deletion based reverse genetics in medicago …...2009/09/16 · a population of 156,000 medicago...

Deletion based reverse genetics in Medicago truncatulaChristian RogersOldroyd GroupJohn Innes CentreNorwich Research Park Colney Norwich NR4 7UH UK Telephone: +44 (0)1603 450000 [email protected]

Breakthrough Technologies; Genome Analysis; Bioinformatics –

Associate Editor C. Robin Buell

Plant Physiology Preview. Published on September 16, 2009, as DOI:10.1104/pp.109.142919

Copyright 2009 by the American Society of Plant Biologists

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Deletion based reverse genetics in Medicago truncatula

Christian Rogers1*, Jiangqi Wen2, Rujin Chen2 and Giles Oldroyd1

1Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park,

Colney Lane, Norwich NR4 7UH.2Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401.



Abstract

The primary goal of reverse genetics, the identification of null mutations in targeted genes, is

achieved through screening large populations of randomly mutagenised 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 (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 which, independent of target size,

exclusively recovers knockout mutants. Deletion TILLING (De-TILLING) employs fast neutron

mutagenesis and a sensitive PCR 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 PCR 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 5 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.

Introduction

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 has also been facilitated by high

throughput characterisation 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

(MPSS) and serial analysis of gene expression (SAGE) 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 (Krysan et. al., 1999; Alonso et al.,

2003; Azpiroz-Leehan and Feldmann, 1997; Sessions et al., 2002; Sussman et al., 2000) or

transposon tagging (Tadege et al., 2008; Parinov et al. 1999; Martienssen, 1998; Parinov and

Sundaresan, 2000; Speulman et al., 1999) which, through the introduction of known

sequences, greatly facilitates gene cloning and allows the high throughput characterisation of

insertion sites. However, these approaches are not feasible in the majority of species where,

although genomic sequence information may be extensive, transformation and tissue culture

based methods are not practical. In these species TILLING is the most widely applied

strategy (McCallum et al., 2000b; Colbert et al., 2001). TILLING is based on the alkylating

agent ethyl methane sulfonate (EMS) which introduces point mutations across the genome.

These can be supported to very high densities without causing lethality. For this reason EMS

mutagenesis has been widely employed in both forward and reverse genetic screens where

relatively small populations can yield multiple mutant alleles of a gene. As a consequence

TILLING mutants possess high numbers of background mutations. TILLING is also relatively

labour intensive with PCR amplification and heteroduplex analysis generally carried out at low

levels of pooling, requiring many hundreds of samples to be screened. In addition, in common

with insertional mutagenesis, the recovery of mutations in genes of less than 1 kb is highly

inefficient, a significant limitation in the light of research highlighting the importance of micro

RNAs and other small transcriptional products.

Fast neutrons are a form of high energy radiation which 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 the current 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 ~ 500 keV to 5 MeV to generate the

short range secondary particles within the cell nucleus which mediate strand breakage

(Palfalvi pers comm.)

Bruggemann et al. (1996) isolated and characterised 20 independent null alleles of the HY4

locus in a forward screen of 300,000 M2 fast neutron irradiated Arabidopsis plants. Deletions

ranging from 300 bp to over 8 kb were identified by Southern analysis although larger or

smaller deletions and more complex rearrangements could not be distinguished using this

method. An indication of the size range of deletions induced by fast neutron bombardment

has also been made through a review of the mutants catalogued by the NSF Arabidopsis



Information Resource (TAIR; www.arabidopsis.org). This revealed 114 fast neutron alleles. Of

the 53 sufficiently characterised, 43 (81%) were deletions and 10 were other types of

mutation including combinations of insertions, deletions, substitution and rearrangements.

The deletions ranged from a single base pair to a 60kb deletion spanning 12 genes.

