Targeting-induced Local Lesions Research Articles

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes)

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon.

A frame shift invoked by a single-base deletion in the gene encoding a cytochrome P450 hydroxylase, CYP81E22, causes the loss of bentazon detoxification function in soybean. Bentazon is an effective herbicide in soybean cultivation applied at post-emergence stages for control of several broadleaf weeds. However, some soybean cultivars are highly sensitive to bentazon and are killed upon application. In this study, the gene related to the high sensitivity of soybean cultivars to bentazon was mapped to chromosome 16, and its location was narrowed down to a 257-kb region where three cytochrome P450 genes were located. In these genes, a single-base deletion of cytosine was detected in the coding region of Glyma.16G149300, CYP81E22, at + 1465bp downstream from the translation start codon, leading to a frame shift in the open reading frame and creating a premature stop codon. This stop codon resulted in the loss of more than half of the P450, and consequently, the remaining molecule failed to form a functioning protein. This single-base deletion was common among the highly sensitive cultivars screened from the soybean mini-core collection and other previously reported highly sensitive cultivars. Furthermore, we screened plant lines from the targeting-induced local lesions in genomes library of the soybean cultivar Enrei based on a modelled 3D structure of CYP81E22. The lines with mutations in Glyma.16G149300 were highly sensitive to bentazon, which provides strong evidence that Glyma.16G149300 is the gene responsible for high sensitivity to bentazon.

An efficient TILLING platform for cultivated tobacco.

Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.

Development of Decreased-Gluten Wheat Enabled by Determination of the Genetic Basis of lys3a Barley.

Celiac disease is the most common food-induced enteropathy in humans, with a prevalence of approximately 1% worldwide. It is induced by digestion-resistant, proline- and glutamine-rich seed storage proteins, collectively referred to as gluten, found in wheat (Triticum aestivum). Related prolamins are present in barley (Hordeum vulgare) and rye (Secale cereale). The incidence of both celiac disease and a related condition called nonceliac gluten sensitivity is increasing. This has prompted efforts to identify methods of lowering gluten in wheat, one of the most important cereal crops. Here, we used bulked segregant RNA sequencing and map-based cloning to identify the genetic lesion underlying a recessive, low-prolamin mutation (lys3a) in diploid barley. We confirmed the mutant identity by complementing the lys3a mutant with a transgenic copy of the wild-type barley gene and then used targeting-induced local lesions in genomes to identify induced single-nucleotide polymorphisms in the three homeologs of the corresponding wheat gene. Combining inactivating mutations in the three subgenomes of hexaploid bread wheat in a single wheat line lowered gliadin and low-molecular-weight glutenin accumulation by 50% to 60% and increased free and protein-bound lysine by 33%.

Discovery of mutations in Chenopodium quinoa Willd through EMS mutagenesis and mutation screening using pre-selection phenotypic data and next-generation sequencing

AbstractQuinoa (Chenopodium quinoaWilld) is a dicotyledonous annual species belonging to the family Amaranthaceae, which is nutritionally well balanced in terms of its oil, protein and carbohydrate content. Targeting-induced local lesions in genomes (the TILLING strategy) was employed to find mutations in acetolactate synthase (AHAS) genes in a mutant quinoa population. TheAHASgenes were targeted because they are common enzyme target sites for five herbicide groups. Ethyl methane sulfonate (EMS) was used to induce mutations in theAHASgenes; it was found that 2% EMS allowed a mutation frequency of one mutation every 203 kilobases to be established. In the mutant population created, a screening strategy using pre-selection phenotypic data and next-generation sequencing (NGS) allowed identification of a mutation that alters the amino acid composition of this species (nucleotide 1231 codon GTT→ATT, Val→Ile); however, this mutation did not result in herbicide resistance. The current work shows that TILLING combined with the high-throughput of NGS technologies and an overlapping pool design provides an efficient and economical method for detecting induced mutations in pools of individuals.

