Effects of mutagenic agents on the DNA sequence in plants

Background:

Modern plant breeders and farmers can exploit a wealth of natural biodiversity, which may be widely broadened through the application of mutation induction techniques. The impact of induced mutation on crop improvement is reflected in the 2316 officially registered varieties (IAEA's database on officially registered mutant varieties, MVD) carrying novel induced variation. Moreover about three-quarters of these are direct mutant varieties derived from treatment with gamma rays, thus highlighting the importance of physical mutagens. All this translates into a tremendous economic impact on agriculture and food production that is currently valued in billions of dollars and millions of cultivated hectares (Ahloowalia et al. in prep.). However, while the agronomic potential of induced mutation is well understood, the precise effects of different mutagenic agents on the DNA sequence in plants have never been described. Furthermore, in recent years novel reverse genetics and gene discovery technologies have spurred renewed interest in induced mutation. For these new applications it is necessary to understand more clearly the types of mutations generated by the different classes of mutagens, and to measure their frequency and distribution along the genome. Today, and for the first time, the technologies are in place to undertake the experiments necessary to gain this understanding.

Mutagenic agents can be classified into three categories: physical (e.g., gamma rays), chemical (e.g., ethyl methane sulphonate) and transposable elements (such as transposons, retrotransposons, T-DNA, retroviruses). At present, limited data are available on the scope of genetic effects induced at the molecular level in plants and on the specificity and relative efficiency of these different categories of agents. These effects involve DNA damage, which results in base pair changes (single/simple nucleotide polymorphisms, SNPs), small insertions and deletions (indels) and chromosomal rearrangements. Even less is known about how induced mutation interact with epigenetic processes, such as methylation, activation of retro-elements, and perturbation of higher order DNA structure.

While breeders have been using mutation induction to broaden the genetic base of germplasm, and have used the mutant lines directly as new varieties or as sources of new variation in cross-breeding programmes, knowledge of the precise nature of the induced mutations was not necessary. Intuitively a conservative level of small base pair rearrangement and deletion was considered to be ideal. Nowadays, the use of mutation techniques has expanded beyond applications in breeding to gene discovery and reverse genetics. These new high-throughput applications require specific classes of mutations that are induced with high efficiency over entire crop plant genomes, and consequently knowledge of the precise nature of induced mutation is becoming an issue.

High-throughput gene discovery methods depend heavily on insertional 'knockout' lines, the now classical 'gene machines', and deletion 'knockout' libraries. Insertional mutagenesis involves inducing increased activity of transposition of known transposable elements (e.g. retrotransposons which tend to transpose into active genes) to produce series of lines in which, in theory, every gene in the genome will have been inactivated by the transposon insertion. These lines can be used to identify genes that cause particular phenotypes or, conversely, can be used to identify gene function by searching for a phenotype associated with the inactivation of a particular known gene. However, insertional mutants have a tendency to be unstable (i.e. excision of the transposon tag, e.g. Ac/Ds binary system, in the next generation might cause the phenotype to revert to the original parent type, or activation of retrotransposon tags through different stresses might multiply insertion events, e.g. during micropropagation). In comparison to insertional mutagenesis, conventional mutation induction (i.e. using physical or chemical agents) provides the advantage of stable mutations.

In theory, the production of deletion libraries involves inducing moderately large deletions, ideally spanning 1 kb to 100 kb in size, in each of a series of lines. These deletions should encompass segments of every gene in the genetic repertoire and should be represented at least by one line in the deletion library. These deletion lines can, when used together with whole genome gene arrays, be used to identify genes responsible for particular phenotypes or to confirm the association of known genes with particular phenotypes.
A novel and important reverse genetics approach is 'targeting induced local lesions in genomes' (TILLING). Here, large numbers of small changes, either DNA base pair substitutions or small deletions spanning no more than a few base pairs, are induced in a series of lines. In these lines gene function can be ascertained by associating a phenotype with changes in a particular gene and novel alleles of known genes can be generated.

Over the coming years, new technologies such as these will have increasing impact in practical plant breeding. However, they will require different types of mutations induced at specific frequencies. In order to tailor the mutation process, there will be a need to understand how specific classes of mutations are generated and distributed over genomes. In the past, this has not been possible because of lack of analytical tools and an inadequate knowledge of both the process of DNA damage and the architecture of plant genomes. In addition, only a restricted number of plant genes were sequenced. Today, high-throughput DNA sequencing methods coupled with bioinformatics and functional genomic approaches provide extensive knowledge on genome architecture. The complete genomic DNA sequence of a model dicotyledonous plant, Arabidopsis, and a model monocotyledon, rice has become available recently. Also scientists find themselves now with an array of methods, mostly developed as molecular marker technologies that can be adapted to quantify changes in DNA sequence. All in all, the stage is set to transfer the science of DNA damage induced by physical and chemical mutagens from human genetics to plant systems. A range of technologies can now be used to quantify both the underlying base rate, over numbers of generations, of spontaneous mutation and the instantaneous effects of mutation agents. Thus scientists now finally find themselves in a position to undertake experiments that can unravel the sorts of mutations induced by different mutagens so that future users of induced mutation may use the technology in a fully informed manner.

Objectives:

The aim is to understand the mechanism of mutation induction in plants and to quantify the types (base pair changes or deletions), frequencies (rates of change relative to mutagens dose) and patterns (induction of changes in different parts of the genome) of changes in DNA induced by a range of physical and chemical mutagens in a range of key crop plant species. Molecular marker, DNA array, and novel reverse genetic methodologies will be used in a unique approach to analyze and survey the induction of mutations elicited in a number of plants of agronomic importance. These results will be used to provide protocols and guidelines important for future use of induced mutation in plant biology and crop improvement. Knowledge obtained will assist Member States in enhancing crop breeding programmes through the application of targeted induced mutation and complementary genomic approaches with the objective of increasing agricultural sustainability, food security and economic stability. This CRP will exploit new developments in DNA analysis and genomics to define types, frequencies, rates and patterns of mutation induced by the different mutagens. This will generate a knowledge base that will guide and assist future users of induced mutation technologies for crop improvement and genomics. Furthermore, it will focus on physical mutagens, such as gamma and fast neutron or X-ray radiation. Selected chemical mutagens will also be used to compare the relative efficiency of both types of mutagenic agents. The effects of these mutagens will be evaluated on genetically homogeneous seed and vegetatively propagated plant material. Specific major objectives include:

  • Determining mechanisms and total levels of DNA damage at the M1 generation, e.g. directly in treated seed in pre- and post-germination assays.
  • Determining types, frequencies, rates and patterns of mutations in M2 generations, over (a) whole genomes and (b) in targeted DNA sequences within genomes.
  • Determining the type and rate of spontaneous mutation over generations in key crop plant groups (e.g., a select plant system, to determine the spontaneous rate as a baseline and as an inherent indicator of genotype mutagenicity).
  • Preparation of protocols and guidelines for the use of particular mutagens for a range of specific applications in crop improvement and genomics.
  • Determining the chemical and molecular basis for differential radiation-sensitivity in different varieties of the same crop species.
  • Quantifying the type and rate of baseline spontaneous mutation, using multi-generation mutation accumulation experiments.

Participants:

Download pdf]

Project Officer:

P.J.L. Lagoda