Strikingly, fast neutron induced deletions spanning megabases have not been identified in

Arabidopsis, or to our knowledge in Medicago or tomato. In contrast deletions of this size

seem to be common in wheat. This may be because hexaploid wheat can support the

removal of essential genes through the presence of duplicate gene copies on homeologous

chromosomes (Roberts, 1999). Fast neutron bombardment therefore provides a non-

transgenic and facile method of mutagenesis creating DNA lesions of a size amenable to

direct PCR detection potentially at high levels of pooling compared to conventional TILLING.

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) who developed a fast neutron based reverse

genetics platform in Arabidopsis known as Delete-a-gene (Li et al., 2001, Li et al., 2002). This

platform employed a PCR strategy for detecting deletions using shortened extension times to

limit amplification of longer wild type sequence, allowing short deletion containing alleles to be

preferentially amplified. Using extension time suppression alone it was demonstrated that this

strategy could be used to selectively amplify a known deletion in pools of 1000 plants.

However, this strategy is limited to detecting deletions which remove a large proportion of the

amplified region. Extension time suppression depends upon a large difference between the

wild type and mutant amplicons. This is an intrinsic weakness because a randomly selected

pair of primers greater than 5kb apart are unlikely to surround a deletion that removes greater

than 80% of the fragment. If smaller deletions could be detected within such amplicons, the

recovery of mutants would be more efficient. The relatively high pooling depths in Delete-a-

gene are only possible when detecting large deletions while the probability of closely flanking

such large deletions with random primers is low. The goal of this study was, therefore, to

develop a method whereby small deletions could be detected in large amplicons at high

pooling depths.

Here we describe the development of a novel reverse genetic strategy in the model legume

Medicago truncatula which exploits a large population of plants harbouring 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 3-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.


http://www.arabidopsis.org/


Results

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 mutagenised 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) by extension

time suppression, but this required targeting large wild type regions which challenged the

processivity of the polymerase under these condition, and large percentage deletions to

enable amplification of the small deletion allele. We tested the efficacy of delete-a-gene

across a range of dilutions and using a range of target sizes. For this analysis we used a

previously-characterized M. truncatula mutant, dmi1-4, with an 18kb deletion removing the 5’

end of the DMI1 gene and upstream region (Ane et al., 2004). PCR templates were prepared

to model the representation of a mutant within a genomic DNA pool. Genomic DNA of dmi1-4

and wild type M. truncatula A17 were combined in ratios up to 1 mutant in 24,000 wild type to

create a set of PCR templates useful for assessing the sensitivity of detection strategies (Fig.

1). Primers that spanned the deletion producing a 0.3kb product were highly efficient allowing

detection of the deletion in a 1 in 8,000 dilution (Fig. 1A). A secondary PCR using nested

primers allowed detection of the deletion up to a 1 in 24,000-fold dilution (Fig 1A). However,

when attempting to discover an unknown deletion it is unlikely that primers would so closely

span such a large deletion. Therefore, we tested the ability to detect the deletion using

primers more distant to the deletion site. Primers spanning the deletion and producing 3 and

8kb targets were far less efficient at detecting the mutation in a dilution series using a single

PCR, however, in a secondary PCR the 3kb target could be detected in a 1:24,000 dilution,

while the 8kb target could be detected in a 1:8,000 dilution (Fig 1B, 1C).

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 mutagenised M. truncatula

using this strategy. We hypothesised 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, that possesses a 435bp deletion. A secondary PCR using nested primers L7, R7, L5

and R5 (Fig. 2A) shows the 1561 bp nsp2-1 amplicon barely able to compete for amplification

with the 1996 bp wild type fragment at a ratio of one mutant in 25 wild type (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 (TMP) 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 1:1000 in the second round

of PCR (Fig. 2B). This is consistent with the pooling depth used in the original C. elegans

study (1:1200) although for a small target of 701 bp a detection sensitivity of 1:5000 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 centred 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

1:4000 (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 30bp 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).