Generation of Mycoplasma hominis gene-targeted mutants by targeting-induced local lesions in genomes (TILLING)

BackgroundMycoplasma hominis is a human urogenital pathogen involved in gynaecological, neonatal and extra-genital infections. However, no versatile genetic tools are currently available to study the pathogenicity of this bacterium. Targeting-Induced Local Lesions IN Genomes (TILLING) is a reverse-genetic method that combines point mutations induced by chemical mutagenesis with a DNA screening technique. We used ethyl methanesulfonate (EMS) that introduces C-G to T-A transition mutations to generate a library of M. hominis mutants. As a proof of concept, mutagenized organisms were screened for mutations in two target genes previously associated with the mycoplasma pathogenicity, the vaa gene encoding an adhesin lipoprotein and the oppA gene encoding the main ectoATPase of the bacterium. The resulting mutants were evaluated using functional assays, an adhesion to HeLa cell assay for vaa-mutants and an ATPase activity test for oppA-mutants.ResultsA 1200-clone library was generated by exposing M. hominis PG21 to 9 mg/mL EMS for 3 h. To identify mutants of interest, targeted gene fragments were amplified, heat-denatured, slowly reannealed and digested with the mismatch-specific endonuclease ENDO1. If multiple alleles were present in the PCR amplicons, these alleles formed heteroduplexes during reannealing that were specifically cleaved by ENDO1 at mismatching positions.A total of four vaa-mutants and two oppA-mutants harbouring missense mutations were obtained and fully sequenced. Zero to eight additional mutations were identified in the genomes of each mutant. The vaa-mutants were tested for adhesion to immobilized HeLa cells but their adhesion was not significantly different from the adhesion of M. hominis PG21. One of the two oppA-mutants that were tested for ATPase activity presented a higher affinity for its ATP substrate than the parental strain.ConclusionFor the first time, we demonstrated that M. hominis gene-targeted mutants could be successfully obtained using this TILLING strategy. In the absence of robust genetic tools for studying M. hominis, the TILLING strategy that can target any gene of the genome could help to elucidate gene functions and to better understand the pathogenesis of this human pathogenic species.

Identification of altered metabolic pathways of γ-irradiated rice mutant via network-based transcriptome analysis.

In order to develop rice mutants for crop improvement, we applied γ-irradiation mutagenesis and selected a rice seed color mutant (MT) in the M14 targeting-induced local lesions in genome lines. This mutant exhibited differences in germination rate, plant height, and root length in seedlings compared to the wild-type plants. We found 1645 different expressed probes of MT by microarray hybridization. To identify the modified metabolic pathways, we conducted integrated genomic analysis such as weighted correlation network analysis with a module detection method of differentially expressed genes (DEGs) in MT on the basis of large-scale microarray transcriptional profiling. These modules are largely divided into three subnetworks and mainly exhibit overrepresented gene ontology functions such as oxidation-related function, ion-binding, and kinase activity (phosphorylation), and the expressional coherences of module genes mainly exhibited in vegetative and maturation stages. Through a metabolic pathway analysis, we detected the significant DEGs involved in the major carbohydrate metabolism (starch degradation), protein degradation (aspartate protease), and signaling in sugars and nutrients. Furthermore, the accumulation of amino acids (asparagine and glutamic acid), sucrose, and starch in MT were affected by gamma rays. Our results provide an effective approach for identification of metabolic pathways associated with useful agronomic traits in mutation breeding.

New starch phenotypes produced by TILLING in barley.

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.

Pea (Pisum sativum L.) in biology prior and after Mendel's discovery

Pea (Pisum sativum L.) has been extensively used in early hybridization studies and it was the model organism of choice for Mendel’s discovery of the laws of inheritance, making pea part of the foundation of modern genetics. Pea has also been used as model for experimental morphology and physiology. However, subsequent progress in pea genomics has lagged behind many other plant species, largely as a consequence of its genome size and low economic significance. The availability of the genome sequences of five legume species (Medicago truncatula, Lotus japonicus, Glycine max, Cajanus cajan and Cicer aerietinum) offers opportunities for genome wide comparison. The combination of a candidate gene and synteny approach has allowed the identification of genes underlying agronomically important traits such as virus resistances and plant architecture. Useful genomic resources already exist and include several types of molecular marker sets as well as both transcriptome and proteome datasets. The advent of greater computational power and access to diverse germplasm collections enable the use of association mapping to identify genetic variation related to desirable agronomic traits. Current genomic knowledge and technologies can facilitate the allele mining for novel traits and their incorporation from wild Pisum sp. into elite domestic backgrounds. Fast neutron and targeting-induced local lesions in genomes (TILLING) pea mutant populations are available for reverse genetics approaches, BAC libraries for positional gene cloning as well as transgenic and in vitro regeneration for proof of function through gene silencing or over-expression. Finally, recently formed International Pea Genome Sequencing Consortium, holds promise to provide the pea genome sequence by 2015, a year of 150 anniversary of Mendel’s work.

