Random and Insertional mutagenesis for functional genomics in plants

Author: Dr. SV Amitha Mithra and Dr. Amolkumar U. Solanke
NRC on Plant Biotechnology, LBS Building, IARI, Pusa Campus, New Delhi

A powerful approach to determine the biological functions of genes is to generate mutants through random (using physical / chemical mutagens) or insertional (transposon /T-DNA based transgenic constructs with reporter genes) mutagenesis and analyze the mutants in comparison to the wild type (WT) for altered phenotypes and biochemical or physiological responses under appropriate environment or screening conditions. In the post- structural genomics era, owing to the availability of structural genomes of some important plant species and availability of techniques such as inverse PCR, TAIL PCR, plasmid rescue, adaptor PCR and genome walking that could amplify regions outside the known sequences use of insertional mutants has become an important tool for functional genomics in plants. Having the flanking sequence information of genes where insertions have occurred in the genome enable cataloguing of the insertions. This serves as one of the invaluable resources for characterizing genes for their biological role. Such insertional mutant resources are readily available for Arabidopsis, rice, maize and tomato. In Arabidopsis alone, more than 20 genes have been functionally characterized using T-DNA insertional mutants.

Random mutagenesis is known since 1927 when HJ Muller, a pioneer in Drosophila genetics, used X-rays to demonstrate that mutations could be induced. Stadler initially used X-rays (1928) and later γ and UV rays to induce mutations in barley. He also initiated a mutation breeding programme which was replicated by researchers across the world to create new mutations and identify useful genotypes. They also characterized the factors affecting the mutagenesis in detail. The first (indirect) mutant cultivar, 'chlorina' was developed in Tobacco by using X rays (Tollenaar 1934-38). The first report of obtaining useful mutations for breeding was powdery mildew resistance in barley using X rays (Frieisleben and Lein, 1944). Ionizing radiations gave rise to more of chromosomal aberrations than gene or point mutations. Use of chemicals for random mutagenesis started only in 1940's with the use of mustard gas by Auerbach in Drosophila (Auerbach and Robson, 1941). However use of random mutagenesis for functional genomics became popular with molecular biologists only after the advent of TILLING (Targeting Induced Local Lesions IN Genomes).

With the advent of Targeting induced local lesions in genomes - TILLING (McCallum et al. 2000), a reverse genetic approach, ethyl methane sulfonate (EMS) based random mutagenesis has gained popularity as it has made high throughput screening for mutations in genes of interest possible. CODDLE (Codons to Optimize Discovery of Deleterious LEsions), a software that helps to identify regions in the gene of interest that are most likely to be mutated with major effects by EMS is freely available (www.proweb.org/coddle/). It works on the principle that EMS creates G to A substitutions at the rate of 1 per 100kb which create either stop codon or splice variants or non-synonymous codons in functional motifs of the gene leading to alteration in gene function, called CODDLE. Owing to these reasons, TILLING has been integrated into many research programs across plant species that are working with EMS based mutant populations.

Till recently, however, the forward genetic approach to identify genes that gave rise to mutant phenotype by EMS treatment had been a tedious one, following the laborious route of developing a biparental mapping population of the mutant with a distant or unrelated genotype and identifying the linked marker, followed by genome walking and cloning of the gene of interest for complementation in the mutant so as to recover the phenotype. The Mutmap and MutMap plus approaches suggested by Abe et al. (2012) and Fekih et al. (2013) respectively have given better and quicker means to do away with the traditional map based cloning and instead use a mutant and WT derived F2 to map the SNP created by EMS in the causal gene for mutation. This latter technique largely depends on the new generation sequencing (NGS) technologies to identify the causal mutation.

Thus random mutagenesis either using only EMS or combining it with some form of physical mutagenesis (using fast neutrons or γ rays) have been developed in rice and tomato, the crop species with reference genomes in the public domain. In rice, IRRI has developed a mutant population in IR64 back- ground using EMS and γ ray mutagens, since they would give point mutations and deletions, respectively. This would be helpful in mapping and assigning function to genes. In India there is a similar effort initiated, wherein EMS mutagenesis is done in Nagina22 background. In tomato the first TILLING population was developed in M82 genetic background (Menda et al. 2004). Later in Microtom and Red Setter background also EMS muatgenesis has been done and populations developed. In India a similar effort has been initiated by Hyderabad University, Hyderabad in Arka Vikas genetic background. All these populations are called TILLING populations referring to their amenability to the reverse genetic approach which makes them more attractive to screen for mutants and their subsequent characterization. With the advent of MutMap and MutMap plus (and similar approaches with transcriptome sequencing) EMS mutagenesis is expected to become more attractive tool for functional genomics.

With cloning, transformation, transgenic technologies, and the availability of reference genomes for rice, Arabidopsis and tomato, insertional mutagenesis (IM) is the favorite approach for functional genomics. However IM is technically more demanding for development of mutant populations than random mutagenesis. Since mutants developed by IM are transgenics, they have to be grown in containment even for experimental purpose. When more number of IM mutants have to be screened it could be difficult to have huge containment facilities. Another important drawback of IM is that it is difficult to saturate the genome with mutation using IM especially T-DNA based IM. Transposon based IM in this regard is more efficient owing to its jumping nature and the jumping could be controlled using appropriate two element systems and selectable markers. However IM cannot create allelic series as they only cause gene disruptions unlike EMS generated mutants. Despite all these shortcomings, what makes IM approach a much sought after one is, its amenability to identify region of insertion (the gene which has been knocked out) in the genome, leading to cataloguing the disruptions based on their physical positions, which serves as a valuable resource for functional genomics globally.

Reference

Abe A, Kosugi S, Yoshida K, Satsume S, Takagi H, Kanzaki H, Matsumura H, Yoshida K, Mitsuoka C, Tamiru M, Innan H, Canon L, Kamoun S and Terauchi R. (2012) Genome sequencing reveals agronomically important loci in rice using MutMap Nat. Biotech. 30, 174-179.

Fekih R, Takagi H, Tamiru M, Abe A, Natsume S, et al. (2013) MutMap+: Genetic Mapping and Mutant Identification without Crossing in Rice. PLoS ONE 8(7): e68529. doi:10.1371/journal.pone.0068529

McCallum, C. M., Comai, L., Greene, E. A., and Henikoff, S. (2000) Targeted screening for induced mutations. Nat. Biotech. 18, 455-457.
Menda N, Semel Y, Peled D, Eshed Y and Zamir D. (2004) In silico screening of a saturated mutation library of tomato. Plant J 38: 861-872

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