Mutagenesis and population structureTo create a population for De-TILLING wild-type M. truncatula seeds were mutagenised 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 mutagenised seed has also been used as an indicator of

mutagenic rate. For a fast neutron mutagenised population of Arabidopsis an albino

frequency of 2% has been equated with around 10 induced deletions per line (Koornneef et

al., 1982). The extrapolation of this to other species must be made with caution. Since the

effective fast neutron dose, usually expressed in Gray (Gy), varies markedly between species

(Koornneef et al., 1982) and the genetic basis of this phenotype may vary. The effective

dosage is also influenced by the water content of seeds and must be adjusted for each new

batch. In our experience the mutagenic effect of fast neutron bombardment, as measured by

M1 survival and albino frequency, varies markedly between seed batches. These measures

should therefore be made for treated seed batches for every new population. A pilot

experiment indicated that a fast neutron dose of 32.5 Gy resulted in between 10% and 30%

survival and represented a maximum usable dose. A later experiment assessed mutagenic

effect following replicated fast neutron doses of 32.5 Gy. M1 survival varied between 35% and

77% (supplementary data). The M1 survival for the seed batches selected for the fast

neutron mutagenised population varied between 21-50%. An assessment of 1440 M1 lines

(288 families) revealed an albino frequency of 2.57% in the M2 progeny (supplementary

data). A more thorough analysis of the mutagenic effect of fast neutron bombardment could

be made through analysis of tiling arrays which would enable us to describe both the size

distribution and number of induced deletions per line. This has not however been carried out

for this study.

The M1 seeds were grown to maturity in groups of 5 plants in a single container and seed

pooled from each container. DNA was isolated from 25 seedlings representing each pool and

this DNA was normalised in a 96-well format. A stack of five 96-well plates was considered a

tower and represented 12,000 M2 seedlings from 2400 M1 plants. 13 towers were produced

to give a total population of 156,000 M2 plants, derived from an original population of 31,200

mutagenised M1 plants. Each tower was pooled in rows, columns and plates to create 25 3-

dimensional (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. Characterisation 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 1261bp 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 Lui et al. (1999). Lui noted a similar number of false positive amplicons of this type



using unmutagenised 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 4 reciprocal half tower pools (HTP) 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 characterise 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 harbouring the

characterised deletion.

Detecting novel deletion mutants in the populationWe searched for deletions in a LysM receptor like kinase. Five restriction sites, unique within

regions of ~2.4 kb, were identified within and closely adjacent to the 1.9kb coding region (Fig.

4A). De-TILLING assays were designed centred upon each restriction site resulting in nested

PCR amplicon sizes of 2.0-2.3 kb. A single deletion allele was detected possessing a 422bp

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 an ERF transcription factor (Vernie et al., 2008) initially identified as being up-

regulated in M. truncatula nodules using microarray analyses (El Yahyaoui et al., 2004) and

suppression subtractive hybridization (SSH) approaches (Godiard et al., 2007). The coding

region of this gene consisted of 2 exons of 85 bp and 503 bp. Because no sequence

downstream of the second exon was available we were limited to designing targets around

the first exon giving us a very small target of only 85 bp. To increase the number of De-

TILLING assays we could design we searched for restriction sites up to 250bp outside the

coding region. We identified 3 targetable restriction sites around which De-TILLING assays of

2.1 kb, 2.4 kb and 2.9 kb were designed. Screening the population revealed a mutant in

Tower 4 for the EcoRI based assay (Fig. 5A). Sequencing of these amplicons revealed a

1570bp deletion entirely removing the exon 1 and the 3’ end of the exon 2. This deletion

completely abolishes the production of the EFD transcript, as verified by quantitative RT-PCR

(Q-RT-PCR) analysis (Vernie et al., 2008). Following screening of the 3D Pools we were able

to recover 2 mutant plants from 50 seeds taken from the identified family, one heterozygous

and one homozygous for the efd1-1 mutation.



We attempted to recover deletions in 14 genes using a minimum of 5 well distributed targets

per locus. Target sizes ranged from 1.2 kb to 3.2 kb with an average target amplicon size of

2335 bp. The sizes of deletions that we detected using the De-TILLING method were 422 bp,

1270 bp, 1570 bp, 1723 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 1). All mutations completely

removed the targeted restriction site and the poison primer binding site.