Genome-wide DNA polymorphism and transcriptome analysis of an early-maturing rice mutant

In order to develop a rice population with improved important traits such as flowering time, we developed 2,911 M2 targeting-induced local lesions in genomes (TILLING) lines by irradiating rice seeds with γ-rays. In all, 15 M3 lines were obtained from 3 different M2 lines that exhibited an early-maturing phenotype: these plants matured approximately 25 days faster than wild-type (WT) plants. To identify genome-wide DNA polymorphisms, we performed whole-genome resequencing of both the plant types, i.e., WT and early-maturing TILLING 1 (EMT1), and obtained mapped reads of 118,488,245 bp (99.53 %) and 128,489,860 bp (99.72 %), respectively; Nipponbare was used as the reference genome. We obtained 63,648 and 147,728 single nucleotide polymorphisms (SNPs) and 33,474 and 31,082 insertions and deletions (InDels) for the WT and EMT1, respectively. Interestingly, there was a higher number of SNPs (2.6-fold) and slightly lower number of InDels (0.9-fold) in EMT1 than in WT. The expression of at least 202 structurally altered genes was changed in EMT1, and functional enrichment analysis of these genes revealed that their molecular functions were related to flower development. These results might provide a critical insight into the regulatory pathways of rice flowering.

Induced mutagenesis in Sugar Beet (Beta vulgaris L.): Obtained results and prospects for use in development of TILLING project

At the Research Institute of Sugar Beets, induced mutagenesis is used for sugar beets as one of the lines in plant selection that have been in development since the 1940s. The following significant scientific and practical results have been obtained: the mutability of the initial material of different genetic origin was tested; the activity of the used physical and chemical mutagens and biologically active substances during various modes of processing was assessed; optimal variants for obtaining mutants with desirable traits were chosen; M2 populations and subsequent generations were created; and the need to follow a definite subsequence and recurrence in work was substantiated. Beet mutants with high root mass, sugarance, and resistance have been obtained that are used in hybridization. The widespread use of methods of the molecular biology in the 1990s decreased the interest in induced mutagenesis. In the 21st century, this interest was revived due to the new method of targeting-induced local lesions in genomes (TILLING), which combines the use of induced mutagenesis with molecular methods of searching for mutations. TILLING is based on the creation of a population treated with mutagens that leads to single nucleotide polymorphism (SNP). This process makes it possible to obtain a set of allele variants in genes that is very important for selection. In different countries, TILLING projects have been developed for many agricultural plants, including sugar beets in Germany. The results obtained by the induced mutagenesis of sugar beets in Russia are of great scientific and practical importance.

Updating the Micro-Tom TILLING platform

The dwarf tomato variety Micro-Tom is regarded as a model system for functional genomics studies in tomato. Various tomato genomic tools in the genetic background of Micro-Tom have been established, such as mutant collections, genome information and a metabolomic database. Recent advances in tomato genome sequencing have brought about a significant need for reverse genetics tools that are accessible to the larger community, because a great number of gene sequences have become available from public databases. To meet the requests from the tomato research community, we have developed the Micro-Tom Targeting-Induced Local Lesions IN Genomes (TILLING) platform, which is comprised of more than 5000 EMS-mutagenized lines. The platform serves as a reverse genetics tool for efficiently identifying mutant alleles in parallel with the development of Micro-Tom mutant collections. The combination of Micro-Tom mutant libraries and the TILLING approach enables researchers to accelerate the isolation of desirable mutants for unraveling gene function or breeding. To upgrade the genomic tool of Micro-Tom, the development of a new mutagenized population is underway. In this paper, the current status of the Micro-Tom TILLING platform and its future prospects are described.

A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants

We developed two mutant populations of oilseed rape (Brassica napus L.) using EMS (ethylmethanesulfonate) as a mutagen. The populations were derived from the spring type line YN01-429 and the winter type cultivar Express 617 encompassing 5,361 and 3,488 M(2) plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8× pooling strategy. Genes of the sinapine biosynthesis pathway were chosen for determining the mutation frequencies and for creating novel genetic variation for rapeseed breeding. The extraction meal of oilseed rape is a rich protein source containing about 40% protein. Its use as an animal feed or human food, however, is limited by antinutritive compounds like sinapine. The targeting-induced local lesions in genomes (TILLING) strategy was applied to identify mutations of major genes of the sinapine biosynthesis pathway. We constructed locus-specific primers for several TILLING amplicons of two sinapine synthesis genes, BnaX.SGT and BnaX.REF1, covering 80-90% of the coding sequences. Screening of both populations revealed 229 and 341 mutations within the BnaX.SGT sequences (135 missense and 13 nonsense mutations) and the BnaX.REF1 sequences (162 missense, 3 nonsense, 8 splice site mutations), respectively. These mutants provide a new resource for breeding low-sinapine oilseed rape. The frequencies of missense and nonsense mutations corresponded to the frequencies of the target codons. Mutation frequencies ranged from 1/12 to 1/22kb for the Express 617 population and from 1/27 to 1/60kb for the YN01-429 population. Our TILLING resource is publicly available. Due to the high mutation frequencies in combination with an 8× pooling strategy, mutants can be routinely identified in a cost-efficient manner. However, primers have to be carefully designed to amplify single sequences from the polyploid rapeseed genome.

