Deleteagene: a fast neutron deletion mutagenesis-based gene knockout system for plants

Deleteagene(trade mark) (Delete-a-gene) is a deletion-based gene knockout system for plants. To obtain deletion mutants for a specific gene, random deletion libraries created by fast neutron mutagenesis are screened by polymerase chain reaction (PCR) using primers flanking the target gene. By adjusting the PCR extension time to preferentially amplify the deletion alleles, deletion mutants can be identified in pools of DNA samples with each sample representing more than a thousand mutant lines. In Arabidopsis, knockout plants for greater than 80% of targeted genes have been obtained from a population of 51 840 lines. A large number of deletion mutants have been identified and multiple deletion alleles are often recovered for targeted loci. In Arabidopsis, the method is very useful for targeting small genes and can be used to find deletion mutants mutating two or three tandem homologous genes. In addition, the method is demonstrated to be effective in rice as a deletion mutant for a rice gene was obtained with a similar approach. Because fast neutron mutagenesis is applicable to all plant genetic systems, Deleteagene(trade mark) has the potential to enable reverse genetics for a wide range of plant species.     

A fast neutron deletion mutagenesis-based reverse genetics system for plants

A new reverse genetics method has been developed to identify and isolate deletion mutants for targeted plant genes. Deletion mutant libraries are generated using fast neutron bombardment. DNA samples extracted from the deletion libraries are used to screen for deletion mutants by polymerase chain reaction (PCR) using specific primers flanking the targeted genes. By adjusting PCR conditions to preferentially amplify the deletion alleles, deletion mutants were identified in pools of DNA samples, each pool containing DNA from 2592 mutant lines. Deletion mutants were obtained for 84% of targeted loci from an Arabidopsis population of 51 840 lines. Using a similar approach, a deletion mutant for a rice gene was identified. Thus we demonstrate that it is possible to apply this method to plant species other than Arabidopsis. As fast neutron mutagenesis is highly efficient, it is practical to develop deletion mutant populations with more complete coverage of the genome than obtained with methods based on insertional mutagenesis. Because fast neutron mutagenesis is applicable to all plant genetic systems, this method has the potential to enable reverse genetics for a wide range of plant species.     

Genome Resilience and Prevalence of Segmental Duplications Following Fast Neutron Irradiation of Soybean

Fast neutron radiation has been used as a mutagen to develop extensive mutant collections. However, the genome-wide structural consequences of fast neutron radiation are not well understood. Here, we examine the genome-wide structural variants observed among 264 soybean [Glycine max (L.) Merrill] plants sampled from a large fast neutron-mutagenized population. While deletion rates were similar to previous reports, surprisingly high rates of segmental duplication were also found throughout the genome. Duplication coverage extended across entire chromosomes and often prevailed at chromosome ends. High-throughput resequencing analysis of selected mutants resolved specific chromosomal events, including the rearrangement junctions for a large deletion, a tandem duplication, and a translocation. Genetic mapping associated a large deletion on chromosome 10 with a quantitative change in seed composition for one mutant. A tandem duplication event, located on chromosome 17 in a second mutant, was found to cosegregate with a short petiole mutant phenotype, and thus may serve as an example of a morphological change attributable to a DNA copy number gain. Overall, this study provides insight into the resilience of the soybean genome, the patterns of structural variation resulting from fast neutron mutagenesis, and the utility of fast neutron-irradiated mutants as a source of novel genetic losses and gains.     