Discussion

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 4000 lines requires

amplification, CelI digestion and analysis of fluorescently labelled PCR products for 500

samples. To reach acceptable levels of cost and efficiency, small, heavily mutagenised

populations are essential. Mutants recovered from these populations will possess a very large

number of non target mutations. For an Arabidopsis TILLING population, conservative

estimates suggest the density of mutations in exons to be ~3 per Mb (Colbert et al., 2001).

The impact of this is around 20 to 25 profoundly affected genes per EMS-mutagenised

genome (Henikoff, and Comai, 2003). This can affect the viability of mutants and confound

and delay downstream analyses. To achieve saturation, fast neutron mutagenised

populations need to be larger than those created using EMS, however, the mutants recovered

will be knockouts and will possess far fewer non target 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 60Gy, Koornneef et al. (1982) estimated an average of

approximately 2500 lines are required to inactivate a gene once. With around 27,000 coding

sequences in the Arabidopsis genome (TAIR8 release, 2008) this implies that around 10-11

genes are randomly deleted per line. The albino rate measured for this Arabidopsis

population (~2%) is similar to that measured for the M. truncatula De-TILLING population

(2.57%). This suggests a similar number of deleted loci per line in the M. truncatula

population. However, we must make this comparison with caution as the basis of the albino

phenotype in M. truncatula is not well defined.

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 2.3kb and ranged from 1.2 kb to 3.2kb. Detected deletions removed

from 18-68% 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 kb – 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: N = In[1-P]/In[1-F]

Screening 13 towers (31,200 M1 plants) enabled us to recover mutants in 4 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 4kb 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) of routinely disrupting more than one gene. While

deletion of single genes is required in the majority of cases, such an approach can be

advantageous for removing tandemly duplicated genes which is discussed below.

Recovering a deletion removing an 85bp exon of the EFD transcription factor demonstrated

the utility of De-TILLING for the targeting of small genes. Mutagenising 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:

P = 1 – [ 1 – ( X / 125000) ] n

where P is the probability of recovering the desired mutant, X is the size of the gene in kilo

base pairs, 125,000 is the approximate size in kb of the Arabidopsis genome and n is the

number of inserts in the mutant library (Krysan et al., 1999). Therefore, for an Arabidopsis

collection containing 100,000 insertions there is only a 55% probability of identifying an

insertion mutant for a 1kb target. For a similar Medicago collection, with a genome size of

400Mb, 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 mutagenise using TILLING or insertional techniques. Insertions are unlikely to be

found within small exons and those which fall within introns or intergenic regions are likely to

have no effect on protein function. TILLING relies on the identification of ~1kb 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 fewer than 10% of the genes tagged in

Arabidopsis are likely to generate a phenotypic change (Meinke et al., 2003). For a mutant

phenotype to become apparent it is sometimes necessary for multiple members of a gene

family to be inactivated. For unlinked loci it is possible to stack insertions or point mutations

within a single line to investigate gene function. However, where homologous genes are

present in tightly linked tandem arrays, recombination becomes extremely improbable and

therefore difficult to achieve. In general over 15% of the identified genes in sequenced plant

genomes are members of tandem-arrayed gene families (Jander et al., 2007). This is slightly

higher in Arabidopsis where about 4000 genes are tandemly repeated as two or more copies

(Arabidopsis Genome Initiative, 2000). In Arabidopsis the recombination rate is estimated to

be around 200 kbp/cM. For two mutations in Arabidopsis separated by 5 kb a homozygous

double mutant would be recovered only once in every 64 million F2 progeny (Jander et al.,

2007). Alternative strategies for recovering double mutants of tandemly homologous genes

are therefore very attractive. Deletions introduced by fast neutron mutagenesis, unlike point

mutations and DNA insertions, have the capacity to remove multiple adjacent genes, however

the Delete-a-gene detection procedures are more suited to this than the De-TILLING methods

described here.