The Magnesium-Chelatase H Subunit Binds Abscisic Acid and Functions in Abscisic Acid Signaling: New Evidence in Arabidopsis

Using a newly developed abscisic acid (ABA)-affinity chromatography technique, we showed that the magnesium-chelatase H subunit ABAR/CHLH (for putative abscisic acid receptor/chelatase H subunit) specifically binds ABA through the C-terminal half but not the N-terminal half. A set of potential agonists/antagonists to ABA, including 2-trans,4-trans-ABA, gibberellin, cytokinin-like regulator 6-benzylaminopurine, auxin indole-3-acetic acid, auxin-like substance naphthalene acetic acid, and jasmonic acid methyl ester, did not bind ABAR/CHLH. A C-terminal C370 truncated ABAR with 369 amino acid residues (631-999) was shown to bind ABA, which may be a core of the ABA-binding domain in the C-terminal half. Consistently, expression of the ABAR/CHLH C-terminal half truncated proteins fused with green fluorescent protein (GFP) in wild-type plants conferred ABA hypersensitivity in all major ABA responses, including seed germination, postgermination growth, and stomatal movement, and the expression of the same truncated proteins fused with GFP in an ABA-insensitive cch mutant of the ABAR/CHLH gene restored the ABA sensitivity of the mutant in all of the ABA responses. However, the effect of expression of the ABAR N-terminal half fused with GFP in the wild-type plants was limited to seedling growth, and the restoring effect of the ABA sensitivity of the cch mutant was limited to seed germination. In addition, we identified two new mutant alleles of ABAR/CHLH from the mutant pool in the Arabidopsis Biological Resource Center via Arabidopsis (Arabidopsis thaliana) Targeting-Induced Local Lesions in Genomes. The abar-2 mutant has a point mutation resulting in the N-terminal Leu-348-->Phe, and the abar-3 mutant has a point mutation resulting in the N-terminal Ser-183-->Phe. The two mutants show altered ABA-related phenotypes in seed germination and postgermination growth but not in stomatal movement. These findings support the idea that ABAR/CHLH is an ABA receptor and reveal that the C-terminal half of ABAR/CHLH plays a central role in ABA signaling, which is consistent with its ABA-binding ability, but the N-terminal half is also functionally required, likely through a regulatory action on the C-terminal half.

TILLING Mutants of Lotus japonicus Reveal That Nitrogen Assimilation and Fixation Can Occur in the Absence of Nodule-Enhanced Sucrose Synthase

In all plant species studied to date, sucrose synthase occurs as multiple isoforms. The specific functions of the different isoforms are for the most part not clear. Six isoforms of sucrose synthase have been identified in the model legume Lotus japonicus, the same number as in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). The genes encoding these isoforms are differentially expressed in all plant organs examined, although one, LjSUS4, is only expressed in flowers. LjSUS1 is the most highly expressed in all plant organs tested, except root nodules, where LjSUS3 accounts for more than 60% of the total SUS transcripts. One gene, LjSUS2, produces two transcripts due to alternative splicing, a feature not observed in other species to date. We have isolated plants carrying ethyl methanesulfonate-induced mutations in several SUS genes by targeting-induced local lesions in genomes reverse genetics and examined the effect of null alleles of two genes, LjSUS1 and LjSUS3, on nodule function. No differences were observed between the mutants and wild-type plants under glasshouse conditions, but there was evidence for a nitrogen-starvation phenotype in the sus3-1 mutant and severe impairment of growth in the sus1-1/sus3-1 double mutant under specific environmental conditions. Nodules of sus3-1 mutant plants retained a capacity for nitrogen fixation under all conditions. Thus, nitrogen fixation can occur in L. japonicus nodules even in the absence of LjSUS3 (the major nodule-induced isoform of SUS), so LjSUS1 must also contribute to the maintenance of nitrogen assimilation.

A protocol for TILLING and Ecotilling in plants and animals

We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.

Detection of quinolone‐resistance genes in Photobacterium damselae subsp. piscicida strains by targeting‐induced local lesions in genomes

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.

High-throughput TILLING for functional genomics.

Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.