Forward and reverse genetics of rapid-cycling Brassica oleracea

Seeds of rapid-cycling Brassica oleracea were mutagenized with the chemical mutagen, ethylmethane sulfonate. The reverse genetics technique, TILLING, was used on a sample population of 1,000 plants, to determine the mutation profile. The spectrum and frequency of mutations induced by ethylmethane sulfonate was similar to that seen in other diploid species such as Arabidopsis thaliana. These data indicate that the mutagenesis was effective and demonstrate that TILLING represents an efficient reverse genetic technique in B. oleracea that will become more valuable as increasing genomic sequence data become available for this species. The extensive duplication in the B. oleracea genome is believed to result in the genetic redundancy that has been important for the evolution of morphological diversity seen in today’s B. oleracea crops (broccoli, Brussels sprouts, cauliflower, cabbage, kale and kohlrabi). However, our forward genetic screens identified 120 mutants in which some aspect of development was affected. Some of these lines have been characterized genetically and in the majority of these, the mutant trait segregates as a recessive allele affecting a single locus. One dominant mutation (curly leaves) and one semi-dominant mutation (dwarf-like) were also identified. Allelism tests of two groups of mutants (glossy and dwarf) revealed that for some loci, multiple independent alleles have been identified. These data indicate that, despite genetic redundancy, mutation of many individual loci in B. oleracea results in distinct phenotypes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Induction and recovery of copy number variation in banana through gamma irradiation and low‐coverage whole‐genome sequencing

Traditional breeding methods are hindered in bananas due to the fact that major cultivars are sterile, parthenocarpic, triploid and thus clonally propagated. This has resulted in a narrow genetic base and limited resilience to biotic and abiotic stresses. Mutagenesis of in vitro propagated bananas is one method to introduce novel alleles and broaden genetic diversity. We previously established a method for the induction and recovery of single nucleotide mutations generated with the chemical mutagen EMS. However, officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic insertions and deletions (indels). Such dosage mutations may be important for generating observable phenotypes in polyploids. In this study, we establish a low‐coverage whole‐genome sequencing approach in triploid bananas to recover large genomic indels caused by treatment with gamma irradiation. We first evaluated the commercially released mutant cultivar ‘Novaria’ and found that it harbours multiple predicted deletions, ranging from 0.3 to 3.8 million base pairs (Mbp). In total, predicted deletions span 189 coding regions. To evaluate the feasibility of generating and maintaining new mutations, we developed a pipeline for mutagenesis and screening for copy number variation in Cavendish bananas using the cultivar ‘Williams’. Putative mutations were recovered in 70% of lines treated with 20 Gy and 60% of the lines treated with 40 Gy. While deletion events predominate, insertions were identified in 20 Gy‐treated material. Based on these results, we believe this approach can be scaled up to support large breeding projects.     

A reverse genetic screen in Drosophila using a deletion-inducing mutagen

We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems.     

Mutagenesis strategies in zebrafish for identifying genes involved in development and disease

It has been nearly a decade since the completion of two large-scale chemical mutagenesis screens in zebrafish, and two years since the completion of a large-scale insertional mutagenesis. In this article, we use the accumulated data from these screens to compare the efficiency of each mutagen to isolate mutants and to identify mutated genes, and argue that the two mutagens target the same set of genes. We then review how both forward genetic screens and reverse genetic techniques, such as morpholinos and TILLING, and transgenics are being used to develop models of human disease.     

<i>In vitro</i> Mutagenesis for the Improvement of Agave Genus

Biotechnological techniques provide a viable alternative to help improve and increase the production of plant species of agricultural and economic importance, which have been affected over the years by climate change, increasing their susceptibility to pests and/or diseases, generating losses in production as well as a decrease in their regenerative and genetic diversity. The application of biotechnological techniques such as in vitro mutagenesis offers a viable option for the generation of crops that are resistant to the different factors caused by abiotic and biotic stress. In vitro mutagenesis has been used in an efficient way to generate genetic changes in different plant species. However, these methods have not been studied thoroughly in crops of agro-industrial interest, such as agave, which represents an economic resource of national importance and are considered as endemic species of Mexico. Therefore, this literary review aimed to focus on the studies that have been used for the genetic improvement of this species via mutagenesis techniques in plants in the agave genus. Therefore, the objective was to set a precedent for future genetic studies that aim to obtain more productive regenerants for various industries, such as food and pharmaceutical. It is also of great interest to compile information from basic research that helps understand and elucidate a model of possible defense mechanisms that are activated in the Agave genus.     

Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals

With the fairly recent advent of inexpensive, rapid sequencing technologies that continue to improve sequencing efficiency and accuracy, many species of animals, plants, and microbes have annotated genomic information publicly available. The focus on genomics has thus been shifting from the collection of whole sequenced genomes to the study of functional genomics. Reverse genetic approaches have been used for many years to advance from sequence data to the resulting phenotype in an effort to deduce the function of a gene in the species of interest. Many of the currently used approaches (RNAi, gene knockout, site-directed mutagenesis, transposon tagging) rely on the creation of transgenic material, the development of which is not always feasible for many plant or animal species. TILLING is a non-transgenic reverse genetics approach that is applicable to all animal and plant species which can be mutagenized, regardless of its mating / pollinating system, ploidy level, or genome size. This approach requires prior DNA sequence information and takes advantage of a mismatch endonuclease to locate and detect induced mutations. Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene. TILLING has proven to be a practical, efficient, and an effective approach for functional genomic studies in numerous plant and animal species. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones. In this review, TILLING and EcoTILLING techniques, beneficial applications and limitations from plant and animal studies are discussed.Key Words: Reverse genetics, functional genomics, TILLING (target induced local lesions in genomes), EcoTILLING (Ecotype TILLING), sequencing, SNP (single nucleotide polymorphism), genetic stocks.     

Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean

Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research.     

Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

Use of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, only floral-dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols, including software for automated data analysis, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1 mutagenesis, combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.     

Ethyl Methanesulfonate as Inductor of Somaclonal Variants in Different Crops

Ethyl methanesulfonate is a chemical mutagen, which is currently being used in plant breeding, to increase genetic variability in genes of agronomic interest, of species useful in agriculture. It primarily causes single base point mutations by inducing guanine alkylation, resulting in GC to AT transitions. Its effect is different between clones of a genotype and between genotypes of the same species. This review presents the results obtained in recent research, where its effect on plant tissues, callus, and cells in suspension has been evaluated. Changes in the phenotypic expression of somaclonal variants were reported, involving morphology, production of secondary metabolites, changes in metabolic routes of resistance, tolerance to stress, increased seed yield, among others. In addition, this review compiles the doses and guidelines to consider before using this mutagen, which can serve as a guide for future trials in deciding the response variables, the type of plant explants and the selection of the study model. Mutant lines have allowed plant breeders to have a collection of plants with different characteristics, in places where the cultivar does not have its center of origin. It is important to note that it is still necessary to continue evaluating the heritability of mutations and their behaviour in the environment where they will be established, in order to obtain new varieties of plants that can be cultivated with uniformity in their genetic response.     

Establishing gene function by mutagenesis in Arabidopsis thaliana

The nuclear genome of Arabidopsis thaliana was sequenced to near completion a few years ago, and ahead lies the challenge of understanding its meaning and discerning its potential. How many genes are there? What are they? What do they do? Computer algorithms combined with genome array technologies have proven efficient in addressing the first two questions as shown in a recent report ( Yamada et al., 2003 ). However, assessing the function of every gene in every cell will require years of careful analyses of the phenotypes caused by mutations in each gene. Current progress in generating large numbers of molecular markers and near‐saturation insertion mutant collections has immensely facilitated functional genomics studies in Arabidopsis. In this review, we focus on how gene function can be revealed through the analysis of mutants by either forward or reverse genetics. These mutants generally fall into two distinct classes. The first class typically includes point mutations or small deletions derived from chemical or fast neutron mutagenesis whereas the second class includes insertions of transferred‐DNA or transposon elements. We describe the current methods that are used to identify the gene corresponding to these mutations, which can then be used as a probe to further dissect its function.     

An Escherichia coli mutant refractory to nitrosoguanidine mutagenesis

Summary A newly-isolated Escherichia coli mutant suffers only about 10% as many mutations as normal strains on exposure to nitrosoguanidine¹. The responsible mutation, inm-1, maps at approximately minute 79 in the current E. coli genetic map. The mutant is normal for overall growth, nitrosoguanidine lethality, spontaneous mutagenesis, ultraviolet light lethality and mutagenesis, ethyl methanesulfonate lethality and mutagenesis, and the adaptive repair induced by alkylating agents. The existence of this mutation proves that nitrosoguanidine mutagenesis is not merely the result of reactions between the chemical and DNA, but requires specific cellular function(s), and underscores the peculiarity of nitrosoguanidine as a mutagen.     

