenesis was assessed by M1 survival (37–48%) and albino rate (2.57%) as calculated as a percentage of M1 treated lines displaying albino phenotypes in the M2 plants. Five M1 plants were grown in soil in a single pot and the seed pods collected in a single pool. These were mechanically threshed and the seeds archived at 4°C. Twenty-five seeds from each M2 family were germinated. The seedlings were freeze dried and the tissue ground.
A 30-mg sample of the tissue from each M2 family was aliquoted for DNA extraction. Extractions were carried out in 96-well format using the DNeasy 96 plant kit (Qiagen) and eluted in 200 µL. DNA was spectrophotometrically quantified and normalized to 50 ng µL–1. The population was structured into towers consisting of five 96-well plates. Samples from each row, column, and plate of each tower were combined to create a 3D pooling structure. The 3D pools were then combined to create four HTPs per tower, which were directly screened using the De-TILLING method. HTPs from each tower were digested with a range of restriction enzymes and stored at –20°C.
A PERL script, known as MtMutDetect.pl, was designed to align a coding and a genomic sequence and, using a user-modifiable list of enzymes, identify restriction sites within and adjacent to exon sequences unique within PCR amplicons of a defined size range. The program then performs automated primer design and returns a five-primer De-TILLING assay centered upon the targeted restriction sites. Poison primers were designed within 30 bp of the targeted restriction site. Parameters can be set to determine the amplicon size, the number of enzymes, and the amount of intron sequence to be included in the identification of targetable restriction sites.
Nested PCR reactions were performed in a total of 50 µL using Taq Polymerase Master Mix (Qiagen), 240 ng of digested genomic HTP DNA, and 10 pmols of each primer. PCR was carried out using an MJ Research tetrad PTC-225 peltier thermocyler over 40 cycles of 30 s at 94°C, 30 s at 55°C, and 2 min and 30 s at 74°C.
Genomic DNAs of A17, dmi1-4, and nsp2-1 were prepared, quantified, and normalized as described above. These were pooled at mutant to wild-type ratios of 1:25, 1:1,000, 1:4,000, 1:8,000, 1:12,000, 1:16,000, 1:20,000, and 1:24,000. Six microliters of each pool (240 ng) was used as template in all amplifications. The following primers were used to amplify dmi1-4 deletion borders: dmi1-4-F (5'-TCTTCTTAATTTCATGTGCATAATTGTCG-3'), dmi1-4 (0.3kb)-R1 (5'-TCAATTTGATGGTGCATAATAGCA-3'), dmi1-4 (0.3kb)-R2 (5'-AGGCAGTAATATGGAATGGACA-3'), dmi1-4 (3kb)-R1 (5'-TCTTCTGTCATTCACCTGAGGCT-3'), dmi1-4 (3kb)-R2 (5'-GTTGATAATAGCACGCTCTTGGT-3'), dmi1-4 (8kb)-R1 (5'-CTGTCACCATTTCTGTATGTGCT-3'), and dmi1-4 (8kb)-R2 (5'-TCTGTATGTGCTGCGATTTTCAC-3'). F and R1 primers were used in the first round PCR, and F and R2 were used in the second round PCR. A De-TILLING assay was designed to detect the nsp2-1 deletion (Fig. 2B). This consisted of two external primer L7 (5'-TTGCATTCACATCAGGTAGGA-3') and R7 (5'-GAGCAATTTGAACCTCTCACG-3'), a poison primer L7B (5'-AATCAAGCCATCATCGAAGC-3') and nested primers L5 (5'-TGACAACAGCGCACATAACA-3') and R5 (5'-AAACCAAAACGCACACACAA-3'; Fig. 2). Amplifications were carried out identical to those described below for De-TILLING screening.
The first-round PCR was analyzed using an e-Gel (Invitrogen) to check for the production of the suppressor fragment. Second-round PCR was identical but scaled to 20 µL, using 2 µL of a 10–2 dilution of the first round PCR products as template and nested primers. The second round products were then analyzed on a 1.2% TBE agarose gel and putative deletion containing fragments recovered using QIAquick Gel extraction kits (Qiagen). The products were then sequenced using the second-round primers and an ABI3730 automated sequencer. The sequences were then compared to the wild type to determine the deletion junction.
The following materials are available in the online version of this article.
We thank Prof. Virginia Walbot for helpful discussions; Paul Bailey for compiling the script of the MtMutDetect.pl De-TILLING assay design software; Joe Palfalvi for providing fast neutron irradiation and useful discussions; and Jonathan Clarke, David Baker, and Bethany McCullagh for providing DNA extraction and sequencing services. We also thank Katy Owen and Gemma Lynes for their extensive contribution to the daily laboratory work in establishing the De-TILLING seed and DNA archives along with glasshouse assistants Ruth Pothecary, Emma Thompson, Paul Ward, Kate Bowdrey, Catherine French, Megan Murray, Richard Birkinshaw, Clare Harden, and Lucy Foulston. Received June 19, 2009; accepted September 15, 2009; published September 16, 2009.
1 This work was supported by the European Union as part of the Grain Legume Integrated Project, by a grant in aid for the Biotechnology and Biological Sciences Research Council, by the Samuel Roberts Noble Foundation, and by the National Science Foundation (grant no. DBI0703285).  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: Christian Rogers (christian.rogers{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.109.142919 * Corresponding author; e-mail christian.rogers{at}bbsrc.ac.uk.
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