Further 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 mutagenised lines with lower

levels of non target 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.

Experimental Protocols

Fast neutron population



Wild type M. truncatula seed (A17, Jemalong) was exposed to fast neutron radiation at a dose

of 32.5-35 Gy at the Atomic Energy Research Institute, (Budapest, Hungary.). The rate of

mutagenesis was assessed by M1 survival (37 to 48%) and albino rate (2.57%) as calculated

as a percentage of M1 treated lines displaying albino phenotypes in the M2 plants. 5 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 40C. 25 seeds from each M2 family

were germinated. The seedlings were freeze dried and the tissue ground.

DNA extraction and pooling

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 DNeasy 96 Plant Kit (Qiagen) and eluted

in 200ul. DNA was spectrophotometrically quantified and normalised to 50ng ul-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 3-dimensional (3D) pooling

structure. 3D pools were then combined to create 4 half tower pools (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 -200C.

Automated De-TILLING assay design.

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 5 primer De-TILLING assay

centred upon the targeted restriction sites. Poison primers were designed within 30bp 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.

PCR conditions

Nested PCR reactions were performed in a total of 50ul using Taq Polymerase Master Mix

(Qiagen), 240ng of digested genomic HTP DNA, 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 940C,

30 s at 550C and 2 min 30 sec at 740C.

dmi1-4 and nsp2-1 detection reconstruction experiments

Genomic DNAs of A17, dmi1-4 and nsp2-1 were prepared, quantified and normalised as

described above. These were pooled at mutant to wild type ratios of 1:25, 1:1000, 1:4000,

1:8000, 1:12000, 1:16000, 1:20,000 and 1:24,000. 6µl of each pool (240ng) 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 a 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.

De-TILLING screening and sequencing analysis

The first round PCR was analysed using an e-Gel (Invitrogen) to check for the production of

the suppressor fragment. Second round PCR was identical but scaled to 20ul, using 2µl of a

10-2 dilution of the first round PCR products as template and nested primers. The second

round products were then analysed 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 wild type to determine the deletion junction.

Acknowledgements

We would like to 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; Jonathan Clarke, David Baker and Bethany

McCullagh for providing DNA extraction and sequencing services. We would also like to 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. This work was funded by the European

Union as part of the Grain Legume Integrated Project (GLIP), by a grant in aid for the BBSRC,

by the Samuel Roberts Noble Foundation and the National Science Foundation (DBI0703285).

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Figure Legends

Figure 1. A reconstruction experiment showing the amplification of the M. truncatula dmi1-4

locus from genomic DNA of wild type M. truncatula (A17). The dmi1-4 mutant, and pools

containing dmi1-1 and wild type DNA at ratios of 1:25 ng to 1:24,000 ng. Nested PCR primers

flank the 18kb deletion by 350bp (A), 3.0kb (B) and 8.0kb (C). Amplification from the wild type

region is suppressed entirely under these conditions allowing amplification of the mutant

product in pools of over 1:24,000 for the 350bp and 3.0kb assay and 1:12,000 genomes for

the 8.0kb assay. 10: primary PCR, 20: secondary PCR.

Figure 2. A reconstruction experiment showing the amplification of the M. truncatula NSP2

locus from genomic DNA of wild type M. truncatula (A17), the nsp2-1 mutant (N2), and pools

containing nsp2-1 and wild type DNA at ratios of 1ng:25 ng (1:25), 1:1000ng (1K), 1:4000ng

(4K), 1:8000ng (8K), 1:12,000ng (12K), 1:16,000ng (16K), 1:20,000ng (20K) and 1:24,000ng

(24K). Only the secondary PCR products are shown in each case. (A) Nested PCR, wild type

alleles are preferentially amplified (B) Poison primer suppression enhances detection of the

mutant allele in pools of up to 1:1000 genomes. (C) Restriction suppression, EcoRV treated

templates allows reliable detection in pools of up to 4000 plants. (D) De-TILLING strategy,

combining poison primer and restriction suppression allows preferential amplification of the

mutant allele in pools containing a 24,000 fold excess of wild type sequences. 10: primary

PCR, 20: secondary PCR.