Induction,rapid fixation and retention of mutations in vegetatively propagated banana

Mutation discovery technologies have enabled the development of reverse genetics for many plant species and allowed sophisticated evaluation of the consequences of mutagenesis. Such methods are relatively straightforward for seed‐propagated plants. To develop a platform suitable for vegetatively propagated species, we treated isolated banana shoot apical meristems with the chemical mutagen ethyl methanesulphonate, recovered plantlets and screened for induced mutations. A high density of GC‐AT transition mutations were recovered, similar to that reported in seed‐propagated polyploids. Through analysis of the inheritance of mutations, we observed that genotypically heterogeneous stem cells resulting from mutagenic treatment are rapidly sorted to fix a single genotype in the meristem. Further, mutant genotypes are stably inherited in subsequent generations. Evaluation of natural nucleotide variation showed the accumulation of potentially deleterious heterozygous alleles, suggesting that mutation induction may uncover recessive traits. This work therefore provides genotypic insights into the fate of totipotent cells after mutagenesis and suggests rapid approaches for mutation‐based functional genomics and improvement of vegetatively propagated crops.     

Plant tagnology

Transposable elements have been used as an effective mutagen and as a tool to clone tagged genes. Insertion of a transposable element into a gene can lead to loss- or gain-of-function, changes in expression pattern, or can have no effect on gene function at all, depending on whether the insertion took place in coding or non-coding regions of the gene. Cloning transposable elements from different plant species has made them available as a tool for the isolation of tagged genes using homologous or heterologous tagging strategies. Based on these transposons, new elements have been engineered bearing reporter genes that can be used for expression analysis of the tagged gene, or resistance genes that can be used to select for knockout insertions. While many genes have been cloned using transposon tagging following traditional forward genetics strategies, gene cloning has ceased to be the rate-limiting step in the process of determining sequence–function relations in several important plant model species. Large-scale insertion mutagenesis and identification of insertion sites following a reverse genetics strategy appears to be the best method for unravelling the biological role of the thousands of genes with unknown functions identified by genome or expressed sequence tag (EST) sequencing projects. Here we review the progress in forward tagging technologies and discuss reverse genetics strategies and their applications in different model species.     

A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction

A simple and fast method for introducing a series of mutations in cloned DNA has been developed. The polymerase chain reaction (PCR) has been used for site-directed mutagenesis. Because mutations can be introduced only within the primer sequences used for PCR, a suitable restriction site in the vicinity of the mutated nucleotide is required to permit recloning. Several methods have been devised to overcome this limitation. Our present method is a modification of the overlap extension method [Ho et al., Gene 77 (1989) 51-57], and has some advantages over this and other published methods. In our method, three common primers and a series of primers specific for various mutations are chemically synthesized. Once the proper oligodeoxyribonucleotides are selected as common primers, each mutation requires only one additional primer. Therefore, this method is very useful for introducing many mutations in various sites of the target DNA. We describe our protocol for the site-directed mutagenesis and an example of the introduction of several mutations in the hen egg-white lysozyme-encoding gene.     

An essential gene mutagenesis screen across the highly conserved piebald deletion region of mouse chromosome 14

The piebald deletion complex is a set of overlapping chromosomal deficiencies on distal mouse chromosome 14. We surveyed the functional genetic content of the piebald deletion region in an essential gene mutagenesis screen of 952 genomes to recover seven lethal mutants. The ENU‐induced mutations were mapped to define genetic intervals using the piebald deletion panel. Lethal mutations included loci required for establishment of the left‐right embryonic axis and a loss‐of‐function allele of Phr1 resulting in respiratory distress at birth. A functional map of the piebald region integrates experimental genetic data from the deletion panel, mutagenesis screen, and the targeted disruption of specific genes. A comparison of several genomic intervals targeted in regional mutagenesis screens suggests that the piebald region is characterized by a low gene density and high essential gene density with a distinct genomic content and organization that supports complex regulatory interactions and promotes evolutionary stability. genesis 47:392–403, 2009. © 2009 Wiley‐Liss, Inc.