Figure 3. Structure of a De-TILLING tower. Each tower consists of 480 pools each of which is

genomic DNA of 25 seedlings taken from the pooled M2 progeny of 5 mutagenised M1 plants.

Each tower is initially pooled into 25 row, column and plate pools. These are used to create a



pair of reciprocal half tower pools (HTP). The De-TILLING population is initially screened

using the 54 HTP representing the 13 towers twice. When a deletion mutant is detected 25

3D pools are screened to locate the mutant to an individual well. The mutant is then

recovered from the identified pool.

Figure 4. Recovering a mutant for a LysM receptor like kinase. (A) Assays were designed

around five restriction site unique within 2-2.3kb regions. The StyI assay (red) detected a

422bp deletion within the population. (B) Two identical PCR products indicate the presence of

a deletion allele within tower 4. Note the spurious PCR products in towers 6 and 7 that are not

real detection events. (C) Amplification from the 3-dimensional pools of tower 4 locates a

single M2 pool containing the mutant. (D) PCR screening of 29 seedlings grown from this

pools allow the lysM1-1 mutant to be recovered.

Figure 5. Detection of the efd-1 mutant. (A) Identical PCR products occurring in tower 4 reveal

the presence of efd-1, an ERF transcription factor mutant possessing a 1571 bp deletion. (B)

Amplification from the 3-dimensional pools reveals the row, column and plate location of the

efd-1 mutant containing M2 pool within tower 4.



Gene NameExons

(length, bp)Assays

Deletions

(% of

amplicon)

ERF1 1 (83) 3 1570 (54%)

HAP2-1 6 (1599) 6 -

HK4 1 (3366) 5 1723 (68%)

LysMorf 1 (1914) 5 422 (18%)

MADS1 4 (725) 10 -

COI1-1 3 (1806) 5 -

Cyc 11 (1334) 6 -

B3 11 (946) 7 -

CLE13 1 (255) 5 -

Della 1 1 (1785) 5 -

ENOD40-1 1 (720) 7 -

MCA8 8 (3246) 7 1270 (68%)

GRAS1 2 (2161) 6 -

GRAS2 1 (1515) 5 -

Table 1. Loci targeted by De-TILLING indicating the range of target sizes and the size of

deletions identified.



Figure 1. A reconstruction experiment showing the amplification of the M. truncatula dmi1-4 locus from genomic DNA of wild type M. truncatula (A17). The dmi1-4 mutant, and pools containing dmi1-1 and wild type DNA at ratios of 1:25 ng to 1:24,000 ng. Nested PCR primers flank the 18kb deletion by 350bp (A), 3.0kb (B) and 8.0kb (C). Amplification from the wild type region is suppressed entirely under these conditions allowing amplification of the mutant product in pools of over 1:24,000 for the 350bp and 3.0kb assay and 1:12,000 genomes for the 8.0kb assay. 10: primary PCR, 20: secondary PCR.

A17 N2 1:25 1K 4K 8K 12K 16K 20K 24K

Admi117.0kb

350bp

10

20

10

20

dmi1

B17.0kb

3.0kb

A17 N2 1:25 1K 4K 8K 12K 16K 20K 24K

10

20

10

20

Cdmi117.0kb

8.0kb

A17 N2 1:25 1K 4K 8K 12K 16K 20K 24K

10

20

10

20



Figure 2. A reconstruction experiment showing the amplification of the M. truncatula NSP2locus from genomic DNA of wild type M. truncatula (A17), the nsp2-1 mutant (N2), and pools containing nsp2-1 and wild type DNA at ratios of 1ng:25 ng (1:25), 1:1000ng (1K), 1:4000ng (4K), 1:8000ng (8K), 1:12,000ng (12K), 1:16,000ng (16K), 1:20,000ng (20K) and 1:24,000ng (24K). Only the secondary PCR products are shown in each case. (A) Nested PCR, wild type alleles are preferentially amplified (B) Poison primer suppression enhances detection of the mutant allele in pools of up to 1:1000 genomes. (C) Restriction suppression, EcoRV treated templates allows reliable detection in pools of up to 4000 plants. (D) De-TILLING strategy, combining poison primer and restriction suppression allows preferential amplification of the mutant allele in pools containing a 24,000 fold excess of wild type sequences. 10: primary PCR, 20: secondary PCR.

A17 N2 1:25 1K 4K 8K 12K 16K 20K 24KA

nsp2

1.0 kb

2.0 kb

1.2 kb

1.5 kb

B

nsp2

L7 L7B R7

L5 R51.0 kb

2.0 kb

1.2 kb

1.5 kb

A17 N2 1:25 1K 4K 8K 12K 16K 20K 24K

CEcoRV

nsp2

1.0 kb

2.0 kb

1.2 kb

1.5 kb

A17 N2 1:25 1K 4K 8K 12K 16K 20K 24K

DEcoRV

nsp2

1.0 kb1.2 kb

1.5 kb

2.0 kb

A17 N2 1:25 1K 4K 8K 12K 16K 20K 24K

435 bp

20

20

20

10

20

20

L7 R7

L5 R5

10

20

L7 L7B R7

L5 R5

10

20

L7 R7

L5 R5

10

20



Figure 3. Structure of a De-TILLING tower. Each tower consists of 480 pools each of which is genomic DNA of 25 seedlings taken from the pooled M2 progeny of 5 mutagenised M1 plants. Each tower is initially pooled into 25 row, column and plate pools. These are used to create a pair of reciprocal half tower pools (HTP). The De-TILLING population is initially screened using the 54 HTP representing the 13 towers twice. When a deletion mutant is detected 25 3D pools are screened to locate the mutant to an individual well. The mutant is then recovered from the identified pool.

(Rows, Columns and Plates)

25 pools

3D POOLS

5 x 96-well plates12,000 M2 plants

TOWER

(Tower x2)4 pools

Half Tower Pools

5 M1 plants

25 M2 plants



23

Figure 4. Recovering a mutant for a LysM receptor like kinase. (A) Assays were designed around five restriction site unique within 2-2.3kb regions. The StyI assay (red) detected a 422bp deletion within the population. (B) Two identical PCR products indicate the presence of a deletion allele within tower 4. Note the spurious PCR products in towers 6 and 7 that are not real detection events. (C) Amplification from the 3-dimensional pools of tower 4 locates a single M2 pool containing the mutant. (D) PCR screening of 29 seedlings grown from this pools allow the lysM1-1 mutant to be recovered.

1.0 kb

2.0 kb1.5 kb

D Individual M2 lines

TOWER2

TOWER3

TOWER4

TOWER7

TOWER6

B

1.0 kb

2.0 kb

1.5 kb

Rows Columns PlatesA C EB D GF H 19 2120 221 3 52 4 76 8 9 1110 12

C

2.0 kb

3.0 kb

1.5 kb

MspIHindIII

NdeIStyI

HindIIIMspI HindIII

EcoRVNdeI

StyI

EcoRV targetMspI target

HindIII target

StyI target

NdeI target

A

23



Figure 5. Detection of the efd-1 mutant. (A) Identical PCR products occurring in tower 4 reveal the presence of efd-1, an ERF transcription factor mutant possessing a 1571 bp deletion. (B) Amplification from the 3-dimensional pools reveals the row, column and plate location of the efd-1mutant containing M2 pool within tower 4.

TOWER2

TOWER3

TOWER4

TOWER7

TOWER6

A

1.0 kb

2.0 kb

1.5 kb

Rows Columns PlatesA C EB D GF H 19 21 2320 221 3 52 4 76 8 9 1110 12

B

1.0 kb

2.0 kb1.5 kb



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